Cc2 Compilations

CONTENTS:
UNIT TEST 1
QUIZZES 1-11
Sir M (LEC VID) NOTES
LABORATORY MANUAL
LECTURE NOTES & QUIZZES
MLS 045 LAB UNIT TEST 1
The exam is good for 1 and a half hour (8:00AM to 9:30AM). Corresponding deductions will be
made for those who submit the exams beyond 9:30AM. Those who will not be able to finish the
exam due to unavoidable circumstances, inform your teacher via messenger regarding the matter.
Good luck!
On which organism or sample was the term enzyme literally associated? *

rice
wheat
meat
yeast
Who gave the word “enzymes” to catalytic molecules? *

Pasteur
Kuhne
Buchner
Harden
Which enzymes are used to diagnose liver diseases? *

ACP & ALP
AST & ALT
AMS & LPS
CK & LDH
Kernicterus is an abnormal accumulation of bilirubin in: *

Heart tissue
Brain tissue
Liver tissue
Kidney tissue
A physician suspects his patient has pancreatitis. Which test(s) would be most
indicative of this disease? *
Creatinine
LD isoenzymes
B-hydroxybutyrate
amylase
When myocardial infarction occurs, the first enzyme to become elevated is: *
CK
LD
AST
ALT
Which vitamin is required for the normal absorption of dietary calcium: *

Vitamin
Vitamin B12
Vitamin C
Vitamin D
It has bromide as its common interferent in its analysis *

Iodide
Chromium
Zinc
Magnesium
The metal deficient in hypochromic microcytic anemia that forms a complex with TPTZ
(sulfonated bathophenanthroline 2,4,6- tripyridyl-S-triazine) is: *
copper
magnesium
iron
cobalt
A person suspected of having dual case of alkalosis would have which of the following
laboratory findings? *
CO2 content and PCO2 elevated, pH decreased
CO2 content decreased and pH elevated
CO2 content PCO2, and pH decreased
CO2 content and pH elevated
Which of these is NOT a liver function test: *

Thymol turbidity test
TP A/G ratio & BCG test
hemoglobin electrophoresis
Lipid profile
Respiratory acidosis is described as an a (n): *

Increase in CO2 content and PCO2 with a decreased pH
Decrease in CO2 content with an increased pH
Increase in CO2 content with an increased pH
Decrease in CO2 content and PCO2 with decreased pH
What is a coenzyme? *
organic molecules derived from vitamins
organic molecules containing metals
transport molecules
all of these
In the bilirubin oxidase method, bilirubin is oxidized to a colorless compound which
is: *
diazotized sulfanilic acid
urobilinogen
biliverdin
bilierythrin
An electrophoretic separation of lactate dehydrogenase isoenzymes that demonstrates

an elevation in LD-1 and LD-2 in a “flipped” pattern is consistent with: *
Myocardial infarction
Viral hepatitis
Pancreatitis
Renal failure
In the determination of lactate dehydrogenase at 340nm, using pyruvate as the

substrate, one actually measures the: *
Decrease in pyruvate
Decrease in NADH
Increase in NADH
Increase in coenzyme
Total lactic dehydrogenase (LD) activity, confirmed to fraction 4 and 5 , is most likely
to be associated with: *
Pulmonary infarction
Hemolytic anemia
Acute viral hepatitis
The aldehyde transport coenzyme is derived from: *

Vitamin A
Vitamin C
Niacin
Thiamine
A critically ill patient becomes comatose. The physician believes the coma is due to
hepatic failure. The assay most helpful in this diagnosis is: *
Ammonia
ALT
AST
GCT
Enzymes that differ in structure and origin but same reaction catalyzed are called: *
Holoenzyme
Apoenzyme
Prosthetic group
Isoenzyme
It is one of the best tests for bilirubin: *

Reitman and Frankel
Isaacson – Jensey
Biuret
Jendrassik – Grof
The polarity and water solubility of bile acids are increased upon conjugated with: *
Glycine
Either glycine or taurine
Neither glycine not taurine
Taurine
In early 19the century, which scientist studied fermentation of sugar to alcohol using a
cell-free extract? *
Edward Buchner
Louis Pasteur
Alfred Joseph
Willy Kuhne
Which method uses sulfanilic acid, HCl and sodium nitrite? *

Jaffe
Diazo
Zimmerman
Lowry
Which of the following parameters using ISE does not require a glass membrane
(oxides of Si, Al and Na) ? *
Sodium
Chloride
pH
Calcium
The transport material associated with bilirubin in the liver is? *

alpha 1-globulin
beta-globulin
albumin
ligandin
Which of the following functions as a transport protein for bilirubin in the blood? *
alpha1-globulin
gamma-globulin
beta-globulin
albumin
If the total bilirubin is 4.3 mg/dL and the conjugated bilirubin is 3.1 mg/dL, the
unconjugated bilirubin is: *
A. 20.52 umol/L
B. 58.14 umol/L
C. 37.62 umol/L
D. 34.20 umol/L
A complete catalytically active enzyme together with its bound enzyme or metal ions is
called? *
Complex enzyme
Apoenzyme
Prosthetic group
Zymogen
Select the polypeptide chain combination designated LD 5. *

M4
M2H2
MH3
H4
A scanning of a CK isoenzyme fractionation revealed two peaks: a slow cathodic peak

(CK-MM) and an intermediate peak (CK-MB). A possible interpretation for this pattern is:
Brain tumor
Muscular dystrophy
Viral hepatitis
Which of the following enzymes catalyzes the conversion of starch to glucose and
maltose? *
Malate dehydrogenase (MD)
Amylase (AMS)
Creatine kinase (CK)
Isocitric dehydrogenase (ICD)
The most widely used methods for bilirubin measurement are those based on the : *
Jaffe reaction
Schales and Schales method
8-hydroxyquinoline reation
Jendrassik Grof method
What metal is the most affected by even the slightest hemolysis?

Calcium
Chloride
Potassium
Sodium
The sum of carbonic acid and bicarbonate in plasma is referred to as? *

standard bicarbonate
total carbon dioxide
buffer base
base excess
What substance gives feces its normal color: *

Uroerythrin
Urochrome
Urobilin
Stercobilin
A coenzyme or metal ion that is very tightly or even covalently bound to the
apoenzyme is called? *
Holoenzyme
Prosthetic group
Apoprotein
None of these
In respiratory acidosis, a compensatory mechanism is the increase in: *

respiration rate
ammonia formation
blood PCO2
plasma bicarbonate concentration
Formation of bile acids is the major mechanism for eliminating: *

Phospholipids
Bilirubin
Cholesterol
Triglycerides
One international unit of enzyme activity is the amount of enzyme that, under specified
reaction conditions of substrate concentration, pH, and temperature, causes utilization of
substrate at the rate of: *
mole/min
millimole/min
micromole/min
nanomole/min
The greatest activities of serum AST and ALT are seen in: *
Primary biliary cirrhosis
Metastatic pancreatic carcinoma
Alcoholic cirrhosis
The expected blood gas results for a patient in chronic renal failure would match: *
Metabolic acidosis
Respiratory acidosis
Metabolic alkalosis
Respiratory alkalosis
Which of the following enzymes are used in the diagnosis of acute hepatitis? *
Amylase (AMS) and lipase (LPS)
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
5’-nucleotidase (5’N) and gamma-glutamyl transferase (GGT)
Aspartate aminotransferase (AST) and creatine kinase (CK)
The sensitive enzymatic indicator for intravascular hemolysis and acute myocardial
infarction is: *
alanine aminotransferase (ALT)
aspartate aminotransferase (AST)
Gamma-glutamyl transferase (GGT)
alkaline phosphatase (ALP)
When determining blood pH, CO2 and O2 concentrations, the best sample is: *
Arterialized capillary blood from finger
Arterial blood
Plasma using heparin
Any of these
A complete catalytically active enzyme together with its bound coenzyme is called? *
Holoenzyme
Prosthetic group
Apoenzyme
None of these
Indirect-reacting bilirubin may be quantified by reacting it initially in which reagent? *

dilute HCl
caffeine-benzoate
dilute sulfuric acid
sodium hydroxide
Zinc protoporphyrin or free erythrocyte protoporphyrin measurement are useful to

assess blood concentrations of: *
Lead
Mercury
Arsenic
Beryllium
A breakdown product of hemoglobin is: *

Lipoprotein
Bilirubin
Hematoxylin
Bence Jones protein
In which year James Sumner isolated and crystallized urease? *

1926
1928
1924
1907
What is an inorganic cofactor? *

Metal ions
Both a and b
Vitamin-derived molecules
None of the above
Increased levels of indirect bilirubin only is seen in: *

Bile duct obstruction
Acute quartan malaria
Pancreatic tumor
Gallstones
What was the name given to enzymes by Louis Pasteur that he based from a process
he himself discovered? *
enzyme
ferment
catalyst
ligand
The determination of total amount and ionized amount may be performed in serum
and plasma: *
Chloride
Sodium
Iron
Calcium
Which of the following is a characteristic shared by lactate dehydrogenase, malate

dehydrogenase, isocitrate dehydrogenase and hydroxybutyrate dehydrogenase? *
They are liver enzymes
They are cardiac enzymes.
They catalyze redox reactions
They are class III enzymes.
Post-hepatic jaundice may be due to: *

Gilbert syndrome
Crigler-Najjar syndrome
Viral hepatitis
Rotor syndrome
In the Jendrassik – Grof method for the determination of serum bilirubin concentration,
quantitation is obtained by measuring the green color of: *
azobilirubin
bilirubin glucuronide
diazobilirubin
urobilinogen
An emphysema patient suffering from fluid accumulation in the alveolar spaces is

likely to be in what state? *
Metabolic acidosis
Metabolic alkalosis
What is the immediate precursor of biliverdin? *

heme
bilirubin
urobilinogen
senescent erythrocytes
Which of the following serum constituents is greatly affected if a blood specimen is left
standing at room temperature for 8 hours before processing? *
Glucose
TAG
Partial pressure of Oxygen
Bilirubin
In ketoacidosis, the blood pH would most likely be affected in what way? *

unchanged from normal
increased
decreased
balanced
At blood pH 7.40 what is the ratio between bicarbonate and carbonic acid? *
15:1
25:1
20:1
30:1
The protein part of holoenzyme is called? *

Apoprotein
Apoenzyme
Coenzyme
Prosthetic group
The most heat labile fraction of alkaline phosphatase is obtained from: *

Liver
Bone
Intestine
Placenta
Type I glycogen storage disease is due to a deficiency in glucose-6-phoshatase in the

liver. This condition is called: *
Gaucher’s disease
von Gierke’s disease
Hers disease
Pompe’s disease
Which fraction is expected to be elevated in alcoholic cirrhosis of the liver? *

ALP
GGT
LDH
ACP
In bilirubin determinations, the purpose of adding a concentrated caffeine solution or

methyl alcohol is to: *
Measure total bilirubin
Dissolve conjugated bilirubin
Precipitate protein
Allow mixing of B1 and B2
Which suffix is added to the name of the substrate or to a word or to a phrase

describing the activity of enzyme, to name an enzyme? *
-Ise
-Ase
–Ic
-Ace
Which of the following enzyme pairs cannot be used in the diagnosis of liver
disorders? *
ALP & LAP
GGT & 5’-NT
LDH & AST
ACP & ALS
Elevated blood levels of ammonia occur in all of the following disorders EXCEPT: *
Reye’s syndrome
renal failure
hepatic cirrhosis
diabetes mellitus
Valinomycin enhances the selectivity of the electrode used to quantitate: *

Sodium
Chloride
Potassium
Calcium
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both

elevated in which of the following disease? *
muscular dystrophy
viral hepatitis
diazobilirubin
urobilinogen
Metabolic alkalosis is described as a (n): *

Increase in CO2 with a decreased pH
Decrease in CO2 content and PCO2 with a decreased pH
Which enzyme CANNOT be used to detect acute myocardial infarction (AMI)? *

ACP
CK
AST
LDH
The most abundant cation in the human body is calcium followed by: *
Sodium
Iron
Magnesium
Potassium
Which of the following tests has NO clinical application in assessing liver function? *
Triglyceride
Amylase
Gamma-glutamyl transferase
Lactate dehydrogenase isoenzymes
The term delta bilirubin refers to: *

Water-soluble bilirubin
Free unconjugated bilirubin
Bilirubin tightly bound to albumin
Direct-reacting bilirubin
If the total bilibirubin is 3.1 mg/dL and the conjugated bilirubin is 2.0 mg/dL.,
unconjugated bilirubin is *
0.5 mg/dL
1.1 mg/dL
2.2 mg/dL
5.1 mg/dL
What is an enzyme cofactor? *

Inorganic ions
Organic molecules
Organic molecules containing metals
All of the choices
Oxidation of urobilinogen by microorganisms will produce: *

porphobilinogen
stercobilinogen
urobilin
protoporphyrin
Which enzyme ratio is the best indicator of acute or chronic hepatitis: *

AST/ALT
CK-MB/total CK ratio
Amylase/lipase ratio
LD1/LD5 ratio
One of the following is used to assess liver function. Which one is it? *
Creatinine Clearance Test
Inulin Clearance test
Para-aminohippurate test
Hippuric Acid Test
What enzyme system is responsible in the oxidation of bilirubin? *

leucine aminopeptidase
bacterial oxidases
glucose-6-phosphate dehydrogenase
carbamoyl phosphate synthetase
The buffering capacity of blood is maintained by a reversible exchange process

between bicarbonate and: *
Sodium
Potassium
Calcium
Chloride
Absorption of vitamin B12 requires the presence of: *

intrinsic factor
gastrin
secretin
folic acid
Which two physiological conditions are characterized by elevated serum alkaline

phosphatase? *
rickets, hyperthyroidism
obstructive jaundice, biliary cirrhosis
growth, third trimester of pregnancy
viral hepatitis, myocardial infarction
Which of the following enzymes are used in the diagnosis of pancreatic

inflammation? *
Aspartate aminotransferase (AST) and leucine aminopeptidase (LAP)
5’-nucleotidase (5’-NT) and gamma-glutamyl transferase (GGT)
alanine aminotransferase (ALT) and lactate dehydrogenase (LD)
Which reagent would not dissolve indirect bilirubin? *

dimethylsulfoxide (DMSO)
methanoic acid
caffeine-benzoate
cetrimide
In which of the following disease states is unconjugated bilirubin NOT a major serum
component? *
Gallstones
HDN
Neonatal jaundice
Erythroblastosis fetalis
The counterion in the chloride shift phenomenon is: *

Sodium
Bicarbonate
Carbon dioxide
Phosphate
Which ion is affected by the use of the chelating agent sequestrene?

Calcium
Iodine
Bicarbonate
Bromide
The cofactor needed in the catalyzed reaction by hexokinase is: *

manganese
copper
calcium
magnesium
Most automated blood gas analyzers directly measure: *

pH and % O2 saturation
pH, PCO2, and PO2
HCO3, PCO2, and PO2
pH, PO2and % O2 saturation
Acetaminophen is particularly toxic to the: *

Liver
Kidney
Heart
Spleen
All of the following methods have been used for the measurement of of serum bilirubin
EXCEPT: *
Bilirubinometer
BSP excretion
Jendrassik-Grof
Bilirubin oxidase
Which of the following is not a function of the liver? *

25-hydroxylation of vitamin D
production of bile acids
storage of iron
production of hippurate and bile
Which of the following is a glycolytic enzyme that catalyzes the cleavage of fructose-1, 6-
diphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate? *
Aldolase
Phosphofructokinase
Pyruvate kinase
Glucose-6-phosphate dehydrogenase
All enzymes are made up of which biomolecules (or, biomolecules)? *

Proteins
RNA
Both protein & RNA
Neither protein nor RNA
Increased total bilirubin due to increased direct bilirubin suggests: *

hemolytic jaundice
neonatal jaundice
obstructive jaundice
The specific protein for the transport of copper present in plasma is: *
Transferrin
Ceruloplasmin
Albumin
Immunoglobulin
Which of the following chemical determinations may be of help in establishing the

presence of seminal fluid? *
Lactic dehydrogenase (LD)
Isocitrate dehydrogenase (ICD)
Acid phosphatase
Alkaline phosphatase
All of the following describe the direct bilirubin EXCEPT: *

insoluble in water
conjugated with glucuronic acid
conjugated in the liver
excreted in jaundiced patients’ urine
In the bloodstream, bilirubin occurs as: *

Delta- bilirubin
Oxidized bilirubin
Bilirubin-glucuronate complex
Beta - bilirubin
This ion can bind calmagite, methylthymol blue and xylidyl blue: *
Manganese
Magnesium
Calcium
Iron
Mesobilirubinogen, urobilinogen and stercobilinogen are collectively known as: *

urobilinogen
bilirubin products
urobilins
mesobilirubins
A serum sample was assayed for bilirubin at 10AM and the result was 12 mg/dL. The
same sample was retested at 3PM. The result now is 8 mg/dL/. The most likely
explanation for this discrepancy is:
The reagent has deteriorated
The sample was exposed to light
A calculation error in the first assay
The sample was not refrigerated
The principle of the tablet test for bilirubin in urine or feces is: *
the reaction between bile and 2,4- dichloronitrobenzene to a yellow color the
liberation of oxygen by bile to oxidize orthotolidine to a blue-purple color
chemical coupling of bilirubin with a diazonium salt to form a purple color
chemical coupling of bile with a diazonium salt to form a brown color
The bicarbonate and carbonic acid ratio is calculated from an equation by: *
Siggaard-Andersen
Gibbs – Donnan
Natelson
Henderson – Hasselbalch
In which year Edward Buchner discovered that yeast extract can cause the
fermentation of sugar to alcohol? *
1888
1896
1900
1907
Reduction of bilirubin occurs in the: *

Colon
Liver
Spleen
Small intestine
Severe vomiting and loss of HCl causes: *

Metabolic acidosis
Metabolic alkalosis
Which pair has clinical utility for AMI detection? *

ALP & LAP
GGT & 5’-NT
LDH & AST
ACP & ALS
Which analyte CANNOT be measured using AAS? *

iodide
calcium
manganese
iron
The bicarbonate ion concentration represents _______ of the Total CO2. *

50%
75%
95%
99%
ALP & LAP concentrations are useful to assess: *
Diabetes mellitus
Hepatobiliary disease
Intestinal malabsorption
Kidney function
MLS 045 LAB Quiz 1

I. DIFFERENTIATION OF BILIRUBIN TESTS: Compare and contrast the four (4) tests for bilirubin by
filling in the table below with the necessary and correct information:
Points of Icterus Index Original Jendrassik & Grof Modified Evelyn &
Differentiation Evelyn-Malloy Method Malloy Method
Method
Reaction The intensity of the Total bilirubin is Bilirubin is formed from Sulfanilic acid reacts
Principle golden yellow color measured by the heme portion of with sodium nitrite
imparted to the adding hemoglobin released to form diazotized
serum by bilirubin is methanol which by aged or damaged sulfanilic acid. In the
measured by permits red blood cells. It is presence of
comparing the reactions of then converted in the accelerator
serum color with indirect (and liver into bilirubin cetrimide,
the color of direct) bilirubin monoglucuronide and conjugated and
bilirubin diglucuronide.
standard solution of with Ehrlich’s unconjugated
Free bilirubin is not
dichromate diazo reagent bilirubin react with
soluble in aqueous
standard.1 unit of and water. diazotized sulfanilic
solution and requires
icterus index has Indirect solubilization by acid to form
been defined as the bilirubin is alcohols or other azobilirubin (total
color of 1:10,000 estimated by solvents to react. bilirubin). In the
solution to subtracting the Reactions carried out absence of
potassium direct bilirubin in these solvents cetrimide, only
dichromate. value from the provide measurements conjugated bilirubin
total level. of total bilirubin. Mono- reacts (direct
ACCORDING TO and diglucuronides of bilirubin).
SIR: NO REACTION bilirubin are soluble in
PRINCIPLE. has test water and
principle but it has measurements
nothing to do with performed in aqueous
the reaction solution measure what
in this form is called
because no reaction
direct bilirubin.
happened. Icterus
In Dialab kit, DMSO and
index is just to
ethylene glycol are
measure or find out solvents for total
how YELLOW the bilirubin assay. Bilirubin
sample is. in these solvents readily
react with diazotized
sulfanilic acid to
produce an intensely
colored diazo dye. The
intensity of color of this
dye in solution is
proportional to the
concentration of direct
or total bilirubin.
Product NONE. no product Total bilirubin Total bilirubin Total bilirubin
Measured measured because Direct bilirubin Direct bilirubin Direct bilirubin
the product is a
result of a chemical
reaction
Color Endpoint Golden yellow Red to reddish- Blue Pink to purple
purple
According to sir: According to sir: blue or

purple-pink green ( but if you base it
on the wavelength, it is
more of blue-violet
Wavelength 415 nm 540 nm 555 nm 550 nm
Used
Light Path 1 cm 1 cm 1 cm 1 cm
Incubation Room temperature 37°C 20-25°C, 37°C 37°C

Temperature
According to sir:
Icterus index
doesn’t need an
incubation because
it is only dilution so
it uses just room
temperature.
Buffer Used Phosphate buffer Alkaline buffer Sodium acetate Cetrimide
solution
Purpose of To maintain the pH, To neutralize Shifts the absorbance Conjugated and
Buffer Used thus neutralizing it spectrum of the unconjugated
azobilirubin to a more bilirubin reacts with
intense blue color that is diazotizedsulfanilic
less subject to acid to form
interfering substances azobilirubin (total
in the sample. bilirubin)
Chromogen Potassium Diazo reagent Diazotinized sufanilic Diazotinized
dichromate acid sulfanilic acid
Composition Serum Dilute Serum/plasma Distilled water
of Blank hydrochloride
acid
Type of Blank Sample blank Reagent blank Sample blank Water blank
Used
Conversion 17.1 17.1 17.1 17.1
Factor
Reagent for Potassium • 2.0 mL of • Sulfanilic acid • Sulfanilic acid
Total Bilirubin dichromate serum • Hydrochloric
Testing (diluted • Ethylene glycol acid
1:10) • Cetrimide
• 3.0mL of • Dimethylsulfoxide
methyl (DMSO)
alcohol
• 0.5 mL of
diazo
blank
• 0.5 mL of
diazo
reagent
Reagent for Potassium • 2mL of Sulfanilic acid • Sulfanilic acid
Direct dichromate serum • Hydrochloric
Bilirubin (1:10) acid
Testing • 3.0 mL of
water
• 0.5 mL of
diazo
blank
• 0.5 mL of
diazo
reagent
Reaction pH 7.4 1.2 10 1.2
Samples serum serum Serum or plasma • Unhemolyzed

Required serum or
heparinized
plasma
• Protected
from light,
samples are
stable for 2
days at RT abd
4 days at 4°C
& even longer
if frozen
Linear Range 0.3 -20 mg/dL 25 mg/dL (0.428 Up to 20 mg/dL Total 0.3- 20 mg/Dl total
mmol/L) bilirubin bilirubin
Reference 0.2-1.0 mg/dL 0.2-1.0 mg/dL 0.2 – 1.0 mg/dL 0.3-1.2 mg/Dl
Range fot (<3.4 uM)
Total Bilirubin
in
Conventional
Unit
Reference 0.0-0.2 mg/dL 0.0-0.2 mg/dL 0.0 – 0.2 mg/dL <0.2 mg/Dl
Range for (5-21 uM)
Direct
Bilirubin in
Conventional
Unit
Reference 0.02-0.8 mg/dL 0.02-0.8 mg/dL 0.2 – 0.8 mg/dL 0.02-0.8 mg/dL
Range for
Indirect
Bilirubin in
Conventional
Unit
Reference 3.42-17.1 µmol/L 3.42-17.1 3.42-17.1 µmol/L 5-21µm
Range for µmol/L
Total Bilirubin
in SI unit
Reference 0.0-3.42 µmol/L 0.0-3.42 µmol/L 0.0-3.42 µmol/L <3.4 µm
Range for
Direct
Bilirubin in SI
unit
Reference 3.42-13.68 µmol/L 3.42-13.68 3.42-13.68 µmol/L 3.42-13.68 µmol/L
Range for µmol/L
Indirect
Bilirubin in SI
unit
Associated Hyperbilirubienmia Gilbert disease Acute or chronic Acute or chronic
Diseases hemolytic anemias hemolytic anemias
Prehepatic jaundice Cringler-Najjar
Syndrome Gilbert Syndrome Gilbert Syndrome
Hepatic jaundice
Acute hepatitis Hepatitis Cirrhosis
Posthepatic
jaundice Hemolytic Cirrhosis Rotor Syndrome
anemias
Rotor Syndrome
Rotor’s
Syndrome
Dubin-Johnson
Syndrome
Viral hepatitis
Cite 3 Testing - The serum used - Reagents - Ethelyne glycol is - Reagent 1 of
Precautions for the icterus index should be harmful if inhaled or in bilirubin total
determination is added exactly in conctact with skin and contains irritant that
absolutely free of the proportions eyes. In case of eye is harmful if inhaled,
visible hemolysis & and orders contact, rinse swallowed or in
chyle listed. thoroughly with plenty contact with skin
- The patient - Deviations of water and seek and eyes.
shouldn’t eat food from the medical advice. - In case of eye
items which procedure may - Bilirubin reagents contact, rinse
containtellow result in protein should not be used if immediately with
pigment for 14-48 precipitation, precipitation forms. plenty of water and
hrs prior to the which may - Avoid exposure of seek medical advice.
drawing of blood invalidate the specimen to light. - Sulfanilic acid may
for the test. results. produce allergenc
- Exposure of - Sera with reactions.
specimen to light bilirubin higher
should be avoided than 50mg%
must be diluted
for accurate
results to be
obtained.
II. DISCUSSION: Explain the following correctly. Explanation should not exceed the lines provided.
1. What is ∆Absorbance? How is it obtained?
Absorbance (O.D) is the term used to describe the monochromate light that is absorbed by the sample.
Delta absorbance is used in conjunction with the calibration data to calculate concentration or activity.
It is obtained by subtracting secondary wavelength from primary wavelength reading.
2. What are the bilirubin products formed upon exposure of bilirubin to light?
The bilirubin products formed upon light exposure are Z, E Bilirubin, E, Z-bilirubin and E, Z-lumirubin.
3. What are the causes of pre-hepatic jaundice? Hepatic jaundice? Post-hepatic jaundice? Cite at least 3
disorders for each type of jaundice.
Pre-hepatic: Increased amount of bilirubin presented to the liver. Disorders: acute hemolytic anemias,
chronic HA, neonatal physiologic jaundice. Hepatic jaundice:Impaired cellular uptake,defective
conjugation or abnormal secretion of bilirubin by the liver cell. Disorders:Gilbert’s,Dubin-Johnson and
Criggler-Najjar Syndrome. Post-Hepatic: impaired excretion due to mechanical obstruction to bile flow.
Disorders: Cholelithiasis, Biliary atresia, Cholangiocarcinoma.
4. What is the role of sodium nitrite?

The role of sodium nitrite is to form a diazonium salt. It is used for conversion of amines into diazo
compounds, a process called “diazotization”. It reacts with sulfanilic acid to form diazotized sulfanilic
acid.
5. Why is total bilirubin determination not enough in that there a need to determine the direct bilirubin &
indirect bilirubin fractions separately?
There is a need to determine the direct bilirubin & indirect bilirubin fractions separately in order to
determine the specific cause of hyperbilirubinemia and identifying the disease as well. Increased direct
bilirubin is a biochemical marker of intrahepatic disruption, bile duct disease and extrahepatic bile duct
obstruction. On the other hand, increased indirect bilirubin is a marker of hemolysis, red cell
degradation and defective hepatocellular uptake for conjugation.
Quiz 1
Colorimetric
Icterus index- no reaction principle, just test principle (only comparison). Comparing the two serum
samples. No chemical reaction.
Evelyn- acidic, pink to purple, 500-550
Jendrassik-alkaline, blue or green, blue & violet (based on wv), >550 Original evelyn:
Modified evelyn: cetrimide
Icterus index- no product
II:
Phosphate buffer- 7.4 (blood) as diluent
1:10
Dilution factor (multiplied to result)
Cuvet, analytical, incubation cell- 1cm or 10mm Incubation temp- 37C, Icterus has none (room temp)
B1-first formed
B2-liver-conjugated, direct
Ph- II- 7.4, others (refer to the manual)
Linear range- what the smallest concentration and highest concentration the test can measure. It can be
lower and higher than the normal value.
No remedy if below. Dilute if above.
Total bilirubin- almost 20uL

Reference ranges- vary but are close to each other among different methods. New methods must be
comparable to the reference method to be available to the public.
0 umol/L- only normal in direct

B1- gives yellow color to serum
B1- prehepatic
B2- hepatic or posthepatic
Delta absorbance- change of absorbance, difference between standard and blank. If zero
absorbance, subtracting is not needed.
Bilirubin products: convert to less harmful & easily excreted so that membranes cannot be penetrated.
Destruction of the rings will result to products with numbers. (check the answers of the ovaries group)
Sodium nitrite: diazotization. Acid will become a salt. Nitrite (2), Azo is nitrogen. Two NO2 will attach to
sulfanilic acid. Azo group (NO2).
Product: diazotized sulfanilic acid
Chromogen: diazotized sulfanilic acid
Hepatobilary- GGT, ALP, 5NT, LAP

ACP- prostate, RBCs
Hepatocytes-parenchymal cells
Class 1: REDO, LDH, SDH
Class 2: Trans or Kinase, AST, ALP, OCT, GGT
Class 3: Phosphatases, ALP, ACP, 5NT, LAP
Paranitrophenylphosphate
ALP will remove 1 phosphate from PNPP = PNP
PNP= no color
PNP= 410/405, yellow, paranitrophenol Vitamin 6- Pyridoxal phosphate- coenzyme of transaminases
Substrates: orthophosphate or monophosphate
(compounds containing 1 phosphate)
Kinetic assay: measuring the concentration as a function of time. Delta OD/Delta A per minute
(parameter)
Old name: transaminase (SGPT & SGOT)

New name: (amino)transferase
ALT- LDH (indicator enzyme), glycolytic

(Embden-meyerhof pathway)
Pyruvate to lactase
AST- Malate dehydrogenase (indicator enzyme),
Kreb’s cycle
AST: RBCs, Liver, Heart

ALT: Primary in liver, more sensitive hepatic marker
De Ritis ratio: AST/ALT
AST is high = >1 ratio, more serious
ALT > AST = liver disease
>1
Viral hepatitis
ALP vs AST: difference is in the location in hepatocytes

ALP: cytosol, cytoplasm
AST: cytosol (CAST), mitochondria (most, MAST)
Conventional
IU = umol/minute/L
SI
Katal = mol/sec/L
Conversion factor: 0.1667 to get the microkatal

per liter
II. DISCUSSION: Explain the following correctly. Explanation should not exceed the lines
provided.
1. What is ∆Absorbance? How is it obtained?
Delta absorbance is the difference in absorbance obtained when there is a

change with the rate of absorbance. It is used in conjunction with the calibration data to
calculate concentration or activity. It is obtained by subtracting secondary wavelength
absorbance reading from primary wavelength reading.
The bilirubin products formed upon light exposure are Z,E-bilirubin, E,Zbilirubin,
and E,Z-lumirubin.
3. What are the causes of pre-hepatic jaundice? Hepatic jaundice? Post-hepatic jaundice?
Cite at least 3 disorders for each type of jaundice.
Prehepatic Jaundice (Hemolytic Jaundice) occurs when the problem causing the
jaundice occurs prior to liver metabolism. It is most commonly caused by an increased
amount of bilirubin being presented to the liver. It can be caused by acute hemolytic
anemias, neonatal physiologic jaundice, chronic hemolytic anemias. Hepatic Jaundice
occurs when the primary problem causing the jaundice resides in the liver (intrinsic liver
defect or disease). Some diseases associated with hepatic jaundice are Criggler-Najar
Syndrome, Gilbert’s disease, and Viral Hepatitis. Posthepatic Jaundice are caused by
physical obstruction of the bile pathway and causes an increased in conjugated bilirubin.
Some examples of the diseases associated with post-hepatic jaundice are Cholelithiasis,
Biliariay cirrhosis and Cholangiocarcinoma.
4. What is the role of sodium nitrite?

It is a reagent for conversion of amines into diazo compounds; or formation of the
diazonium salt, a process called “diazotization”. It reacts with Sulfanilic acid to form
diazotized sulfanilic acid.
5. Why is total bilirubin determination not enough in that there a need to determine the
direct bilirubin & indirect bilirubin fractions separately?
Total bilirubin level is not enough to know the condition of the patient,
knowing the values of direct or indirect biluribin can make diagnosis easier and
more specific than having to consider various clinical diseases. Obtaining
information about direct and indirect bilirubin can also give us information of
where the defect is located, whether it is prehepatic, hepatic or post-hepatic type
of disease.
MLS 045 LAB Quiz 2

Choose the enzymes used to diagnose parenchymal liver disorders. * - can be diagnosed by the
enzymes produced by the hepatocytes
AST & ALT
AST only
ALT only
ALP & ACP
ALP only
ACP only
GGT
5'-NT
LDH
SDH
OCT
De Ritis ratio
LAP
Choose the enzymes used to diagnose hepatobiliary diseases. *
AST & ALT
AST only
ALT only
ALP & ACP
ALP only
ACP only
GGT
5'-NT
LDH
SDH
OCT
De Ritis ratio
LAP
NOTE: ACP is for prostate or rbc disorders but all the rest aside sa 4 na hepatobiliary are parenchymal
Choose among these hepatic enzymes that belong to Class 3 enzymes. * Phosphatases
AST & ALT
AST only
ALT only
ALP & ACP
ALP only
ACP only
GGT
5'-NT
LDH
SDH
OCT
De Ritis ratio
LAP ( Leucine Amino Peptidase)
Choose among these hepatic enzymes that belong to Class 1 enzymes. * redox enzymes(
oxidoreductases)
AST & ALT
AST only
ALT only
ALP & ACP
ALP only
ACP only
GGT
5'-NT
LDH
SDH
OCT
De Ritis ratio
LAP
Choose among these hepatic enzymes that belong to Class 2 enzymes. * Those with trans or kinase
AST & ALT
AST only
ALT only
ALP & ACP
ALP only
ACP only
GGT ( Gamma Glutamyl Transferase or Transpeptidase)
5'-NT
LDH
SDH
OCT ( Ornithine Carbamoil Transferase)
De Ritis ratio
LAP
What coenzyme is required in reactions involving transaminases? * coenzymes are derived from
vitamins; P5P is derived from vitamin B6
pyridoxal-phosphate (PLP) or P5P
If the chromogen used for ALP assay is PNPP, what 1) wavelength, 2) product measured, and 3)
product color are expected? * PNPP( PARANITROPHENYL PHOSPHATE)
Alkaline phosphatase will cleave PNPP to produce PNP and P . So alkaline phosphatase will remove
1 phosphate
MLS 045 LAB Quiz 3
Heart tissue
Brain tissue
Liver tissue
Kidney tissue
All of the following methods have been used for the measurement of of serum bilirubin
EXCEPT: *
Bilirubinometer
BSP excretion
Jendrassik-Grof
Bilirubin oxidase

Arterial blood
Any of these
Metabolic acidosis
Metabolic alkalosis
insoluble in water
The buffering capacity of blood is maintained by a reversible exchange process between

bicarbonate and: *
Sodium
Potassium
Calcium
Chloride

Holoenzyme
Apoenzyme
Prosthetic group
Isoenzyme
pH, PCO2, and PO2
HCO3, PCO2, and PO2
alpha 1-globulin
beta-globulin
albumin
ligandin
M4
M2H2
MH3
H4
RATIONALE: Two different polypeptide chains, designated H (heart) and M
(muscle), combined in five arrangements to yield the five major isoenzymes each
under separate genetic control (Mackawa & Kanno, 1989). The five major fractions
each comprise four subunits, which are LD-1 (H4), LD-2 (H3M), LD-3 (H2M2), LD-
4 (HM3) and LD-5 (M4). LD-6 had been identified in the sera of severely ill patients
its identify remain uncertain (Moss & Henderson 1999)

AST/ALT – de ritis ratio

LD1/LD5 ratio
In the bilirubin oxidase method, bilirubin is oxidized to a colorless compound which is: *

urobilinogen
biliverdin
bilierythrin
What is an enzyme cofactor?

Inorganic ions
Organic molecules
All of the choices
One international unit of enzyme activity is the amount of enzyme that, under specified
reaction conditions of substrate concentration, pH, and temperature, causes utilization of
substrate at the rate of: *
1 mole/min
millimole/min
1 micromole/min
1 nanomole/min
Which two physiological conditions are characterized by elevated serum alkaline

phosphatase? *
Transferrin
Ceruloplasmin
Albumin
Immunoglobulin
A physician suspects his patient has pancreatitis. Which test(s) would be most indicative of
this disease? *
Creatinine
LD isoenzymes
B-hydroxybutyrate
amylase
A person suspected of having dual case of alkalosis would have which of the following
laboratory findings? *


Uroerythrin
Urochrome
Urobilin
Stercobilin
Which of the following enzymes catalyzes the conversion of starch to glucose and maltose? *

Amylase (AMS)
The determination of total amount and ionized amount may be performed in serum and
plasma: *
Chloride
Sodium
Iron
Calcium
ACP
CK
AST
LDH
Lipoprotein
Bilirubin
Hematoxylin
Bence Jones protein
Vitamin
Vitamin B12
Vitamin C
Vitamin D

respiration rate
ammonia formation
blood PCO2

Colon
Liver
Spleen
Small intestine - wrong
urobilinogen
bilirubin products
urobilins
mesobilirubins

transport molecules
all of these
If the total bilirubin is 4.3 mg/dL and the conjugated bilirubin is 3.1 mg/dL, the unconjugated
bilirubin is: *
4.3-3.1 = ANSWER x 17.1
A. 20.52 umol/L
B. 58.14 umol/L mg/dl conversion factor is answer x 17.1
C. 37.62 umol/L
D. 34.20 umol/L
Which of the following is a characteristic shared by lactate dehydrogenase, malate

dehydrogenase, isocitrate dehydrogenase and hydroxybutyrate dehydrogenase? *

Jaffe reaction
In the Jendrassik – Grof method for the determination of serum bilirubin concentration,
quantitation is obtained by measuring the green color of: *
azobilirubin
diazobilirubin
urobilinogen
Which of the following enzyme pairs cannot be used in the diagnosis of liver disorders? *
ALP & LAP (leucine aminopeptidase)

GGT & 5’-NT
LDH & AST
ACP & ALS ( acetolactate synthase)

Lipid profile
Zinc protoporphyrin or free erythrocyte protoporphyrin measurement are useful to assess

blood concentrations of: *
Lead
Mercury
Arsenic
Beryllium

ACP & ALP

AST & ALT
AMS & LPS
CK & LDH
Sodium
Iron
Magnesium
Potassium
alpha1-globulin
gamma-globulin
beta-globulin
albumin

Liver
Bone
Intestine
Placenta
Labile- unstable to heat
Creatinine Clearance Tes – GFR( glomerular filtration rate)

Hippuric Acid Test
CK
LD
AST
ALT
What was the name given to enzymes by Louis Pasteur that he based from a process he
himself discovered? *
enzyme
ferment
catalyst
ligand
Pasteur
Kuhne
Buchner
Harden
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both elevated in
which of the following disease? *
muscular dystrophy
viral hepatitis
diazobilirubin
urobilinogen
Gilbert syndrome
Viral hepatitis
Rotor syndrome
In which year Edward Buchner discovered that yeast extract can cause the fermentation of
sugar to alcohol? *
1888
1896
1900
1907
Delta- bilirubin
Oxidized bilirubin
Beta - bilirubin
If the total bilibirubin is 3.1 mg/dL and the conjugated bilirubin is 2.0 mg/dL., unconjugated
bilirubin is *
0.5 mg/dL
1.1 mg/dL
2.2 mg/dL
5.1 mg/dL

Same with DNA polymerase – it needs magnesium
manganese
copper
calcium
magnesium
Which of the following chemical determinations may be of help in establishing the presence
of seminal fluid? *

Acid phosphatase
Sodium
Bicarbonate
Carbon dioxide
Phosphate
Reye’s syndrome
renal failure
hepatic cirrhosis
diabetes mellitus
intrinsic factor
gastrin
secretin
folic acid
Vitamin A
Vitamin C
Niacin
Thiamine
Metal ions
Both a and b
None of the above
What metal is the most affected by even the slightest hemolysis? *
Calcium
Chloride
Potassium
Sodium
dilute HCl
caffeine-benzoate
sodium hydroxide
hemolytic jaundice
neonatal jaundice
Total lactic dehydrogenase (LD) activity, confirmed to fraction 4 and 5 , is most likely to be
associated with: *
Hemolytic anemia
Which of the following serum constituents is greatly affected if a blood specimen is left
standing at room temperature for 8 hours before processing? *
Glucose – wrong
TAG
Bilirubin
Note: without partial pressure of oxygen, use bilirubin
Metabolic acidosis
Metabolic alkalosis
Reitman and Frankel

Isaacson – Jensey
Biuret
Jendrassik – Grof
Holoenzyme
Prosthetic group
Apoenzyme
None of these
A scanning of a CK isoenzyme fractionation revealed two peaks: a slow cathodic peak (CK-
MM) and an intermediate peak (CK-MB). A possible interpretation for this pattern is: *
Brain tumor
Muscular dystrophy
Viral hepatitis
Jaffe
Diazo
Zimmerman
Lowry
In early 19the century, which scientist studied fermentation of sugar to alcohol using a cell-
free extract? *
Edward Buchner
Louis Pasteur
Alfred Joseph
Willy Kuhne
1926
1928
1924
1907
ALP & LAP

GGT & 5’-NT
LDH & AST
ACP & ALS
A critically ill patient becomes comatose. The physician believes the coma is due to hepatic
failure. The assay most helpful in this diagnosis is: *
Ammonia
ALT
AST
GCT
Phospholipids
Bilirubin
Cholesterol
Triglycerides
In which of the following disease states is unconjugated bilirubin NOT a major serum
component? *
Gallstones
HDN
Neonatal jaundice
Which suffix is added to the name of the substrate or to a word or to a phrase describing the
activity of enzyme, to name an enzyme? *
-Ise
-Ase
–Ic
-Ace
porphobilinogen
stercobilinogen
urobilin
protoporphyrin
Manganese
Magnesium
Calcium
Iron
bacterial oxidases
In the determination of lactate dehydrogenase at 340nm, using pyruvate as the substrate,
one actually measures the: *
Decrease in NADH
Increase in NADH
Apoprotein
Apoenzyme
Coenzyme
Prosthetic group
heme
bilirubin
urobilinogen
the reaction between bile and 2,4- dichloronitrobenzene to a yellow color

the liberation of oxygen by bile to oxidize orthotolidine to a blue -purple color
iodide
calcium
manganese
iron
storage of iron

Alcoholic cirrhosis

Pancreatic tumor
Gallstones
Which of the following enzymes are used in the diagnosis of pancreatic inflammation? *

methanoic acid
caffeine-benzoate
cetrimide
Type I glycogen storage disease is due to a deficiency in glucose-6-phoshatase in the liver.

This condition is called: *
Gaucher’s disease
Hers disease
Pompe’s disease
In bilirubin determinations, the purpose of adding a concentrated caffeine solution or methyl
alcohol is to: *

Precipitate protein
The metal deficient in hypochromic microcytic anemia that forms a complex with TPTZ
(sulfonated bathophenanthroline 2,4,6- tripyridyl-S-triazine) is: *
copper
magnesium
iron
cobalt

buffer base
base excess
Which of the following parameters using ISE does not require a glass membrane (oxides of
Si, Al and Na) ? *
Sodium
Chloride
pH
Calcium
An emphysema patient suffering from fluid accumulation in the alveolar spaces is likely to be
in what state? *
Metabolic acidosis
Metabolic alkalosis
Proteins
RNA
Both protein & RNA
Diabetes mellitus
Kidney function
Sodium
Chloride
Potassium
Calcium
An electrophoretic separation of lactate dehydrogenase isoenzymes that demonstrates an
elevation in LD-1 and LD-2 in a “flipped” pattern is consistent with: *
Viral hepatitis
Pancreatitis
Renal failure
A serum sample was assayed for bilirubin at 10AM and the result was 12 mg/dL. The same
sample was retested at 3PM. The result now is 8 mg/dL/. The most likely explanation for this
discrepancy is: *

Siggaard-Andersen
Gibbs – Donnan
Natelson
Acetaminophen is particularly toxic to the:
Liver
Kidney
Heart
Spleen
Which ion is affected by the use of the chelating agent sequestrene? *
Calcium
Iodine
Bicarbonate
Bromide
A coenzyme or metal ion that is very tightly or even covalently bound to the apoenzyme is
called? *
Holoenzyme
Prosthetic group
Apoprotein
None of these
Aldolase
Phosphofructokinase
Pyruvate kinase
Glycine
Taurine
50%
75%
95%
99%
A complete catalytically active enzyme together with its bound enzyme or metal ions is
called? *
Complex enzyme
Apoenzyme
Prosthetic group
Zymogen
The sensitive enzymatic indicator for intravascular hemolysis and acute myocardial infarction
is: *
Triglyceride
Amylase

increased
decreased
balanced
ALP
GGT
LDH
ACP
Iodide
Chromium
Zinc
Magnesium
15:1
25:1
20:1
30:
rice
wheat
meat
yeast
Elevated serotonin metabolites may indicate which of the following? *
Idiopathic hypertension
Intestinal cancer
Pheochromocytoma
Diabetes mellitus
Patient has a TSH result that is normal. This is maybe due to: *
Tertiary hypothyroidism
Primary aldosteronism
Secondary thyroid hypofunction
None of these
The major controlling gland for calcium homeostasis is played by *
Thyroid gland
PTG
Osseous tissues
Hypothalamus
Which of these hormones are especially related in the delicate balance of production and
utilization of glucose in the body? *
Growth hormone and cortisol

Glucagon and thyroxine
Somatostatin & glucagon
Epinephrine and thyroid hormones
SOMATOSTATIN is also known as GHIH( growth hormone inhibiting hormone)

ADH acts on the: *
Pancreas
Adrenal cortex
Adrenal medulla
Kidneys
The major nonspecific binding protein for hormones is: *
TBPA (thyroid-binding prealbumin)

TBG (thyroid binding globulin)
Albumin
CRP
Which of the following is the major glucocorticoid? *
cortisol
aldosterone
testosterone
corticosterone
NOTE: Glucocorticoids are part of the feedback mechanism in the immune system,
which reduces certain aspects of immune function, such as inflammation. They are
therefore used in medicine to treat diseases caused by an overactive immune system,
such as allergies, asthma, autoimmune diseases, and sepsis.
Melatonin & serotonin are produced by the: *
Pineal gland
Hypothalamus
Pituitary gland
Thyroid gland
NOTE : Serotonin is the key hormone that stabilizes our mood, feelings of well-being,
and happiness. This hormone impacts your entire body. It enables brain cells and other
nervous system cells to communicate with each other.
In secondary hypothyroidism, the TRH level is: *
Normal
Unknown
Decreased
Increased
NOTE: TRH- Thyrotropin releasing hormone
The euthyroid patient’s radioactive count in RT3U test is expectedly: *
Significantly lower
Higher
Normal
Slightly below than normal
Persistent hypoglycemia is seen in which of the following conditions?1. insulinoma 2.
acromegaly 3. hyperthyroidism 4. Cushing’s disease *
1 and 2
1 only
1 and 3
1,2,and 3
One of the following is NOT a tropic hormone. Which one is it? *
Somatotropin
ADH
LH
ACTH
Majority of the thyroid hormones in blood are bound with: *
Thyroxine –binding albumin

Disodium phenyl phosphate
Phenolphthalein phosphate
Glycerophosphate
Why is it important to time samples for cortisol level: *
Actually, there is no timing

None of the above
Because maximal secretion of cortisol has a diurnal pattern
The best period to get is from 10 PM to 2 AM
Epinephrine and dopamine are secreted by the: *
Adrenal glands
Pituitary gland
Pancreas
Kidneys
The reference method for the assay of catecholamines is *
GC
ELISA
RIA
HPLC
In thyrotoxicosis, RIA assay results for thyroid hormones is: *
Increased
Normal
Slightly decreased
Markedly decreased
The parent compound of steroid hormones is *
acetate
cholesterol
sitosterol
triglycerides
The major transport protein of sex hormones is *
albumin
TBG
transcortin
SHBG
The Zimmerman determination of 17-ketosteroid is based on reaction with: *
Ehrlich’s reagent
m-dinitrobenzene
Acetic anhydride
Potassium ferricyanide
At low or absent levels, which one of the following hormones has the ability to produce
hyperglycemia: *
Insulin
Glucagon
Parathyroid hormone
Thyroid hormone
EPO is produced by the kidneys to respond to which target tissues: *
Pituitary gland
Osseous tissues
Adrenal cortex
Muscle tissues
Which hormone-function dyad is incorrect? *
Progesterone – maintains levels of estrogen in the luteal phase

hypothalamus – releases hCG during gestation
FSH – stimulates meiosis in ovaries; release of the ovum
LH – development of the ovum into a corpus luteum
NOTE: hCG is produced by the placenta
Which of the following is a metabolite of serotonin: *
17-ketosteroids
Vanillylmandelic acid
Tryptophan
5’-HIAA
Which of the parameters is the gold standard for thyroid function testing? 1. T4 2. T3 3. Resin
T3 Uptake 4. TSH *
1,2 and 3
1,2,3 and 4
1 and 2
4 only
The major function of thyroid gland is: *
Increase the BMR and Calcium in blood

Thermogenesis & calorigenesis
Negative feedbacking to pituitary gland secretion
Production of thyroid hormones
Free drug levels can generally be determined by analyzing what body fluid? *
PFF of plasma
plasma ultrafiltrate
urine
whole blood
hCG level monitoring is used in: *
Dwarfism
Cushing’s disease
Thyrotoxicosis
Pregnancy
Which of the following methods would yield reliable quantification of ethanol in the presence
of other alcohols? *
reaction with permanganate and a chromotropic acid

Alcohol dehydrogenase reaction
Gas liquid chromatography
Conway diffusion followed by dichromate reaction
Assessments of primary & secondary hypothyroidism include the following EXCEPT: *
T3
T4
TSH
calcitonin
The best time to collect a blood sample for cortisol measurement is *
8 am
12 noon
12 midnight
4 pm
All of the following statements are true regarding drug distribution patterns except *
Renal clearance of drugs is faster in newborn than adults.

Drug metabolism is more rapid for 6-year old children than for adults.
Drug metabolism often changes during pubescence.
Drug metabolism is slower in newborn than adults.
The pituitary growth hormone is also known as the *
Calcitonin
Somatotropin
Insulin
Thyroxine
The principal cations’ concentration in the serum is regulated by: *
thyroxine
aldosterone
insulin
parathyroid hormone
In primary hyperthyroidism, the patient’s value of of blood TSH is: *
Decreased
None of the above
Increased
Normal
The use of iodized salt serves to provide iodine for the synthesis of: *
Growth hormone
Insulin
Sex hormones
Thyroid hormone
Reinsch’s test is used to screen urine for toxic concentrations of all of the following except *
mercury
cyanide
bismuth
arsenic
The reference method for the assay of steroid hormones is *
GC
RIA
ELISA
HPLC
Because of infertility problems, a physician would like to determine when a woman ovulates.
The physician orders serial assays of plasma progesterone. From these assays the physician
can tell when ovulation occurs because *
after ovulation, progesterone rapidly increases
right before ovulation progesterone spikes
right before ovulation progesterone rapidly increases
after ovulation progesterone rapidly decreases
The method used to quantitate androgens except testosterone is *
Kober
Porter-Silber
Pisano
Zimmermann
The assessment of thyroid function that employs RIA includes: *
All of these
TRH stimulation
TSH
T3U
Which of these have receptors for ADH? *
Cardiac muscles
Adrenals
Renal tubules
Mammary gland
The following are true regarding the Zimmerman reaction except *
not used to assess estrogen production

the reaction requires urine samples
red-purple product is measured at 520nm
dinitrophenylhydrazine is the chromogen
The hormone which controls the reabsorption of sodium in the kidneys: *
Is a hyponatremic hormone.
Is derived from cholesterol.
Is produced by the pancreas.
Is involved in the RAA and EPO systems.
The determination of estrogens by the Kober reaction requires which clinical sample? *
12-hour urine
24-hour urine
pooled urine
random urine
The main thyroid hormone is *
Calcitonin
ACTH
T3
Thyroxine
The predominant estrogen in
women is *
estrone
progesterone
estriol
estradiol
The metabolically active thyroid hormone is produced in the: *
Bloodstream & thyroid gland

Thyroid gland & liver
Thyroid & parathyroid glands
Pituitay & pineal glands
When is the best time to get samples for plasma testosterone testing? *
8 PM
12 AM
12 PM
8 AM
Glucagon is produced by: *
G-cell of the pancreas

A-cell of the pancreas
D- cell of the pancreas
B-cell of the pancreas
The functional plasma thyroxine ( T4) is: *
Bound to globulin
Bound to albumin
Free
Bound to prealbumin
The Kober method is performed to *
establish the cause of hypocortisolism

establish the cause of hypercortisolism
assess androgen production by adrenals
assess estrogen production by adrenals
A patient shows the following data: T4 concentration of 8 ug/dL and T3 uptake of 30%. What
is the condition? *
Hypothyroidism
Euthyroidism with high TBG
Hyperthyroidism
Euthyroidism
Persistent hypoglycemia is seen in which of the following conditions? 1. insulinoma 2.
galactosemia 3. Hypothyroidism 4. Addison’s disease *
1 and 2
1 and 3
1,2,and 3
1,2,3,and 4
The most potent estrogen which is considered the true ovarian hormone is *
16-epiestriol
estrone
estriol
estradiol
The predominant estrogen during pregnancy is *
estradiol
progesterone
estriol
estrone
Which of the reactions measures urinary estrogens? *
Zimmerman
Porter-Silber
Murphy-Pattee
Kober
In which of the following are the catecholamines classified? *
Amino acid derivatives

Fatty acid derivatives
Steroid hormones
Peptide hormone
Patient has a TSH result markedly elevated. This maybe due to: *
Tertiary hyperthyroidism
Primary hypothyroidism
Secondary hypothyroidism
Graves disease
PRL is produced in: *
Adenohypophysis
Pancreas
Adrenal medulla
Adrenal cortex
basophilic stippling in RBCs; produce hypochromic anemia *
lead
methanol
carbon monoxide
ethylene glycol
ethanol
presence of CaOx crystals in urine; anti-freeze *
ethanol
methanol
lead
carbon monoxide
ethylene glycol
the most common toxic substance *
ethylene glycol
methanol
carbon monoxide
ethanol
lead
Wood alcohol; produces metabolic acidosis *
lead
ethylene glycol
ethanol
carbon monoxide
methanol
measurement of carboxyhemoglobin; cherry red face *
methanol
ethanol
ethylene glycol
lead
carbon monoxide
May result to hemosiderosis *
organophosphate
arsenic
Iron
mercury
cyanide
assay of cholinesterases *
mercury
organophosphate
arsenic
Iron
cyanide
breath with odor of bitter almonds *
arsenic
cyanide
mercury
Iron
organophosphate
garlic odor of breath; positive Reinsch test *
mercury
arsenic
cyanide
Iron
organophosphate
antidote used is dimercaprol or penicillamine *
Iron
cyanide
mercury
arsenic
organophosphate
prostatic CA *
Acid phosphatase & PSA

Bence-Jones protein
human chorionic gonadotropin
alpha-feto protein
carcinoembryonic antigen
colon CA *

Bence-Jones protein
alpha-feto protein
liver CA *
Bence-Jones protein
alpha-feto protein
endometriosis and ovarian CA *

Bence-Jones protein
alpha-feto protein
multiple myeloma *

Bence-Jones protein
alpha-feto protein
Calcitonin *
Pancreatic, GIT
Ovarian, Breast
Brain, lung, colon, GIT, breast
Neuroblastoma, pheochromocytoma
Breast only
Medullary thyroid
Ovarian, endometrial
CA-125 *
Breast only
Medullary thyroid
Pancreatic, GIT
Ovarian, Breast
CA-27, CA-29 *
Medullary thyroid
Breast only
Pancreatic, GIT
Ovarian, Breast
CA-19-9, CA 19-5, CA 242, CA-50 *
Breast only
Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
CA-15-3, CA549, MCA *
Breast only
Medullary thyroid
Pancreatic, GIT
Ovarian, Breast
RB 1 *
lymphoma, leukemia
Chronic myeloid leukemia (CML)
Wilm’s tumor
breast, liver, bladder,sarcomas
retinoblastoma, osteosarcoma
bladder, melanoma, glioblastoma
breast only
p53 *
Wilm’s tumor
lymphoma, leukemia
breast only
c-abl/bcr *

breast only
Wilm’s tumor
lymphoma, leukemia
K-ras, bcl-2 *

breast only
lymphoma, leukemia
Wilm’s tumor
P16 E-cadherin, BRCA2, RB1 A *

Wilm’s tumor
lymphoma, leukemia
breast only
hCG *
Neurohypophysis
Placenta
Adenohypophysis
Liver
Thymus
Ovaries
Thyroid gland
Somatomedins *
Adenohypophysis
Liver
Placenta
Thyroid gland
Neurohypophysis
Thymus
Ovaries
Vasopressin *
Thymus
Liver
Ovaries
Adenohypophysis
Thyroid gland
Neurohypophysis
Placenta
T3 & T4 *
Liver
Ovaries
Thymus
Thyroid gland
Placenta
Neurohypophysis
Adenohypophysis
Estradiol *
Liver
Placenta
Thyroid gland
Ovaries
Thymus
Adenohypophysis
Neurohypophysis
Liver cirrhosis *
euthyroidism
hypothyroidism
hyperthyroidism
Decrease RT3U *
euthyroidism
hyperthyroidism
hypothyroidism
Iodine deficiency *
hyperthyroidism
euthyroidism
hypothyroidism
Renal failure *
euthyroidism
hyperthyroidism
hypothyroidism
Starvation *
hypothyroidism
euthyroidism
hyperthyroidism
Increased TBG *
hypothyroidism
euthyroidism
hyperthyroidism
Increased RT3U *
hypothyroidism
hyperthyroidism
euthyroidism
Decreased TBG *
hypothyroidism
hyperthyroidism
euthyroidism
Thyroid cancer *
euthyroidism
hypothyroidism
hyperthyroidism
Cystic fibrosis *
euthyroidism
hypothyroidism
hyperthyroidismts
THC *
Tranquilizers
Opiates
Dopaminergic stimulants
Sedative-hypnotics
Hallucinogens
Benzoylecgonine *
Sedative-hypnotics
Tranquilizers
Opiates
Phencyclidine *
Sedative-hypnotics
Hallucinogens
Tranquilizers
Opiates
Naloxone *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens
Amobarbital *
Hallucinogens
Opiates
Sedative-hypnotics
Tranquilizers
Heroin *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens
Methadone *
Opiates
Sedative-hypnotics
Tranquilizers
Hallucinogens
Phenobarbital *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens
Cocaine *
Opiates
Hallucinogens
Tranquilizers
Sedative-hypnotics
Oxazepam *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
MLS 045 LAB Quiz 4
What is the reference range of iron in serum in conventional units & SI units? What is the
conversion factor for iron? *
···/5
Conventional Unit: 65 -165 ug/dL (men), 4 -160 ug/dL(women) SI Unit : 11.6 -29.5 umol/L (Men),
5
8. -28.6 umol/L (women). Conversion Factor : 0.179
1
Correct answer
6 -170 mcg/dl; 10.74 -30.43 umol/L; 0.179
0
The titrant used in the Clark & Collip method for the measurement of calcium is
_______________________. *
···/2
Potassium permanganate
Correct answer
potassium permanganate
Which electrolyte can be measured via a turbidimetric method with tetraphenylboron? *

1/1
Chloride Calcium
Potassium
Sodium
Phosphorus
Iron
Magnesium
Monovalent electrolytes INCLUDE the following: *

1/1
Chloride
Nitrogen Calcium
Potassium Mercury
Ferrous ion Cupric ion
Iodine
All of the 8 choices
7 out of the 8 choices
3
out
of
the 8 choices
only 2 out of all of the choices

Which electrolyte can be measured via a titrimetric method with potassium permanganate? *
1/1
Chloride
Calcium
Potassium Sodium
Phosphorus
Iron
Magnesium
The chromogen used in the turbidimetric assay for potassium is

___________________________________. *
···/2
sodium potassium cobaltinitrite
Correct answer
none, turbidimetric assay is NOT colorimetric
AAS method can measure all of the following EXCEPT: *

0/1
Chloride
Nitrogen
Calcium
Potassium Mercury
Ferrous ion
Cupric ion
Iodine
4 out of the 8 choices 3 out of the 8
choices only 2 out of all of the choices
Correct answer
What is the reference range of sodium in serum in conventional units & SI units? What is the
conversion factor for sodium? *
···/5
Conventional units: 135 – 15 mEq/L SI units: 135 – 155 mmol/L Conversion Factor: 1
5
Correct answer
135-155 mEq/L; 135-155 mmol/L; 1

The chromogen in the Fiske-Subbarrow method for phosphorus is
______________________. *
···/2
aminonaphtholsulfonic acid
Correct answer
ammonium molybdate
The most common interfering substance for chloride is

________________________________. *
2/2
bromide
Group No. & Group Name *
Which monovalent electrolyte can be assayed using the Cotlov titrator? * 1/1
Chloride
Calcium
Potassium Sodium
Phosphorus
Iron (II) species
Magnesium
choices only 2 out of all the choices
What is the reference range of potassium in serum in conventional units & SI units? What is the
conversion factor for potassium? *
···/5
Conventional units: 3.8 - 5. mEq/L SI units: 3.8 - 5. mmol/L Conversion Factor: 1

0 0
Correct answer
135-2. -5. mEq/L; 2. -5. mmol/L; 1

4 3 4 3
The protein transporter of copper ions in plasma is ______________________. *

2/2
ceruloplasmin
Which electrolyte can be regulated by ANF? *
0/1
Chloride Calcium
Potassium
Sodium
Phosphorus
Iron
Magnesium
Correct answer only 2 out of all the
choices
What is the reference range of calcium in serum in conventional units & SI units? What is the
conversion factor for calcium? *
···/5
Conventional units: 8.5 – 11.0 mg/dL SI units: 2.13 – 2.75 mmol/L Conversion Factor: 0.25
Correct answer
8.5-11 mg/dl; 2.125-2.75 mmol/L; 0.25
What is the reference range of inorganic phosphorus in serum in conventional units & SI units?
What is the conversion factor for inorganic phosphorus? *
···/5
Conventional units: 2.6 – 4. mg/dL SI units: .87 – 1.50 mmol/L Conversion Factor : 0.3329
5
Correct answer
2. -4. mg/dl; 0.84 -1.45 mmol/L; 0.323
6 5
The major cause of spurious hyperkalemia is ______________________. *

···/2
Prolonged application of tourniquet increases muscular activity (repeated or excessive clenching of
fist prior to and during drawing of blood.
Correct answer
hemolysis
Which electrolyte can be an effectively participate in redox reactions? *

1/1
Chloride Calcium
Potassium Sodium
Phosphorus
Iron
Magnesium
Which TRACE METAL has an electrochemical means of detection in serum samples? *

1/1
Chloride
Nitrogen gas Calcium
Potassium Mercury
Ferrous ion
Cupric ion
Iodine
3
out
of
the 8 choices
only 2 out of all of the choices
The removal of interfering Mg ions in the calcium assay is made possible using
__________________________. *
···/2
8-hydroxyquinolone
Correct answer
8-hydroxyquinoline
Which electrolyte can be regulated by PTH? *

1/1
Chloride Calcium
Potassium Sodium
Phosphorus
Iron
Magnesium
Which divalent electrolyte can be assayed in serum using a dye-binding method? *

1/1
Chloride Calcium
Potassium Sodium
Phosphorus
Iron (II) species
Magnesium
3
out
of
the 7 choices
only 2 out of all the choices
The indicator used in Schales & Schales method for chloride determination in serum is
____________________________. *
2/2
diphenylcarbazone
The most important 1st step in the determination of TIBC is

____________________________. *
···/2
TIBC is very sensitive to contamination, therefore, any glassware must be thoroughly cleaned
(especially prior to the first use) (iron free).
Correct answer
addition of known amount of iron
What is the reference range of chloride in serum in conventional units & SI units? What is the
conversion factor for chloride? *
···/5
Conventional units: 96 – 10 mEq /L SI units: 96 – 106 mmol/L Conversion Factor: 1

6
Correct answer
9 -106 mEq/L; 9 -10 mmol/L; 1
6 6 6
The hormones that balance Na & K concentration in blood are __________________ &
_______________. *
···/4
aldosterone and antidiuretic hormone
Correct answer
aldosterone & atrial natriuretic hormone
The protein transporter of ferric ions in plasma is ______________________. *

2/2
transferrin
Which monovalent electrolyte can be assayed using the Cotlov titrator? * 1/1
Chloride
Calcium
Potassium Sodium
Phosphorus
Iron (II) species
Magnesium
What is the reference range of magnesium in serum in conventional units & SI units? What is
the conversion factor for magnesium? *
···/5
Conventional Unit: 1.3 - 2. mEq/L, SI Unit: 0.65 - 1.05 mmol/L, Conversion factor : 0.5
1
Correct answer
1. -2. mg/dl; 0.74 -1. mmol/L; 0.4114
8 6 1
MLS 045 LAB Quiz 5

I. DIFFERENTIATION OF TESTS ON PANCREATIC ENZYME MARKERS: Compare and contrast the
three (3) tests below by filling in the table below with the necessary and correct information:
Points of Caraway Method for AMS Kinetic IFCC Method Shihabi, et al. Method
Differentiation AMS for LPS
Reaction Principle Amylase splits the Amylase splits the Serum lipase
polysaccharide starch substrate 4,6-ethylidene- hydrolyzes the olive
into the disaccharide (G7)-p-nitrophenyl-(G1)-α- oil emulsion.
maltose and a residue, D-maltoheptaoside (EPS- The decrease in
limit dextrin, resulting G7) to produce turbidity at 400 nm
in a loss of color. This oligosaccharides, then the after incubation is
color change at 640nm α-glucosidase hydrolyzes proportional to the
is directly proportional the oligosaccharides lipase activity in the
to amylase producing glucose and p-
specimen.
concentration. nitrophenol (PNP).
Product Starch p-nitrophenol Diglyceride and fatty

Measured acid
Substrate used Starch paste 4,6-ethylidene-G7-p- Olive oil in alcohol
nitrophenyl-G1-α-D-
maltopheptaoside (EPS-
G7)
Parameter U/L (U/dL, ukat/L) U/L (ukat/L) U/L, (ukat/L)
reported
Color Endpoint colorless colorless none
Wavelength 640 nm 405 nm 400 nm

Used
Light Path 1 cm 1 cm 1 cm
Incubation 37˚C 37˚C 37˚C

Temperature
Buffer Used Phosphate buffer Pipes buffer, pH 6.90 Tris buffer, pH 9.0
Purpose of Buffer Helps maintain a Helps maintain a constant pH Helps maintain a

Used constant pH level level constant pH level
Chromogen 0.01M Iodine reagent p-nitrophenyl None
Composition of Starch reagent, iodine NaCl, CaCl,Pipes buffer, p- Olive oil in alcohol, tris
Blank reagent, distilled nitrophenylmaltoheptaoside, buffer,distilled water
water Glucoamylase, a-glucosidase
Type of Blank Reagent blank Reagent blank Reagent blank
Used
Conversion Factor 0.01667 0.01667 0.01667 (To convert
IU/L to Cherry-Crandall
The approximate units, divide IU/L by 70)
conversion factor
between Somogyi units
and IU is 1.85
Purposes of 5 Acid Sodium Chloride - Pipes buffer- to maintain Tris buffer, pH 9.0 (69
reagents activator that increases pH at 6.90 mM) - buffer used to
the enzyme pKa thus maintain the pH.
increasing its Vmax
initiating the cleavage of
the substrate chain
Starch reagent, pH 7.0 - NaCl - activators and the Olive oil in alcohol (0.8%
buffer used to maintain sources of chloride ions (w/v)) – substrate used
the pH optimum in lipase activity
0.01M Iodine reagent – CaCl- for activation and Co-lipase – aid in lipase
used as chromogen stabilization of a-amylase hydrolysis
Starch paste – substrate α-Glucosidase (≥2 kU/L) - Bile salts - aid in lipase
used in the method indicator enzyme; enzymes hydrolysis
that hydrolyze the
oligosaccharides from the
breakdown of the substrate.
Phosphomolybdic acid – p- Triolein – can used as

used as preservative in nitrophenylmaltoheptaoside- substrate for LPS assay
Caraway method used as substrate for amylase
Type of Test Amyloclastic Method Chromogenic (colorimetric Turbidimetric
test)
Reaction pH pH 7.0 pH 6.90 pH 9.0
Samples Required serum or heparinized Serum/heparinized plasma serum

plasma
Linear Range Up to 500 U/dL up to 1985 U/L for up to 280 IU/L
substrate start and up to
1594 U/L for sample start
Reference Range 60-160 somogyi Adults Females: Serum/ Adults:
in Conventional units/dL plasma: <100 U/L Urine: 10 – 150 IU/L (more
Unit <100 U/L Males: than 60 years old is
Serum/plasma: <100 U/L expected to have 18-
Urine: <491 U/L 180 IU/L)
Reference Range Adults Females: Adults Females: Serum/ Adults:

in SI Unit Serum/ plasma: <100 plasma: <1.667 ukatal/L 0.143-2.143 ccu/L
U/L Urine: <100 U/L Urine: <1.667 ukatal/L >60 y.old: 0.257-2.571
Males: Serum/plasma: Males: Serum/plasma: ccu/L
<100 U/L Urine: <491 <1.667 ukatal/L Urine:
U/L <8.18 ukatal/L
Associated Serum amylase levels Acute pancreatitis; Acute pancreatitis;

Diseases are elevated in acute Other disorders causing an Elevations have
pancreatitis, obstruction elevated serum AMS level been reported in cases
of pancreatic ducts include salivary gland of penetrating
(carcinoma, stone, lesions, such as mumps and duodenal ulcers and
stricture, duct sphincter parotitis, and other intra- perforated peptic
spasm after morphine), abdominal diseases, such as ulcers, intestinal
mumps; occasionally perforated peptic ulcer, obstruction, and acute
elevated in the presence intestinal obstruction, cholecystitis.
of renal insufficiency, cholecystitis,
diabetic acidosis, and ruptured ectopic
with inflammation of the pregnancy, mesenteric
pancrease from a infarction,
perforating peptic ulcer. and acute appendicitis. In
Serum amylase levels addition, elevations have
are decreased in acute been
and chronic hepatitis, reported in renal
pancreatic insufficiency, insufficiency and diabetic
occasionally in toxemia ketoacidosis.
of pregnancy, and in
barbiturate poisoning
Cite 3 Testing 1. Highly icteric or 1. Saliva and skin contain 1. Lipase activity in
Precautions lipemic sera may a-amylase therefore serum is stable at
produce false never pipette reagents RT for 1 week;
values in this by mouth and avoid sera may be
method so that skin contact with the stored for 3 weeks
serum blanks are reagents. at 4-8̊C and for
recommended 2. The reagents contain several months if
2. Most sodium azide (0.95 g/L) frozen.
anticoagulants, as preservative. Do not 2. Hemolysis should
such as EDTA, swallow! Avoid contact be avoided
sodium fluoride, with skin and mucous because
citrate, and membranes. hemoglobin
oxalates 3. Little loss of activity inhibits the
reportedly occurs at room activity of serum
produce erratic temperature for 1 week LPS, causing
results due to or at 4°C for 2 months. falsely low values.
binding of calcium 3. Bacterial
ions which are contamination of
required for the specimens
maximum may result in an
activation. increase in lipase
3. Abnormally low activity.
concentrations of
protein in the
sample reportedly
produce erratic
results.
6. What is ∆Absorbance/minute? How is it obtained?

*∆Absorbance/minute measures the rate of change in absorbance per minute. You must estimate the
absorbance change vs time of your assay mixture, that is, the absorbance change per minute of the reaction
(ΔA/min). The change in absorbance per minute (ΔA/min) is proportional to the micromoles of NADH
oxidized and in turn to micromoles of substrate transformed per minute. Spectrophotometry which uses
a secondary wavelength absorbance reading which is the absorbance of the sample subtracted from a
primary wavelength absorbance of the blank sample reading to obtain a delta absorbance reading (Ad)
per minute.
7. Explain the sensitivity of AMS and specificity of LPS as enzyme markers for pancreatic disorders.
*Serum amylase compared with lipase, returns more quickly to values below the upper limit of normal
and it cannot be used alone for a reliable diagnosis of AP. Serum lipase is more sensitive/specific than
amylase and remains elevated longer than amylase after disease presentation.
8. Compare & contrast the peak times for AMS and LPS in serum when pancreatitis occurs in a patient?
*After an attack of acute pancreatitis, serum LPS activity increases within 4 to 8 hours, peaks at about 24
hours, and decreases over 8 to 14 days. Concentrations often remain elevated longer than those of
AMY. In acute pancreatitis, arise in serum AMY activity occurs within 5 to 8 hours of symptom onset and
peaks at 12-72 hours. Activities return to normal by the third or fourth day.
9. What are the cofactors needed for AMS & LPS assays in serum?
*For AMS assays in serum, Ca+2 is needed as a cofactor. Cofactors are generally not required for lipase
activity, but divalent cations like calcium often stimulate enzyme activity. This effect has been
suggested to be due to the formation of long chain fatty acid calcium salts.
Serum amylase (AMS)
- NaCl and MgCl2
Serum Lipase (LPS)

- Calcium
- Co-Enzyme produced in the pancreas, salivary glands, gastric, pulmonary and intestinal
mucosa.
10. What is macroamylasemia? Give its clinical importance and show the procedure on how to detect its
presence.
*Macroamylasemia is a condition characterized by persistent elevation of serum amylase activity with
no apparent clinical symptoms of pancreatic disease. It is a condition that results when the AMY
molecule combines with immunoglobulins to form a complex that is too large to be filtered across the
glomerulus. It maybe an early marker of pancreatic disease. A blood test will show high levels of
amylase. However, macroamylasemia can look similar to acute pancreatitis, which also causes high
levels of amylase in the blood. Measuring amylase levels in the urine can help tell
macroamylasemia apart from acute pancreatitis.
MLS 045 LAB Quiz 6
HORMONES
Specific Name Functional Group Unique R
Prostaglandin A2 Hydroxyl Cyclopentanone
Carboxyl
Alkene
Ketone
B
Norepinephrine Hydroxyl Catechol
Alkene
Phenol
Amine
Catechol
C
Serotonin Amine Amine
Alcohol
Alkene
Phenol
D
Thyroxine (T4) Amine Halide
Ether
Alcohol
Phenol
Alkene
Carboxyl
Halide
E
Thromboxane A2 Halide Halide
Carboxyl
Ether
Alkane
Phenol
F
Calcitrol Alcohol Hydroxyl
Hydroxyl
Alkene
G. Epinephrine
Functional Group: hydroxyl, alcohol, amine
Unique R: amino
H. Acetylcholine
Functional Groups: alcohol, acyloxy, quaternary ammonium
Unique R: ethylene
Thyroxine
Functional group: carboxyl, phenol, hydroxyl, halide, amine,
Unique R: Halide
J. Dopamine
Functional group: Hydroxyl, amine, benzene
Unique R: Catecholamine
K. Vasopressin
Functional Group: Amides, mercapto
Unique R: phenylalanine, arginine
L. Leukotrienes B4
Functional Group: Alkene, carboxyl, hydroxyl
Unique R: Hydroxyl
STEROIDS
Steroids
1. Specific name: Androstenedione

Functional group: Hydroxyl, Alkene, Carbonyl
Unique R nucleus: Cyclopenatano-perhydro-phenanthrene (Aldrostane)
2. Specific name: Aldosterone

Functional group: Aldehyde, hydroxyl, ketone, alkene
Unique R nucleus:Cyclopenatano-perhydro-phenanthrene (Pregnane)
3. Specific name: Testosterone
Functional group: ketone, alkene, and secondary alcohol groups

Unique R nucleus:Cyclopenatano-perhydro-phenanthrene (Aldrostane)
4. Specific name: Estradiol
Functional group: hydroxyl groups, one at the C3 position and the other at the 17B position, as
wekk as three double bonds in the A ring
Unique R nucleus:Cyclopenatano-perhydro-phenanthrene (Estrane)
5. Specific name: Estriol
Functional group: hydroxyl groups, one at the C3 position and the other at the 17B position, as
wekk as three double bonds in the A ring
Unique R nucleus:Cyclopenatano-perhydro-phenanthrene (Estrane)
Progesterone
Functional group: Ketone
Unique R nucleus: cyclopentanoperhydro-phenanthrene (Pregnane)
Dehydroepiandrosterone (DHEA)
Functional group: Ketone
Unique R nucleus: cyclopentanoperhydro-phenanthrene (Androstane)
8. Cortisol
Functional group: Carbonyl, Alkene, Methyl group
Unique R nucleus: cyclopentanoperhydro-phenanthrene (Pregnane)
9. Estrone
Functional group: Ketone. hydroxyl
Unique R nucleus: cyclopentanoperhydro-phenanthrene (Estrane)
10. Estrone
Functional group: Ketone, hydroxyl
Unique R nucleus: cyclopentanoperhydro-phenanthrene (Estrane)
MLS 045 LAB Quiz 7
Osseous tissues
Thyroid gland
Hypothalamus
PTG
Persistent hypoglycemia is seen in which of the following conditions? 1. insulinoma 2.

galactosemia 3. Hypothyroidism 4. Addison’s disease *
1,2,3,and 4
1 and 2
1 and 3
1,2,and 3

Somatotropin
Insulin
Calcitonin
Thyroxine

T3
TSH
T4
calcitonin


Kidneys
Adrenal glands
Pituitary gland
Pancreas

Increased
Normal
Decreased
None of the above
8 AM
12 PM
8 PM
12 AM
Correct answer
12 AM

None of the above

Hypothalamus
Pineal gland
Pituitary gland
Thyroid gland

insulin
thyroxine
parathyroid hormone
aldosterone
Which of these hormones are especially related in the delicate balance of production
and utilization of glucose in the body? *

Porter-Silber
Murphy-Pattee
Zimmerman
Kober
Pancreas
Adenohypophysis
Adrenal cortex
Adrenal medulla


ADH acts on the: *

Pancreas
Kidneys
Adrenal cortex
Adrenal medulla
hyperglycemia: *
Glucagon
Thyroid hormone
Insulin
Parathyroid hormone

Cardiac muscles
Adrenals
Renal tubules
Mammary gland

LH
ADH
Somatotropin
ACTH

T3
Thyroxine
ACTH
Calcitonin

Pheochromocytoma
Diabetes mellitus
Intestinal cancer

Normal
Decreased
Increased
Unknown

Muscle tissues
Adrenal cortex
Pituitary gland
Osseous tissues

17-ketosteroids
5’-HIAA
Tryptophan
None of these

Acetic anhydride
m-dinitrobenzene
Ehrlich’s reagent
Persistent hypoglycemia is seen in which of the following conditions?1. insulinoma 2.

acromegaly 3. hyperthyroidism 4. Cushing’s disease *
1 only
1 and 2
1 and 3
1,2,and 3

Albumin

CRP

Thyrotoxicosis
Pregnancy
Cushing’s disease
Dwarfism
A patient shows the following data: T4 concentration of 8 ug/dL and T3 uptake of 30%.
What is the condition? *
Hyperthyroidism
Euthyroidism
Hypothyroidism
Which of the parameters is the gold standard for thyroid function testing? 1. T4 2. T3
3. Resin T3 Uptake 4. TSH *
1 and 2
1,2,3 and 4
1,2 and 3
4 only

Glycerophosphate

Bound to globulin
Bound to prealbumin
Free
Bound to albumin

TSH
TRH stimulation
T3U
All of these


Graves disease
Sex hormones
Insulin
Thyroid hormone
Growth hormone

Markedly decreased
Increased
Normal
Slightly decreased

Significantly lower
Normal

Higher

Steroid hormones
Peptide hormone



acetate
cholesterol
sitosterol
triglycerides

GC
RIA
ELISA
HPLC

Porter-Silber
Pisano
Zimmermann
Kober

SHBG
albumin
transcortin
TBG

12 midnight
8 am
12 noon
4 pm






estrone
estradiol
estriol
progesterone
The predominant estrogen in post-menopausal women is *

estrone
estradiol
estriol
progesterone
estriol
estradiol
estrone
16-epiestriol
Because of infertility problems, a physician would like to determine when a woman

ovulates. The physician orders serial assays of plasma progesterone. From these
assays the physician can tell when ovulation occurs because *


aldosterone
corticosterone
cortisol
testosterone

GC
RIA
ELISA
HPLC
random urine
12-hour urine
24-hour urine
pooled urine
whole blood
urine
PFF of plasma
Reinsch’s test is used to screen urine for toxic concentrations of all of the following
except *
bismuth
arsenic
mercury
cyanide
Which of the following methods would yield reliable quantification of ethanol in the
presence of other alcohols? *
Matching Type
5 of 5 points

ethanol
methanol
ethylene glycol
lead
carbon monoxide

ethanol
methanol
ethylene glycol
lead
carbon monoxide

ethanol
methanol
ethylene glycol
lead
carbon monoxide

ethanol
methanol
ethylene glycol
lead
carbon monoxide

ethanol
methanol
ethylene glycol
lead
carbon monoxide
Matching Type
5 of 5 points
arsenic
mercury
cyanide
Iron
organophosphate

arsenic
mercury
cyanide
Iron
organophosphate

arsenic
mercury
cyanide
Iron
organophosphate

arsenic
mercury
cyanide
Iron
organophosphate

arsenic
mercury
cyanide
Iron
organophosphate
Tumor Markers and associated Diseases

5 of 5 points
multiple myeloma *
alpha-feto protein
Bence-Jones protein

alpha-feto protein
Bence-Jones protein
liver CA *
alpha-feto protein
Bence-Jones protein
colon CA *
alpha-feto protein
Bence-Jones protein
prostatic CA *
alpha-feto protein
Bence-Jones protein
Protein Markers of Tumor Types

5 of 5 points
CA-125 *
Breast only
Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
CA-15-3, CA549, MCA *
Breast only
Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
CA-19-9, CA 19-5, CA 242, CA-50 *

Breast only
Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
CA-27, CA-29 *
Breast only

Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
Calcitonin *
Breast only
Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
Genes and its cancer types

5 of 5 points
K-ras, bcl-2 *
breast only
lymphoma, leukemia
Wilm’s tumor

breast only

lymphoma, leukemia
Wilm’s tumor
p53 *
breast only
lymphoma, leukemia
Wilm’s tumor
c-abl/bcr *
breast only
lymphoma, leukemia
Wilm’s tumor
RB 1 *
breast only
lymphoma, leukemia
Wilm’s tumor
Hormone Origins
5 of 5 points
Vasopressin *
Thymus
Thyroid gland
Ovaries
Adenohypophysis
Neurohypophysis
Placenta
Liver
T3 & T4 *
Thymus
Thyroid gland
Ovaries
Adenohypophysis
Neurohypophysis
Placenta
Liver
Estradiol *
Thymus
Thyroid gland
Ovaries
Adenohypophysis
Neurohypophysis
Placenta
Liver
hCG *
Thymus
Thyroid gland
Ovaries
Adenohypophysis
Neurohypophysis
Placenta
Liver
Somatomedins *
Thymus
Thyroid gland
Ovaries
Adenohypophysis
Neurohypophysis
Placenta
Liver
Thyroid Hormone Levels and Disease

10 of 10 points
Liver cirrhosis *
hypothyroidism
hyperthyroidism
euthyroidism
Renal failure *
hypothyroidism
hyperthyroidism
euthyroidism
Starvation *
hypothyroidism
hyperthyroidism
euthyroidism
Cystic fibrosis *
hypothyroidism
hyperthyroidism
euthyroidism
Increased RT3U *
hypothyroidism
hyperthyroidism
euthyroidism
Decreased TBG *
hypothyroidism
hyperthyroidism
euthyroidism
Iodine deficiency *
hypothyroidism
hyperthyroidism
euthyroidism
Thyroid cancer *
hypothyroidism
hyperthyroidism
euthyroidism
Decrease RT3U *
hypothyroidism
hyperthyroidism
euthyroidism
Increased TBG *
hypothyroidism
hyperthyroidism
euthyroidism
Drugs of abuse and their classes

10 of 10 points
Heroin *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Amobarbital *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Methadone *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Cocaine *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Phencyclidine *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Naloxone *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Phenobarbital *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
THC *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
Benzoylecgonine *
Sedative-hypnotics
Opiates
Tranquilizers
Oxazepam *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers
MLS 045 LAB Quiz 8

Make a 5-step procedure on how to properly collect a 24-hour urine sample. *
Provide patient with written instructions and explain the collection procedure. 2. Issue the
proper collection container and preservative. 3. Day 1: 7 a.m patient voids and discards
specimen. 4. Patient writes the exact time on the sample label and places the label on the
container. 5. Patient collects all urine for the next 24 hours.
What are the preparatory requirements for OGTT? Cite at least 5. *
Patients should be in a position to wait at the laboratory for at least 2-3 hours, since 5 or more
blood samples are collected at the interval of 30 minutes. 2. Normal to high carbohydrate intake for
3 days before the test 3. should be fasting for atleast 10 hours but not more than 16 hours 4. Just
before tolerance, patients should refrain from exercise, eating, drinking(patinets may drink water)
and smoking. 5. Certain medications can interfere with test results, and the phlebotomist should
ask the patient about medications before beginning the test.
Draw a single OGTT graph with correct labels of x- & y-axes showing 5 separate
curves depicting hypoglycemia, normal, borderline or pre-diabetic, moderate DM &
severe DM. *
answers sent to teacher
Give the reagents used for the Porter-Silber reaction with the following purposes:
HYDROLYSIS, EXTRACTION, PURIFICATION & ESTIMATION. *
Chloroform, AR - extraction
Ethanol - purification
Sulfuric acid - estimation/ m β-Glucuronidase. - hydrolysis
Give the reagents used for the Zimmermann reaction with the following purposes:
Hydrolysis: Glacial acetic acid and 3 ml concentrated hydrochloric acid Extraction: Ethylene
dichloride, redistilled. Purification: m-Dinitrobenzene, 1.16 g/100 ml purified ethanol. Estimation:
Potassium hydroxide-ethanol solution, Ethanol, 70 percent (v/v).
Give the reagents used for the Kober reaction with the following purposes:
Hydrolysis: Hydrochloric acid Extraction: diethyl ether Purification: carbonate buffer Estimation:
hydroquinone
Give the reagents used for the Pisano, et al method with the following purposes:
Hydrolysis: acidified urine (Hydrochloric acid) with ethyl acetate. Extraction: aqueous potassium
carbonate solution Purification: sodium metaperiodate Estimation: toluene
What hormones will give a POSITIVE test for Porter-Silber reaction? *
17-hydroxycorticosteroids (17-OHCS) (cortisol (hydrocortisone), prednisolone, dexamethasone,

and corticosterone, aldosterone)
What hormones will give a POSITIVE test for Zimmermann reaction? *
17-ketosteroids (Androstenedione,Androstanedione,Androsterone,Dehydroepiandrosterone
(DHEA),Epiandrosterone,Epietiocholanolone,Etiocholanolone)
Enumerate the hormones that will give a POSITIVE test for Kober reaction. *
Estrogen (estrone, estradiol, estriol)
Enumerate the hormones that will give a POSITIVE test for Pisano, et al. method. *
Catecholamine (dopamine, norepinephrine, and epinephrine )

MLS 045 LAB Quiz 9
Phosphorus
a spot test for each of the 14 volatile poisons in your laboratory manual in
Exercise no. 40 and you compare and contrast them below by filling in the
table with the necessary and correct information according to the column
headings:
Volatile Poison Spot Test Method Reagents Used Observable Positive
Result
Phosphoru
s Ammoniu – few gtts. conc. HNO3 yellow ppt. of NH4
m phosphomolybdat
AND NH4 molybdate. e
molybdate test
Hydrocyanic acid tartaric acid and guaiac on heating, the paper

copper that is exposed to the
fumes becomes blue
Schonbein-
or bluish green.
Pagenstecher test
Phenol, Millon’s
Lysol, reagent red color
Cresol,
Creosote, Millon’s test
Creolin, &
Pyrogallol
. Chloroform Add 1 or 2 drops of penetrating

repulsiv odo
aniline, few ml. of e r of
Hoffmann’s aqueous or phenylisocyanide.
Phenylisocyanide alcoholic potassium
test hydroxide solution.
Ether 1. Dichromate test Few mm K2Cr2O7 + –greenish color of

4 gtts H2SO4 = CHCl3
Chromic
acid
H2O
CHCl3
Nessler’s yellowis
Chloral Hydrate Nessler’s test reagent h red
precipitate which
afte
change r a
1
while to a dirty
yellowish green.
. Iodoform sodium phenolate a red substance

solution(mixture of shall be dissolved
Lusgarten’s test 20 grams of phenol
wit 4
h 0 grams of
sodium hydroxide
and 70 grams of
H2O), few drops of
dilute alcohol.
Aniline few gtts. CHCl3 and repulsive odor or

Phenylisocyanide KOH phenylisocyande
iod lemo
Ethyl Alcohol Lieben’s iodoform o potassium n yellow
precipitat
test iodide solution e of
(I2+KO
H) – iodoform
aqueou
s.
Methyl
Alcohol 1. Trichloroactic acid, A purple ring at the
(wood alcohol): Potassium interference
Cu-Oxidation permanganat
e,
test Sodium bisulfite,
Chromotropic acid
and Sulfuric acid,
concentrated.
Acetone Lieben’s Iodine + KOH Iodoform (note odor

Iodoform test and crystals).
Carbon Disulfide PbAc2 + KOH and with PbAc2 – no

hea effect
Lead acetate test t (distinction ; with KOH –
between CS2 & H2S- black ppt (PbS)
no reaction
2
between PbAc2 and
CS2).
Formaldehyde Recorcinol test 5% resorcinol red color

(aqueous) + 40%
NaOH
Thymol Piria’s test H2SO4 + beautiful violet-red

unknown + 10 vol H2O color
+ white Pb + ferric
chloride solution
GOOD LUCK!!!
3
MLS 045 LAB Quiz 10
I. DIFFERENTIATION OF TESTS ON NONVOLATILE POISONS, METALS &

NONMETALS: Choose a spot test for each of the 14 nonvolatile poisons, 3 metals & 3
nonmetals in your laboratory manual and you compare and contrast them below by filling
in the table with the necessary and correct information according to the column headings:
Substance Tested Spot Test Method Reagents Used Observable Positive
Result
Picric acid Isopurpuric acid test Picric acid and saturated Deep red color
aqueous potassium (Potassium
cyanide solution isopurpurate)
Acetanilide Indophenol test Acetanilide Upper layer- indigo-

Fuming hydrochloric blue color
acid Under layer- Red color
Saturated, aqueous
Carbolic acid solution
Ammonium hydroxide
solution
Phenacetin Oxidation test Phenacetin Yellow color to
Concentrated gradually turning green
Hydrochloric acid
Chromic acid solution,
Water
Salicylic Acid Ferric-chloride test Ferric chloride solution Blue-violet color
Alcoholic solution of If the solution is very
salicylic acid dilute:
- Red-violet color
Aspirin Ferric-chloride test ferric chloride solution Blue-violet color
If solution is very
dilute (red-violet)
Veronal Million reagent or white, gelatinous

Millon’s test soluble of yellow precipitate
mercuric oxide in 2 ml.
of nitric acid
1
Antipyrine ferric chloride solution Deep red color
Ferric chloride test
Dilute sulfuric acid
changes the red to a
pale yellow color
Caffeine Strong chlorine water Red-red brown

Amalic acid test
If added with
ammonium hydroxide,
it will produce purple
violet
Nicotine Ether, Iodine Roussin’s crystals

(ruby red needles
Roussi’s test having a dark blue
reflex will crystallize)
Strychnine concentrated sulfuric evanescent blue or

acid, potassium blue-violet color
Sulfuric Acid- dichromate
Dichromate test
Atropine fuming nitric acid, evanescent violet color

Vitali’s test potassium hydroxide in at once changing to a
absolute alcohol fine red will appear
Cocaine 5% of chromic acid crystalline orange

solution, or potassium precipitate
Chromic acid test dichromate solution,
concentrated
hydrochloric acid
Quinine Dilute sulfuric acid, fine blue fluorescence
Fluorescent test ether solution
Codeine Concentrated nitric acid yellow color
Nitric acid test

Reinsch test - HCl, concentrated, AR In the presence of
- Copper spiral arsenic or selenium,
2
ARSENIC the surface of the
copper will be gray to
black.
Colorimetric -Diphenylthiocarbazone Lead forms a red
LEAD analysis (dithizone) complex with
diphenylthicarbazon
- Nitric acid e
-HCl
-NH4OH
-Cyanide
- Citrate
THALLIUM Colorimetric -HCl (concentrated,AR) A positive tests imparts
- Bromine
analysis water a blue to violet color to
- Sulfosalicylic acid (20 the benzene.
g/100 ml in water)
- Methyl violet (AR, 20
g/100 ml in water)
BROMIDES Colorimetric Trichloroacetic Brown color is very
analysis stable in acid solution
acid, 10 g/100 ml
and can be read
aqueous solution quantitatively at 440
Gol nm
d (auric)
chloride solution
Trichloroacetic
(1
acid 0 g/100
ml) – sodium
chloride (0.06
g/10
0 ml)
mixture
Standard
s
✓
Stock, 10
mg/ml
Dilute standard, 0.5
mg/
ml
FLUORIDE Colorimetric Perchloric acid, o A developing
analysis
70 g/100 g color with a
Silv
er cerium or
perchlorate, AR lanthanum
3
Sodium hydroxide, 0.50 complex with
mola
alizari
r
n
complexone
Magenta or blue color
BORON Colorimetric Hydrochloric o Brownish-red
analysis o
acid, color f the
concentrated, acidified urine
AR on the wet or
Turmeric paper, dry paper
commercially Green-black or blue
color after exposure to
available, AR ammonia fumes
Ammonium hydroxide,
concentrated, AR
GOOD LUCK!!!
MLS 045 LAB Quiz 11
Answer the following questions correctly regarding the SD BIOLINE MET/THC Test Kit:
It is used to detect metabolic disorders involving carbs. *
1/1
insulin
17-hydroxycorticosteroids
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Cocaine *
1/1
Organic volatile poison
Inorganic volatile poison
Organic nonvolatile poison
COMPILED BY:cam cabase

Inorganic nonvolatile poison
The SD BIOLINE MET/THC Test Kit is: *
1/1
Qualitative
Quantitative
chloral hydrate *
1/1
Strychnine *
1/1
MeOH *
1/1
The chromogen used is hydroquinone. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Carbon disulfide *
1/1
The standard used is estradiol-17beta. *
1/1
insulin via OGTT
17-ketosteroids

urine estrogens
VMA
all of the above
none of the above
Iodoform *
1/1
It uses ethyl acetate as extractant. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Creosote *
1/1
If the control band (C) is not visible within the result window after performing the test, the result
is considered ____________________________. *
1/1
invalid
negative
MET-positive
THC-positive
dual (MET- & THC-) positive
It reflects purely gonadal function. *
2/2
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Ethanol *

1/1
Which hormone is indirectly measured? *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
1/1
Presumptive
Confirmatory
It reflects dysfunction of the adrenal glands. *
4/4
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It is tested on 24-hour urine samples. *
4/4
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It can be detected by the Zimmerman reaction. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above

none of the above
It requires dietary restrictions prior to testing. *
0/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Correct answer
all of the above
Phosphorus *
0/1
Correct answer
Veronal *
1/1
Formalin *
1/1
Phenylhydrazine-sulfuric acid mixture is used as reagent blank. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Aspirin *
1/1

Chloroform *
1/1
The presence of only the control band (C) within the result window indicates a/an
_________________ result. *
1/1
invalid
negative
MET-positive
THC-positive
dual (MET- & THC-) positive
Caffeine *
1/1
A reddish-purple product is obtained. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It is a type of colorimetric test. *
3/3
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It is increased in Cushing's syndrome. *

3/3
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
The test is affected by high glucose concentration in the sample. *
4/4
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It is elevated in blood and urine in pheochromocytoma. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
The SD BIOLINE MET/THC Test Kit uses this type of URINE sample. *
1/1
24-hour
12-hour
early morning midstream catch
random
timed
nocturnal
It requires blood and random urine samples. *
0/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Correct answer

insulin via OGTT
The presence of three colored bands (1, 2 and C) within the result window, regardless of the
order in which the bands appear, indicates a/an _______________ result. *
1/1
invalid
negative
positive
It is a UV spectrophotometric assay. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Thymol *
1/1
1/1
Immunometric
Immunoassay
It may indicate hypofunction of the associated gland. *
0/3
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Correct answers
insulin
urine estrogens
VMA
It requires plotting glucose values in blood against the time. *
1/1
insulin via OGTT

17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It requires a 75-gram oral glucose load. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
The presence of both the THC test band (2) and the control band (C) within the result window,
regardless of which band appears first, indicates a/an ____________________________________
result. *
1/1
invalid
negative
MET-positive
THC-positive
both MET- & THC-positive
The product measured is reddish brown in color. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
There is a need for two wavelengths in reading absorbance. *
0/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Correct answer
17-ketosteroids
HCN *

1/1
It is an example of a dynamic test. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Ether-ethanol mixture serves as the extractant. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
1/1
Chromatographic
Electrophoretic
It requires the oxidation of the analyte to vanillin. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
It requires an alkaline medium for color reaction. *
1/1
insulin
17-ketosteroids
urine estrogens
VMA
all of the above

none of the above
Acetone *
1/1
Diethylether is used as extractant. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Pyrogallol *
1/1
Phenacetin *
1/1
The SD BIOLINE MET/THC Test Kit is a: *
1/1
One-step test
Multi-step test
The presence of both the MET test band (1) and the control band (C) within the result window,
regardless of which band appears first, indicates a/an ____________________________ result. *
1/1
invalid
negative
MET-positive
THC-positive
both MET- & THC-positive
It is easily affected by drugs taken by the patient. *
4/4

insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
Nicotine *
1/1
The standard used in testing is DHEA. *
1/1
insulin via OGTT
17-ketosteroids
urine estrogens
VMA
all of the above
none of the above
SIR MARIO LEC VID. #1

DIFFERENTIATION OF BILIRUBIN TESTS
- It is called INSERT because it is INSERTED into the reagent kit. Once you opened the reagent kit,
you will see bottles duly labeled as standard, monoreagent/ several reagents separate from one
another. You will see inserted paper usually folded several times. It contains all the information
that you need regarding the test kit/reagent kit.
PRINCIPLE - it shows the chemical reaction. A substance tested reacts with a combination of
reagent particularly with the CHROMOGEN, if it is a colorimetric method. If it is a colorimetric
method, the method depends on the color reaction . The most important reaction principle is the
LAST enzyme.
ICTERUS INDEX – doesn’t have a reaction principle. It has test principle but it has nothing to do
with the reaction because no reaction happened. Icterus index is just to measure or find out how
YELLOW the sample is.

STANDARD( ICTERUS INDEX) – diluted Dichromate solution( orange color but the dilution is
1:10,000 that is why it appears yellow) . So if the sample appears more yellow than the standard,
meaning there is a possibility that the patient has jaundice.
ICTERUS INDEX- is just an indication of the intense yellowishness of the sample; it is a screening
test for jaundice. It is just comparing the 2 yellow solutions; diluted dichromate solution and
serum sample of the patient.
EVELYN MALLOY AND JENDRASIK AND GROFF- they have the SAME chemical reaction but
different Ph that is why their color is different.
EVELYN MALLOY- acidic
Jendrassik and Groff – Alkaline
ORIGINAL evelyn malloy have some common reagents with the MODIFIED. However, the modified
uses some reagent that cannot be found in the original
ORIGINAL EVELYN MALLOY- when measuring with total, it uses ALCOHOL which will dissolve B1
and B2 that is why it measures the TOTAL
MODIFIED- it uses cetrimide
What are the chemicals used to dissolve B1 and B2?

- DMSO( dimethylsulfoxide) , cetrimide, caffeine- benzoate ( JG method)
PRINCIPLE- has something to do with the chemical reaction which is the basis for the
measurement of particular product involving the substance that you are going to test.
ICTERUS INDEX- no product measured because the product is a result of a chemical reaction. So
the answer should be NONE.
Original Evelyn-Malloy and Modified Evelyn Malloy – the same; PINK – PURPLE; RED is also correct
because it is near to the color scheme.
Jendrassik- Groff – blue or green depending on the other mixture. There is actually no particular
color indicated in the manual. But when you look at the nanometer ( wavelength) you can identify
what color it is.
COLOR ENDPOINT:
ICTERUS INDEX- Yellow because it only uses DILUTION, the diluents used is PHOSPHATE BUFFER
solution with a particular Ph ( 7.4) because that is the normal Ph of blood
Because we used a DILUTED sample ( 1:10) the formula to solve icterus index is multiplied by 10.
This is a concept that you need to bear in mind. Whenever you have a diluted sample, the dilution
factor should be multiplied before releasing the result.
ORIGINAL AND MODIFIED EVELYN-MALLOY – Purple- pink
JENDRASSIK AND GROFF – blue or green ( but if you base it on the wavelength, it is more of blue-
violet) because it is 500 something. So the violet has a blue material because violet is a
combination of red and blue . You cannot have a violet without a blue .
WAVELENGTH USED :
Icterus Index- Yellow ( 410 or 405 nm)

Original and Modified Evelyn – pink – purple ( 500-550 nm)
Jendrassik and Groff- blue ( if the product has already blue color it will exceed 550 nm)
LIGHT PATH – all the same because they used the cuvette( analytical cell or absorption cell
is the basis of the light path), the thickness of the solution. Actually based on the diameter
of the cuvette. The most common standard universal diameter for a cuvette is 10 mm or 1
cm.
INCUBATION TEMPERATURE – mostly 37 degree Celsius but the Icterus index doesn’t need an
incubation because it is only dilution so it uses just room temperature.So if there is no indicated
temperature, then most of the time that is RT or room temperature.
BUFFER USED:
Icterus Index: Phosphate buffer
Note: The buffer is provided to maintain a particular Ph. So if ever you are adding a reagent that
has different Ph so the buffer will act on it and maintain a particular Ph
REAGENT FOR TOTAL BILIRUBIN TESTING:
For TOTAL BILIRUBIN TESTING, the needed reagents are those in which the B1 and B2 are both
soluble .
B1- first bilirubin formed ( In the bloodstream)
B2- liver conjugated bilirubin
REAGENT FOR DIRECT BILIRUBIN TESTING
Direct bilirubin is also known as B2 so the reagent should be soluble in water. Water-based
aqueous solution. So B1 will not be dissolved in a reagent which is water-based that is why the
water-based reaegtn will only dissolve the B2 and will eventually make the color reaction possible
for the B2.
REACTION PH
7.4 – icterus index
Evelyn Malloy – should be acidic. Right after a buffer you can see a number and that is the Ph that
is maintained by the buffer
SAMPLES REQUIRED- you can see this on the test kit like serum or plasma
LINEAR RANGE- some have NONE
What is the purpose of the linear range?
- It will give you an idea what the smallest concentration and the highest concentration that the
test can measure involving a particular substance. Notice that in the linear range, the normal
value is fitting and sometimes exceeding the normal value so we can also measure the decreased
, lower than the normal value and higher than the normal value. Too much high value can still be
given a solution by DILUTING and then multiply by the DILUTION FACTOR. But if below the normal
value OR BELOW THE LINEAR RANGE ANG SUBSTANCE TESTED, there is NO REMEDY . But if the
substance concentration exceeds the linear range, like for example the patient has hepatic failure
so the bilirubin will exceed the highest principal concentration in a linear range so there is no
problem because we can just dilute it so that the color is fitting to the concentration and then
later on multiply by the reciprocal of the dilution.
REFERENCE RANGE FOR TOTAL BILIRUBIN IN COVENTIONAL UNIT

- Usually the total bilirubin is near 20 microliter. The conversion factor is 17 so the mg/dL is mga 1
point something
EVENTHOUGH THERE ARE DIFFERENT METHODS USED IN TESTING THE SAME SUBSTANCE , THE
REFERENCE RANGES ARE STILL ALMOST THE SAME. DI SILA MAGKAKALAYO whether
conventional or SI unit. Bakit hindi sila magkakalayo? Because when you want to invent a new
method for that substance, you have to look for the reference method for that substance.
Is 0 micromole/L bilirubin normal?
WHICH OF THE FOLLOWING VALUES IS NORMAL?
For total bilirubin – it is not normal
For direct- it is normal
For indirect- no ( because it is B1) look at your serum it is colored yellow.
What gives the normal yellow color to the serum ?
- It is actually the B1
ASSOCIATED DISEASES:
Icterus Index- Jaundice
Diseases of direct and indirect are different tapos sa total it depends.
Why is it there is a need for the physicians to know the direct and indirect . Why is the total not
sufficient? Because there is a difference in the set of diseases for B1 and B2 . B1 elevation is due
to pre-hepatic jaundice( the total bilirubin is elevated because of the B1 tapos the direct is normal)
But if the total has higher value than normal, B1 is normal while B2 is abnormal) this is due to
hepatic or post- hepatic jaundice
CITE 3 TESTING PRECAUTIONS
Most important precaution for bilirubin it should be away from light because it is light- sensitive
or place it in an amber colored solution.Refer to MSDS( Material Safety Data Sheet)
Triangle symbol is Capital Delta. The small delta is d na styling.

1. What is Delta Absorbance? How is it obtained?
- Delta is actually the change in absorbance. So how do you know the change in absorbance? You
just deduct the two absorbance values . So if you have 2 absorbance values, the higher value you
put as minuend and the lower value should be the subtrahend. So CHANGE in absorbance . It is
obtained by getting the difference between the 2 absorbance values .
- You always have 2 absorbance values because all the tubes are read against blank( So you
determine the absorbance of the blank and the absorbance of the tube) . But if you have zeroed
the absorbance, you no longer have the problem because any value minus 0 is the value
Jaundice babies are undergoing light therapy to convert bilirubin into less harmful products . Less
harmful in a way that they cannot anymore penetrate the membranes of the tissues and go to the
brain and cause this kernicterus.
WHY DOES BILIRUBIN CAN EASILY PENETRATE THE BRAIN OF CHILDREN WITH JAUNDICE?
- The jaundice is brought about by incompatibilities in blood ( ABO and RH incompatibilities) ;
erythroblastosis fetalis . If the baby has kernicterus, it will have mental retardation or death . You
convert bilirubin into products which are easily excreted in the urine. Products( bilirubin

molecules that have numbers because the rings of bilirubin are cut by light . Note: The light that
we are providing to the child is not harmful to the child but it is able to destroy the rings of the
bilirubin . The destruction of the rings of bilirubin will cause the production of the products with
numbers . The rings are opened so those that are no longer ring shaped are no longer harmful . It
will be easily excreted eventually after a few days so that the child is normal looking
3. What are the cause of pre-hepatic, hepatic and post-hepatic jaundice? Cite at least 3 disorders of
each type of jaundice
CAUSES OF PRE-HEPATIC JAUNDICE – increased hemolysis like Malaria and enzyme deficiency
disorder like G6PD deficiency that will cause hemolysis intravascular, injuries, parasitic infection
that causes hemolysis; blood flukes , drug interactions that cause hemolysis, blood incompatibility
, production of bilirubin, B1 will increase causing pre-hepatic jaundice
HEPATIC JAUNDICE – Viral, bacterial, PARASITIC, PROTOZOAL diseases affecting the liver and
drug toxicity like ACETAMINOPHEN and some antibiotics can also affect the liver so it can affect
the conjugation process .
Note : in pre-hepatic jaundice, the liver is normal. But in hepatic jaundice, the liver is abnormal
Post- hepatic- bloodstream is normal, liver is normal but after the liver there is obstruction like
stones , surgical errors, tumors from the nearby organs like the stomach , pancreas , or intestine
4. What is the role of sodium nitrite? NaNO2
- Whatever the method for bilirubin, colorimetric, there is usually a Sodium nitrite like Jendrassik
and Groff, Evelyn Malloy
- Its role is DIAZOTIZATION of SULFANILIC ACID. ( How does sodium nitrite DIAZOTIZE sulfanilic
acid?)Sodium nitrite will react with sulfanilic acid and the acid will become a salt because there is
a nitrite attaching to it. How many nitrites? 2
- NOTE: Di is 2 and Azo is nitrogen . So 2 NO2 molecules will attach to sulfanilic acid to form a
diazonium salt
- AZO group is NO2 . Any substance with NO2 is azo containing substance . so it will form sulfanilic
nitrite or diazotized sulfanilic acid
WHAT IS THE CHROMOGEN FOR BILIRUBIN TESTING?

- Diazotized sulfanilic acid
If the total bilirubin is increased, we need to know what caused the increase of total bilirubin
SOUTHWESTERN UNIVERSITY PHINMA

COLLEGE OF MEDICAL TECHNOLOGY
Quiz 6 in MLS 045L
Group Name: __________________________________
Group No. ______________________
Members Present: ____________________________________
________________________________________
________________________________________
________________________________________

________________________________________
I. DIFFERENTIATION OF TESTS ON CARDIAC ENZYME MARKERS: Compare and contrast the

two (2) tests below by filling in the table below with the necessary and correct information:
Points of CK-NAC UV LDH-L IFCC
Differentiation
Reaction Principle Creatine kinase (Ck) catalyzes the LDH catalyzes the oxidation of L-
conversion of creatine phosphate and lactate to pyruvate with the
ADP to creatine and ATP. The ATP and mediation of NAD+ as a hydrogen
glucose are converted to ADP and acceptor.
glucose-6-phosphate by hexokinase.
Glucose-6-phosphate dehydrogenase Forward reaction
oxidizes the glucose-6-phosphate and
reduces NAD+. Reaction Catalyzed: hydrogen transfer
enzyme that catalyzes the oxidation of L-
lactate to pyruvate with the mediation
of NAD as hydrogen acceptor or as
coenzyme
LDH
L-lactate + NAD+ --------------------→
Pyruvate + NADH + H+
Product Measured NADH Pyruvate
Notes: Sir M: Because it is kinase, it

will attach the phosphate to creatine
. Product measured is NADH. If NAD
becomes NADH, there is increase in
absorbance because we have an
additional one bond in which
hydrogen is bonded to NAD.
*If NADH to NAD, this is decreasing
because the bond is cut. DECREASE
IN A BOND WILL CAUSE DECREASE
IN ABSORBANCE.
Substrate used Creatine phosphate L-lactate
Notes: L and P type. If L yan siya,

that is Lactate. If P, that is pyruvate.
L type Is LACTATE to PYRUVATE so
the substrate is usually lactate.
Parameter IU/L LDH activity (U/L)

reported
NOTES: applicable for all tests:

ACTIVITY UNITS or ukatal/L
Color Endpoint NONE/ not applicable NONE ( look at the wavelength, that
is 340 nm) if the wavelength is
below 400, that is UV.
Wavelength 340nm 340nm
Used
Light Path 1cm or 10 mm 1cm or 10mm
Incubation 37°C 37°C

Temperature
NOTES: Usually the incubation
tempis 37
Buffer Used Imidazole N-methyl-D-Glucamine
Purpose of Buffer To maintain pH of the reaction To maintain pH of the reaction

Used
PS! Make sure you mention the
particular Ph. Example: to maintain
pH at 7.5 etc.
Chromogen None None
Composition of Distilled water Distilled H2O: 40ul

Blank Mono reagent: 100ul
Notes: just copy what is being put

inside the tube for the blank plus
distilled water usually or if the blank
is air, so none.
Type of Blank Used Reagent blank Reagent blank
Conversion Factor 16.67 0.01667
NOTE: The factor na libo-libo is not a

conversion factor but it is a factor
used out of the standardized formula
for a fixed substrate.
Purposes of 5 N-acetylcysteine- activates creatinine N-methyl-D-Glucamine

reagents kinase - Maintains pH of the reaction
EDTA-Na2 = preservative NAD+
- Hydrogen acceptor; coenzyme

AMP Sodium azide
- inhibits adenylate kinase (AK) - preservative
Glucose = oxidation reagent for L-lactate = substrate

intermediate reaction
Imidazole
- used as buffer; maintains pH of
the reaction
Type of Test Photometric, increasing reaction, UV, kinetic, increasing reaction,

kinetic, UV enzymatic, CK-NAC LDH-L IFCC method
method
SIR M: Kinetic, colorimetric

Reaction pH 6.8 9.4
Samples Required Serum Serum, EDTA-plasma, heparinized

plasma
Linear Range Extends up to 1, 200 IU/L Up to 1512 U/L on Hitachi 911
Reference Range in Serum: 25-192 IU/L at 37°C Female: < 247 U/L
Conventional Unit 10-109 IU/L at 30°C Male: < 248 U/L
Reference Range 417-3200 nkat/L at 37 degrees C Female: <4.12 ukatal/L

in SI Unit 167-1817 nkat/L at 30 degrees C Male: <4.13 ukatal/L
Associated 1. Myocardial infarction 1. Myocardial infarction

Diseases 2. Duchenne’s muscular 2. Anemias: pernicious,
dystrophy hemolytic, megaloblastic
3. Hypothyroidism 3. Leukemia
4. Pulmonary infarction 4. Renal infarction
5. Reye’s syndrome 5. Hepatitis and hepatic cancer
6. Strenuous exercise and 6. Muscular dystrophy
intramuscular injections 7. Delirium tremens
7. Cerebral vascular accident 8. Malignancy
8. Rocky mountain spotted fever
9. Carbon monoxide poisoning Notes: sir M: LDH is not limited to
Cardiac Disease because LDH can be
NOTES: SIR M: CK can be found in 3 found in all organs that is why the
organs. The brain, heart and skeletal electrophoresis of LDH is very
muscles.So those are the associated important because it will form 5
diseases but the major use of CK is sa fractions that have different
heart talaga, Myocardial infarction. characteristic and sources/origins.

LDH is very common in TRANSFER
RATE EXUDATE(?) to distinguish
between the 2.
Cite 3 Testing 1. Do not use components beyond 1. The reagents contain sodium
Precautions the expiration date. azide as preservatives. Avoid
2. Avoid contact with reagents as it contact with skin and mucous
causes skin irritation. membranes.
3. Moderate or severely hemolyzed 2. Samples of patient with
specimens can liberate adenylate gammopathy might give false
kinase, ATP, and G-6-P which may results.
affect the lag phase and side 3. Results should always be
reactions of the CK assay system. assessed with the patient’s
medical history, clinical
examinations and other findings
for diagnostic purposes.
11. What is ∆Absorbance/minute given the following data?

OD1 after 1 min = 0.456; OD2 after another min = 0.487; OD3 after another min = 0.500
((A3-A2)+(A2-A1))/2
= ((0.500-0.487)+(0.487-0.456))/2
= (0.013+0.031)/2
= (0.044)/2
= 0.022
Notes by sir M: This is an INCREASING reaction( since absorbance values are increased) .
So OD2-OD1 then after OD3-OD2. So you already have 2 delta OD’s . Delta OD is difference . So
get the Delta OD difference, that is the Delta OD per minute.
• Delta OD1 is 0.031
• Delta OD2 is 0.013
• Add OD1 and OD2
• 0.031 + 0.013 = 0.044 divided by 2
• 0.022
• Since 0.022 is your delta OD per minute, this will be the one that will be multiplied to the factor to
get your international unit per liter.

• To convert IU/L , you multiply it by 0.01667
• You will get ukatal/L
•
12. Differentiate the L-type & P-type of LDH testing.

- Both Forward (lactate [L-type]) and Reverse (pyruvate [P-type]) reactions have been used in clinical
assays. The rate of the Reverse reaction is approximately three times faster, allowing smaller
sample volumes and shorter reaction times. However, compared to Forward reaction, it is more
susceptible to substrate exhaustion and loss of linearity hence, IFCC developed Forward reaction
as the reference assay. The optimal pH for the Forward reaction is 8.3 to 8.9; for the Reverse
reaction, it is 7.1 to 7.4.
.
Notes (Sir M) : L is lactate and P is pyruvate . Either the product or the substrate used for LDH
testing.
13. Compare & contrast the peak times for CK and LDH in serum when AMI occurs in a patient?
-CK is released within 12 hours after symptom onset of AMI, peaks in serum at 24–36 hours, and
returns to normal in 48–72 hours while LDH levels begin to rise within 12 to 24 hours, reach peak
levels within 48 to 72 hours, and may remain elevated for 10 days.
NOTES (SIR M) : CK after acute MI. or a heart attack ( approx. 6-8 hours it will elevate) so it is
more sensitive than LDH which is after 2 days OR 72 HRS. (3 DAYS) pa mag peak.
4. What are the cofactors needed for CK & LDH assays in serum?
The cofactors need for creatine kinase assay are Adenosine triphosphate (ADP) and Nicotinamide
adenine dinucleotide (NAD+). Cofactor needed for LDH assay is Nicotinamide adenine
dinucleotide (NAD+).
Notes ( Sir M) : Usually, if phosphatases or Kinases, magnesium is there ( Magnesium chloride)

For LDH, the cofactor is METAL, ZINC
5. What is LD flipped pattern? Give its clinical importance.

In conditions involving cardiac necrosis (AMI) and intravascular hemolysis, the serum levels of LDH-
1 will increase to a point at which they are present in greater concentration than LDH-2, resulting
in a condition known as the LDH flipped pattern (LDH-1 >LDH-2) This flipped pattern is suggestive
of myocardial infarction.

Notes sir M: This is for AMI detection in which LD2 which is normally higher than LD1 is flipped in
which LD1 is already higher than LD2.
.
6. What is the formula used in solving for the factor utilized in order to solve the enzyme activity
expressed in conventional units?
𝚫 𝐀𝐛𝐬/𝐦𝐢𝐧 𝐱 𝐓𝐕 𝐱 𝟏𝟎𝟎𝟎
= IU/L
𝑏𝑥Ɛ𝑥𝑆𝑉
Where: ΔA/min = average absorbance change per minute

TV = total reaction volume
1000 = conversion of IU/mL to IU/L
b = light path in cm.
ε = millimolar absorptivity of NADH
SV = sample volume in mL
What
are
the
parameters in order to look for the conversion factor?which will you multiply to delta OD per
minute so we can get the international units per liter.
*To convert IU/L to ukat/L multiply by 0.01667

*To convert IU/L to nkatal/L multiply by 16.67

E2(?)- Prostaglandin(PG)
Identify the hormone:
Give the specific name:
Functional group:
1.carboxylate ion ( Because there is no
hydrogen, it will become a negatively
charged ion) that is why it becomes
more reactive;
2.OH or hydroxyl

3.keto group( carbonyl= the C =(
double bond O na nasa ring ( RCOR)
Double bonds are considered

functional group
Note: Prostaglandin is very reactive because it has a

lot of functional group. CH3 is not a functional group
it is just a hydrocarbon. CH3 is nonpolar and
nonfunctional.
: The more functional groups there are in a 1 molecule
is a very potent chemical.
: Prostaglandins are hidden in the cell membrane. It is
derived from a fatty acid which has 20 CARBONS( This
is what we call eicosanoid.
: How do you know that this is an Eicosanoid. Eicosa
is 20. This is derived from a 20 fatty carbon acid
known as ARA or arachidonic acid
: Clue: Prostaglandin looks like a HAIRPIN
Unique R group:

Read this site for more understanding of
prostaglandin:
https://aztecspirulina.weebly.com/spirulinablog/pros
taglandins
https://www.sciencedirect.com/topics/biochemistry-
genetics-and-molecular-biology/eicosanoids

2. Norepinephrine
Notes: This is an AMINE. This is derived from an

AMINO ACID. This is derived from TYROSINE.
Norepinephrine- Tyrosine-derived hormone
ALWAYS REMEMBER:
Norepinephrine, Epinephrine and Dopamine are
collectively called CATECHOLAMINES
Is the ring a functional group?- No. The double bond
is the functional group NOT THE RING. If there are 3
double bonds, you can write it as HEXAGON and then
you just put a circle in the center because the pi(?)

electrons move; it stabilizes in the ring that’s why
STABILITY is OPPOSITE TO REACTIVITY. So that ring is
not a reactive group. FUNCTIONAL GROUPS SHOULD
BE REACTIVE GROUPS
So the reactive groups in NOREPINEPHRINE are: the
3 OH. The 2 OH’s attached to the ring makes the ring
a CATECHOL ring . The norepinehrine and
epinephrine have OH in the
catechol ( may 3 ka OH tanan
tapos ang DOPAMINE no OH
extra like 2 ra(?))
2. AMINO GROUP – this is a
functional group ( clue :
Norepinephrine starts with
letter N and the amino group starts with letter N)
Note: It is actually very close to
Tyrosine but the only difference is
that Tyrosine doesn’t have 2 OH ( isa
lang tapos my COOH because
TYROSINE is an amino acid . COOH is
cleaved to be replaced by Hydrogen
NOTE: Catecholamines are NERVOUS
SYSTEM hormones and they are quick – acting, they

are usually hypertensive( sila ang nagpapa increase
nang blood pressure)
3.
What hormone is
this?
-serotonin
( Note : Melatonin has a different structure. It has an
additional functional group) The source/ the origin of
the hormone gives them the skeleton. The only
difference is that sometimes we add additional
functional group that’s why it becomes more reactive.
Remember that hormones are very reactive
chemicals; small amount gives massive effects
What amino acid is this derived from?
- This is derived from tryptophan

Note: the 2 rings are very characteristic with an indole
that has a 5 member
What are the tryptophan-derived hormones?
These are the TONINS. Like SeroTONIN, MelaTONIN.
Serotonin- Mood- influencer. If you have HIGH

SEROTONIN, you are in good mood . But if this is
VERY LOW, this can trigger suicidal tendencies. This
also belongs to NEUROTRANSMITTERS.
NOTE: Your nervous system is BEHAVIOURAL. It
affects the change in behavior and it is also SUDDEN.
So because this is derived from TRYPTOPHAN, the
foods that you need to eat in order to increase your
serotonin levels to have a HAPPY MOOD.One
example for Tryptophan source is MEAT. You need to
take complete protein foods because it contains all
the 20 amino acids
Tyrosine- Catecholamine sources ( it also influences
the mood as well as blood pressure)
A very popular metabolite that goes into the urine is
the urinary 5-HIAA ( which comes from SEROTONIN)
. Measuring 5’ HIAA in the urine will give us an idea
will give us an idea of the over/underproduction( but

usually overproduction) of 5’HIAA . It is NORMAL that
there is NO 5’HIAA in the urine but if it is
overproduced, it is a sign that you have TUMORS in
your body.
WHAT PRODUCES SEROTONIN in which if this organ
has cancer , our serotonin levels will increase in our
blood and body fluid?
- 1. Brain 2. Intestine ( That is why serotonin is a ____
test for cancers)

Note: This is the
easiest to identify. If
you see a hormone
with IODINE . this is
T4 (Thyroxine)
because there are 4
iodines. If there are
only 3 iodines, then
this would be T3.
This is still another hormone that comes from
TYROSINE.
How many Tyrosine MOLECULES do you need in
order to have one molecule of T4?
-2 Tyrosine molecules. Tyrosine molecule has a
benzene ring. Since there are 2 benzene rings,
meaning there are 2 tyrosine molecules that fuse .
One COOH and water is removed . That is why the
remaining is O . The connecting piece between 2
tyrosine molecules is O .
That’s a functional group. What functional group(COC

or ROR )
- Ether group (ROR) , Amino, carboxylate anion,
hydroxyl,

What are the functional groups here?
-Similar to Epinephrine. It has Amino . NH2 is Amino
and NH3 is an Amino that accepts Hydrogen ions
that is why 3 hydrogens and positive charge. The
positivity will actually provide reactivity . That COO-
is the one that donates the HYDROGEN that is why
it became carboxylate anion which is a reactive
group also
That is why Thyroid hormone is for (?)_____

increasing temperature, increasing blood glucose.
Note the IODINE in T4 is NOT REACTIVE because it
is NOT IODIDE.IODIDE is reactive because it has
minus/ charge .

-This is also an ARACHIDONIC ACID derived
hormone. If you count the number of carbons, there
are 20 of them.
- There is an OXANE ring. ( This is already a clue) So
the ending should be OXANE . So the structure is
THROMBOXANE. As the name suggests: Thrombo-
it has something to do with blood clotting .
ARACHIDONIC ACID – ESSENTIAL fatty acid ; it is
not produced in the human body and it should be
obtained from FOOD( deep sea creatures , plant
sources that have many polyunsaturated fatty acids
– sources of eicosanoids)

FUNCTIONAL GROUPS: carboxylate ions, hydroxyl,
and double bonds .
F. VITAMIN D3 (1,25 dihydroxycholecalciferol –

Those are the 2 carbons that have hydroxyl; ang
dalawang magkatabi, 1 and 3 and sa may buntot
banda, that’s 25; the OL ending, that’s number 3) .
Vitamin D3 has something to do with CALCIUM
ABSORPTION.
If you are Vitamin D deficient, you have rickets or
osteomalacia, these are bone diseases
Note: This is a vitamin-derived hormone. This is
cholesterol-derived. The 4 cholesterol rings in
which one is opened. When cholesterol is made into

vitamin, the functional groups should be exposed.
That is why the ring is broken and the double bonds
appear .
FUNCTIONAL GROUPS: 3 OH,( The 3 OHs are

actually clue to the name of the hormone , kaya ang
pangalan may 3) and double bonds.
Note: This is
Tyrosine
derived.There is a CATECHOL ring, so this is a

catecholamine.
This hormone is EPINEPHRINE. The difference
between Norepinephrine and Epinephrine is
Methyl. The hydrogen of amino and H2(?) is
substituted with methyl. The hormone

NOREPINEPHRINE was produced first before the
EPINEPHRINE.
In order for norepinephrine to become epinephrine,
the process is METHYLATION. Catechol O methyl
transferase, this is the enzyme used because this is
the one that will attach the methyl to the amino
group. The amino has lost its power because the
amino becomes imino . Amino is NH2 reactive and
imino is not. Nabawasan nang functional group ang
epinephrine .
Is the ring here stable?
Note: Epinephrine is MORE POTENT than
NOREPINEPHRINE. Norepinephrine is just a
precursor of Epinephrine. It is really the
EPINEPHRINE that increases the blood pressure. But
if there is no norepinephrine, there is also no
epinephrine.
In short, Norepinephrine WALAY METHYL while ang

Epinephrine nay Methyl (CH3)

Look at the
+ positive
charge, this makes it a reactive group. The N that
has 3 methyl groups, is called choline . This
hormone is called ACETYLCHOLINE ( CH3COO-
acetate) so this is an ACETYL GROUP.

Are thos elements with ION(IONIC ELEMENTS
POLAR OR NON POLAR?
- They are POLAR. But it is surrounded by non-polar
methyls
FUNCTIONAL GROUPS: Carbonyl and positively
charged Nitrogen . Kasi naka tago ang positive,
parang magiging nonpolar and non-polar is effective.
Small molecule+ nonpolar can traverse the
membrane .
Acetylcholine is inhibited because if we have many
acetylcholine, it will block the RECEPTORS; that is why
we will have paralysis . Cholinesterase is an enzyme
that converts ACETYLCHOLINE into ACETIC ACID and
CHOLINE; so that acetylcholine will not accumulate
which can cause paralysis . To prevent the
accumulation of ACETYLCHOLINE, we need to break
them down so the enzyme is needed to prevent
paralysis. Acetylcholine blocks the nerve endings. So
it can cause paralysis.
Acetylcholine is a neurotransmitter together with the
catecholamines .

Hormone: T3 ( Triiodothyronine) The 2 tyrosines , if
fused will become THYRONINE.
FUNCTIONAL GROUPS: OH, COOH AND AMINO.

J.
Tyrosine-
derived
hormone
. This is a
catecholamine because there is a CATECHOL ring. But

there is NO EXTRA OH .
HORMONE NAME: Dopamine.
Note: Dopamine is precursor to Norepinephrine. First
is Tyrosine. The very first neurotransmitter that is a
CATECHOLAMINE that is produced is DOPAMINE.
And if that H is replaced with OH, it will become
NOREPINEPHRINE. If one hydrogen of Nitrogen is
replaced with CH3, it will become
Epinephrine.Dopamine is also reactive
FUNCTIONAL GROUP: 2 OH and then AMINO GROUP.
Tyrosine-Dopamine-Norepinephrine-Epinephrine

K. This is a Nonapeptide. If you count the building
blocks na Amino acids, there are 9 of them .
What are examples of nonapeptides?
-Oxytocin and Vasopressin( a.k.a ADH )
Hormone Name: Anti-diuretic Hormone / Vasopressin
What is the difference between Oxytocin and ADH?

The only difference is Phenylalanine. Instead of
Phenylalanine, in Oxytocin, it is ISOLEUCINE(ILE)
There is a disulfide bond which is 2 cysteine. If these

2 cysteine is medyo malapit lapit sa isa’t isa it will form
a disulfide bond which is the VERY STRONGEST bond

in the body. This formation of a disulfide bond causes
a KEY SHAPE formation( it looks like a susi) in which
if it is broken, it will no longer be that functional. This
acts on the tubules of the kidneys. It is produced in
the posterior pituitary gland and it will act on the
kidneys by reabsorbing water because it is
ANTIDIURESIS so it prevents water loss.
NOTE : ADH and OXYTOCIN ( posterior pituitary) the
rest, ANTERIOR na
C double bond O is CARBONYL. Amino can be
positively charged like the branching in Arginine
ARGININE-VASOPRESSIN (AGV) because the arginine

there is VERY REACTIVE.

Note: This is an Eicosanoid . The striking feature is the
continuous and alternationg single, double bond.
That is why this is a TRIENE( Three double bonds that
are continuous) .
HORMONE NAME: LEUKOTRIENE
NOTE: Leukotrienes are vasodilating . They are
antidote to those who have difficulty in breathing like
ASTHMATICS; the one that is sprayed; fast-acting ; it
dilates the airways and then relaxes the exchange of
gases . This is also a 20-carbon fatty acid ( eicosanoid)
Functional groups: OH, COO-(Carboxylate ion) ,

double bonds

STEROIDS
NOTE: Example of steroids are androgen,

mineralocorticoids, glucocorticoids
QUESTION: How would you know that it is a steroid?

It is derived from what functional group? What lipid
source?( All of these came from one molecule , and
that molecule gives the structure) The 4 rings are
suggestive that it is derived from CHOLESTEROL.
-If you are going to label the rings, that would be
letters A,B,C,D . Start from the bottom A, followed by
B, and C and lastly, the only 5-membered ring is D.

If the A ring has one double bond, then that is
ANDROGEN. If the A ring has 3 double bonds,
meaning it is stable, then it is ESTROGEN.
If 2 methyls(CH3) are attached to Carbons 10 and 13-
that is ANDROGEN
If 1 methyl(CH3) attached to carbon 13 (above, near
the carbonyl) , that is ESTROGEN .
STEROIDS:
1. Steroid name: Androstenedione
(2 ketones, that’s why the ending is –dione)

Ketones can be found in carbon number 3 and 17.
That is why this is called 17-ketosteroid
Test used for 17-ketosteroid- Zimmerman
All the steroids with carbonyl( double bond O) in
Carbon 17 are called 17-ketosteroids and the test for
17-ketosteroids is ZIMMERMAN reaction.
NOTE: There is also 1 group that has 17 but this is 17-
hydroxycorticosteroid but there is OH in carbon 17.
Note: This androstenedione will give us a POSITIVE
ZIMMERMAN reaction.

2. The carbon 17 no longer has keto group . But above
the carbon 17, which is Carbon 20 . Note: MALI ANG
18 , that should be 13. The 18 IS the carbon that is
attached to 13 and that is ALDEHYDE . This is a clue
that this is an ALDEHYDE.
STEROID NAME: ALDOSTERONE. The –one from
Carbon number 3 and carbon number 20. Aldehyde is
actually the most functionally reactive group here,
that is why it is called as ALDOSTERONE. This is a
mineralocorticoid able to regulate the sodium and
potassium levels in blood. This is HYPERNATREMIC
but HYPOKALEMIC HORMONE. It increases sodium
because it is a mineralocorticoid
FUNCTIONAL GROUPS: double bond, if the double

bond is not alternating, that is unstable or reactive.But
if 3 double bonds, that is stable so you can just draw
a circle inside and Carbonyl group ( C double bond O)

and 2 hydroxyls (OH) attached to carbon 11 and
carbon 21 as well as OH of 17
NOTE: Aldosterone is a member of 17-
hydroxycorticosteroids because there is a hydroxyl
group on carbon 17. This gives a positive reaction
with PORTER-SILBER TEST
3.Testosterone – androgenic hormone

Clue : In the “A” ring, there is only 1 double bond,
there are 2 methyls attached to carbons 10 and 13.
Carbonyl on carbon number 3. And then carbon
number 4 and 5 is the location of the double bond.

OH on carbon number 17. If the ketone group that
connects on carbon number 3 becomes OH, it will
become more reactive, this is called
dihydroxytestosterone.
Testosterone – major male hormone.
4.Estradiol – look at the A ring. ( Clue, mas maarte
ang mga babae that is why marami silang double

bond) .
Diol- there are 2 OH. Carbon number 3 and 17.
There are a lot of hormones that are members of 17-
hydroxycorticosteroids. They have hydroxyls on
carbon number 17.

Notice the methyl attached to carbon number 13.
The methyl carbon is number 18. There is methyl
attached to carbon number 10 and the A ring is
stable, alternating single, double bonds.
5. Still ESTRADIOL
If ever it will become estriol, where is the 3rd OH
located? C16
Estriol has 3OH attached at carbon 3, 17 and 16.

6.
Progesterone
*the structure is border of estrogen and androgen.
Why? It is a female hormone but it has 2 methyls that
looks like androgen and it doesn’t have an A ring. It
has double bond like the androgen. It has a carbonyl,
C double bond O. Carbon 3 and 20 . It looks like
aldosterone but it doesn’t have aldehyde group
attached to carbon 13, instead it has methyl.
*This is the major hormone elevated in response to
LH ( Luteinizing hormone) during the luteal phase of
menstrual cycle. Luteal phase ( after the
menstruation)

During menstruation, the estrogen that is increased is
ESTRADIOL, after the 14th day, you will ovulate, after
ovulation, no sperm arrived, progesterone will
replace estradiol. ( Note: Estrogen is the major
female hormone) .
*Progesterone – Luteal phase hormone.
7. DHEA- dehydroepiandrosterone
(Clue, no triple or double bond in the A ring, so this is
definitely an androgen) .
*one has something to do with the keto group at
carbon 17. This will give a ZIMMERMAN REACTION
POSITIVE, because this is a 17-ketosteroid.

* Testosterone will not give a positive Zimmerman
because it doesn’t contain a keto group in carbon 17
but instead it has hydroxyl so this is 17-
hydroxycorticosteroid
*This is PORTER- SILBER POSITIVE kaysa mag
Zimmerman.
*dehydro- meaning there is removal of
hydrogen.Removal of hydrogen in carbon number 5
and 6. Kanina the double bond is 4 and 5, ngayon sa 5
and 6 na, yan yung nabawasan nang hydrogen.
*If you remove hydrogen, double bonds appear.
*If you want to remove the double bond, you
hydrogenate the chemical. Mag CH3 or CH2, wala na
yang double bond.
*When we hydrogenate the fat, we will make the fat
not healthy but if we remove hydrogens, fat will
become polyunsaturated and it is easier to
metabolize, so it is not prone to clogging arteries.
*DHEA- is also precursor to other hormones such as
testosterone and androstenedione.
* The 2 CH3 are still there because this is an
androgen

* The A ring is not unsaturated. If it is unsaturated, it
is estrogen but if it is saturated or only one double
bond, it is Androgen.
* Functional group : OH ( hydroxyl) at carbon number
3 and carbonyl (keto) at C17.
8.
CORTISOL
*It looks like progesterone but it has extra OH at
carbon 11 so this is an alcohol, and the name has ol
ending.
*The striking feature of cortisol is the hydroxyl
attached at C11.
*It is also a 17-hydroxycorticosteroid, Porter-Silber
positive.

* Characteristic of cortisol- it increases glucose, it is a
glucocorticoid, produced in the adrenal gland
particularly in the cortex ( because it is cortico) .
*Carbon number 4 and 5 has a double bond reaction.
*Carbonyls=2
*Hydroxyls= 3
9.
9. ESTRONE ( E1)
*This is estrogen because A ring of the sterane
nucleus is stable. It is unsaturated.
* If it is estrogen, usually, you count the number of
carbons. It only has 18 carbons. If it is androgen, it
has 19 carbons. 19 because may methyl pa sa baba.

Dalawa yung methyl that is attached to carbon 10
and 13. Estrogen is 18 lang because methyl is
attached to carbon 13 only, walang naka attach sa
carbon 10, kaya 18 lang ang carbon.
* E2 – Estradiol
*E3- Estriol
*Estrone- keto group found in carbon 17.
* OH at carbon number 3.
*E1 estrONE( clue: napag iiwanan kasi MENOPAUSE
NA) . Keto group at carbon 17
10. ESTRONE

QUIZ #5
• What type of amylase test is caraway?
If
cholesterol, there are 27 carbons but steroids,
hanggang 21 lang usually.
20-21- mineralocorticoid, corticosteroid,
progesterone
18 and 19 carbons- androgens and estrogens
18 carbons- estrogen

19 carbons- androgens because of the methyl
attached
27 carbons- cholesterol
28-30 – steroid din but they are derivatives of
cholesterol.
The very common na tinintingnan na carbon nang
cholesterol is 17. Wala nang ibang number, d
namimention.
R NUCLEUS- R is the hydrocarbon. For the cholesterol,

the sterol nucleus, the ABCD ring. Hindi siya
nawawala. Kahit anong steroid, andyan ang apat
nayan. This makes the steroids unique.
What makes the EICOSANOID unique?

• The 20 carbon
The R of the peptides are the sequence of the Amino
Acids.
*Nucleus siya kasi andyan siya palagi tapos iba iba
lang yung naka attach.
*Example for Tyrosine ring, catechol ring is always
there.

For tryptophan, yung dalawang ring R is the R
nucleus. Mga Melatonin and Serotonin.
Isang 6 sided ring tapos 5 sided ring na may
NITROGEN is the indole ring.
Caraway Method – it is chromometric, parang

binibreak down ni Amylase ang carbohydrate
substrate into glucose units so the substrate is fixed.
How much time do you need to completely break up
the carbohydrate with the help of the amylase to its
building blocks? - Chronometric
Chronometric- the time. “Chrono- means time”;
measurement of time.
( If chromometric, that is COLOR, dapat may COLOR
REACTION.
As amylase breaks down the substrate, color appears.
That’s how we know that marami ang amylase sa
sample because there are a lot of color of produced.
*Saccharolytic- it has something to do with lysis or
the breakdown of the substrate with changes in the
color usually IODINE. Starch color with iodine is blue-
black but if starch is broken down into glucose, it will
no longer color blue-black if you put iodine so that’s

how we know that starch has been lysed by the
enzyme amylase.
The words descriptive of the method speaks of what
the reaction principle of the method– principle or the
measurement of the method.
AMYLASE KINETIC IFCC- the fact that it is kinetic it is

delta OD per minute.
HOW TO SOLVE THE DELTA OD PER MINUTE?
SHIHABI et. Al method – it has something to do with

lipase activity wherein the fatty acid in the olive oil ( a
triglyceride) is broken down by lipase into glycerol
and fatty acid (oleic acid).
Shihabi has a color/dye.
PRODUCT MEASURED
IU/L or ukatal/L – these are parameters for reporting

katal activity.
QUIZ 6

CARDIAC ENZYME MARKERS
- These are the enzymes produced in our heart.
- CK is more sensitive cardiac marker than LDH.
- P.S. if the principle is UV, there is NO CHROMOGEN
used and NO COLOR produced.
- We have a chemical reaction but the product
produced does not have a color endpoint but how do
we know that there are changes in absorbance? The
molecule has undergone decrease or increase in
absorbance but not due to the color but it has
something to do with the ACQUISITION of the NEW
FUNCTIONAL GROUP OR A NEW BOND FORMED OR
BOND THAT IS CLEAVED. That is why we have this so
called reaction that is increasing or decreasing.
- If decreasing, a huge molecule is cleaved by the
enzyme that is why it becomes smaller.
- For UV, the changes can ONLY BE DETECTED by the
MACHINE. Our eyes cannot detect it
- If the reaction is increasing, the enzyme facilitated a
product that binds with the reagent that we included
that is why increase in absorbance.
- LDH has 2 types of reaction: the L and the P
- IFCC stands for International Federation of Clinical
Chemistry and Laboratory Medicine.

LIPASE:
*If nauna ang sample tapos nahuli ang substrate that
is substrate start.
*Kapag nauna ang substrate or reagents kaysa sa
sample that is sample start.
*U/L – conventional unit
*If L-type lactate ang substrate, pyruvate ang product
also an increasing reaction because NAD becomes
NADH. *Meaning the OD increases from OD1 to OD2
to OD3.
*If P type, pyruvate to lactate that is decreasing
because NADH becomes NAD. So OD1 is higher than
OD2 and OD3.
QUIZ 8
1. Make a 5-step procedure on how to properly collect a
24-hour urine sample.

*Be particular with the first step. If you begin with
the container, describe the container that it should
be wide mouthed, malaki, clean and dry and with
preservative, then empty the bladder . There should
be time like for example 6 am or 5 am, empty the
bladder then collect all the successive samples until
24- hour but in between it should be refrigerated.
Everytime you collect get it in the refrigerator,
urinate , void in the container with the preservative,
mix very well return to the refrigerator until you will
urinate again. It is critical to be put into the
refrigerator or container on ice. For the last, you
include the last urine .
*You can write any as long as it is complete. The
refrigeration, the preservation, containers
description,emptying the bladder first and the last
collected urine.EVEN AFTER 24 HOURS, umihi siya
tapos malapit sa 24 hrs., it should be included.
*The 24-hour collection is problematic because it is
not perfectly 24-hours. Because you cannot force
yourself to urinate at the same time after 24 hrs.
unless if the patient is catheterized because at 6
o’clock, pwede mo nang putulin or alisin ang

catheterized na part but usually it is not removed
because it is difficult to insert a catheter, the patient
is prone to infection. Minsan nakakalimutan ang 24
hour collection may mga ihi na tulo tulo pa tapos
bumawas nayan.
2. What are the preparatory requirements for OGTT?

Cite at least 5.
• No smoking
• No exercise
• Heavy carbohydrate meal 5 days before the test.
• If Hypoglycemia, you should have a point below 50
mg/Dl
• You cannot say that it is hypoglycemia unless
below 50.
• Normal blood glucose level is 70-110 mg/Dl
• It is not okay that you do not have fasting.
• We have oral glucose load that is 75 g. Before you
drink it, determine first your fasting glucose level.
So if you have HYPOglycemia, the STARTING point
should be BELOW 50.

• If it is a normal curve, fasting should be in a normal
range . that should be 70-100 tapos after 1 hour it
will peak but it SHOULD NOT EXCEED 200(mga 150-
180 lang)
• The glucose you are taking in 75 g so meaning it is
75,000 grams. The load of glucose that you take in
will trigger you to urinate.
• When you do OGTT plotting, the patient after 3
hours or 2 hours, it will urinate because of too
much solute( glucose that you are taking in) .
• If you are not diabetic, the blood sugar in your
urine should be negative. If positive after a load,
there is a possibility that you are in borderline.
• If borderline, you already have 200.
• Moderate Diabetes, you don’t have a plot that
reaches 300.
• Severe diabetes, it should exceed 300 mg/Dl
• Fasting blood glucose level should be below
50mg/Dl.
• If your blood sugar does not exceed 180, but your
urine is positive for sugar, then INVALIDATE your
test.( Glucose renal threshold 160-180 mg/dl)
because it contains Vitamin C interference because
d mo nalampasan ang renal threshold.

• If may isang point sa graph na nalagpasan ang 180
(for blood glucose) , positive na ang urine for
glucose because you already surpassed the renal
threshold.
• If pre-diabetic the highest blood pressure you have
measured is after 1 ½ hours or 90 minutes na
malapit ka sa 200 , lumampas ka sa 180. So the
urine will become positive. You have positive urine
test for glucose within the 2-hour OGTT or 3-hour
OGTT.
• MODERATE DM, wala pang 2 hours, lumampas
kana sa 200. But you do not exceed 300.
• Severe, as early as 1 hour kasi ang start mo , your
fasting glucose level is 200 tapos inom kappa 75 g
glucose, lumampas na sa 300.
3. Draw a single OGTT graph with correct labels of x-and

y- axes showing 5 separate curves depicting
hypoglycemia, normal, borderline or pre-diabetic,
moderate DM and severe DM.

OGTT is a test for insulin activity which is a hormone.
4. Give the reagents used for the Porter-Silber reaction
with the following purposes:
HYDROLYSIS,EXTRACTION, PURIFICATION AND
ESTIMATION.
5. Give the reagents used for the Zimmermann reaction

with the following purposes: HYDROLYSIS,
EXTRACTION, PURIFICATION AND ESTIMATION.
6. Give the reagents used for the Kober reaction with

the following purposes: HYDROLYSIS,EXTRACTION,
PURIFICATION AND ESTIMATION.
NOTE:
HEPE – mnemonic device in measuring hormones in
24-hour urine. Usually in sample analysis is 24-hour
urine sample that is why in the first activity in
endocrinology part

*Hydrolysis- you can use an enzyme or you can use
an acid.
*What are being hydrolyzed? The hormones that
are bound to glucuronic acid sa sulfate; naka bind
sa conjugating substances. Remember, ang mga
naka conjugate na materials are non-polar like
bilirubin that is conjugated in the liver with
glucuronic acid (B2) similarly because our steroids
are cholesterol-derived which is a lipid nonpolar,
dapat I hydrolyze ang transport polar parts like
glucuronic kaya sa procedure, like Porter-Silber,
glucuronidase(hydrolysis reagent; hydrolysant) .
*EXTRACTION: It uses a solvent kung saan miscible
ang steroid . Choose organic materials like alcohol,
acetone, ether. Usually they are the extractant of
lipid-soluble substances na ang steroids natin doon
na belong. Organic solvents that are miscible with
nonpolar steroids.
*PURIFICATION: there are many substances that
interfere with the reaction. To remove interfering
substances. Alkalizing or the addition of this
alkaline earth . Maadsorb ang interfering
substances. Yan yung purification para ang matira

nalang is purified substance that we wanted to
measure. And will react with our porter-silber
reagents.
*ESTIMATION: this refers to the colored-product.

Porter-Silber – yellow
Zimmerman- (red-violet(?))
Kober-Reaction – reddish-brown?
Has something to do with the reaction of Vaniline?
ENUMERATE THE HORMONES THAT WILL GIVE A

POSITIVE TEST FOR
1. Kober reaction – estrogens
2. Pisano, et al. method – Catecholamines ( those that
have catechol ring)
3. Porter-Silber reaction- chemicals that have
HYDROXYL group in Carbon 17 of sterane nucleus
4. Zimmermann reaction – 17-ketosteroids( look for
chemicals that have keto or carbonyl group sa carbon
17 nang structure nila; ANDROGENS AND
ESTROGENIC precursors.

Laboratory SOP and Safety Guidelines
You will be doing many laboratory activities which require the use of hazardous chemicals. Safety
in the laboratory is the #1 priority for students and teachers. To ensure a safe laboratory, a list of
rules has been developed and provided to you. These rules must be followed at all times. Your
success will depend on your attitude and conduct. If you work with an attitude of rushing through,
you will profit but little. An interest in your work, an understanding of its purpose and a clear
interpretation of your results are necessary factors for a good laboratory course. The clinical
chemistry laboratory is a safe place to experiment if you are careful. You must assume
responsibility of the safety of yourself and your co-lab workers. The following are some safety
and procedural rules to help guide you in protecting yourself and others from injury in the
laboratory.
(A) General Guidelines
1. Conduct yourself in a responsible manner at all times in the laboratory.

2. Be familiar with your lab assignment before you come to lab. Follow all written and verbal
instructions carefully. If you do not understand a direction or part of a procedure, ask the
teacher before proceeding.
3. Never work alone. No student may work in the laboratory without an instructor present.
4. When first entering a laboratory room, do not touch any equipment, chemicals, or other
materials in the laboratory area until you are instructed to do so.
5. Do not eat food, drink beverages, or chew gum in the laboratory. Do not use laboratory
glassware as containers for food or beverages.
6. Perform only those experiments authorized by the instructor. Never do anything in the
laboratory that is not called for in the laboratory procedures or by your instructor. Carefully
follow all instructions, both written and oral. Unauthorized experiments are prohibited.
7. Safety goggles and gowns must be worn whenever you work in lab. Gloves should be worn
whenever you use chemicals that cause skin irritations or need to handle hot equipment.
8. Observe good housekeeping practices. Work areas should be kept clean and tidy at all times.
Bring only your laboratory instructions, worksheets, and/or reports to the work area. Other
materials (books, purses, backpacks, etc.) should be kept away from the working area.
9. Know the locations and operating procedures of all safety equipment including the first aid kit,
and fire extinguisher. Know where the exits are located.
10. Be alert and proceed with caution at all times in the laboratory. Notify the instructor
immediately of any unsafe conditions you observe.
11. Dispose of all chemical waste properly. Never mix chemicals in sink drains. Sinks are to be used
only for water and those solutions designated by the instructor. Solid chemicals, metals,

matches, filter paper, and all other insoluble materials are to be disposed of in the proper waste
containers, not in the sink. Check the label of all waste containers twice before
2
adding your chemical waste to the container. Cracked or broken glass should be placed in the
special container for “Broken Glass.”
12. Labels and equipment instructions must be read carefully before use. Set up and use the
prescribed apparatus as directed in the laboratory instructions provided by your teacher.
13. Keep hands away from your face, eyes, mouth, and body while using chemicals. Wash your
hands with soap and water after performing all experiments. Clean (with detergent powder),
rinse, and dry all work surfaces and equipment at the end of the experiment.
14. Experiments must be personally monitored at all times. You will be assigned a laboratory
station at which to work. Do not wander around the room, distract other students, or interfere
with the laboratory experiments of others.
15. Students are never permitted in the stockroom, storage rooms or preparation areas unless
given specific permission by their instructor.
16. Know what to do if there is a fire drill during a laboratory period; containers must be closed,
gas valves turned off, fume hoods turned off, and any electrical equipment turned off.
17. If you spill acid or any other corrosive chemical on your skin or clothes immediately wash area
with large amounts of water (remember that small amounts of water may be worse that no
water at all). After this get the teacher’s attention. The spill kit will be used for spills on floor
or counter-top.
18. At the end of the laboratory session see that: a) main gas outlet valve is shut off b) the water
is turned off c) desk top, floor area, and sink are clean d) all equipment is cool, clean, and
arranged.
(B) Clothing
19. Any time chemicals, heat, or glassware are used, students will wear laboratory goggles. There
will be no exceptions to this rule! Contact lenses should not be worn in the laboratory unless
you have permission from your instructor.
20. Dress properly during a laboratory activity. Long hair, dangling jewelry, and loose or baggy
clothing are a hazard in the laboratory. Long hair must be tied back and dangling jewelry and
loose or baggy clothing must be secured. Shoes must completely cover the foot. No sandals
are allowed.
(C) Accidents and Injuries

21. Report any accident (spill, breakage, etc.) or injury (cut, burn, etc.) to the instructor immediately,
no matter how trivial it may appear.
22. If you or your lab partner are hurt, immediately get the instructor's attention. Everyone should
turn off burners and prepare to help if needed.
3
23. If a chemical should splash in your eye(s), immediately flush with running water for at least 20
minutes. Notify the instructor immediately.
(D) Handling Chemicals
24. All chemicals in the laboratory are to be considered dangerous. Do not touch, taste, or smell
any chemical unless specifically instructed to do so. The proper technique for smelling chemical
fumes (when instructed to do so by the teacher) is to gently fan the air above the chemical
toward your face. Breathe normally.
25. Check the label on chemical bottles twice before removing any of the contents. Take only as
much chemical as you need. Smaller amounts often work better than larger amounts. Label
all containers and massing papers holding dry chemicals.
26. Never return unused chemicals to their original containers.
27. Never use mouth suction to fill a pipet. Use a pipet bulb or pipet filler or rubber aspirator.
28. Acids must be handled with extreme care. ALWAYS ADD ACID SLOWLY TO WATER, with
slow stirring and swirling, being careful of the heat produced, particularly with sulfuric acid.
29. Handle flammable hazardous liquids over a pan to contain spills. Never dispense flammable
liquids anywhere near an open flame or source of heat.
30. Never take chemicals or other materials from the laboratory area.
31. Take great care when transferring acids and other chemicals from one part of the laboratory to
another. Hold them securely and in the method demonstrated by the teacher as you walk.
(E) Handling Glassware and Equipment
32. Inserting and removing glass tubing from rubber stoppers can be dangerous. Always lubricate
glassware (tubing, thistle tubes, thermometers, etc.) before attempting to insert it in a stopper.
Always protect your hands with towels or cotton gloves when inserting glass tubing into, or
removing it from, a rubber stopper. If a piece of glassware becomes "frozen" in a stopper, take
it to your instructor for removal.
33. When removing an electrical plug from its socket, grasp the plug, not the electrical cord. Hands
must be completely dry before touching an electrical switch, plug, or outlet.
34. Examine glassware before each use. Never use chipped or cracked glassware. Never use dirty
glassware. Do not immerse hot glassware in cold water; it may shatter.

35. Report damaged electrical equipment immediately. Look for things such as frayed cords,
exposed wires, and loose connections. Do not use damaged electrical equipment.
36. If you do not understand how to use a piece of equipment, ask the instructor for help.
(F) Heating Substances

4
37. SHOULD THE BUNSEN BURNER GO OUT, IMMEDIATELY TURN OFF THE GAS AT THE
GAS OUTLET VALVE. If you wish to turn off the burner, do so by turning off the gas at the
gas outlet valve first, then close the needle valve and barrel. Never reach over an exposed
flame. Light gas burners only as instructed by the teacher.
38. Never leave a lit burner unattended. Never leave anything that is being heated or is visibly
reacting unattended. Always turn the burner or hot plate off when not in use.
39. You will be instructed in the proper method of heating and boiling liquids in test tubes. Do
not point the open end of a test tube being heated at yourself or anyone else.
40. Heated metals, glass, and ceramics remain very hot for a long time. They should be set aside
to cool and then picked up with caution. Use tongs or heat-protective gloves if necessary.
Determine if an object is hot by bringing the back of your hand close to it prior to grasping it.
I, ______________________________________, have completely read and understood the

Laboratory SOP and Safety rules and guidelines. I also hereby promise to abide by
these rules and guidelines for the entire duration of MT 103 course under the
supervision of ______________________________________.
Signed on ________________________ , of the ________________ semester of academic year
________________.
___________________________________
Signature

5
LIVER FUNCTION TESTS
There is a hefty number of tests that can be performed to assess liver disorders because of the
many metabolic activities that the liver does. Most commonly, laboratory tests that assess the
levels of bilirubin in body fluids are performed to diagnose liver diseases. The type of bilirubin
that is present in clinical samples are also determined to verify whether the jaundice of a patient
is pre-hepatic, hepatic, post-hepatic or a combination. Measurement of glucose and other
carbohydrates, variety of lipids, and total protein as well as specific types of protein also are liver
function tests. In this portion, the more common hepatic enzyme markers are given.
Exercise No. 1
Bilirubin by Malloy & Evelyn Method (Hilado, 1996)
I. Objective: To learn the method and technic of determining bilirubin level in serum.
II. Materials and Reagents:
Serologic pipet, 1 ml, 5 ml, 10 ml

Volumetric pipet, 1 ml, 2 ml
Test tubes, 10 x 100 mm
Diazo Reagent
Bilirubin Standard
Methyl Alcohol, absolute, reagent grade
Serum
III. Procedure:
Principle: Total bilirubin is measured by adding methanol which permits reactions of indirect (and
direct) bilirubin with Ehrlich’s diazo reagent and water. Indirect bilirubin is estimated by
subtracting the direct bilirubin value from the total level.
1. Dilute 1 ml of serum to 10 ml. with distilled water.

2. Prepare four test tubes as follows:
A. 3.0 ml of water and 0.5 ml of diazo blank
B. 3.0 ml of water and 0.5 ml of diazo reagent
C. 3.0 ml of methyl alcohol and 0.5 ml of diazo blank
D. 3.0 ml of methyl alcohol and 0.5 ml of diazo reagent
3. Determination of the Total Bilirubin level.

Add 2.0 ml of serum (diluted 1:10) to tubes C and D. Mix and read absorbance after 30
minutes.
4. Determination of the Direct (conjugated) Bilirubin level.

6
Add 2.0 ml of serum (diluted 1:10) to tubes A and B. Mix and read absorbance after
exactly 1 minute.
5. Optical Density is determined at 540 in any standard photometer.
Calculation:
a.) O.D. of Unknown – O.D. of Blank x Conc. of The Standard = mg % bilirubin
O.D. of Standard
b) A standard curve may be prepared and the results read directly from the curve. The
optical density for this reading is that of the unknown minus the blank (see
standardization)
NOTE: Sera with bilirubin levels higher than 50 mg% must be diluted for accurate results to be
obtained.
Standardization:
1. Bilirubin Stock Solution (10 mg per 100 ml)
Dissolve 10 mg pure bilirubin in approximately 50 ml of chloroform with slow heating
on a hot plate. After cooling to room temperature the solution is diluted with
chloroform to exactly 100 ml and stored in a brown bottle in a refrigerator.
2. Standards
To give this a standard equivalent to a serum with a concentration of 2 mg per 100 ml
(diluted to 10), the stock solution is diluted 1:50 with chloroform. To make an
equivalent to 4 mg per 100 ml, the stock solution is diluted 1:25 and to give equivalent
to 10 mg per 100 ml, the stock solution is diluted 1:10 and so forth.
3. Results are expressed in mg of total bilirubin (procedure 3) and of direct bilirubin

(procedure 4). Indirect bilirubin may be calculated as the “total” minus “direct”
Precautions: Reagents should be added exactly in the proportions and orders listed. Deviations
from the procedure may result in protein precipitation, which may invalidate the results.
Normal Values:
Total Bilirubin: 0.2-1.0 mg%

Direct Bilirubin: 0.0-0.2 mg%
Indirect Bilirubin: 0.2-0.8 mg%
Preparation of Reagents:
1. Methyl alcohol, absolute, reagent grade
2. Diazo Blank Solution (dilute hydrochloride acid)
3. Diazo Reagent
a. Solution A
7
To 985 ml of distilled water, add 15 ml of hydrochloric acid. Then add 1 gm of

sulfanilic acid. Stir until dissolved.
b. Solution B
Dissolve 0.5 gm sodium nitrite in 100 ml of distilled water. Refrigerate.
c. To prepare the Diazo Reagent:
Add 3 ml of solution B to 10 ml of Solution A. Mix. This should be prepared daily.
Observation and Results:

Readings O.D.
A. Direct Blank
B. Direct Test
C. Total Blank
D. Total Test
Calculation:
mg% Direct Bilirubin
mg% Total Bilirubin
mg% Indirect Bilirubin
Remarks: ______________________________________________________________
________________________________________________________________
Guide Questions:
1) What type of bilirubin is elevated in the following hepatic disorders:
1.1. Gilbert’s disease
1.2. Crigler-Najjar syndrome
1.3. Acute hepatitis

1.4. Hemolytic anemias
1.5. Rotor’s syndrome
1.6. Dubin-Johnson syndrome
1.7. Hepatolenticular degeneration
1.8. Cholelithiasis
1.9. Viral hepatitis
1.10. Biliary cirrhosis
2) What laboratory tests may be performed to assess each of the following liver tasks?
2.1. Excretory function
2.2. Storage function
2.3. Immune function
8
2.4. Carbohydrate metabolism

2.5. Protein metabolism
2.6. Lipid metabolism
Exercise No. 2
Icterus Index by Meulengracht (Hilado, 1996)
I. Objective: To learn the method and technic of determining serum icterus index.
II. Materials and Reagent:

Serologic pipet, 20 ml
Volumetric pipet, 1 ml
Test tubes, 10 x 100 nm
Potassium dichromate Standard
Serum
III. Procedure:
Principle: The intensity of the golden yellow color imparted to the serum by bilirubin is measured
by comparing the serum color with the color of standard solution of dichromate standard. One
unit of Icterus Index has been defined as the color of a 1:10,000 solution to potassium dichromate.
1. Transfer 1 ml of unhemolyzed serum to a test tube and add 9ml of phosphate buffer
solution, pH 7.4
2. Invert several times to mix. Transfer to a cuvet and read OD at 415 nm.

3. If the reading is high, transfer to 1 ml of the mixture to another test tube and dilute to
10 with distilled water. Mix and read using the same wavelength.
4. Read the working potassium dichromate standard setting the instrument at zero against
distilled water.
Calculations:
A. O.D. of Unknown x 10 = Icterus Index units
O.D. of Standard
Notes:
1. It is essential that the serum used for the Icterus Index determination is absolutely free
of visible hemolysis and chyle.
2. The patient should not eat food items which contain yellow pigment for 14 to 48 hours
prior to the drawing of blood for the test.
Normal Values:
Icterus Index: 4-6 Units
9
1. Standard Stock Solution
Dissolve 1 gm of potassium dichromate in 90 ml of distilled water. Add 0.1 ml of
concentrated sulfuric acid and dilute to 100 ml. Keep in brown bottle.
2. Standard Working Solution

Dilute 10 ml of Stock Standard Solution to 1L with distilled water. This makes a
1:10,000 solution of potassium dichromate. The intensity of the yellow color of this
solution represents one Icterus Index Unit.
3. Phosphate Buffer Solution, pH 7.4

Into a flask, place 23.16 gm monopotassium dihydrogen phosphate. Add 400 ml
distilled water and dissolve. Add 4.8 gm of sodium hydroxide reagent grade. Dissolve.
Check pH and adjust if necessary. Transfer with rinsings to a 500 ml volumetric flask.
Dilute to the graduation mark. Store in refrigerator.

Readings: O.D.
Unknown
Standard
Calculations:
Units of Icterus Index = ____________________________

Remarks: _________________________________________________________________
___________________________________________________________________
Guide Questions:
1) What is the clinical significance of the Icterus Index?
2) What are some of the causes of positive bias in Icterus Index determination? How
about sources of negative bias?
Exercise No. 3
BILIRUBIN DIRECT/TOTAL (Dialab) (Dialab product insert, 2007)
Diagnostic reagent for quantitative in vitro determination of direct and total bilirubin in human
serum or plasma on photometric systems.
TEST PRINCIPLE
10
Bilirubin is formed from the heme portion of hemoglobin released by aged or damaged red blood
cells. It is then converted in the liver into bilirubin monoglucuronide and bilirubin diglucuronide.
Free bilirubin is not soluble in aqueous solution and requires solubilization by alcohols or other
solvents to react. Reactions carried out in these solvents provide measurements of total bilirubin.
Mono- and diglucuronides of bilirubin are soluble in water and measurements performed in
aqueous solution measure what in this form is called direct bilirubin.
In Dialab kit, DMSO and ethylene glycol are solvents for total bilirubin assay. Bilirubin in these
solvents readily react with diazotized sulfanilic acid to produce an intensely colored diazo dye.
The intensity of color of this dye in solution is proportional to the concentration of direct or total
bilirubin.
TEST PARAMETERS
Method: Colorimetric, Increasing reaction, Endpoint, Jendrassik-Grof method
Wavelength: 555 nm
Temperature: 20-25˚C, 37˚C
Sample: serum or plasma
Linearity: up to 20 mg/dL Total bilirubin

REAGENT COMPOSITION
Components Concentration
Bilirubin Direct, Reagent 1
Sulfanilic acid 32.2 mmol/L
Bilirubin Total, Reagent 2
Sulfanilic acid 32.2 mmol/L
Ethylene glycol
Dimethylsulfoxide (DMSO)
Reagent 2
Sodium nitrite 109 mmol/L
REAGENT PREPARATION
Substrate start: Reagents are ready for use.
Sample start: Working reagent is made by mixing 150 parts of reagent 1 with 1
part of reagent 2. It is stable for 8 hours at 20-25˚C in amber
bottles.
REAGENT STABILITY & STORAGE

Conditions: protect from light; close immediately after use
Storage: 2-8˚C
11
Stability: up to the expiration date
INTERFERING SUBSTANCES No
interference up to:
Hemoglobin………………………1000 mg/dL
MANUAL TEST PROCEDURE

Bring reagents and samples to room temperature.
SAMPLE START
Pipet into Sample Sample Calibrator Calibrator
test tubes blank Blank
Reagent 1 2000 uL - 2000 uL -
Working - 2000 uL - 2000 uL
reagent
Sample 200 uL 200 uL - -
Calibrator - - 200 uL 200 uL

Mix without delay. Incubate for 3 minutes at 30˚C or 2 minutes at 37˚C. Read
absorbance of each test against the respective blank.
SUBSTRATE START
Pipet into Sample Sample Calibrator Calibrator
test tubes blank Blank
Reagent 1 2000 uL 2000 uL 2000 uL 2000 uL
Sample 200 uL 200 uL - -
Calibrator - - 200 uL 200 uL
Reagent 2 - 20 uL - 20 uL
Mix without delay. Incubate for 3 minutes at 30˚C or 2 minutes at 37˚C. Read
absorbance of each test against the respective blank.
CALCULATION (light path 1 cm)
With Calibrator
Bilirubin (mg/dL) = ∆A Sample X Concentration of Calibrator (mg/dl)
∆A Calibrator
With Factor
Bilirubin (mg/dL) = ∆A Sample X Factor
Factor = 12.9
NOTE: The factor has to be checked by a calibration serum and adapted if necessary.
UNIT CONVERSION
mg/dL X 17.1 = umol/L
REFERENCE RANGE
Conjugated (direct) bilirubin 0.0 – 0.2 mg/dL
Unconjugated bilirubin 0.2 – 0.8 mg/dL
Total bilirubin 0.2 – 1.0 mg/dL
12
WARNINGS AND PRECAUTIONS

Reagent 1 of bilirubin total contains ethylene glycol that is harmful if inhaled,
swallowed or in contact with skin and eyes. In case of eye contact, rinse immediately
with plenty of water and seek medical advice.
Exercise No. 4
BILIRUBIN DIRECT/TOTAL (EliTech Clinical Systems product insert, 2012)

Diagnostic reagent for quantitative in vitro determination of direct and total bilirubin in human
serum or plasma on photometric systems. For professionals only.
CLINICAL SIGNIFICANCE
Approximately 80-85% of the bilirubin produced is formed from the heme portion of hemoglobin
released by aged or damaged red blood cells in the reticuloendothelial system. Bilirubin, bound
to albumin, is transported into the liver where it is rapidly conjugated with glucuronide to increase
its solubility. Then it is excreted into the biliary canaliculi , and hydrolyzed in the gastrointestinal
tract.
Unconjugated bilirubin serum concentration is elevated in cases of overproduction of bilirubin
(acute or chronic hemolytic anemias) and in case of disorders of bilirubin metabolism and
transport defects (impaired uptake of liver cells: Gilbert’s syndrome; defects in the conjugation
reaction: Crigler-Najjar syndrome). Reduced excretion (hepatocellular damage, hepatitis, cirrhosis;
Dubin-Johnson and Rotor syndrome) and obstruction to the flow of bile (most often produced
by gallstones or by tumours) induce an important elevation of conjugated bilirubin and in a minor
extent an increase of conjugated bilirubin (conjugated hyperbilirubinemia).
TEST PRINCIPLE
Sulfanilic acid reacts with sodium nitrite to form diazotized sulfanilic acid. In the presence of
accelerator cetrimide, conjugated and unconjugated bilirubin react with diazotized sulfanilic acid
to form azobilirubin (total bilirubin). In the absence of cetrimide, only conjugated bilirubin reacts
(direct bilirubin).
Sulfanilic acid + NaNO2 ------------------------- Diazotized sulfanilic acid
Bilirubin + Diazotized sulfanilate ------------------ acid Azobilirubin
TEST PARAMETERS
Method: Colorimetric, Endpoint, Modified Evelyn-Malloy method
Wavelength: 550 nm
Temperature: 37˚C
Sample: unhemolysed serum or heparinized plasma ( properly filled according to

manufacturer)
13
Protected from light, samples are stable for 2 days at RT and 4 days at 4oC and even
longer if frozen.
Linearity: 0.3 to 20 mg/dL (5.1-342.1 uM) Total bilirubin
REAGENT COMPOSITION

Bilirubin Direct, Reagent 1
Sulfanilic acid 29 mmol/L
Hydrochloric acid 67 mmol/L
Bilirubin Total, Reagent 1
Sulfanilic acid 29 mmol/L
Hydrochloric acid 67 mmol/L
Cetrimide 37 mmol/L
Reagent 2: Total/Direct
Sodium nitrite 5.8 mmol/L
REAGENT PREPARATION
Reagents are ready for use.

Storage: 2-8˚C

SAMPLE START
Pipet into Total Total Direct Direct
test tubes Bilirubin Bilirubin Bilirubin Bilirubin
Sample Standard Sample Standard
Reagent 1 1600 uL 1600 uL - -
Total
Reagent 1 - - 1600 uL 1600 uL
Direct
Sample 200 uL - 200 uL -
Calibrator - 200 uL - 200 uL
Mix without delay. Incubate for 5 minutes at 37˚C. Read A 1 of each test against the
water blank.
Reagent 2 Add 400 uL into each tube.
Mix and read A2 after 5 minutes of incubation.
∆A = A2 – A1
Bilirubin (mg/dL) = ∆A Sample X Concentration of Calibrator (mg/dl)
∆A Calibrator
14

UNIT CONVERSION mg/dL X
17.10 = umol/L
REFERENCE RANGE
Conjugated (direct) bilirubin < 0.2 mg/dL (<3.4 uM)
Total bilirubin 0.3 – 1.2 mg/dL (5-21 uM)

Reagent 1 of bilirubin total contains irritant that is harmful if inhaled, swallowed or in
contact with skin and eyes. In case of eye contact, rinse immediately with plenty of
water and seek medical advice. Sulfanilic acid may produce allergic reactions.
15
Serum Transaminases
This is one of the most important representatives of a group of enzymes, the transferases or
transaminases, which catalyzes the transfer of an amino group from alanine to a a-ketoglutamic
acid forming glutamic and pyruvic acid.
As a liver specific enzyme ALAT is only significantly elevated in hepatobiliary diseases. A parallel
measurement of ALAT and ASAT is therefore applied to distinguish liver form heart or skeletal
muscle damages. The ASAT/ALAT ratio is used for differential diagnosis in liver diseases. While
ratios <1 indicate mild liver damage, ratios >1 are associated with severe, often chronic liver
diseases.
Exercise No. 5
Reitman & Frankel Method for ALT/sGPT (Annino, 1964)
I. Objectives: To learn the technics in performing two methods of determining serum

ALT/sGPT.

Test tubes, 10.0mL
Thermometer
Water bath
Pipets; 2.0mL; 5.0mL; 10.0mL
Cuvets
Spectrophotometer
Substrate, ALAT

Phosphate buffer, pH 7.4
Color reagent
0.4N Sodium hydroxide
Normal Control Serum
III. Procedure: Reitman and Frankel Method
Principle: Alanine aminotransferase catalyzes the transfer of an amino group from alanine to
aketoglutaric acid forming pyruvic and glutamic acid. The pyruvic acid reacts with dinitrophenyl
hydrazine in the presence of an alkaline to form a brown color, which is measured
spectrophotometrically.
1. Into each of two 10.0mL test tubes, measure 1.0mL of substrate. Label one tube “Blank” and
the other “Test.”
2. Warm both tubes in a 37 deg Celsius water bath for 5 minutes.
16
3. Take out both tubes from the water bath. To the blank, pipet 0.2mL distilled water and to
the test, pipet 0.2mL serum. Swirl to mix and return to the water bath. Incubate for 30
minutes.
4. Remove the tubes from the water bath and add 0.5mL color reagent. Mix and allow it to
stand for 20 minutes.
5. Add 5.0mL 0.4N Sodium hydroxide to both tubes. Stopper and invert to mix.
6. Allow to stand for 5 minutes.
7. Set the “blank” at 60% T and read the transmittance of the “test.”
Calculation.
Determine value of “test” from the calibration curve. If the value is above 200 units repeat the
testing using 0.2mL of a 1:5 dilution. Multiply the result by 5.
Preparation of Standard Curve.
Dissolve 20mg of pure sodium pyruvate (pyruvate standard) in 100mL phosphate buffer (pH7.4)
Set-up tube as follows:
Standard (mL) ALAT Substrate (mL) Water (mL) ALAT units
0 1.0 0.2 Blank
0.1 0.9 0.2 28
0.2 0.8 0.2 57
0.3 0.7 0.2 97

0.4 0.6 0.2
To each tube add 1.0mL color reagent. Allow to stand for 20 minutes. Add 10mL 0.4N sodium
hydroxide. Invert to mix. Let it stand for 5 minutes. Set the blank at 60% and read %T of each tube
at 505nm. Plot %T versus concentration in a graphing paper.
Reference Values: Up to 30U/mL
Results:
Exercise No. 6
Optimized UV Method for ALT/sGPT (Annino, 1964)
Principle: L-Alanine + 2-Oxoglutarate ALAT L-Glutamate + Pyruvate
Pyruvate + NADH + H+ LDH D-lactate + NAD+
Addition of pyridoxal-5-phosphate (P-5-P) stabilizes the transaminase and avoids falsely low
values in samples containing insufficient endogenous P-5-P such as in patients with myocardial
infarction, liver disease and intensive care patients.
17
Reagents: R1: TRIS pH 7.15 100mmol/L

L-Alanine 500mmol/L
Lactate dehydrogenase about 1700U/L
R2: 2-Oxoglutarate 15mmol/L
NADH 0.18mmol/L
Pyridoxal-5-Phosphate
Good’s buffer pH 9.6 0.7mmol/L Pyridoxal-5-phosphate 0.09mmol/L
Storage Instruction and Reagent Stability:
The reagents are stable up to the end of the indicated month of expiry, if stored at 2-8 deg Celsius,
protected from light and contamination is avoided. Do not freeze reagents. Reagent Preparation:
Substrate start
The reagents are ready to use.
For the determination with pyridoxal-5-phosphate, mix 1 part of p-5-p with 100 parts of reagent
1. Example 100uL P-5-P + 10mL of Reagent 1.
Stability after mixing is 6 days stored at 2-8 deg Celsius or 24 hours stored at room temperature.

Sample start
Without P-5-P
Mix 4 parts of R1 + 1 part R2. Example 20mL R1 + 5mL R2 = monoreagent
Stability after mixing is 4 weeks stored at 2-8 deg Celsius or 5 days stored at room temperature.
Note: The monoreagent must be protected from light.
Sample: Serum, heparin or EDTA plasma (Lost of activity within three days)
Procedure: Optimized UV-test according to IFCC (International Federation of Clinical Chemistry

and Laboratory Medicine)
1. Prepare three 5mL test tubes and label “blank”,“control,” “test.”

2. Make a monoreagent by measuring 500uL of R2 and 2000uL of R1 into a test tube. Swirl
to mix and let it stand for 10 minutes.
3. Measure two 1000uL monoreagent and put them into the “control” and “test” tubes.
4. Add 100uL of control serum into the “control” tube. And add 100uL of test serum to the
“test” tube.
5. Swirl and let it stand for 1 minute, and then feed into the microlab 300. Read at 340nm.
18
Test tubes ALAT Value

Blank
Control
Test
Calculations:
Absorbance x factor = ALAT activity (U/L)
Remarks:
Guide Questions:
1. Mention other methods for ALT determination. Explain the principle of the method.
2. Show the reaction involved in the assay of ALT.
3. Explain the significance of the ALT assay.

Serum AST/sGOT
It is a tissue enzyme that catalyzes the transfer of amino and keto groups between a-amino acids
and a-keto acids; hence, it is a transferase. Methods for determination of this enzyme employ
aspartic acid and oxalacetic acid. In the classic method the oxalacetic acid formed oxideizes the
coenzyme diphosphopyridine nucleotide (reduce form of DPNH) in the presence of malic
dehydrogenase. The DPNH, which absorbs ultraviolet light at 340nm is converted to DPN, which
does not absorb ultraviolet light; therefore the conversion results in a lowering of absorbance as
measured in the ultraviolet region. This procedure is tedious and timeconsuming, employs
unstable reagents and requires the use of an ultraviolet spectrophotometer.
In a simpler procedure the oxalacetic acid is decarboxylated with aniline citrate to form pyruvic
acid. The pyruvic acid then combines with dinitrophenylhydrazine to form a
pyruvatedinitrophenylhydrazone, which is then extracted with toluene and measured
colorimetrically in an alkaline medium.
The simplest version is the method that involves in the direct conmbination of oxalacetic acid with
dinitrophenylhydrazine and measurement of the color in an alkaline solution.
Although the ultraviolet procedure is the reference method, the colorimetric method eliminates
the need for an ultraviolet spectrophotometer and lends itself more readily to multiple analyses
while giving results which compare favorably with the ultraviolet technique.
19
Exercise No. 7
Reitman and Frankel Method for AST/sGOT (Annino, 1964)

AST/sGOT.

Test tubes, 10.0mL
Thermometer
Water bath
Pipets; 2.0mL; 5.0mL; 10.0mL
Cuvets
Spectrophotometer
Substrate, ASAT
Phosphate buffer, pH 7.4

Color reagent
Normal Control Serum
Principle: Aspartate aminotransferase catalyzes the transfer of an amino group from aspartic acid
to a-ketoglutaric acid forming oxalacetic acid and glutamic acid. The oxalacetic acid reacts with
dinitrophenylhydrazine in the presence of an alkaline to form a brown color, which is measured
spectrophotometrically.
III. Procedure:
1. Into each of two 10.0mL test tubes, measure 1.0mL of substrate. Label one tube “Blank” and
the other “Test.”
2. Warm both tubes in a 37 deg Celsius water bath for 5 minutes.
3. Take out both tubes from the water bath. To the blank, pipet 0.2mL distilled water and to
the test, pipet 0.2mL serum. Swirl to mix and return to the water bath. Incubate for 1 hour.
4. Remove the tubes from the water bath and add 0.5mL color reagent. Mix and allow it to
stand for 20 minutes.
5. Add 5.0mL 0.4N Sodium hydroxide to both tubes. Stopper and invert to mix.
6. Allow to stand for 5 minutes.
7. Set the “blank” at 60% T and read the transmittance of the “test.”
Calculation.
Determine value of “test” from the calibration curve. If the value is above 200 units repeat the
testing using 0.2mL of a 1:5 dilution. Multiply the result by 5.
Preparation of Standard Curve.

20
Dissolve 20.0mg of pure sodium pyruvate (pyruvate standard) in 100mL phosphate buffer
(pH7.4)
Set-up tube as follows
Standard (mL) ASAT Substrate (mL) Water (mL) ALAT units
0 1 0.2 Blank
0.1 0.9 0.2 24
0.2 0.8 0.2 61
0.3 0.7 0.2 114
0.4 0.6 0.2 190
To each tube add 1.0mL color reagent. Allow to stand for 20 minutes. Add 10mL 0.4N sodium
hydroxide. Invert to mix. Let it stand for 5 minutes. Set the blank at 60% and read %T of each tube
at 505nm. Plot %T versus concentration in a graphing paper.

Reference Values: Up to 30U/mL
Exercise No. 8
Optimized UV Method for AST/sGOT (Dialab product insert, 2000)
Principle: L-Alanine + 2-Oxoglutarate ASAT L-Glutamate + Oxalacetate
Oxalacetate + NADH + H+ MDH L-Malate + NAD+

Addition of pyridoxal-5-phosphate (P-5-P) stabilizes the transaminase and avoids falsely low
values in samples containing insufficient endogenous P-5-P such as in patients with myocardial
infarction, liver disease and intensive care patients.
Reagents: R1: TRIS pH 7.15 100mmol/L

L-Aspartate 500mmol/L
Malate dehydrogenase
Lactate dehydrogenase about 1700U/L
R2: 2-Oxoglutarate 15mmol/L
NADH 0.18mmol/L
Pyridoxal-5-Phosphate
Good’s buffer pH 9.6 0.7mmol/L
Pyridoxal-5-phosphate 0.09mmol/L
Storage Instruction and Reagent Stability.

The reagents are stable up to the end of the indicated month of expiry, if stored at 2-8 deg Celsius,
protected from light and contamination is avoided. Do not freeze reagents.
Reagent Preparation.
Substrate start
The reagents are ready to use.
21
For the determination with pyridoxal-5-phosphate, mix 1 part of p-5-p with 100 parts of reagent
1. Example 100uL P-5-P + 10mL of Reagent 1.
Stability after mixing is 6 days stored at 2-8 deg Celsius or 24 hours stored at room temperature.
Sample start
Without P-5-P
Mix 4 parts of R1 + 1 part R2. Example 20mL R1 + 5mL R2 = monoreagent

Stability after mixing is 4 weeks stored at 2-8 deg Celsius or 5 days stored at room temperature.
Note: The monoreagent must be protected from light.
Sample: Serum, heparin or EDTA plasma (Lost of activity within three days)
Procedure: Optimized UV-test according to IFCC (International Federation of Clinical

Chemistry and Laboratory Medicine)
“test” tube.
Test tubes ASAT Value

Blank
Control
Test
Calculations.
Absorbance x factor = ASAT activity (U/L)
Remarks:
Guide Questions:
1. Mention other methods for ASAT determination. How do they differ from the method
given?
2. What precautions must do one observe when performing the assay?
3. Discuss the effect of using hemolyzed serum sample in the test.
4. What condition/s is/are this test important? What is it normal values?
Exercise No. 9
22
GLUTAMATE-PYRUVATE TRANSAMINASE (GPT) (Dialab product insert, 2006)

Diagnostic reagent for quantitative in vitro determination of GPT (ALT) in human serum or plasma
on photometric systems.
TEST PRINCIPLE
NADH (reduced NAD) is oxidized to NAD+ (nicotinamide adenine dinucleotide) the resulting
decrease in absorbance at 340 nm is directly proportional to the activity of GPT in the sample.
GPT
L-alanine + 2-oxoglutarate -------------------- Pyruvate + L-glutamate
LDH
Pyruvate + NADH + H+ -------------------- L-lactate + NAD+
This is a modified formulation for the assay of GPT, as recommended by IFCC (International
Federation of Clinical Chemistry). The IFCC reference method includes pyridoxal phosphate (PP).
PP functions as coenzyme in AA (amino acid) transfer, therefore addition of PP results in increased
enzyme activity. It avoids falsely low values in samples containing insufficient endogenous PP, e.g.
from patients with myocardial infarction, liver disease and intensive care patients.
TEST PARAMETERS
Method: UV, Kinetic, Decreasing reaction, Modified IFCC method
Wavelength: 340 nm, Hg 334 nm, Hg 365 nm
Temperature: 25˚C, 30˚C, 37˚C
Sample: serum, EDTA-plasma, heparinized plasma
Linearity: up to 600 U/L on Hitachi 911
Sensitivity: LOD (limit of detection) of 4 U/L

REAGENT COMPOSITION
Components Final Concentration
Reagent 1
Tris, pH 7.5 100 mmol/L
L-alanine 500 mmol/L
LDH ≥1800 U/L
Reagent 2
2-oxoglutarate 15 mmol/L
NADH 0.18 mmol/L
REAGENT PREPARATION
part of reagent 2. It is stable for 4 weeks at 2-8˚C and 5 days at 15-
25˚C.
23

Conditions: protect from light; close immediately after use; do not freeze
Storage: 2-8˚C
Minimum allowable absorbance of the working reagent measured at 340 nm against water
as reference is 1.6.
SAMPLE STABILITY & STORAGE

Loss of Activity: at 2-8˚C is <10% within 3 days
at 15-25˚C is < 17% within 3 days
Stability: at -20˚C is at least 3 months Discard contaminated
specimens.
interference up to:
Ascorbic acid……………………..30 mg/dL
Bilirubin…………………………….40 mg/dL
Triglycerides……………………2000 mg/dL

SUBSTRATE START
Pipet into test tubes 25 or 30˚C 37˚C
Reagent 1 2000 uL 2000 uL
Sample 400 uL 200 uL
Mix. Incubate for 5 minutes. Then add:
Mix without delay. Read initial absorbance against air after 1
minute and start a timer. Read absorbance again after exactly 1, 2
and 3 minutes.
SAMPLE START
Working Reagent 2000 uL 2000 uL
and 3 minutes.

GPT (U/L) = ∆A/ minute X Factor
24
Factors:
Substrate start 25 or 30˚C 37˚C
At 340 nm 1151 2143
At 334 nm 1173 2184
At 365 nm 2132 3971
Sample start 25 or 30˚C 37˚C
At 340 nm 952 1745
At 334 nm 971 1780
At 365 nm 1765 3235
UNIT CONVERSION
U/L X 0.01667 = ukatal/L
REFERENCE RANGE (U/L)
Without addition of pyridoxal phosphate
25˚C 30˚C 37˚C

males <22 <29 <41
females <17 <22 <31
With addition of pyridoxal phosphate
25˚C 30˚C 37˚C

males - 7-36 10-50
females - 7-25 10-35
Results:
Remarks:
______________________________________________________________________________________________________
____________________________________________

Exercise No. 10
GLUTAMATE-OXALOACETATE TRANSAMINASE (GOT) (Dialab product insert, 2006)
Diagnostic reagent for quantitative in vitro determination of GOT (AST) in human serum or plasma
TEST PRINCIPLE
NADH (reduced NAD) is oxidized to NAD+ (nicotinamide adenine dinucleotide) the resulting
decrease in absorbance at 340 nm is directly proportional to the activity of GOT in the sample.
GOT
25
L-aspartate + 2-oxoglutarate-------------------- Oxaloacetate + L-glutamate

MDH
Oxaloacetate + NADH + H+ -------------------- L-malate + NAD+
This is a modified formulation for the assay of GOT, as recommended by IFCC (International
Federation of Clinical Chemistry). The IFCC reference method includes pyridoxal phosphate (PP).
PP functions as coenzyme in AA (amino acid) transfer, therefore addition of PP results in increased
enzyme activity. It avoids falsely low values in samples containing insufficient endogenous PP, e.g.
from patients with myocardial infarction, liver disease and intensive care patients.
TEST PARAMETERS
Method: UV, Kinetic, Decreasing reaction, Modified IFCC method
Temperature: 25˚C, 30˚C, 37˚C

REAGENT COMPOSITION
Reagent 1
Tris, pH 7.8 80 mmol/L
L-aspartate 240 mmol/L
MDH ≥600 U/L
LDH ≥1200 U/L
Reagent 2
2-oxoglutarate 12 mmol/L

NADH 0.18 mmol/L
REAGENT PREPARATION
part of reagent 2. It is stable for 4 weeks at 2-8˚C and 5 days at
15-25˚C.
unit
Storage: 2-8˚C
Minimum allowable absorbance of the working reagent measured at 340 nm against water
as reference is 1.6.
26

Loss of Activity: at 2-8˚C is <10% within 3 days
at 15-25˚C is < 17% within 3 days
Stability: at -20˚C is at least 3 months Discard contaminated
specimens.
interference up to:
Very high interference with Hemoglobin
SUBSTRATE START
Mix. Incubate for 5 minutes. Then add:
and 3 minutes.

SAMPLE START (NOTE: Do not use sample start with pyridoxal phosphate)
and 3 minutes.

GOT (U/L) = ∆A/ minute X Factor
Factors:
Substrate start 25 or 30˚C 37˚C
At 340 nm 1151 2143
At 334 nm 1173 2184
At 365 nm 2132 3971
Sample start 25 or 30˚C 37˚C
At 340 nm 952 1745
27
At 334 nm 971 1780

At 365 nm 1765 3235
UNIT CONVERSION

Without addition of pyridoxal phosphate
25˚C 30˚C 37˚C

males <18 <25 <37
females <15 <21 <31
With addition of pyridoxal phosphate
25˚C 30˚C 37˚C

males - 7-34 10-50
females - 7-24 10-35
Result:

Remarks:____________________________________________________________________________________________
_________________________________
Alkaline Phosphatase
Alkaline phosphatase (ALP), a hydrolytic enzyme acting optimally at alkaline pH, exists in blood in
numerous distinct forms which originate mainly form bone and liver, but also from other tissues
as kidney, placenta, intestine, testes, thymus, lungs and tumors.
There are suitable methods for its determination that depend upon the liberation (hydrolysis) and
measurement of a simpler compound from a phosphoric ester under controlled conditions. In
one method the inorganic phosphate liberated from sodium glycerol-phosphate is measured;
another method depends upon the liberation of phenolphthalein from sodium phenolphthalein
phosphate; while in still another method, the quantity of beta-naphthol hydrolyzed from sodium
beta-naphthyl phosphate is taken as an indirect measure of phosphatase activity.
Physiologic increases are found during bone growth in childhood and in pregnancy, while
pathological increases are largely associated with hepatobiliary and bone diseases. In
hepatobiliary disease they indicate obstruction of the bile duct as in cholestasis caused by gall
stones, tumors or inflammation. Elevated activities are also observed in infectious hepatitis. In
bone diseases elevated ALP activities originate from increased osteoblastic activity as in Paget’s
disease, osteomalacia (rickets), bone metastases and hyperparathyroidism.
28
Exercise No. 11
Bessey, Lowry & Brock Method for ALP (EliTech product insert, 2000)
I. Objectives: To learn the technics in performing two methods of determining serum alkaline
phosphatase.

Test tubes
Pipets, volumetric; 0.05mL, 5.0mL, 10.0mL
Water bath
Cuvets
Spectrophotometer

Alkaline buffer pH 10.5
p-Nitrophenyl phosphate substrate
Stock p-Nitrophenyl standard, 10.0mmol/L
Concentrated hydrochloric acid
Alkaline phosphatase reagent Set (ELITECH)
Principle: Alkaline phosphatase hydrolyzes p-nitrophenyl phosphate to form inorganic phosphate

and p-nitrophenol. In the presence of an alkali, p-nitrophenol forms a yellow color which is
measured spectrophotometrically. One Bessey Lowry unit of alkaline phosphatase activity is the
amount of enzyme which will release one micromol of substrate per milliliter of serum per hour
at pH 10.5.
III. Procedure:
1. Prepare two test tubes mark “blank” and the other “test”. Place 0.5mL of the buffer substrate
mixture into each tube. Warm for 5 minutes in a 37oC water bath.
2. Pipet 0.05mL distilled water into the tubed marked “blank” and 0.05mL to the “test.”
3. Incubate both tubes for exactly 30 minutes.
4. Remove both tubes out from the water bath and immediately add 5.0mL 0.02N sodium
hydroxide. Mix by inverting the test tubes.
5. Read the absorbance of the test at 410nm setting the spectrophotometer to zero with the
“blank”. Add two drops 0.1mL of concentrated hydrochloric acid to both tubes and repeat
the absorbance reading the second absorbance reading from the first.
6. Refer the corrected reading to the calibration curve to obtain the units of alkaline
phosphatase activity.
Reference Values:
0.7-2.7 BLB units
29
11.7-45.1mU/mL
Results:
Remarks:__________________________________________________________

Exercise No. 12
Kinetic Assay for ALP (Hilado, 1996)
Reaction Principle: p-Nitrophenylphosphate + water –ALP Phosphate + p-Nitrophenol
Reagents: R1: 2-amino-2-methyl-1-propanol pH 10.5 0.35mol/L

Magnesium sulfate 2.0mmol/L Zinc sulfate 1.0mmol/L HEDTA
2.0mmol/L
R2: p-Nitrophenylphosphate 16.0mmol/L
Storage Instructions and Reagent Stability.

The reagents are stable up to the end of the indicated month of expiry, if stored at 2-8 deg Celsius.
Do not contaminate at room temperature. Do not freeze reagents. Reagent 2 must be protected
from light.
Sample: Serum or heparin plasma
Procedure:
“test” tube.
Test tubes ALP Value

Blank
Control
Test
30
Calculations.
Absorbance x factor = ALP activity (U/L)
Remarks: _________________________________________________________

Guide Questions:
1. Compare the two method of alkaline phosphatase determination.
2. What are the advantages and disadvantages of each of the methods?
3. Mention other methods of alkaline phosphatase assay. Discuss their principle.
Exercise No. 13
ALKALINE PHOSPHATASE (ALP) (Dialab product insert, 2011)
Diagnostic reagent for quantitative in vitro determination of ALP in human serum or plasma on
photometric systems.
TEST PRINCIPLE
Under alkaline condition, colorless p-nitrophenol is converted to 4-nitrophenoxide, which
develops a very intense yellow color. Increase of absorbance is proportional to the activity of ALP
in the sample.
ALP
p-nitrophenylphosphate + HOH -------------------- p-nitrophenol + phosphate
TEST PARAMETERS
Method: Colorimetric, Kinetic, Increasing reaction, Optimized DGKC
Wavelength: 405 nm (400-420 nm)
Temperature: 37˚C
Sample: serum, heparinized plasma
REAGENT COMPOSITION
Reagent 1
Diethanolamine, pH 9.8 1.2 mol/L
Magnesium chloride 0.6 mmol/L Reagent 2 p-
nitrophenylphosphate 50 mmol/L 31
REAGENT PREPARATION

Sample start: Working reagent is made by mixing 4 parts of reagent 1 with 1 part of
reagent
2. It is stable for 4 weeks at 2-8˚C and 5 days at 15-25˚C.

Storage: 2-8˚C

Stability: at 4-8˚C 7 days
at 20-25˚C 7 days
at -20˚C 2 months
Discard contaminated specimens.
interference up to:
Hemoglobin……………………..150 mg/dL

SUBSTRATE START
Pipet into test tubes 37˚C
Reagent 1 2000 uL
Sample 200 uL
Mix. Incubate for 1 minute. Then add:

Reagent 2 500 uL

and 3 minutes.

SAMPLE START
Pipet into test 37˚C
tubes
32
Working Reagent 2000 uL
Sample 40 uL

and 3 minutes.

ALP (U/L) = ∆A/ minute X Factor
o
Factors: (37 C)
Substrate start 3433
Sample start 2757
UNIT CONVERSION
Adults (37oC) <258 U/L

Children: Females <448 U/L
Males <935 U/L
Result:
Remarks:
______________________________________________________________________________________________
____________________________

33
Electrolytes
Electrolytes are usually indicated for water and pH balance. The common electrolytes like sodium
and potassium are determined using colorimetric, turbidimetric, flame emission technic, and
electrochemical methods. The trace metals are measured using the atomic absorption
spectroscopy.
Exercise No. 14
SODIUM (A.L.S. Biochemicals product insert, 2001)
Colorimetric determination of sodium in human serum or plasma on photometric systems.
TEST PRINCIPLE
The method is based on modifications of those first described by Maruna and Trinder in which
sodium is precipitated as the triple salt, sodium magnesium uranyl acetate, with the excess
uranium then being reacted with ferrocyanide, producing a chromophore whose absorbance
varies inversely as the concentration of sodium in the test specimen.
Sodium is the major cation of extracellular fluid. It plays a central role in the maintenance of the
normal distribution of water and the osmotic pressure in the various fluid compartments. The
main source of body sodium is sodium chloride contained in ingested foods. Only about 1/3 of
the total body’s sodium is contained in the skeleton since most of it is contained in the
extracellular fluids.
Hyponatremia (low serum sodium level) is found in a variety of conditions including the following:
severe polyuria, metabolic acidosis, Addison’s disease, diarrhea, and renal tubular disease.
Hypernatremia (increased serum sodium level) is found in the following conditions:
hyperadrenalism, severe dehydration, diabetic coma after therapy with insulin, and excess
treatment with sodium salts.

TEST PARAMETERS
Method: Colorimetric, Modified Maruna & Trinder method
Wavelength: 550 nm
Temperature: 20-25˚C (Room temperature)
Linearity: up to 200 mEq/L
Sensitivity: 0.5 mEq/L based on an instrument resolution of A = 0.001

Comparison: A comparison between this procedure and flame photometric analysis produced
a
34
regression equation of y = 0.69x + 4.5 with a coefficient correlation of 0.92
REAGENT COMPOSITION
Filtrate reagent
Uranyl acetate 2.1 mM
Magnesium acetate 20 mM
Both acetates are dissolved in ethyl alcohol.
Acid reagent
Diluted acetic acid
Color reagent
Potassium ferrocyanide
Nonreactive stabilizers & fillers
Sodium calibrator
Sodium chloride solution 150 mEq/L
REAGENT DETERIORATION
If turbidity has occurred , there may be contamination.

Storage: 15-30˚C (room temperature)
Stability: up to the expiration date indicated on the label
SAMPLE STABILITY

Freshly drawn serum is the specimen of choice and a 50uL (0.05 mL) amount is required. Plasma
from non-sodium containing anticoagulants (e.g. lithium, calcium, magnesium or heparin) is an
acceptable alternative.
Sodium is stable for at least 24 hours at room temperature and 2 weeks when refrigerated.

A) FILTRATE PREPARATION
1) Label test tubes: Blank, Standard, Control, & Unknown 2) Pipette 1.0 mL of the filtrate
reagent to all tubes.
3) Add 50 uL of sample to Unknown; 50uL calibrator to Standard, 50 uL control serum to
Control, and 50 uL distilled water to Blank.
4) Shake all tubes vigorously.
5) Centrifuge tubes at high speed (1500G) for 10 minutes and proceed to the next
procedure taking care not to disturb the precipitate.
(Alternative volumes: 50 uL sample to 2.5 mL filtrate reagent)
35
B) COLOR DEVELOPMENT
1) Label test tubes corresponding to the above Filtrate tubes.
2) Pipette 1.0 mL acid reagent to all tubes.
3) Add 50 uL of supernatant to respective tubes and mix.
4) Add 50 uL color reagent to all tube and mix.
5) Zero spectrophotometer with distilled water at 550 nm.
6) Read and record absorbance of all tubes.
(Alternative volumes: 2.5 mL acid reagent & 0.1 mL supernatant & 0.1 mL color reagent)

Sodium (mEq/L) = ( A Blank – A Unknown) X Concentration of Calibrator
(A Blank – A Standard)
UNIT CONVERSION
mEq/L X 1 = mmol/L
REFERENCE RANGE
135 – 155 mEq/L
Result:

Remarks:____________________________________________________________________________________________
___________________________________
PROCEDURAL LIMITATIONS
When preparing filtrates, inadequate shaking or centrifugation will cause falsely lowered test
results. Blood calcium, chloride, and potassium levels of up to 3x normal reportedly exert no
adverse influence on the procedure; phosphorus levels exceeding 5x normal likewise present no
problems.

The reagent is for in vitro diagnostic use only. Do not pipet the solution by mouth. Avoid
ingestion/contact. Specimens should be considered infectious and handled appropriately.
Exercise No. 15
POTASSIUM (A.L.S. Biochemicals product insert, 2001)
Colorimetric determination of potassium in human serum or plasma on photometric systems.
TEST PRINCIPLE
36
The amount of potassium is determined by using sodium tetraphenylboron in a specifically

prepared mixture to produce a colloidal suspension. The turbidity of which is proportional to
potassium concentration in the range of 2-7 mEq/L.
Sodium is the major cation of intracellular fluid. It is also an important constituent of the
extracellular fluid due to its influence on muscle activity. Its intracellular function parallels that of
its extracellular function namely, influencing acid-base balance and osmotic pressure, including
water retention.
Hypokalemia (low serum potassium level) is found in a variety of conditions including the
following: malnutrition, negative nitrogen balance, gastrointestinal fluid losses and hyperactivity
of adrenal cortex. Hyperkalemia (increased serum potassium level) is found in the following
conditions: renal failure, dehydration shock or adrenal insufficiency.
TEST PARAMETERS
Method: Colorimetric & turbidimetric method
Wavelength: 500 nm

Linearity: 2-7 mEq/L
Comparison: A comparison between this procedure and a similar method produced a
regression equation of y = 1.06x + 0.37 with a coefficient correlation of 0.99
REAGENT COMPOSITION
Potassium reagent
Sodium tetraphenylboron 2.1 mM
Preservatives and thickening agents.
Potassium calibrator
Potassium salt solution 4 mEq/L
If turbidity has occurred , there may be contamination.

SAMPLE STABILITY
Freshly drawn serum is the specimen of choice. Potassium in serum is stable for at least 2 weeks
at 2-8˚C. Specimens for serum potassium analysis should be free from hemolysis since the high
concentration of potassium released from red cells significantly increase the serum levels and this
invalidates the test results. Blood specimens should also be separated from red cells shortly 37
after collection to prevent any leakage of potassium from the intracellular to the extracellular fluid.
Plasma from anticoagulants not containing potassium is ideal.

1) Label test tubes: Blank, Standard, Control, & Unknown.

2) Pipette 2.0 mL of the potassium reagent to all tubes.
3) Add 0.02 mL of sample to Unknown; 0.02 mL calibrator to Standard, 0.02 mL control serum
to Control, but the reagent blank requires only the potassium reagent. Mix and let stand for
3 minutes at RT.
4) After 3 minutes, set the the wavelength to 500 nm and zero the instrument with reagent
blank.

NOTE: If the spectrophotometer being used requires 2.5 mL reagent, use 0.02 mL sample to 2.5 mL
of reagent.

Potassium (mEq/L) = A Unknown X Concentration of Calibrator
A Standard
UNIT CONVERSION
mEq/L X 1 = mmol/L
REFERENCE RANGE
2.4 – 5.3 mEq/L
Results:
Remarks:
______________________________________________________________________________________________________
______________________________________

ingestion/contact. Specimens should be considered infectious and handled appropriately. Sodium
tetraphenylboron is a corrosive substance. Flush with plenty of water if contact occurs.
INTERFERENCES
Turbid or icteric serum produce falsely elevated results. Bilirubin above 40 mg/dL and BUN above
80 mg/dL will produce elevated results. Hemolyzed sera produce elevated results. Sera containing
high levels of ammonia should be avoided.
38
Exercise No. 16
CHLORIDE (A.L.S. Biochemicals, undated)
Colorimetric determination of chloride in human serum and plasma on photometric systems.
TEST PRINCIPLE
Chloride displaces thiocyanate from mercury forming nonionized mercuric chloride. Thiocyanate
reacts with ferric ions to form red-colored complex which can be measured at 480 nm. The color
intensity is proportional to the chloride concentration.

Chloride represents the major extracellular anion and helps to balance the electrical neutrality of
body fluids. It plays an important role in maintaining proper hydration and osmotic pressure. Low
serum chloride values occur with extensive burns, excessive vomiting, internal obstruction,
nephritis, metabolic acidosis, Addisonian crisis and diarrhea. High serum chloride levels are seen
in dehydration, hyperventilation, congestive heart failure, urinary obstruction, and excessive
treatment with chloride.
TEST PARAMETERS
Method: Colorimetric method
Wavelength: 480 nm
Linearity: 70-140 mEq/L
REAGENT COMPOSITION
Chloride reagent
Mercuric thiocyanate 1.0 mM
Ferric nitrate in dilute acid solution 38 mM
Chloride blank
Sodium chloride 25 mEq/L
Methanol
Chloride calibrator
Sodium chloride 100 mEq/L
Methanol
The chloride reagent should be clear yellow fluid and the rest of the reagents should be colorless.
If turbidity has occurred , there may be contamination and should not be used.

39
Conditions: protect from light the chloride reagent; close immediately after use
SAMPLE STABILITY

Freshly drawn serum is the specimen of choice. Specimens for serum chloride analysis should be
free from hemolysis and lipemia. Blood specimens should also be separated from red cells shortly
after collection. Do not take blood from a limb receiving infusion. Chloride is stable for 7 days at
2-8˚C and 3 months if frozen. Store in tightly sealed containers.

1) Label test tubes: Blank, Standard, Control, & Unknown.

2) Pipette 4.0 mL of the chloride reagent to all tubes.
3) Add 0.02 mL of sample to Unknown; 0.02 mL calibrator to Standard, and 0.02 mL control
serum to Control. Chloride blank is ready to use. Mix and let stand for 5 minutes at RT.
4) After 5 minutes, set the wavelength to 480 nm and zero the instrument with chloride blank.

Chloride (mEq/L) = A Unknown X Concentration of Calibrator
A Standard
UNIT CONVERSION
mEq/L X 1 = mmol/L
REFERENCE RANGE
96 – 106 mEq/L
Results:
Remarks:____________________________________________________________________________________________
___________________________________

Chloride reagent contains mercury that is harmful if swallowed. If ingested perform gastric lavage
and call a physician. All reagents contain methanol that is toxic when inhaled.
40
INTERFERENCES

Exercise care not to touch pipette tips with fingers since chloride contamination may result.
Exposure of samples and reagents to hydrochloric acid fumes will cause falsely high values.
Patients receiving bromide may show falsely elevated chloride levels. Young et al have reviewed
drug effects on serum chloride.
Exercise No. 17
Schales & Schales Method for Chloride (Hilado, 1996)
I. Objective: To learn the method and technic of determining chloride in serum.

Microburst
Erlenmeyer Flask, 25 ml
Diphenylcarbazone indicator
Mercuric Nitrate Solution
Sodium Chloride Standard Solution
10% Sodium Tungstate
2/3 N Sulfuric Acid
III. Procedure:
Principle: Mercuric chloride is very slightly dissociated as standard mercuric nitrate

solution is added, the chloride ion unites with mercury forming mercuric chloride. On
addition of excess mercury, a purple color is formed with the indicator.
1. Prepare a Folin-Wu filtrate from serum.

2. Pipet 2 ml of the filtrate into a 25 ml Erlenmeyer flask marked “test”. Add 2-3 drops
diphenylcarbazone indicator.
3. Prepare a “standard” tube by pipetting 2 ml Standard Chloride Solution into another
Erlenmeyer flask and adding 2-3 drops of the indicator into it.
4. Titrate both the “Test” and the “Standard” with mercuric nitrate solution from a
microburet. The clear solution becomes an intense violet on the addition of the first
drop of excess mercuric nitrate (end point).
Calculation:
Since 2ml of a 1:10 dilution of the filtrate and 2 ml of a 10 mEq/L standard are titrated, this is
equivalent to titrating 2 ml of serum and 2 ml of a 100 mEq/L Standard. Hence:

41
ml titration of filtrate x 100 = mEq/L Chloride

ml titration of standard
The chloride concentration is sometimes expressed in terms of mg/100 (as sodium chloride).
NOTE: The serum may also be titrated directly. Add 0.2 ml serum to 1.8 ml water in a flask, add
indicator and titrate. When the first drops of mercuric nitrate are added, the solution will turn
blue. As the titration is continued the blue color will disappear to a pale pink, with the blue color
appearing again at the end point. The end point may not be quite so sharp as with the titration
of the filtrate. For this quantity of serum the calculations is the same as previously given.
Preparation of Reagents.
1. Mercuric Nitrate Solution
Dissolve 1.7 gm reagent grade mercuric nitrate and 2.6 ml concentrated nitric acid in 200
ml water, then dilute to 1 liter. This solution is quite stable.
2. Diphenylcarbazone indicator
Dissolve 100 mg s-diphenylcarbazone in 100 ml 95% ethyl alcohol. Store in a brown bottle
in the refrigerator. This solution slowly deteriorates, especially on exposure to light, and
should be prepared fresh monthly.
3. Sodium Tungstate and Sulfuric Acid

These are the same as used for the preparation of Folin-Wu filtrate
4. Chloride Standard
Dry reagent grade sodium chloride at 120 C for several hours. Weigh out exactly 584.5
mg of the salt and dissolve in water to make 1 liter. This solution contains 10 mEq/L
chloride.
Normal Values:
Serum Chloride: 98 –109 mEq/L

ml of Mercuric Nitrate used for Standard ________________________
ml of Mercuric Nitrate used for Test ________________________
Results:
mEq/L Chloride = _______________________________
Remarks: _________________________________________________________

42
Exercise No. 18
CALCIUM (A.L.S. Biochemicals, undated)
Colorimetric determination of calcium in human serum and urine.
TEST PRINCIPLE
O-cresolphthalein complexone binds calcium in an alkaline medium to form a purple colored
complex. Magnesium interference is minimized by the addition of 8-hydroxyquinoline. Stabilizers
are included to produce a low blank absorbance and increased working reagent stability. A
reaction accelerator assures reaction completion in less than one minute.
CLINICAL CIGNIFICANCE
Calcium in serum is distributed about equally as the ionized form and the protein bound
nonionized form. The ionized form plays a physiologically active role in blood coagulation and
enzyme activation. Increased levels of calcium are seen in multiple myeloma, bone neoplasms,
and hyperparathyroidism. Decreased levels are seen in steatorrhea, rickets, nephroses, and
hypoparathyroidism. A reciprocal relationship appears to exist between calcium and phosphorus
in serum.
There are several methods used for calcium assay but the one which offers maximum sensitivity
and specificity with ease of handling involves the use of O-cresolphthalein complexone to bind
calcium . The ALS Calcium procedure is based on a modification of this method. A reaction
accelerator has been added to assure reaction completion in less than a minute.
TEST PARAMETERS
Method: Colorimetric OCP method
Wavelength: 570 nm
Incubation Time & Temperature: 1 minute at 15-30˚C (Room temperature)
Sample volume: 0.05 mL serum or urine
Color reagent volume: 2.0 mL
Base reagent volume: 2.0 mL
Total volume: 4.05 mL
Linearity: extends up to 15 mg/dL
Specimens exceeding this value should be diluted using 0.9% sodium chloride
solution and reassayed. Multiply the test result by the dilution factor to obtain the
final answer.

Sensitivity: 0.02 mg/dL per 0.001 absorbance unit
REAGENT COMPOSITION
Calcium Color Reagent
43
O-cresolphthalein complexone 0.082 mM

8-hydroxyquinoline 17.2 mM
Stabilizers and reaction accelerators
Calcium Base Reagent
Diethylamine 0.51 M
Potassium cyanide 7.7 mM
Calcium calibrator
Calcium carbonate in dilute HCl 10.0 mg/dL
The calcium color reagent should be clear yellow fluid and the rest of the reagents should be clear
and colorless. If turbidity or darkening in color, there may be contamination and should not be
used. The reagent blank should have an OD of 0.250 at 570nm.
SAMPLE STABILITY
Freshly drawn serum is the specimen of choice. No special additives for serum are needed. An
aliquot of urine collection should be acidified to pH=4.0 with concentration HCl before testing.
Mix the urine well before sampling to assure an even suspension of any precipitated calcium.
Calcium in serum and urine is stable for 1 week at 2-8˚C and 1 year if frozen. Store in tightly
sealed containers.

Preparation of the Working Reagent:

Use a plastic container or acid washed glass container. Add one part Ca color reagent to one part
Ca base reagent. Swirl to mix. Use within one hour of preparation. The Working Reagent
contains a small amount of cyanide and should not be mixed with acid. Discard by flushing
with water.
1) Label test tubes: Reagent Blank, Standard, Control, & Sample.

2) Pipette 4.0 mL of the Calcium working reagent to all tubes.
3) Add 0.05 mL of deionized water to Blank; 0.05 mL of urine/serum sample to Sample; 0.05
mL calibrator to Standard, and 0.05 mL control serum to Control. Chloride blank is ready
to use. Mix and let stand for 1 minute at RT.
4) Set the wavelength to 570 nm and zero the instrument with reagent blank.

44
SERUM: Calcium (mg/dL) = A Sample X Concentration of Calibrator (10.0 mg/dL)

A Standard
URINE:
mg Calcium/24 Hr = A Sample x concentration of Calibrator x 2 x 10dL/L urine volume( L/24 Hr)
A Standard
UNIT CONVERSION mg/dL X 0.25

= mmol/L
REFERENCE RANGE
Serum: 8.5-11.0 mg/dL
Urine: 50-150 mg/24 Hr
Result:
Remarks:
______________________________________________________________________________________________
________________________________________________

Calcium color reagent, base reagent and calibrator thus avoid in contact with eyes, skin and
clothing. The calcium base reagent contains cyanide and should not be reacted with acid.
INTERFERENCES

Specimens from patients receiving EDTA or bromsulfonphthalein (BSP) should not be used.
Exercise No. 19
Clark & Collip Method for Calcium (Hilado, 1996)
I. Objective: To learn the method and technic of determining calcium in serum.

Volumetric Pipet, 2 ml
45
Graduated centrifuge tube, 15 ml

Thermometer, 100oC
Water bath
Serum
Ammonium oxalate, 4%
Ammonium hydroxide, 2%
Sulfuric acid, 1N
Sulfuric acid, concentrated
Potassium permanganate, 0.01N
Sodium oxalate, 0.01N
III. Procedure:
Principle: Calcium from serum is precipitated directly as oxalate. The calcium is titrated with
standard permanganate in the presence of sulfuric acid.
1. This test should always be made in duplicate. If serum is limited run one half amounts.
Prepare four 15 ml graduated centrifuge tubes as follows:
Two Tubes for Test Two Tubes for Blank

2 ml serum 4 ml distilled water
2 ml distilled water 1 ml 4% ammonium oxalate
1 ml 4% ammonium oxalate
2. Thoroughly mix the contents by holding the tubes at the mouth and tapping the lower
ends.
3. Stopper and allow to stand for 30 minutes or more.
4. Mix the contents by tapping the sides.

5. Centrifuge for 5 minutes at high speed.
6. Decant off all the liquid and while the tube is still inverted drain for 5 minutes, resting
the mouth of the tube on filter paper.
7. Add 3 ml of 2% ammonium hydroxide.
8. Mix contents well by tapping the tube.
9. Centrifuge for 5 minutes at high speed.
10. Decant off all the liquid and allow the tube to drain over absorbent paper for 15 minutes.
11. Dissolve the crystals in 2 ml of approximately 1N sulfuric acid blowing from the pipet
directly onto the precipitate.
12. Prepare 2 tubes for the Standard by placing into each 2 ml of 0.01N Sodium oxalate and
0.1 ml concentrated sulfuric acid.
13. Place all tubes in a boiling water bath for 1 minute, or until all of the precipitate has
dissolved.
46
14. Maintain the temperature between 70 oC and 75oC and titrate with 0.01N potassium
permanganate to a definite pink color, persisting for at least 1 minute, which is seen by
looking down through the tube rather than through the sides of the tube.
Notes:
1. In titrating, the permanganate should be added very slowly at the beginning, as it takes
a little time for the reaction to start and oxygen will be lost if permanganic acid
accumulates. The second drop should not be added until the pink color given by the
first drop has disappeared.
2. The titration temperature is important and should be 70–75oC at the start and not lower
than 60oC at the end, otherwise too much permanganate will be used.
3. The centrifuge tube may be conveniently held in the water bath with a test tube holder
and the contents may be stirred by giving it a gentle whipping motion.
4. The endpoint is to be taken as the faintest persisting pink color that can be recognized
when looking down the tube against a white background. At this point no pink color
is recognized if one looks through the tube.
Calculation:
1 ml of 0.01N KmnO4 is equivalent to 0.02 mg calcium.
(X-B) x 0.02 x 100 = mg% Calcium if KmnO4 is exactly 0.01N

2
where: X = titration of Unknown
B = titration of Blank
OR:
mg% Calcium = (ml KmnO4 used to titrate serum – ml KmnO4
used to titrate Blank) x Normality of KmnO4

0.01 (Normality of sodium oxalate)
Preparation of Reagents.
1. Ammonium oxalate 4%. Dissolve 4 gm of ammonium oxalate in distilled water and
dilute to 100 ml. Filter before using.
2. Sulfuric acid, 1N. To a 1 liter volumetric flask partly filled with distilled water add
with caution 30 ml of concentrated sulfuric acid. Dilute to mark with distilled water.
Mix well.
3. Ammonium Hydroxide, 2% or 0.35N. Dilute 2 ml of concentrated ammonium
hydroxide to 100 ml with distilled water.
4. Sodium oxalate, 0.1N. Accurately weigh 6.701 gm of pure, dry, reagent grade sodium
oxalate. Transfer with 1N sulfuric acid through a funnel into a 1 liter volumetric flask,
cool and dilute to mark with 1N sulfuric acid. Mix thoroughly. This solution is stable
for several months.
5. Sodium Oxalate, 0.01N. Pipet exactly 10 ml of the 0.01N sodium oxalate to a 100 ml
volumetric flask and dilute to mark with 1N sulfuric acid. This solution is used for the
standardization of 0.01N permanganate solution. Even though it is stable, it should be
prepared weekly.
47
6. Potassium Permanganate, 0.1N stock solution. Dissolve 4.2 gm of reagent grade

potassium permanganate crystals in water. Dilute to 1100 ml in a graduated cylinder
(use a 1 liter cylinder plus 100 ml of water). Allow to stand in the dark for 2 to 3 days
to insure complete solution and stabilization. Filter through glass wool or with gentle
suction through a 3-inch Buchner funnel lined with asbestos. Keep in refrigerator.
Standardization of Stock Permanganate. Pipet 25 ml of 0.1N sodium oxalate into a
white, porcelain casserole. Add 1 ml of concentrated sulfuric acid. Warm to about
70oC and titrate with permanganate solution just described in the biuret. The first pink
is the end point. Let % equal the milliliters of permanganate needed to titrate 25 ml
of 0.1N sodium oxalate. To a 1L volumetric flask add 40 (25-T) ml of water and dilute
to the mark with permanganate solution. This is exactly 0.1N permanganate. Titrate
again for accuracy. Store in a brown bottle in the dark.
7. Potassium Permanganate, 0.01N. Pipet exactly 10 ml of the 0.1N potassium
permanganate into a 100 ml volumetric flask. Dilute to the mark with distilled water
and mix. This solution changes its strength with dilution and or standing; it should be
titrated each time before a calcium determination is done.
Standardization: A 2 ml buret graduated to 0.01 ml or 0.02 ml should be used. Pipet
2 ml of 0.01N sodium oxalate into a heavy duty centrifuge tube. Heat in a boiling
water bath for one minute. Titrate with a 0.01N permanganate solution to the first
pink color (against a white background) look through the end of the tube rather than
the side when determining the end point. Place back in boiling water to make sure
the solution is hot when nearing the end point. Use the following formula, when

calculating the correction factor, to correct for variation in strength of the potassium
permanganate:
2
F = ml of permanganate used
The titration should require 1.9 to 2.1 ml of permanganate; if not, prepare fresh 0.01N
potassium permanganate.
Normal Values: 9 – 11 mg%
Observation and Result:
ml KmnO4 used for Blank Test tube #1_______

Test tube #2_______
ml KmnO4 used for Unknown Test tube #1_______

Test tube #2_______
ml KmnO4 used for Standard Test tube #1_______

Test tube #2_______
48
Remarks: ________________________________________________________________
___________________________________________________________________
Exercise No. 20
MAGNESIUM (Dialab product insert, 2010)
Diagnostic reagent for quantitative in vitro determination of magnesium in human serum, plasma,
cerebrospinal fluid or urine on photometric systems..
TEST PRINCIPLE
Magnesium reacts with xylidyl blue to form a colored compound in alkaline solution. The intensity
of the color formed is proportional to the magnesium concentration in the sample. Interference
with calcium is achieved by the use of GEDTA.
TEST PARAMETERS
Method: Colorimetric, Increasing reaction, Endpoint, Xylidyl blue method
Wavelength: 520 nm (500-550 nm), Hg 546 nm

Sample: serum or heparinized plasma, CSF, urine
Linearity: up to 5 mg/dL
Sensitivity: LOD is 0.05 mg/dL
REAGENT COMPOSITION
Xylidyl blue 110 umol/L
Ethanolamine, pH 11.0 1 mol/L
GEDTA 60 umol/L
REAGENT PREPARATION
Reagents are ready for use.

Storage: 2-8˚C
SAMPLE PREPARATION
Urine: Acidify urine with some drops of concentrated HCl to pH 3-4 then dilute 1+4 with
distilled water.
INTERFERING SUBSTANCES
No interference up to:
49
Hemoglobin interferes because they are released from RBCs.

Ascorbic acid……………………….………………………30 mg/dL
Bilirubin……………………………………………………….40 mg/dL
TAG………………………………………………………….2000 mg/dL
Calcium……………………………………………………….25 mg/dL

Pipet into tubes Blank Standard Sample
Reagent 2000 uL 2000 uL 2000 uL
Sample 20 uL
Standard 20 uL

Mix. Incubate for 5 minutes at RT or 37oC. Measure A of Standard amnd Sample against
Blank within 60 minutes.

Serum/Plasma:
Magnesium (mg/dL) = ∆A Sample X Concentration of Calibrator (2 mg/dl)
∆A Calibrator
Urine:
Magnesium (mg/dL) = ∆A Sample X Concentration of Calibrator (2 mg/dl) x 5
∆A Calibrator
0.4114 = mmol/L
Result:
Remarks:____________________________________________________________________________________________
___________________________________
REFERENCE RANGE
Serum or Reference Ranges
Plasma
Neonates 1.2-2.6 mg/dL
Children 1.5-2.3 mg/dL
Females 1.9-2.5 mg/dL
Males 1.8-2.6 mg/dL
CSF 2.1-3.3 mg/dL
Urine 73-122 mg/24 Hr
Exercise No. 21
50
PHOSPHORUS INORGANIC (Dialab product insert, 2007)
Diagnostic reagent for quantitative in vitro determination of phosphorus in human serum, plasma
or urine on photometric systems.
TEST PRINCIPLE

In acid medium phosphate reacts with ammonium molybdate to form a yellow phosphorus
molybdate complex. The complex’ absorption is maximal at 340 nm. Absorption is proportional
to the concentration of inorganic phosphate in the sample.
TEST PARAMETERS
Method: UV, Increasing reaction, Endpoint, Phosphomolybdate
Wavelength: 340 nm, Hg 334nm, Hg365nm
Sample: serum, heparinized plasma, urine
Linearity: up to 15 mg/dL
Sensitivity: LOD is 0.7 mg.dL
REAGENT COMPOSITION
Ammonium molybdate 0.4 mmol/L
Sulfuric acid 210 mmol/L
Detergent
REAGENT PREPARATION
part of reagent 2. It is stable for 8 hours at 20-25˚C in amber
bottles.

Storage: 2-25˚C
SAMPLE PREPARATION, STABILITY, & STORAGE

Urine: For collection of 24 hr urine add 10 mL of HCl (10 g/dL) into the collection bottle to avoid
phosphate precipitations. Dilute urine 1+20 with distilled water before determination and
multiply the result by 21.
Stability: in serum/plasma: at 4-25oC 7 days
o
at -20 C 3 months
In urine (pH <5): at RT 2 days
51

interference up to:
TAG………………………………….800 mg/dL
Pipet into tubes Blank Standard Sample
Reagent 2000 uL 2000 uL 2000 uL
Sample 20 uL
Standard 20 uL
Mix. Incubate for 5 minutes at RT or 37oC. Measure A of Standard amnd Sample against
Reagent Blank within 60 minutes.

Serum/Plasma:
Phosphorus (mg/dL) = ∆A Sample X Concentration of Calibrator (5 mg/dl)
∆A Calibrator
Urine:
Phosphorus (mg/dL) = ∆A Sample X Concentration of Calibrator (5 mg/dl) x 21
∆A Calibrator
0.3229 = mmol/L
Result:
Remarks:
______________________________________________________________________________________________
________________________________________________
REFERENCE RANGES
Adults (Serum/Plasma)…………………………………….2.6-4.5 mg/dL
Urine……………………………………………………….…….0.4-1.3 g/24Hr

52
Exercise No. 22
Fiske & Subbarrow for Inorganic Phosphate (Hilado, 1996)
I. Objective: To learn the method and technic of determining inorganic phosphate in serum.

Volumetric pipet, 1 ml, 5 ml
Erlenmeyer flask, 25 ml
Glass stoppered graduated cylinder, 10 ml
Beaker, 25 ml
Ashless filter paper (Whatman #44)
Serum
Trichloroacetic acid, 30%
Trichloroacetic acid 5%
Phosphorus standard
Molybdate reagent
Aminoaphtholsulfonic acid solution
III. Procedure:
Principle: The proteins of the serum are precipitated with trichloroacetic acid. The protein-free
filtrate is treated with an acid molybdate solution which combines with the inorganic phosphorus
to form phosphomolybdic acid. The phosphomolybdic acid is reduced by the addition of amino-
naphtholsulfonic acid reagent to produce a blue color, the intensity of which is proportional to
the amount of phosphate present.
1. Measure 9 ml of water into a 25 ml Erlenmeyer flask.

2. Add 1 ml of serum and 2 ml of 30% trichloroacetic acid
3. Mix well and filter through Ashless filter paper (Whatman #44)
4. Transfer 4 ml of filtrate to a 10ml glass-stoppered graduated cylinder. Add 4 ml of 5%
trichloroacetic acid.

5. Prepare a standard by pipeting 3 ml of working standard containing 0.015 mg of
phosphorus and 5 ml of % trichloroacetic acid into a glass stopped graduated cylinder.
6. Prepare a blank by pipeting 8 ml of 5% trichloroacetic acid into another glass stoppered
cylinder.
53
7. To all tubes add 1 ml of molybdate reagent.

8. Mix and add 0.4 ml of aminoaphtholsulfonic acid.
9. Dilute with water to 10 ml.
10. Allow to stand exactly 10 minutes and read in the photometer at 640 nm.
Notes:
1. The length of time which elapses between the development of the color and the reading
in the photometer is very important
2. Set the photometer at zero with water and read the blank, unknown and standard
against water. Then, subtract the reading of the blank from the readings of the unknown
and standard.
3. The serum should be separated from the clot as soon as possible. The separated serum
may be stored in the refrigerator for several times.
Calculation:
RU
RS x conc. of Standard = mg% Phosphorus
1. 30% Trichloroacetic acid
Dissolve 30 mg of reagent-grade phosphorus-free trichloroacetic acid in water and
dilute to 100 ml. This solution is stable indefinitely.
2. 5% Trichloroacetic Acid
Dilute 1 volume of 30% trichloroacetic acid with 5 volumes of water.
3. Molybdate reagent
Dissolve 25 g of reagent grade ammonium molybdate in about 200 ml of water. Place
this solution with washing in a 1L volumetric flask. Add 300 ml of 10N sulfuric acid.
Dilute to volume and mix. This solution is stable indefinitely.
4. 10N Sulfuric Acid
Carefully add 450 ml of concentrated sulfuric acid to 1300 ml of water. To check, dilute
10 ml of this solution to 100 ml in a volumetric flask. Mix and titrate 10 ml portions
with Standard 1.000N Sodium Hydroxide. From the results, adjust the original solution,
if necessary to make it exactly 10 N.
5. Aminonaphtholsulfonic Acid Reagent
Place 195 ml of 15% Sodium Bisulfite Solution (see below) in a glass-stoppered
cylinder. Add 0.5 g of 1, 2, 4- aminonaptholsulfunic Acid. Add 5 ml of 20% Sodium

Sulfite (see below). Stopper and shake until the powder is dissolved. If solution is not
complete, add more sodium sulfite, 1 ml at a time, with shaking, but avoid an excess.
Transfer the solution to a brown glass bottle and store in the cold. This solution is
stable for about for weeks.
6. 15% Sodium Bisulfite
To 30 g of Reagent- grade sodium Bisulfite in a beaker add 200ml. Of water from a
graduated cylinder. Stir to dissolve. If turbid, place in a well stoppered bottle for
several days and filter. Keep well stoppered. Prepare fresh for each use.
54
7. 20% Sodium Sulfite

Dissolve 20 g of reagent-grade anhydrous Sodium Sulfite in water. Dilute to 100 ml.
And filter if necessary. Keep in a well stoppered bottle.
8. Phosphate Standard, Stock Solution
Dissolve exactly 0.439 mg of pure dry monopotassium phosphate in water in a 1 liter
volumetric flask. Add 10 ml. Of 10N Sulfuric/Acid and dilute to volume with water Mix.
This solution contains 0.5 mg. of phosphorus in 5 ml. It is stable indefinitely.
9. Phosphate Standard, Working Solution
Transfer 5 ml. of stock phosphate standard to a 100 ml volumetric flask. Add 16.7 ml of
30% trichloroacetic acid and dilute to volume with water. This solution contains
0.015 mg of phosphorus in 3 ml.
Normal values:
Adults 2.5 to 4.3 mg%
Infants and Children 4.0 to 7.0 mg%
Results:
Remarks:
______________________________________________________________________________________________________
____________________________________________________________________
Exercise No. 23
Iron-Binding Capacity (Hilado, 1996)
Iron is one of the vital minerals in the body that indicates effectiveness of the systemic transport
of blood gases and good functioning of enzymes. It is clinically important because it decreases
during infection, malnutrition, and anemia. The latter is the most frequently monitored in the
laboratory.
I. Objective: To learn the method and technic of determining serum iron-binding capacity.

Principle: The iron in serum is bound to the protein transferrin (siderophilin). Normally, this
protein is about one third saturated with iron (serum iron). The additional quantity of iron which
can be taken up by the transferrin is termed the unsaturated iron- binding capacity (UIBC). The
total quantity of iron in serum after the complete saturation of the transferrin is the total
ironbinding capacity (TIBC).
UIBC. To determine the UIBC, an excess of iron is added to the serum. Under favorable conditions
(pH 8.3) the transferrin becomes saturated with iron within a short period of time. After reduction,
the unbound iron is converted into a red compound by a specific iron reagent (sulfonated
bathophenanthroline) and determined photometrically. The difference between the iron added
and the unbound iron after saturation is the UIBC.
55
TIBC. The sum of the UIBC and iron concentration of the serum equals the total iron binding
capacity.
II. Materials & Reagents:

Serum (Stability of 7 days at 4OC/ 4 days at 15-25OC)
1. Buffer
(1.0 mol/1 tris (hydroxymethyl) aminomethane, pH 8.3
2. Reduction Solution
(30 mmol/1 methylaminophenol sulfate, 162 mmol/I sodium hydrogen sulfite)
3. Color Reagent
(1.7 mmol/1 bathophenanthrolinedisulfonic acid, disodium salt)
4. Iron Standard Solution
(2.5 mg/dL)
III. Procedure:
Note: Before use, the glassware must be thoroughly cleaned (especially prior to the first use).
Preferably, leave the glassware overnight in chromosulfuric acid or in the case of stubborn
impurities heat in a 5% solution of EXTRAN, for one hour at 95 OC, and rinse thoroughly with
distilled water.
Only one blank is needed for each test series.

A. Maximum Absorbance: 535 nm
B. Light Path: 1 cm
C. Wavelength: Between 530 and 550 nm

1. Pipette into the test tubes marked “Serum Blank”, “Sample or Unknown” and
Reagent Blank”
Serum Sample Reagent
Blank Blank
Serum 1.0 ml 1.0 ml -----

Buffer (1) 0.5 ml 0.5 ml 0.5 ml
Standard Sol. (4) 0.2 ml 0.2 ml -----
J. Mix well, cover the test tubes, and place in a water bath, and place in a water bath
at 37OC or 45oC for 10 minutes.
3. Pipette into the same tubes the following solutions:

Serum Sample Reagent
Blank Blank
56
Reduction Sol. (2) 0.2 ml 0.2 ml 0.2 ml

Distilled water 0.2 ml ----- 1.0 ml
Color Reagent ----- 0.2 ml 0.2 ml
4. Mix well, cover the tubes and place in a water bath at 37’C for 180 minutes or in a water
bath at 45’C for 90 minutes. Cool to room temperature
5. Measure the absorbance of the Sample or Unknown against the Reagent Blank and the
absorbance of the serum Blank Against Distilled Water.
Calculations:
For UIBC
535 nm: UIBC = (0.905- Abs. Sample + Abs. Serum Blank) X 552 ug/dl
546 nm: UIBC= (0.838 – Abs. Sample + Abs. Serum Blk ) X 596 ug/dl
For TIBC
Total IBC = UIBC + iron concentration of the serum
Normal Values:

Men UIBC = 200-300 ug/dL
= 36-54 umol/1
TIBC = 300-400 ug/dL

= 54-72 umol/dL
Women UIBC = 150-250 ug/dL
= 24-45 umol /1
TIBC = 250-350 ug/dL

= 45-63 umol/1
Observation and Result:
Readings O.D.
Serum Blank
Sample or Unknown
Calculations:
57
Remarks: _____________________________________________________________
_____________________________________________________________

ENZYMES
Enzymes are proteinoid substances that catalyze essential biochemical reactions. They act as true
catalysts in that they alter the rates of these chemical reactions without being used up. Their
activity is quite specific. They are found in cells and all tissues; serum to which gain access from
injured cells, cells undergone stress. Application of the science of enzymes to the diagnosis and
treatment of disease accounts for about 25% in clinical laboratories.
Enzyme activities and concentration in serum become elevated by: in disease states, caused in
increased membrane permeability; caused by increase rates of intracellular synthesis; and 58
subsequent diffusion of enzymes. Enzymes found in serum will help physicians diagnose certain
disease and aid in the monitoring of the disease condition.
Mechanism of abnormal serum enzyme levels: Increase serum levels that may be due to increased
release of enzyme from source, necrosis and increased membrane permeability; increased size of
tissue source of enzyme; impaired excretion of enzyme; or increased enzyme synthesis.
These enzymatic test included in the exercises are those that are frequently done in the clinical
laboratory setting. These are most often ordered by requesting physicians to rule out or confirm
suspected pathological conditions that have occurred in by the patient. Most of the methods
presented are quantitative colorimetric procedures adaptable to laboratory instrument that are
made available for student use. It is important that students as beginners are exposed to the
standard methods to learn more of the basic principles and techniques performed in the clinical
chemistry set-up in the laboratory.
At the end of the exercises, the students will be expected to:

1. Perform quantitative determination of enzyme levels in serum.
2. Explain the principles and reactions involved in each method performed.
3. Follow the current technique used when performing the procedure.
4. Explain the clinical significance of normal and abnormal values.
Amylase & Lipase
Amylase is found chiefly found on the saliva and in pancreatic tissue. Normally, small amounts of
amylase are present in the blood, but with various forms of pancreatic disturbances such as
pancreatitis in which large amounts of amylase are secreted into the blood by the pancreas.
Methods for the determination of amylase depend on the ability of this enzyme to catalyze the
hydrolysis of starch (amylum) to simple sugars. Amylase is a sensitive marker for pancreatitis.
Lipase, on the other hand, is chiefly a pancreatic enzyme that acts on lipids in the duodenum. Its
clinical significance is as a specific marker for pancreatic disorders.
Exercise No. 24
Caraway Method for Amylase (Hilado, 1996)

amylase.
59

Spectrophotometer
Water bath
Timer
Volumetric flask, 50mL
Serologic pipets, 0.1mL, 1.0mL, 5.0mL, 10.0mL
Volumetric pipets, 2.0mL, 5.0mL, 10.0mL
Test tubes
Cuvets
Glass funnel
Erlenmeyer flasks, 50.0mL
Starch reagent, pH 7.0

0.01M Iodine reagent Starch paste
Acid Sodium Chloride
5% Copper sulfate
6% Sodium tungstate
Glucose stock standard
Alkaline copper reagent
Phosphomolybdic acid
Principle: Amylase splits the polysaccharide starch into the disaccharide maltose and a residue,
limit dextrin, resulting in a loss of color. This color change at 640nm is directly proportional to
amylase concentration.
Starch + Iodine –a-amylase a-Maltose + limit dextrin
III. Procedure:
1. Pipet 5.0mL of starch reagent into each of the 50ml volumetric flask marked “test” and
“blank.”
2. Incubate test flask at 37% for 5 minutes. “Blank” need not be incubated.
3. Pipet 0.1mL of serum into “test” flask. Mix by swirling. Incubate for exactly 25 minutes.
Note: Avoid contamination with saliva.
4. After 7.5 minutes, remove “test” flasks from incubator and immediately pipet 5.0mL of
iodine reagent into “test” and “blank” flasks.
5. Immediately add distilled water to each flask until 50mL mark is reached. Mix contents by
inversion.
6. Set spetrophotometer to zero at 640nm with distilled water. Transfer a portion of each
solution into appropriate cuvet and record absorbance.
60
Result:
Remarks:________________________________________________________________________________________
________________________________
Calculation:

Amylase units/dL = Ab - At x 800
Ab
Where:
Ab =
absorbance of blank
At = absorbance of test
800 = maximum activity of enzyme needed to reduce all the starch present
under conditions of the procedure
Exercise No. 25
Kinetic Assay for Amylase
Principle: The a-amylase hydrolyzes the blocked p-nitrophenylmaltoheptaoside producing

glucose polymers and p-nitrophenol oligosaccharides with shorter chain. These are further
hydrolyzed by glucoamylase and a-glucosidase to release p-nitrophenol. The increase in
absorbance at 405nm is proportional to the a-amylase activity.
Reagents: R1: Pipes buffer, pH6.90 50mmol/L

Sodium chloride 40mmol/L
Calcium chloride 350mmol/L
R2: p-nitrophenylmaltoheptaoside (blocked) 1.0mmol/L
Glucoamylase > 3,000U/L
a-glucosidase >10,000U/L
Storage Instructions and Reagent Stability.

The reagents are stable up to the end of the indicated month of expiry, if stored at 2-8 deg
Celsius.
Sample: Serum or heparin plasma
Procedure:
61
“test” tube.

Test tubes Amylase Value
Blank
Control
Test
Calculation.
Absorbance x factor = Amylase activity (U/L)
Results:
Remarks: __________________________________________________________
Guide Questions:
1. What are the common sources of error in amylase testing?
2. Give the differences among these methods of amylase determination:
amylometric, amyloclastic, saccharogenic, and chronometric methods.
3. What are some of the substrates used in modern methods of amylase testing?
4. Aside from serum, what other specimens can be indicated for amylase assay?
Exercise No. 26
AMYLASE (AMS) (Dialab product insert, 2011)
Diagnostic reagent for quantitative in vitro determination of AMS in human serum, plasma or urine
TEST PRINCIPLE
Amylase splits the substrate 4,6-ethylidene-(G7)-p-nitrophenyl-(G1)-α-D-maltoheptaoside
(EPSG7) to produce oligosaccharides, then the α-glucosidase hydrolyzes the oligosaccharides
producing glucose and p-nitrophenol (PNP). The result of the sequential hydrolysis is free PNP
whose absorbance is measured at 405 nm.
α-
Amylase
5 EPS-G7 + HOH ------------- 2-ethylidene-G5 + 2 G2-PNP + 2Ethylidene-G4 + 2 G3PNP +
Ethylidene-G3 + G4-PNP α-Glucosidase
2 G2-PNP + 2 G3PNP + G4-PNP + 14HOH ---------------- 5 p-nitrophenol + 14 Glucose 62

G = glucose
TEST PARAMETERS
Method: Colorimetric, Kinetic, Increasing reaction, Modified IFCC
Wavelength: 405 nm
Temperature: 37˚C
Sample: serum, EDTA/heparinized plasma, urine
Linearity: up to 1985 U/L for substrate start and up to 1594 U/L for sample start

REAGENT COMPOSITION
Reagent 1
Good’s buffer, pH 7.15 0.1 mol/L
Magnesium chloride 10 mmol/L
NaCl 50 mmol/L
α-Glucosidase ≥2 kU/L
Reagent 2
Good’s buffer, pH 7.15 0.1 mol/L
EPS-G7 1.6 mmol/L
REAGENT PREPARATION
reagent 2. It is stable for 4 weeks at 2-8˚C and 5 days at 15-25˚C.

Storage: 2-8˚C
WORKING REAGENT STABILITY

Stability: at 2-8˚C 6 months at
15-25˚C 4 weeks Discard
contaminated specimens.

Stability in serum/plasma:
At 20-25 deg C 7 days

At 4-8 deg C 7 days
63
At -20 deg C 1 year

Freeze only once!
Stability in urine:
At -20 deg C 3 weeks
No interference up to:
Hemoglobin interferes even at minimal concentration.
Ascorbic acid……………………….30 mg/dL
Triglycerides……………………..1000 mg/dL

SUBSTRATE START
Pipet into test tubes Blank/Sample/Standard
Reagent 1 2000 uL
Sample/Standard 40 uL
Mix. Incubate for 1 minute. Then add:

Reagent 2 500 uL

minutes and start a timer. Read absorbance again after exactly 1, 2
and 3 minutes.
SAMPLE START
Pipet into test tubes Blank/Sample/Standard
Working Reagent 2000 uL
Sample/Standard 40 uL

minutes and start a timer. Read absorbance again after exactly 1, 2
and 3 minutes.

AMS (U/L) = ∆A/ minute X Factor
Factors: (37oC) Substrate start Sample start
Serum/Plasma 5670 4554 Urine 11250 9018 64
UNIT CONVERSION

Adults (37oC) Serum/Plasma Urine
Females <100 U/L <100 U/L
Males <100 U/L <491 U/L
Result:
Remarks:
______________________________________________________________________________________________________
______________________________
Exercise No. 27
LIPASE (LPS) (A.L.S. Biochemicals product insert, 2001)
Turbidimetric determination of pancreatic lipase in human serum on photometric systems.
INTRODUCTION
Lipase is defined as that group of enzymes, which hydrolyze the glycerol esters of long-chain fatty
acids. The measurement of lipase activity in serum and other body fluids is to evaluate conditions
associated with pancreas. Voget et al. proposed an oil emulsion in measuring the rate of change
in turbidity over a specific unit of time. Later, Shihabi et al. modified the previous method and
eliminated some of interferences in which this method is based on.
TEST PRINCIPLE
Serum lipase hydrolyzes the olive oil emulsion. The decrease in turbidity at 400 nm after incubation
is proportional to the lipase activity in the specimen.
Lipase
TAG + HOH ------------------- mono- + di-glycerides + fatty acids

TEST PARAMETERS
Method: Turbidimetric, Shihabi et al. method
Wavelength: 400 nm (390-420 nm)
Temperature: 37˚C
Sample: serum
Linearity: up to 280 IU/L
Sensitivity: 5.4 IU/L based on an instrument resolution of A = 0.001
Comparison: A comparison between this procedure and a similar photometric analysis produced
a regression equation of y = 0.94x + 9.13 (N=59) with a coefficient correlation of 0.98
REAGENT COMPOSITION
65
Substrate
Olive oil in alcohol 0.8% (w/v)
Tris buffer, pH 9.0 69 mM
REAGENT PREPARATION
Add lipase buffer to a 50 mL Erlenmeyer flask. Add 25 mL distilled water and swirl to
dissolve. Pipette 1 mL of well-mixed olive oil substrate into buffer solution. NOTE: The OD of the
emulsion prior to use must be greater than 1.0. Due to variations in regional temperatures, the
OD may be less than 1.0. If this occurs, add 0.5-1.0 mL more substrate until OD is greater than
1.0.

Ureconstituted reagent should be stored at RT (15-30˚C). Reconstituted reagent is stable
for 7 days refrigerated (2-8oC) and tightly capped.
SAMPLE STABILITY
Freshly drawn serum is the specimen of choice. Lipase activity in serum is stable at RT for 1 week;
sera may be stored for 3 weeks at 4-8oC and for several months if frozen. Bacterial contamination
of the specimens may result in an increase in lipase activity.
Hemolyzed serum should not be used. According to Young et al., number of drugs and
substances affect lipase activity.

1) Reconstitute lipase reagent according to instructions.
2) Label test tubes: Blank, Standard, Control, & Unknown (Sample)
3) Pipet 3.0 mL of reagent into appropriate test tubes and prewar at 37 oC for
at least 5 minutes.
5) Read and record absorbance of blank and place back in heating bath.
6) Using timed intervals, add 0.1 mL of sample to to each tube and read
OD1.Return each tube to water bath after initial reading.
7) Exactly 5 minutes after the OD1 determination, using the same timed
intervals, remove each tube from the water bath and mix. Read OD of blank
and each of the sample tubes against distilled water.
PROCEDURAL NOTES
66
If the OD of the blank is a negative value, consider it zero. Elevated blank cates i.e., (0.005) and
above may be caused by olive oil coating on cuvette surface. Periodically rinse with
acetone followed by water flush. Turbid samples should be diluted with distilled water
(1:5). Multiply final result by the dilution factor. Use fresh sera, when possible, for greatest
accuracy.
CALIBRATION
The lipase activity in the sample is calculated based on the millimolar absorptivity of olive
oil (3.15 in working solution).

The enzyme activity is expressed in International units (IU). One IU is defined as the
amount of enzyme that catalyzes the transformation of one micromole of substrate per
minute under decreed conditions.
IU/L = Corrected ΔOD/5 minutes x 1953

Initial OD of Blank
Corrected ΔOD = ΔOD Unknown - ΔOD Blank
DERIVATION OF FACTOR
Corrected ΔOD/5 minutes x 315 x 3.1 = 315 x 3.1 =___9765___ = 1953
Initial OD of Blank x 0.1 0.1 ΔOD/min x 5
Where: 315 = concentration of olive oil in umol/L in working solution

3.1 = volume of reaction mixture
0.1 = volume of sample in mL
ΔOD/min x 5 = conversion of ΔOD/min
UNIT CONVERSION
To convert IU/L to Cherry-Crandall units, divide IU/L by 70.
REFERENCE RANGE
Adults: 10 – 150 IU/L (more than 60 years old is expected to have 18-180 IU/L)
Result:
Remarks:____________________________________________________________________________________________
___________________________________
67
CARDIAC ENZYMES
Some enzymes like CK, LDH, HBD and AST are good markers of heart muscle injury like in the
case of myocardial infarction. Other protein markers include myoglobin and cardiac troponins I
and T.
Exercise No. 28
CREATINE KINASE (CK) (A.L.S. Biochemicals product insert, 2000)
Quantitative determination of creatine kinase in human serum.
INTRODUCTION
Creatine kinase plays an important role in energy-storing mechanism of tissue by catalyzing the
reversible reaction between creatine and ATP to form creatine phosphate and ADP. CK is
distributed in various organs; highest activities (in decreasing order) are skeletal muscles, heart,
and brain. Thus, determination of CK is an aid in diagnosing muscular dystrophy and other
diseases of the skeletal muscles, myocardial infarction, hypothyroidism, renal diseases, and/or
dysfunction.
The early procedure for determining CK was based on the rate of ATP production. A modified
method was described by Nielson by adding a sulfhydryl compound and AMP to assure maximum
CK activity and inhibit adenylate kinase activity. Optimized conditions for measuring CK were
published by Szasz in 1976 as well as the Scandinavian committee on enzyme. The above
procedure was modified again in 1979 to include EDTA. The present reagent is a modification of
the above revision.

TEST PRINCIPLE
CK catalyzes the conversion of creatine phosphate and ADP to creatine and ATP. The ATP and
glucose are converted to ADP and glucose-6-phosphate by hexokinase. Glucose-6-phosphate
dehydrogenase oxidizes the glucose-6-phosphate and reduces NAD+. The rate of NADH
formation, measured at 340 nm is directly proportional to serum CK activity.
CK
Creatine phosphate + ADP ------------------ Creatine + ATP
Hexokinase
ATP + Glucose ----------------- ADP + Glucose-6-phosphate
G-6-PD
+
Glucose-6-phosphate + NAD --------------- 6-phosphogluconate + NADH + H+
REAGENT COMPOSITION
When reconstituted as directed, the reagent for CK contains the following:
D-glucose 20 mM
Magnesium ions 10 mM
68
AMP 50 mM
N-acetylcysteine 20 mM
Creatine phosphate 30 mM
ADP 2 mM
Oxidized NADP 2 mM
G6PD (EC 1.1.1.49) 3000 U/L
Hexokinase (EC 2.7.1.1.) 3000 U/L
EDTA 2 mM
Buffer 100 mM
TEST PARAMETERS
Method: UV, Increasing reaction, CK-NAC method
Wavelength: 340 nm
Incubation Temperature: 37˚C
Linearity: extends up to 1,200 IU/L

The damp and clumped appearance of reagents indicate deterioration and should be discarded.
If the reconstituted CK reagent without added sample has an OD of greater than 0.70 at 340nm
versus reagent grade water, the reagent is considered to be unsatisfactory for use and should be
discarded.

Storage: 2-8˚C prior to reconstitution
Stability: up to the expiration date indicated on the label; after reconstitution the
reagent is stable up to 24 hours at RT and 21 days at refrigerator temperature.
SAMPLE STABILITY
Freshly drawn blood from non-traumatic venipuncture is ideal. No special additives for serum are
needed. Centrifuge and remove serum immediately. Serum is stable for 4 hours at RT., 8-12 hours
at 4degC, and 2-3 days when frozen. Hemolysed specimens should not be used because of side
reactions that may occur due to adenylate kinase, adenosine triphosphate, and G6PD liberated
from cells.

1) Reconstitute reagent according to instruction.
2) Pipet 1 mL of reagent into appropriate tubes and pre-warm at 37oC for 4 minutes.
4) Add 0.25 mL of sample to the reagent, mix, and incubate at 37deg C for 2 minutes.
69
5) After 2 minutes, read and record the OD. Return tubes to 37 deg C and repeat readings
every minute for the next 2 minutes.
6) Calculate the average OD per minute (ΔOD/min).
7) The ΔOD/min multiplied by the factor 6592 will yield results in IU/L.
8) Samples with values above 1200 IU/L should be diluted 1:1 with saline, re-assayed, and
the results multiplied by 2.

IU/L = Δ A/min X TV x 1000 = Δ A/min X 1.025 x 1000 = Δ A/min X 6592
b x ε x SV 1 x 6.22 x 0.025
Where: ΔA/min = average absorbance change per minute

TV = total reaction volume (1.025)
1000 = conversion of IU/mL to IU/L
b = light path in cm.

ε = millimolar absorptivity of NADH (6.22)
SV = sample volume in mL (0.025)
If 3 mL reagent and 0.1 mL of samples are used, then IU/L = ΔA/min x 4984.
UNIT CONVERSION
SI units: To convert to SI units (nKat/L) multiply IU/L by 16.67.
REFERENCE RANGE
Serum: 25-192 IU/L at 37 deg C
10-109 IU/L at 30 deg C
Result:
Remarks:
______________________________________________________________________________________________
________________________________________________
PROCEDURE LIMITATIONS
1) Some inhibitors of CK activity are as follows:
a) Excessive ions like magnesium, chloride and sulfates
b) Most heavy earth metals like zinc, copper and manganese
c) Iodoacetate and other sulfhydryl binding groups
d) Excess ADP, citrate, fluoride, L-thyroxine
e) Excess uric acid
70
2) This procedure measures total CK activity irrespective of its tissue or organ of origin
3) Lower than expected CK values have been reported in samples having high ALP activity.
Exercise No. 29
LACTATE DEHYDROGENASE (LDH) (Dialab product insert, 2008)
Diagnostic reagent for quantitative in vitro determination of LDH in human serum or plasma on
photometric systems.

TEST PRINCIPLE
LDH is an enzyme, consisting of five different isoenzymes that catalyze the interconversion of
Llactate and pyruvate. LDH is present in the cytoplasm of all human tissues with higher
concentrations in liver, heart, and skeletal muscles and lower in RBCs, pancreas, kidney, and
stomach. Increased LDH activities are found in a variety of pathological conditions such as
myocardial infarction, liver diseases, blood diseases, cancer or muscle diseases. However, because
of the lack of organ specificity, determination of its isoenzymes or other enzymes such as ALP or
GOT/GPT is necessary in differential diagnosis.
LDH
L-lactate + NAD+ -------------------- Pyruvate + NADH + H+
TEST PARAMETERS
Method: UV, Kinetic, Increasing reaction, LDH-L IFCC method
Temperature: 37˚C
REAGENT COMPOSITION
Reagent 1
N-methyl-D-Glucamine, pH 9.4 325 mmol/L
L-lactate 50 mmol/L
Reagent 2
71
NAD+ 10 mmol/L
REAGENT PREPARATION
reagent 2. It is stable for 12 hours at 2-8˚C and 2 hours at 15-25˚C.

Storage: 2-8˚C

Do not freeze the reagents.
Loss of Activity: at 2-8˚C is
<2% within 2 days at 15-25˚C is < 8%
within 2 hours
Discard contaminated specimens. Do not use hemolysed samples.
interference up to:

SUBSTRATE START
Pipet into test tubes Blank Sample/Stan
dard
RGW 40 uL -
Sample/Standard - 40 uL
Mix. Incubate for 1-5 minutes. Then add:
and 3 minutes. Determine ΔA/min = [ΔA/min sample] – [ΔA/min
blank] during the linear part of the assay.
SAMPLE START
Pipet into test tubes Blank Sample/Stan
dard
72

RGW 40 uL -
Sample/standard - 40 uL

and 3 minutes. Determine ΔA/min = [ΔA/min sample] – [ΔA/min
blank] during the linear part of the assay.

LDH (U/L) = ∆A/ minute X Factor
Factors:
Substrate start 37˚C
At 340 nm 10080
At 334 nm 10275
At 365 nm 18675
Sample start 37˚C

At 340 nm 8095
At 334 nm 8250
At 365 nm 15000
UNIT CONVERSION

Adults Female: <247 U/L Male: <248 U/L
Result:
Remarks:____________________________________________________________________________________________
_________________________________________
73

ENDOCRINOLOGY
Exercise No. 30
24-HOUR URINE (dU) COLLECTION
Timed urine samples are requested for some laboratory tests that require more amount of urine
and thus a higher possibility of measuring a particular substance that is usually produced in the
body during pathologic states or monitoring substances after they are introduced in the body.
Hormone and drug or dye metabolites are conventionally assessed using 24-hour urine collection
as well as completeness of urine collection, and diagnosis of oliguria or polyuria.
Objectives:
1. To collect properly a 24-hour urine and aliquot the collection in preparation for
hormone assays.
2. To accurately assess macroscopically the 24-hour urine collected.
Materials: 1-liter or 2-liter wide-mouthed polyethylene bottles with screw caps

Cooling equipment or apparatus
15 ml. Glacial HAc
Procedure: 1. Discard the 1st morning urine to signal the start of the 24-hour collection.
2. Collect all the succeeding urine samples thereafter and mix evenly with the acid
preservative and maintain refrigerated or on ice the whole time.
3. Stop the collection after the 24th hour but include the urine during the last hour.
Comments: 1. Keep the urine container tightly capped every after collection.
2. Follow food restrictions before the 24-hour urine collection.
Results of Macroscopic Examination:

Color: _____________________________ Clarity:
_____________________________ pH:
_____________________________
Specific gravity:_____________________________
Volume: _____________________________
Aliquots made:_____________________________

74
Exercise No. 31
ORAL GLUCOSE TOLERANCE TEST
Patients with mild or diet-controlled diabetes may have fasting blood glucose levels within the
normal range, but be unable to produce sufficient insulin for prompt metabolism of ingested
carbohydrate. As a result, blood glucose rises to abnormally high levels and the return to normal
is delayed. In other words, the patient has decreased tolerance for glucose. Therefore, glucose
tolerance tests are most helpful in establishing a diagnosis of a mild case of diabetes.
When a standard dose of 75 g of glucose is given orally, absorption occurs rapidly and the blood
glucose concentration increases. This stimulates the pancreas to produce more insulin, with the
result that after 30 to 60 minutes the blood glucose level begins to decrease. Since there now
exists more insulin than necessary, the blood glucose tends to drop below the fasting level after
2 hours, and then returns to normal levels. Values refer to serum or plasma glucose
concentrations.
The significance and interpretation of glucose tolerance tests have been reviewed by Duff et al.
The major problem is to define criteria that provide both sensitivity and specificity. Sensitivity is
the extent to which the test identifies diseased individuals. Specificity on the other hand, is the
extent to which non-diseased individuals are classified as normal. When limits are set too low we
achieve good sensitivity but poor specificity. Conversely, with high limits we lose sensitivity but
attain good specificity.
Criteria for the diagnosis of diabetes have been evaluated by the Committee Statistics of the
American Diabetes Association and are shown in Table 1. The Wilkerson Point System provides
the same interpretation as the criteria adopted by the United States Public Health Service. Other
investigators are of the opinion that plasma values of more than 175 mg/100 ml after 1 hour and
more than 129 mg/100 ml after 2 hours should be considered abnormal.
Table 1. Various Criteria for the Standard Oral Glucose Tolerance Test
Hour Whole Blood* Plasma* Points

Wilkerson Point System 0 110 130 1
1 170 195 ½
2 120 140 ½
3 110 130 1

Values equal to or more than those listed are given points as shown. Two
points or more are judged diagnostic of diabetes.
Fajans-Conn Criteria 1 160 185
1½ 140 165
2 120 140
All levels must be equal to or greater than values shown at the times
specified to make a diagnosis of diabetes. Criteria apply to ambulatory
individuals under the age of 50.
75
University Group Diabetes Program

The subject is judged diabetic if the sum of values obtained at 0, 1, 2, and 3 hours
equals 500 or more for whole blood, or 600 or more for plasma.
*Values for whole blood of plasma glucose are given in mg/100 ml.
Attempts have been made to correlate results of glucose tolerance tests on diabetics with
fundamental defect in insulin secretion. Ketosis-prone diabetics show low to zero levels of plasma
insulin, both fasting and after glucose administration. The most consistent abnormality in
diabetics appears to be blunting of the early insulin peak in the first few minutes after intravenous
glucose injection. However, 15 to 20 percent of “normal” patients also have absence of the early
insulin peak. Whether this group is really normal or potentially diabetic is not clear.
Growth hormone inhibits glycolysis and glucose uptake by muscle cells, and causes a rise in blood
glucose. Growth hormone secretion is stimulated by hypoglycemia. Hence, growth hormone and
insulin levels tend to vary inversely. As glucose is absorbed from the gastrointestinal tract, blood
glucose levels rise. Feedback control normally results in a 10- to 15fold rise in insulin levels and
almost complete disappearance of growth hormone from the plasma. This insures storage of
glycogen. After 2 to 4 hours, growth hormone rises to near basal levels, and those of insulin fall,
although remaining at several times those of the initial concentrations. If there is a relative excess
of insulin at this time, hypoglycemia may occur. If fasting continues, insulin almost disappears
from the plasma and growth hormone rises to very high levels. This stimulates oxidation of fat
and release of free fatty acids while minimizing metabolism of glucose and protecting glycogen
stores for potential stress situations. Both insulin and growth hormone assays in plasma may be
requested of the laboratory as aids in the interpretation of glucose tolerance tests.
The severe diabetic is strongly disposed to develop ketosis and pass into diabetic coma. In
diabetic ketosis, plasma glucose levels, derived mostly from gluconeogenesis, are usually
significantly elevated. Resultant severe glycosuria produces osmotic diuresis with fluid loss and
depletion of electrolytes. Vomiting is frequently present and adds to the fluid and electrolyte
depletion. Ketosis develops as a result of reduced glucose metabolism. Increased utilization of
depot fat results in increased release of free fatty acids which in turn form acidic ketone bodies.
These react with part of the plasma bicarbonate, resulting in a lower blood pH, a lower plasma

bicarbonate, and a decrease in urinary pH. The developing metabolic acidosis stimulates the
respiratory center and breathing becomes deeper followed by a lowering of pCO 2. In summary,
the major findings in diabetic ketosis are hyperglycemia and glycosuria, hyperventilation,
dehydration (hemoconcentration and mild uremia), ketone bodies in blood and urine, low plasma
bicarbonate, low blood pH, and hyperkalemia.
Severe symptoms may develop also in hypoglycemia, i.e., with plasma glucose levels below 40
mg/100 ml. The clinical symptoms are related to the rate of decrease of plasma glucose levels; if
levels have dropped rapidly, a person may appear clinically hypoglycemic with higher glucose
levels. If the levels have fallen gradually, the individual may show no symptoms, even with a
plasma glucose as low as 30 mg/100 ml. Cerebral metabolism is dependent on an adequate 76
supply of glucose from the blood, and symptoms of hypoglycemia resemble those of cerebral
anoxia. These include fainting, dizziness, or lethargy which may progress rapidly into coma. If
untreated, death or permanent cerebral damage may result. Rapid restoration of blood glucose
concentration is essential.
A number of conditions may cause or precipitate hypoglycemia. Among these are overdosage
with insulin, drug administration (sulfonylureas, phenformin, antihistamines), functional
hypoglycemia (sensitivity to glucose), depleted glycogen stores in the liver, ingestion of large
amounts of alcohol, islet cell tumors (insulinoma) or islet cell hyperplasia, galactosemia, and
glycogen storage disease.
Plasma glucose levels in newborn infants are typically less than those for adults. In the lowbirth-
weight neonate, hypoglycemia may be defined as levels of whole blood glucose below 20 mg/100
ml. In the full-sized infant, blood glucose levels less than 30 mg/100 ml in the first 48 h of life
and less than 40 to 50 mg/100 ml thereafter may be considered hypoglycemic.
Objectives: 1. To perform correctly the standard OGTT procedure.

2. To evaluate accurately the patient curve obtained by plotting glucose levels
measured at different time times after the baseline glucose level
determination.
3. To perform glucose measurements using blood and urine concurrently.
Procedure: 1. Determine the baseline blood glucose level of the patient after a 8-hour fast.
2. Let the patient take orally the 75g. glucose load within 5 minutes.
3. Determine serially the blood glucose levels 1 hour, 1½-hours, 2-hours, and 3-
hours after the glucose load.
4. Urine glucose should also be determined concurrently.
5. Plot the glucose concentrations versus the collection times.
6. Interpret the curve as hypoglycemic, borderline diabetic, mild diabetic,

moderate diabetic or severe diabetic.
COMMENTS ON THE PROCEDURE
Occasionally the final solution may show some turbidity. This can occur if the cooling bath is too
cold, but usually is encountered with serum or plasma specimens having a high lipid content. If
the mixture is cloudy, sometimes one repeats the test on a protein-free filtrate. The color follows
Beer’s law with most spectrophotomers but this should always be checked for a given instrument
by analyzing standards ranging from 100 to 500 mg/100 ml. Sufficient reagent is present to
permit simple dilution of the final reaction mixture with acetic acid for values up to 2000 mg/100
ml.
Moderate hemolysis does not interfere significantly. Each 100 mg/100 ml of hemoglobin increases
the apparent glucose concentration by 2 mg/100 ml. Bilirubin does not react under 77
the above conditions, and interference is negligible. In some modifications of this method,
undiluted serum is added directly to the reagent. More intense color is obtained with glucose
under these relatively anhydrous conditions, but the interference from bilirubin becomes
significant because of its conversion to the green pigment biliverdin.
EDTA in concentrations greater than 1 mg/ml and sodium fluoride at leves grater than 5 mg/ml
in the specimen will cause some increase in color. Thymol preservative should be avoided since
this inhibits color formation. Dextran, used as plasma expander, produces turbidity in the reaction
and leads to falsely elevated values.
Normal ranges for the o-toluidine method are essentially the same as those for the glucose
oxidase and hexokinase procedures, since all three methods give similar results. In patients with
uremia, however, higher values are obtained by the o-toluidine method, though they are not as
high as those obtained with the ferricyanide method.
Results:
Time (Hours) Blood Glucose Concentration (in mg/dl & mmol/L)
Baseline (8-hour fast) ______________________________
1 hour after glucose load ______________________________
1 and 1/2 hours after load ______________________________
2 hours after glucose load ______________________________
3 hours after glucose load ______________________________
Urine glucose in mg/dl Sample 1 _______________ Sample 2 _______________
Paste the graph (Glucose concentration vs. Time) with the curve duly interpreted beside this
page.

Exercise No. 32
URINARY CORTICOSTEROIDS
DETERMINATION OF URINARY CORTICOSTEROIDS BY A MODIFIED PORTER-SILBER

METHOD
PRINCIPLE
In 1950 Porter and Silber described a color reaction based upon the formation of a yellow pigment
(absorption maximum at 410 nm) when certain corticosteroids react with phenylhydrazine in the
presence of alcohol and sulfuric acid. They demonstrated that this color reaction is given primarily
with corticosteroids that possess a dihydroxyacetone side chain. Corticosteroids with this
configuration include cortisol, cortisone, 11-deocycortisol, and their tetrahydro derivatives.
78
The basic steps of the procedure consist of hydrolysis of conjugates by β-glucoronidase;

extraction with chloroform; washing the chloroform extract with dilute alkali to remove estrogens,
bile acids, and interfering chromoggens; and color reaction with alcoholic phenylhydrazine-
sulfuric acid reagent.
Objectives: 1. To perform correctly a conventional method of measuring urinary

corticosteroids.
2. To calculate accurately corticosteroid levels in conventional units as mg/dl &
mg/d.
REAGENTS
1. Chloroform, AR. The solvent should be freshly distilled from anhydrous potassium carbonate
(K2CO3). Store freshly distilled chloroform in an amber bottle. To prevent formation of
phosgene, 1 percent ethanol should be added.
2. Sodium hydroxide, 0.1 mol/l. Dissolve 4 g of sodium hydroxide pellets in 1 liter of distilled
water.
3. Ethanol, purified. Absolute ethanol is purified as follows: To 1 liter of absolute ethanol, add
2 g 2,4-dinitrophenylhydrazine hydrochloride and 0.5 ml concentrated hydrochloric acid (HCl).
Let stand for approximately 48 h. Distill through a 10 inch Vigreaux column, discarding the
first and last 100 ml. Redistill through the same column, again discarding the first and last

100 ml. The purity is determined by mixing this ethanol with the phenylhydrazine-sulfuric acid
reagent. No color should develop on standing overnight at room temperature.
4. Sulfuric acid, 64 percent (v/v). To 360 ml distilled water slowly add 640 ml of concentrated
sulfuric acid, AR, with constant swirling. Prepare only in a Pyrex container (2 liter Erlenmeyer
flask) immersed in an ice water bath; the solution becomes extremely hot.
5. Alcoholic-sulfuric acid reagent (blank reagent). Mix 100 ml 64 percent sulfuric acid with 50 ml
absolute ethanol. The reagent is stable indefinitely.
6. Phenylhydrazine hydrochloride, recrystallized. A commercially available, chemically pure of

phenylhydrazine hydrochloride is purified further as follows: add 100 g of phenylhydrazine
hydrochloride to 500 ml of warm water at 70 oC. Add 1 g activated charcoal. Heat 1 liter
ethanol to boiling and add to the dissolved phenylhydrazine in the water. Quickly filter while
hot through Whatman No. 2 filter paper. Cool the filtrate in the refrigerator and collect the
crystals in a sintered glass filter with medium porosity. Repeat the procedure of
recrystallization, 79
dissolving the crystals in proportionally less water. Wash the last collection of crystals with cold
ethanol and dry thoroughly. Store in a tightly stoppered brown bottle in a dessicator over
anhydrous calcium chloride. The purified material should have a melting point of 240o to 243oC.
7. Alcoholic phenylhydrazine-sulfuric acid reagent. Dissolve 50 mg recrystallized

phenylhydrazine hydrochloride in 50 ml alcoholic-sulfuric acid reagent. The reagent should
be prepared fresh before use.
8. β-Glucuronidase. The optimal pH and buffer to be used will vary with the source of the
enzyme. Beef liver β-glucuronidase (Ketodase, Warner-Chilcott Laboratories, Morris Plains,
N.J.) is incubated in the presence of 0.1 mol/l acetate buffer at pH 5. Bacterial βglucuronidase
(Sigma Chemical Company, St. Louis, MO.) is incubated in 0.1 mol/l phosphate buffer at pH
6.8. Prepare the enzyme solution is the concentration of 1000 units/ml. This should be
prepared fresh before use.
9. Buffer solutions. Phosphate buffer, pH 6.8, 0.5 mol/l. To 500 ml 1.0 mol/l solution of KH 2PO4
(68.0 g dissolved in 500 ml) add 1 mol/l NaOH to bring the pH to 6.8. Adjust the solution to
a final volume of 1 liter.
Acetate buffer, 1.0 mol/l, pH 5. Dissolve 95 g of sodium acetate 3H 2O and 17.2 ml glacial acetic
acid in water, and dilute to a volume of 1 liter.
10. Steroid standard, stock solution. Transfer 25 mg cortisol or tetrahydrocortisone to a 250 ml

volumetric flask, and dilute to the mark with absolute ethanol. This stock standard solution
contains 100 g/ml.

11. Working standard solution. Transfer 5 ml of stock standard solution to a 100 ml volmetric
flask, and dilute to the mark with distilled water. This working standard solution contains 5
g/ml.
COLLECTION OF SPECIMEN
Collection of complete 24 h urine specimen is very important. Creatinine determined is believed

to be a fair check for completeness of the specimen. The urine may be stored without any
preservative in the refrigerator for a few days. Alternatively, the addition of 1 g boric acid per liter
of urine will preserve the specimen at room temperature without any bacterial decomposition of
the steroids.
PROCEDURE
Hydrolysis, Extraction, and Washing
80
1. Transfer 10 ml urine to a 250 ml glass-stoppered cylinder. Adjust the pH of the urine to 6.8
using indicator paper. Add 1 ml β-glucurindase (bacterial) solution (1000 units), 2 ml 0.5 mol/l
phosphate buffer, and 0.1 ml chloroform.
2. In a similar manner, prepare the water bland and standards using 10 ml of distilled water and
10 ml of working standard solution, respectively, instead of urine.
3. Mix the samples well and incubate at 37oC for 18 to 24 h.
4. To each tube add approximately 3 g ammonium sulfate, and mix. Add 100 ml chloroform to
each glass-stoppered cylinder and mix the contents by repeated inversion for 30 s. Let the
cylinders stand for 5 min in order to separate the aqueous and the organic phases.
5. Remove the aqueous supernatants by aspiration.
6. Add 10 ml of 0.1 mol/l NaOH to each cylinder and shake for 30 s. Allow to stand for 5 min.
Aspirate off the alkali layer.
7. In a similar manner wash the chloroform extracts twice with 10 ml of distilled water.
Porter-Silber Reaction

1. Transfer 40 ml aliquots of each chloroform extract to properly labeled 50 ml glass-stoppered
centrifuge tubes as follows:
Blank-Blank Phenyl- Standard- Standard- Test-Blank Test-
Blank Blank Phenyl Phenyl
40 ml blank 40 ml blank 40 ml standard 40 ml standard 40 ml urine 40 ml urine
extract extract extract extract extract extract
+ + + + + +
5 ml blank 5 ml 5 ml blank 5 ml 5 ml blank 5 ml

phenylhydrazine phenylhydrazine phenyl-
reagent reagent reagent reagent reagent
hydrazine
(alcoh. reagent
H2SO4)
81
2. Tightly stopper all tubes, shake vigorously for 30 s and allow to stand for 15 to 20 min.
Alternatively, centrifuge the tubes at 2000 rpm for 10 min.
3. Transfer approximately 2.5 ml of the supernatant phase from each tube into corresponding
labeled 10 X 75 mm Coleman cuvets.
4. Incubate the tubes in a water bath at 60 oC for 30 min, or overnight in the dark at room
temperature.
5. Measure the absorbance (A) with a spectrophotometer at wavelength 410 nm as follows: Adjust
the photometer to zero absorbance using the blank-blank, and read the standard and test
blanks. Similarly, set the phenyl blank at zero absorbance and read the standard and test phenyl
tubes.
CALCULATION
The standard sample contains 0.05 mg of cortisol. Incorporating this value and the
appropriate dilution factor (10) to calculate the concentration of corticosteroids/100 ml of
urine, the following equation is derived:

Corticosteroids (mg/100 ml) = A test – A test blank X 10 X 0.05
A standard – A standard blank
= A test – A test blank X 0.05

A standard – A standard blank
The excretion of corticorsteroids per diem (d) is calculated as follows:
Corticorsteroids = conc (mg/100 ml) X urine volume (ml/d)

(mg/d) 100
COMMENTS
Acid hydrolysis is unsuitable because the free corticorsteroids are liable in a strongly acidic
medium. The metabolites of cortisol contain numerous hydroxyl and keto groups, making
them relatively hydrophilic. The use of a polar organic solvent such as chloroform insures
quantitative extraction of these steroids from hydrolized urine. To remove acidic components
and phenols including estorgens, the solvent extract is washed with dilute 82
alkali. The use a strong alkali (stronger than 0.1 mol/l) destroys the corticosteroids. The alkali-
washed extract, termed the neutral fraction, contains metabolites of cortisol and of all other
steroids excreted as glucuronides, as well as any other neutral lipid-soluble materials of urine.
The selectivity of the color reaction toward the steroids with dihydroxyacetone side chains
obviates the need for further purification. The impurities present in the extract form
nonspecific brown chromogens in the presence of sulfuric acid. The use of “urine blank”
corrects for such background interference.
Various nonsteroidal substances, including acetone, fructose, and dehydroascorbic acid, also
form a colored complex with the Porter-Siber reagent. In addition, the following drugs and
their metabolites have reported to cause interference with the colorimetric estimation:
iodides, paraldehyde, chroal hydrate, Furadantin, bilirubin, colchicine, coffee, most sulfa drugs,
chlorophenothiazines, spirolactones, quinine, and Darvon. Administration of these drugs
should be withheld for several days prior to determination of corticosteroids.
NORMAL VALUES
Children (up to 1 year): 0.5 – 1.0 mg/d

Adult male: 3 – 10 mg/d
Adult female: 2-8 mg/d
REFERENCE

Sunderman, F.W., Jr.: In Lipids and the Steroid Hormones in Clinical Medicine. F. W. Sunderman
and F.W. Sunderman, Jr., Eds. Philadelphia, J.B. Lippincott Co., 1960, p. 162
Results: Urinary corticosteroids in mg/dl: __________________________

Urinary corticosteroids in mg/d: __________________________
83
Exercise No. 33
TOTAL 17-KETOGENIC STEROIDS
PRINCIPLE
In 1952 Norymberski reported that sodium bismuthate oxidizes several groups of
17hydroxycorticosteroids to 17-ketosteroids, which can then be measured by Zimmermann
reaction (see determination of total 17-ketosteroids). He termed these steroids “17-ketogenic
steroids.”
Group I includes cortisol, cortisone, their tetrahydro derivatives, 11-deoxycortisol (compound S),
and tetrahydro S; group II includes cortols and cortolones; group III constitutes pregnanetriol and
its 11-oxygenated derivatives; group IV includes 17-hydroxyprogesterone and
17hydroxypregnanolone.
It should be noted that the first two groups consist of active corticosteroids (cortisol, cortisone)
and their metabolites, whereas groups III and IV comprise mainly the metabolites of the
precursors of active corticosteroids (e.g., 17-hydroxyprogesterone). The excretion of the latter is
quantitatively very significant in certain forms of the adrenogenital syndrome. Sodium bismuthate
does not oxidize the 17-hydroxy compounds containing a ketone at C-20 and a methyl group at
C-21 as shown in IV. In later modifications, a reduction step, using sodium borohydride prior to
bismuthate oxidation, was introduced. This made it possible to measure the metabolites
containing a 21-deoxy keto side chain (e.g., 17-hydroxyprenanolone together with the

compounds included in groups I, II, and III). Following borohydride reduction, the 17hydroxy-20-
keto-21-deoxy steroids are reduced to 17,20-dihydrodroxy-21-deoxysteroids, and naturally
occurring urinary 17-keto steroids are reduced to C19 17-hydroxysteroids. Subsequent treatment
of the urine with sodium bismuthate produces 17-ketosteroids from all four groups of C21 17-
hydroxysteroids. Since sodium bismuthate does not reoxidize the C19 17hydroxysteroids, the 17-
ketosteroids originally present in the urine become negative to the Zimmermann reaction. As a
result, a determination of 17-ketosteroids after borohydride reduction and sodium bismuthate
oxidation provides a direct measure of the total C21 17hydroxycorticosteroids.
Objectives: 1. To perform correctly a conventional method of measuring urinary total 17-

hydroxycorticosteroids.
2. To calculate accurately 17-hydroxycorticosteroid levels in conventional units in
mg/d.
REAGENTS
1. Ethylene dichloride. Distill commercially available AR solvent from sodium carbonate (2 g/l) in
an all-glass distilling apparatus. Collect the fraction distilling between 83o and 84oC.
84
2. Sodium bismuthate, Merck, AR.
3. Sodium bisulfite solution, 5 g/100 ml. Prepare fresh use.
4. Sodium borohydride (Metal Hydrides, Inc. Beverly, MA).
5. Tes-Tape (Eli Lily & Co., Indianapolis, IN).
Other reagents are as those described for urinary 17-ketosteroids determination.
APPARATUS
Special glassware: glass-stoppered heavy-walled centrifuge tubes of 35 ml and 50 ml capacity.
Mechanical shaker: Burrel, wrist-action shaker.
PROCEDURE:
1. Test urine with pH paper. If alkaline, acidify with glacial acetic acid (to dissolve phosphate
precipitate if present).
2. Using Test-Tape, determine the approximate concentration of glucose in the sample. If the
specimen contains less than 0.5 g glucose/100 ml, proceed to step 3. If the specimen contains
more than 0.5 glucose/100 ml, separate the glucose from the steroids as follows: Transfer 20
ml of urine to a glass-stoppered centrifuge tube, add 10 g ammonium sulfate, and mix to

dissolve the salt. Extract three time with 20 ml portions of solvent (ether ethanol, 3:1).
Evaporate the combined extracts to dryness under nitrogen in a water bath at 50 oC. Add 10 ml
ethanol to the residue and warm the solution in hot water to dissolve the steroids. (Ignore the
insoluble material.) Cool, centrifuge, and transfer two 4 ml of aliquots (equivalent to 8 ml urine)
of the supernatant fluid (for duplicate analysis) to 50 ml centrifuge tubes. Evaporate the ethanol
to dryness. Redissolve the residue in 0.5 ml methanol and dilute to 8 ml with water. Proceed
to step 3, beginning with addition of sodium borohydride.
Reduction, Oxidation, Hydrolysis and Extraction
3. Place 8 ml of urine in a 125 ml Erlenmeyer flask. Add 100 mg sodium borohydride. Check the
pH. If the pH is not over 8, add an additional 25 mg borohydride. Let it stand for 2 h or
overnight at room temperature. (Preferably, instead of adding solid borohydride, 0.8 ml of
10 g/100 ml of freshly prepared solution of sodium borohydride may be used.)
4. Add 8 ml glacial acetic acid and allow to stand for 15 min. (The acid decomposes the excess
borohydride.)
85
5. Transfer to a 50 ml centrifuge tube. Add 2 g sodium bismuthate. Stopper and shake

mechanically for 30 min away from direct sunlight. (The sample may be covered with a heavy
black cloth during the treatment with bismuthate.) Add 2 g fresh sodium bismuthate and shake
for an additional 15 min. Leave the samples overnight at room temperature. The following
morning shake the tubes for 15 min.
6. Centrifuge for 10 min at 2000 rpm, and transfer 6.0 ml of the supernatant fluid to 35 ml glass-
stoppered centrifuge tubes containing 1.5 ml of freshly prepared sodium bisulfate solution.
Mix the solution and allow to stand for 5 min.
7. Add 5 ml distilled water and 3.6 ml concentrated hydrochloric acid. Let stand for 15 min.
8. Place in a boiling water bath for 10 min. Remove and cool the samples in a cold water bath.
9. Add 12 ml ethylene dichloride and shake mechanically for 15 min. Centrifuge for 2 min at 2000
rpm.
10. Aspirate off the upper phase as completely as possible without losing any organic solvent.

11. Add to the organic extract 25 to 30 pellets of sodium hyroxide. Place in shaking machine for
15 min, centrifuge, and filter through 7 cm Whatman No. 1 filter paper into a test tube.
12. Transfer 4 ml of filtrate (= 1 ml of urine) to a test tube and evaporate to dryness under nitrogen
in a water bath at 50 to 55o C. (In the case of 24 h collection of large volume, use 8 ml of
filtrate.)
Color Reaction
13. Perform the Zimmermann color reaction and measure the absorbance as described in the
method for total 17-ketosteroid determination.
CALCULATIONS
Total 17-ketogenic steroids (mg/d) = corrected A of sample x 0.05 x total urine volume (ml)
corrected A of standard
COMMENTS
86
Since the 17-ketosteroids formed from the 17-hydroxycorticosteroids are fairly stable in a hot acid
medium, the hydrolysis of steroid conjugates can now be performed with acid as opposed to the
enzymatic hydrolysis used in the direct method based on the Porter-Silber reaction. The presence
of glucose in urine interferes with the bismuthate oxidation. All urine specimens should therefore
be routinely tested with Tes-Tape, and the glucose removed before the determination is begun.
The most suitable means to rid the sample of the glucose appears to be the procedure outlined
in step 2. Errors due to the presence of glucose may, however, also be avoided by increasing the
amount of sodium bismuthate (1 g for each gram of glucose above 0.5 g/100 ml). The presence
in urine of varying amounts of reducible substances other than glucose makes the use of a large
excess of borohydride necessary. Addition of sufficient borohydride is indicated by effervescence
on the addition of acetic acid (step 4). The absence of effervescence is suggestive of an insufficient
amount of borohydride, which may yield misleading results because of the incomplete reduction
of different ketone groups. Instead of sodium bismuthate, the oxidizing agent sodium meta
periodate (10 vol percent of 10 g/100 ml solution in 0.1 mol/l NaOH) may also be used. The
advantage of using this reagent lies in the fact that in addition to oxidizing 17-
hydroxycorticosteroids to 17-ketosteroids, it oxidizes glucuronides to the free steroids or to their
formates, which are easily hydrolyzed in alkaline solution; thus, the need for acid hydrolysis is
eliminated. Necessary precautions and drug interference in the color reaction will be discussed
elsewhere (see determination of 17-ketosteroids).

Since a greater number of metabolites of cortisol (cortol and cortolone) are estimated by the
ketogenic method, the normal urinary excretion values are generally higher than those obtained
by the Porter-Silver method. This method also yields high values in the adrenogenital syndrome
because of the presence of excessive amounts of urinary metabolites of the cortisol precursors,
which go undetermined by the latter procedure.
NORMAL VALUES
Children (up to 1 year): < 1 mg/d

Children (1-10 years): 2.3 – 3.8 mg/d
Adult male: 5-23 mg/d
Adult female: 3-15 mg/d
REFERENCE
Sobel, C.S., Golub, O.J., Henry, R.J., Jacobs, S.L., and Basu, G.K.: J. Clin. Endocrinol, 18: 208, 1958.
Results: Total 17-hydroxycorticosteroids in mg/d: ________________________
87
Exercise No. 34
PLASMA CORTICOSTEROIDS
THE ESTIMATION OF CORTICOSTEROIDS IN PLASMA
The main purpose of the estimation of corticosteroids in blood or urine is to evaluate the rate of
secretion of cortisol by the adrenal cortex as well as the actual level of the hormone to which the
tissues are exposed. While the estimation of the urinary excretion of metabolites renders indirect
information regarding the overall activity of the gland (i.e., secretion rate), the blood estimation
appears to be more useful to ascertain whether the tissues are exposed to proper amounts of
cortisol. It should be noted in this connection that the urinary excretion may be elevated by the
increased rate of production and metabolism of the hormones without the physiological level in
the blood being enhanced. For example, in obesity and hyperthyroidism, the urinary excretion of
17-hydroxycorticosteroids is elevated even though the plasma level of cortisol is within the normal
range. The measurement of plasma cortisol is of value in studying the existence of normal diurnal
variation and in obtaining quick information regarding the response to functional tests employing
stimulation and suppression of the adrenals.

Cortisol represents almost 80 percent of the total 17-hydroxycorticosteroids in the blood, and the
majority circulates in its original form along with small amounts of unconjugated reduced
derivatives. The biologically active unconjugated cortisol in the plasma is bound to some extent
by albumin and to an -globulin derived mainly from the liver. This latter protein is called
transcortin, or corticosterone-binding globulin (CBG). While the precise function of such protein
binding is still obscure, it is generally suggested that this mechanism assures a ready source of
available circulating hormone and protects it from inactivation and conjugation in the liver. The
concentration of CBG in the plasma rises during pregnancy and during estrogen therapy, with a
concomitant increase of total 17-hydroxycorticosteroids in plasma. However, since the amount
of free hormone, unassociated with protein, remains at physiological levels, there are no untoward
effects. It should be noted there that the unbound portion is biologically most significant, because
this is the amount which is available for immediate physiological action. Customary procedures
involving organic solvent extraction or protein precipitation estimate both free and protein-bound
corticosteroids. Thus, with the procedure discussed, total unconjugated cortisol is measured.
Some authors have suggested that determination of urinary cortisol levels may be a more reliable
index of adrenocortical hyperfunction. It is reasoned that normally, only about 1 percent of the
total amount of cortisol secreted appears unchanged in urine, since steroids, by virtue of their
ketone group at C-17. While the conjugation of all these steroids may occur with sulfuric acid
and glucuronic acid, the glucuronide is predominant in androsterone, etiocholanolone, and 11-
oxygenated 17-ketosteroids; dehydroepiandrosterone is present exclusively as the sulfate
conjugate.
88
Exercise No. 35
URINARY 17-KETOSTEROIDS
The 17-ketosteroids are metabolites of precursors secreted by the adrenals, by the testes, and
possibly to some extent by the ovaries. In men, approximately one-third of the total urinary
17ketosteroids represent the metabolites of testosterone secreted by the testes, whereas most of
the remaining two-thirds are derived from the steroids produced by the adrenals. In women, who
usually excrete smaller quantities than men, the total 17-ketosteroids are derived almost
exclusively from the adrenals.
The bulk of the urinary 17-ketosteroids consists of androsterone, epiandrosterone,

etiocholanolone, dehydroepiandrosterone, 11-keto- and 11β-hydroxyandrosterone, and 11keto-
and 11β-hydroxyetiocholanolone. It should be recalled that dehydroepiandrosterone and 11-
oxygenated 17-ketosteroids are the products of adrenals only, while the others also give from the

precursors (androstenedione, testosterone) elaborated by the gonads. Thus, the main purpose
of the quantitation of these steroid metabolites is to assess gonadal and adrenal function.
Decreased values of 17-ketosteroids are generally obtained in primary hypogonadism

(Klinefelter’s syndrome, castration), secondary hypogonadism (panhypopituitarism), and primary
hypoadrenalism (Addison’s disease, especially in women). Increased values are obtained in
testicular tumors (interstitial cell tumor, chorioepithelioma), adrenal hyperplasia and adrenal
carcinoma.
The level of total 17-ketosteroids in urine is not a good index of androgen production by the
gonads. Urinary 17-ketosteroids are primarily derived from adrenal precursors
(dehydroepiandroseterone and androstenedione), which have little or no biological activity,
androgenic or otherwise, is implied. For example, etiocholanolone has no androgenic activity,
and testosterone (which is potent androgen) is not a 17-ketosteroid. Thus, in individual patients,
the determination of urinary 17-ketosteroids does not indicate whether testosterone production
is normal, excessive, or deficient. Direct measurement of circulating plasma testosterone in males
and the production rate of testosterone in females (see plasma testosterone, clinical significance)
represent the most accurate index of androgen production. Since suitable methods for plasma
testosterone estimation have become recently available, the measurements of total urinary 17-
ketosteroids for the evaluation of androgen production should be discontinued. However, the
relevance of the 17-ketosteroids in adrenal disease and the popularity of this test in clinical
laboratories require the description and discussion of a detailed procedure.
There are a number of chemical methods available for the estimation of total 17-ketosteroids.
The final quantitation in most of these is based on the color reaction originally described by
Zimmermann. The method described by Drekter et al., with modifications by Sobel et al., has
been shown to be most adequate for routine clinical use and is given below.
89
PRINCIPLE
The 17-ketosteroids are excreted as water-soluble conjugates of glucuronic acid and sulfuric acid.
Cleavage of these conjugates with acid is followed by extraction, washing with alkali, and finally
the color reaction. Estrone, which is a 17-ketosteroid, is removed by alkali treatment because of
its phenolic nature and thus is eliminated prior to the colorimetric reaction of the “neutral” 17-
ketosteroids. The reaction is based on the treatment of 17-ketosteroids with metadinitrobenzene
in alcoholic alkali to produce a reddish-purple color with maximum absorption at 520 nm. Marlow
has demonstrated that the development of color depends on the presence of an active methylene
group adjacent to a carbonyl group, most likely giving the following product:
When the ketone group is situated at other positions (e.g., 4-3-keto in testosterone,
progesterone, cortisol) the color development is less intense and the absorption maxima differ.

The quantitation is carried out by the comparison of the color density of the sample with that of
a known amount of pure standard, such as dehydroepiandrosterone.
Objectives: 1. To perform correctly a conventional method of measuring urinary 17-

ketosteroids.
2. To calculate accurately 17-ketosteroid levels in urine in conventional units as
mg/d.
REAGENTS
1. Ethanol, purified.
2. Ethanol, 70 percent (v/v). Dilute 700 ml of purified ethanol to 1 liter with distilled water.
3. Ethylene dichloride, redistilled.

4. Potassium hydroxide, AR grade. Prepare a saturated aqueous solution.
5. Potassium hydroxide-ethanol solution. Add 1 vol of saturated solution of potassium hydroxide

to 4 vol of purified ethanol. Centrifuge and use the supernatant. Prepare this reagent just
before use.
6. m-Dinitrobenzene, 1.16 g/100 ml purified ethanol. Purify the commercially available material
as follows: Dissolve 30 g of the substance in a minimal volume of ethanol by warming in a
steam bath. Cool, add 30 ml of 20 g/100 ml solution hydroxide in water and allow to stand for
30 min. Add 3 vol of distilled water with mixing and let it stand 15 min. Filter off the crystalline
precipitate on a Buchner funnel. Wash the crystals on the funnel with distilled water and suck
dry. Redissolve the crystals in a minimal volume of ethanol and add
90
3 vol distilled water as before. Wait for 15 min and filter on a Buchner funnel. Wash the
crystals with distilled water and suck as dry as possible. Transfer crystals to a petri dish and
dehydrate in a desiccator over anhydrous calcium chloride. Store the final product in an
amber bottle.
7. Dehydroepinadrosterone (DHEA) standard. Dissolve 10 mg of DHEA in 100 ml of purified

ethanol. This solution contains 100 g/ml.
PROCEDURE
1. Test urine with pH paper. If alkaline, acidify with glacial acetic acid to dissolve phosphate
precipitate if any is present.

Hyrolysis, Extraction, and Washing
2. Transfer 8 ml of urine to a 35 ml glass-stoppered centrifuge tube. Add 2 ml of glacial acetic

acid and 3 ml concentrated hydrochloric acid.
3. Stopper the tube and place it in a 100oC bath for 10 min. Cool it under cold, running tap water.
4. Add 10 ml of ethylene dichloride and place in a shaking machine for 15 min.
5. Centrifuge for 2 min at 2000 rpm and aspirate off the urine as completely as possible.
6. Add to the solvent 25 to 30 pellets of sodium hydroxide and place in shaking machine for 15
min. Alternatively, the extract may be washed with 10 g/100 ml NaOH solution followed by
two water washes. Centrifuge as before and filter the solvent through Whatman No. 1 filter
paper.
7. Transfer 2.5 ml of the filtrate (equivalent to 2 ml urine) to a test tube and evaporate to dryness
under nitrogen in a water bath at 50 to 55 oC. (If very low concentrations are expected, use 5
ml of the filtrate.)
Color Reaction and Spectrophotometer Reading
8. Perform the Zimmermann reaction as follows: (a) To a blank tube, the sample tube, and a
standard tube containing 50 g DHEA, add 0.2 ml of m-dinitrobenzene solution. (b) Add 0.2
ml of freshly prepared alcoholic potassium hydroxide solution and mix. (c) Place the tubes in
91
a water bath at 25oC in the dark for 30 min. (d) To each tube add 5 ml of 70 percent ethanol
and mix.
9. Measure the absorbance of the standard and the sample in a spectrophotomer or a colorimetric
at 480, 520 and 560 nm, setting the instrument at 100 percent transmission with the blank
solution.
CALCULATION
Calculate the corrected optional density of the standard and sample using the following formula:
Corrected absorbance = (A520) - (A480 +(A560)

2

Calculate 24 h excretion of 17-ketosteroids as follows:
mg 17-ketosteroids = corrected A of sample x 0.05 x 24 h urine volume (ml)

corrected A of standard 2
COMMENTS
Most of the urinary 17-ketosteroids are excreted as sulfate and glucuronide conjugates which are
hydrolyzed by strong acid and heat. The duration of hydrolysis is very critical. Less than 10
minutes will cause incomplete hydrolysis and more than 10 minutes will lead to gradual
destruction of the steroids and formation of an increased amount of nonsteroidal chromogens.
Addition of glacial acetic acid helps to minimize the formation of nonspecific chromogens during
the hydrolytic procedure, particularly in the case of an alkaline urine specimen. Although solvents
such as benzene, carbon tetrachloride, and ether are suitable for extraction, ethylene dichloride
is aptly suitable because it can extract the steroid hormones from hydrolyzed urine more
quantitatively at a relatively low ratio of the solvent to urine. This is technically advantageous for
a routine laboratory method since it avoids the need for handling and evaporating large quantities
of solvent.
According to Drekter et al., the treatment of the extract with pellets of sodium hydroxide is
superior to the customary treatment with aqueous sodium hydroxide because solid NaOH
removes phenols and other urinary pigments more completely.
For the Zimmermann reaction two different alkaline reagents have been in common use, namely,
aqueous and alcoholic KOH. The former yields colors of much less intensity than the alcoholic
reagent and the latter has the disadvantage of being unstable. As suggested by Sobel et al., the
saturated solution of KOH is stable and yields very low blank absorbance. According to the same
authors, the time, temperature, and dilution with 70 percent ethanol give maximum 92
color development and stability. To avoid undue effects in the color reaction, the ethanol must
be of highest quality and purified according to the procedure given in the text. However, in spite
of meticulous care in the preparation of reagents and in the color of development there is always
the formation of nonspecific background chromophors arising from other ketonic steroids and
nonsteroid ketones. The reading of the absorbance at three wavelengths and the use of the
correction formula serve to eliminate the effect of such background interference in the estimation.
The correction is based on the assumption that the absorbances of nonspecific materials at the
three chosen wavelengths lie on a straight line. In other methods the preparation of a urine blank
and subtraction of its reading from the sample is supposed to serve the same purpose. The
following drugs and their metabolites in urine are known to yield spurious results causing either
under- or overestimation of 17-ketosteroids: ascorbic acid, Doriden, morphine, meprobamate,
and penicillin G.

NORMAL VALUES
Up to 1 year less than 1 mg/d

1-4 years less than 2 mg/d
5-8 years less than 3 mg/d
8-12 years 3 to 10 mg/d
13-16 years 5 to 12 mg/d
Young adult male 9 to 22 mg/d
Adult male 8 to 20 mg/d
Adult female 6 to 15 mg/d
The excretion values are the same for both sexes throughout childhood. After about age 60, the
rate of excretion declines progressively in both sexes.
Results: Total urinary 17-ketosteroids in mg/d:_____________________________
Exercise No. 36
URINARY TOTAL ESTROGENS
PRINCIPLE
The following basic steps are involved: acid hydrolysis, extraction with diethyl ether, washing the
ether extract with carbonate buffer, separation into phenolic and neutral steroids by partition
between sodium hydroxide solution and the organic extract, reextraction of phenolic sterids with
diethyl ether from aqueous solution, development of the Kober color followed by extraction of
the color complex with chloroform containing 2 percent -nitrophenol, and measurement of the
fluorescence of the organic phase in a fluorometer using 530 nm as the wavelength for excitation
and 550 nm as the wavelength for emission. The amount of estrogens is calculated by comparing
the intensity of fluorescence of the sample with that obtained from standard mixtures (estrone,
estradiol-17β, and estriol) of known concentration.
93
Objectives: 1. To perform correctly a conventional method of measuring urinary total

estrogens.
2. To calculate accurately total estrogens levelsin urine in conventional units as
μg/d.
REAGENTS
All reagents should be of AR grade.

1. Hydrochloric acid, concentrated.
2. Diethyl ether, freshly distilled in all-glass apparatus.
3. Sodium carbonate buffer, pH 10.5, prepared by mixing 150 ml of 20 percent (w/v) sodium
hydroxide solution with 1 liter of 8 percent (w/v) sodium hydroxide solution with 1 liter of 8
percent (w/v) sodium bicarbonate.
4. Light petroleum ether (b.p. 40 to 60oC), redistilled in all-glass apparatus.
5. Sodium bicarbonate, 8% (w/v) solution.
6. Sodium hydroxide, 1 mol/l. Dissolve 40 g sodium hydroxide pellets in 1 liter of distilled water.
7. Sodium sulfate, anhydrous.
8. Ethanol, absolute, purified (see corticosteroid estimation).
9. Hydroquinone, recrystallized from ethanol, 4 g/100 ml ethanol.
10. -Nitrophenol, recrystallized from benzene; dissolve 2 g -nitrophenol in 98 ml of chloroform

containing 1% (v/v) ethanol.
11. Sulfuric acid, concentrated.
12. Chloroform, freshly distilled in all-glass apparatus.
13. Standard solution: Weigh 8 mg each of estrone and estriol, and 4 mg of estradiol 17β and
dissolve in 100 ml absolute ethanol to 100 ml in a volumetric flask. (This working standard
94
contains 0.008 g of estrone and estriol and 0.004 g estradiol-17β/0.1 ml.) The stock solution
should be stored at 4oC.
PROCEDURE
Collect the specimen of urine, and test the urine for glucose as described for the total 17ketogenic
steroids. If the glucose concentration is more than 0.5 g/100 ml, follow the procedure as

described there, using 1% of the total volume of urine. Dilute the glucose-free residue to 25 ml
with distilled water and proceed as follows.
Hydrolysis and Extraction. Transfer 1% of the total volume of urine into a 250 ml with distilled
water. Add several glass beads (to prevent bumping) and heat to boiling under a reflux a 250 ml
round-bottom flask and dilute to 25 ml with distilled water. Add several glass beads (to prevent
bumping) and heat to boiling under a reflux condenser. Add 5 ml of concentrated hydrochloric
acid through the condenser and continue boiling for 30 min. Cool the flask rapidly under running
tap water and transfer the contents to a separatory funnel. Extract the hydrolyzed urine once with
25 ml of ether and twice with the half the volume (12.5 ml) of ether. Then shake the combined
ether layers with 10 ml of sodium carbonate buffer (pH 10.5) solution. Discard the aqueous layer.
Separation of Phenolic Steroids. Add 50 ml of petroleum ether to the ether extract in the
separatory funnel. Extract the organic solvent mixture two times with 25 ml of 1 molar NaOH,
collecting the alkali layer in an Erlenmeyer flask. Partly neutralize the alkaline solution by adding
solid NaHCO3 in portions until the pH is 10. Transfer the aqueous solution into a separatory
funnel. Extract the solution three times with ether—once with an equal volume (50 ml) and twice
with half the volume (25 ml). Then shake the combined ether layers with 20 ml of sodium
bicarbonate (8 g/100 ml). Discard the aqueous layer. Wash the ether extract with 10 ml of distilled
water and drain off the water as completely as possible. Transfer the ether extract to an
Erlenmeyer flask containing 5 g anhydrous sodium sulfate. Rinse the separatory funnel with a few
ml of fresh ether and add to the flask. Filter the ether extract through a Whatman No. 1 filter
paper into a 250 ml round-bottom flask. Evaporate the ether to dryness at 30 oC under reduced
pressure.
Fluorometry. Dissolve the residue in the flask in 5 ml of absolute ethanol. Transfer 2 ml aliquots
to two glass-stoppered tubes. Prepare standard tube (in duplicate) containing 0.1 ml (= 0.02 g
total steroids; estrone = 0.008 g, estradiol = 0.004 g, and estriol = 0.008 g) of working
standard solution and a blank tube containing pure ethanol. Add 0.5 ml of 4% hydroquinone
solution to each tube, and evaporate to dryness under nitrogen in water bath at 50 oC. Place the
tubes in an ice-water bath. Add 0.4 ml of distilled water and 0.75 ml of concentrated sulfuric acid.
Stopper the tubes and heat in a boiling water bath for 40 min with occasional shaking. Place the
tubes in an ice bath. Add to the tubes 1.5 ml of distilled water and mix thoroughly. Allow the
tubes to stand in ice not less than 5 min and not more than 25 min. Add 2.5 ml of 2% -
nitrophenol in chloroform. Stopper the tubes and shake vigorously for 30 s. Centrifuge the tubes
for 3 min at 2500 rpm. Aspirate off the upper layer. Transfer the 95
organic phase into the cuvets. Read the fluorescence at 550 nm following excitation at 530 nm.
Set the instrument to zero absorbance with the blank, and read the standards and the samples.
CALCULATION

If 1% of the total volume of urine is used, then
A g of total estrogens/d = reading of sample x 0.02 x 5/2 x 100

reading of standard
COMMENTS
Since the bulk of the estrogens are excreted as water-soluble conjugates of glucuronic and sulfuric
acids, hydrolysis is necessary to allow extraction of the steroids with an organic solvent such as
ether. As a matter of expediency, acid hydrolysis is generally used. However, during acid
hydrolysis the presence of excess glucose, hydrochlorothiazide, and a wide variety of
formaldehyde-generating drugs (e.g., methenamine mandelate) can seriously decrease the
recovery of estrogens, particularly estriol. The interference is also observed in the presence of
phenolphthalein, cascara, senna, and diethylstilberstrol. The acid hydrolysis of urine from patients
with liver disease results in negative values for estrogens. In the presence of significant amounts
of glucose, the destruction may be greater than 50%, and is over 95% in the presence of sucrose,
fructose, and inulin. A change of specific gravity of urines from 1.010 to 1.020 also causes a linear
decrease from 90% to 60% in the recovery of estriol. In recent years, urinary estriol determinations
in pregnant women have been increasingly employed as an index of the fetal well-being. Thus, it
is important that changes in estriol values truly reflect changes in excretion and not losses incurred
during hydrolysis. Measures suggested to eliminate the effects of interfering substances include
dilution of the urine, isolation of estrogen conjugates by ammonium sulfate precipitation, solvent
extraction, gel infiltration, and extraction of conjugates by neutral polysterene resin.
Washing the specimen with sodium carbonate buffer (pH 10.5) removes strongly acidic
components. As described before, estrogens are slightly acidic in nature because of the presence
of phenolic hydrolxyl group at C-3. This acidic property has been utilized for the separation of
phenolic estrogens and neutral steroids. Shaking the organic solvent with sodium hydroxide
solution moves the estrogens into the alkali layer while the neutral steroids (17ketosteroids,
pregnanediol, corticosteroids, etc.) stay in the organic phase. Petroleum ether is added to achieve
better recovery of some of the estrogens (estrone, estradiol-17β) which would otherwise be left
in some quantity in the ether layer when partitioned with sodium hydroxide solution. Since
estrogens are quite soluble at a pH above 11, the adjustment of the pH to below
10.5 is very important for quantitative extraction of estrogens with ether.
The addition of hydroquinone protects the estrogen from oxidation and facilitates the formation
of the Kober-color complex. The directions for addition of exact aliquots of different reagents for
color development, the duration of heating, and the extraction of the color complex with 96
nitrophenol solution after the specified length of time should be followed as closely as possible.
The tubes should be kept in ice at all times. The intensity of fluorescence decreases with time;
thus, the fluorometric reading should be completed within half an hour after extraction of the
color complex. Since the relative intensities of fluorescence of the three estrogens—estrone,

estradiol-17β, and estriol—are different, determination of the total estrogen content without a
separation of the individual estrogens may result in some error. The use of a standard solution
of three estrogens mixed in a ratio generally excreted in the urine of a normal nonpregnant
woman (estrone, estradiol-17β, estriol, 2/ ½ ) reduces the possibility of such error.
NORMAL VALUES
The excretion of estrogens in children is very low, being generally less than 1 g/d. In men, a
constant amount of estrogens excreted derives from the adrenals and probably also from the
testes. The average is approximately 11 g, with a range of from 5 to 18 g/d.
Normal menstrual cycle: Brown has studied extensively the excretion of estrone, estradiol-17β,
and estriol during normal menstrual cycles. The following are the excretion values of total
estrogens, computed from his data:
Onset of menstruation: 4-25 g (mean 13 g)/d

Ovulation peak: 28-29 g (mean 56 g)/d
Luteal peak: 22-105 g (mean 43 g)/d
Menopausal women: 1.4-19.6 g (mean 6.4 g)/d
Results: Total urinary estrogens in g/d: ___________________________
97

Exercise No. 37
URINARY VANILMANDELIC ACID (3-Methoxy-4-Hydroxy-mandelic Acid)
Since urinary VMA is quantitative the most important metabolite of catecholamines (epinephrine
and norepinehrine), it has served as a useful index of endogenous production of these amines.
Various methods depending on chromatography, isotope dilution, and colorimetry have been
used for its quantitative measurement. Among them, colorimetric estimations based on the
oxidation of VMA to vanillin have found widest application in routine clinical laboratories. Since
the first introduction of such a procedure, a great number of methods with various modifications
have appeared in the literature. The method of Pisano et al., which is considered to be the most
simple and best suitable for routine clinical use, is described.
PRINCIPLE
VMA, along with other phenolic acids, is extracted from acidified urine with ethyl acetate. It is
then extracted from the organic solvent with aqueous potassium carbonate solution. The
potassium carbonate extract is treated with sodium metaperiodate to oxidate VMA to vanillin. To
separate it from contaminating urinary phenolic acids, vanillin is selectively extracted into toluene
and its determined spectrophotometrically at a wavelength of 360 nm.
Objectives: 1. To perform correctly a conventional method of measuring urinary VMA.

2. To calculate accurately vanilmandelic acid levels in urine in conventional units
as mg/d.
REAGENTS
All reagents should be of AR quality.
1. Hydrochloric acid, 6 mol/l. Slowly add 500 ml concentrated HCl to a liter volumetric flask
containing approximately 300 ml water, and dilute to mark with water. Water to be used for
the preparation of all reagents should be distilled twice in all-glass apparatus.
2. Sodium chloride.
3. Ethyl acetate.
4. Potassium carbonate, 1 mol/l. Dissolve 138 g potassium carbonate in 1 liter of distilled water.
5. Sodium metaperiodate, 2 g/100 ml distilled water. Make fresh weekly and store in an amber
bottle.

98
6. Sodium metabisulfate, 10 g/100 ml distilled water.
7. Acetic acid, 5 mol/l. Dilute 286 ml glacial acetic acid with distilled water to 1 liter.
8. Phosphate buffer, 1 mol/l, pH 7.5 Solution A: Dissolve 178 g disodium phosphate

(Na2HPO42H2O) in distilled water and dilute to 1 liter. Store in a refrigerator. Solution B:
Dissolve 27.22 g potassium dihydrogen phosphate (KH 2PO4) in 200 ml distilled water. Mix
168.2 ml of solution A with 31.8 ml of solution B. Check pH meter and make any necessary
adjustment to obtain a pH of 7.5.
9. Hydrochloric acid, 0.01 ml/l. Dilute 0.83 ml of concentrated HCl to 1 liter with distilled water.
10. Standard solutions. Stock solution (1 mg/ml): Accurately weigh 100 mg of VMA and dissolve
in 100 ml of 0.01 mol/HCl in a volumetric flask. The solution is stable approximately three
months under refrigeration. Working solution (10 g/ml): Dilute 1 ml of the stock solution to
100 ml with 0.01 mol/HCl. Prepare fresh before use.
COLLECTION OF SPECIMEN
To preclude false elevations of urinary VMA, the intake of chocolate, coffee, bananas, foods
containing vanilla, citrus fruits, and drugs such as aspiring and antihypertensive agents (e.g.,
Aldomet) must be restricted three days prior to and during collection of the urine specimen. The
pH of the urine should be kept approximately 2 during the collection by placing 10 ml of 6 mol/l
HCl into a suitable container (dark-brown bottle). After measurement of the total volume, 100 ml
aliquots may be stored at 4oC for subsequent analysis. The specimen so preserved is stable for
several weeks.
It has recently been reported that no dietary restrictions during urine collection are necessary if
the VMA method based on the oxidation of VMA to vanillin is used. However, for other methods
which employ a reaction of the phenolic acids with diazotized -nitroaniline, a rigid control of
diet and drugs is still necessary.
PROCEDURE
Pipet 0.2% of the 24 h volume into 50 ml glass-stoppered (or screw-cap) centrifuge tubes marked
previously as “tests”, internal standards” and “unoxidized blanks” in duplicate. To the internal
standard tubes add 1 ml of the working standard. Dilute the contents of all these tubes to 5.5 ml
with distilled water, and further acidify with 0.5 ml of 6 mol/l hydrocholoric acid. Add a saturating

amount of sodium chloride (approximately 3 g), mix and extract with 30 ml of ethyl acetate by
shaking on a mechanical shaker for 30 min. Centrifuge for 5 min. Transfer 25 ml of the organic
extract (upper layer) to a second glass-stoppered centrifuge tube containing 1.5 ml 99
of 1 mol/l potassium carbonate. Shake mechanically for 3 min and centrifuge for 5 min. Pipet 1
ml of the carbonate phase (lower layer) to a third glass-stoppered centrifuge tube. To the test
and standard tubes, add 0.1 ml of 2 g/100 ml sodium metaperiodate, mix and stopper loosely;
place all tubes including the tubes marked “unoxidized blank” (metaperiodate solution is omitted
at this stage) into a water bath of 50oC for 30 min. At the end of the incubation period, remove
the tubes and cool to room temperature. To the unoxidized blanks, add 0.1 ml of sodium
metaperiodate and mix. Without delay add to all tubes 0.1 ml of metabisulfite solution to reduce
residual periodate. Neutralize with 0.3 ml of 5 mol/l acetic acid, and add 0.6 ml of 1 mol/l
phosphate buffer at pH 7.5. (The pH can be checked at this point by adding one drop of aqueous
cresol red (0.04 g/100 ml). The solution should be yellow, indicating a pH of less than 8.8). Shake
mechanically for 3 min with 20 ml of toluene to extract vanillin, the oxidized product of VMA.
Centrifuge for 5 min, and transfer 15 ml of the toluene extract into a fourth glassstoppered
centrifuge tube containing 4.0 ml of 1 mol/l potassium carbonate. Shake mechanically for 3 min
and centrifuge for 5 min. transfer the carbonate layer containing vanillin into a microcuvet, and
determine the absorbance at 360 nm against a water blank.
CALCULATION
mg VMA/d = At – Ab x 10 x 100 = At – Ab x 5
Ast – At 1000 0.2 Ast - At
where Ab = absorbance of “unoxidized” urine blank

At = absorbance of test
Ast = absorbance of internal standard (standard + test)
COMMENTS
Necessary care in the collection and preservation of the urine specimen as outlined in the text is
very important. Diets and drugs contributing to the excretion of related phenoxy acids which may
be oxidized to vanillin will yield falsely elevated results. However, as a precautionary measure, it
is advisable to prepare an unoxidized blank for every sample to correct for the presence of vanillin
in urine even when the dietary restrictions prior to and during collection of the specimen have
been followed. The absorbance may be measured against the unoxidized blank instead of the
water blank, and in that case the need for subtraction of the absorbance of the urine blank from
the absorbance of the test samples is obviated. The internal standard (addition of a known
amount of VMA to the test specimen) compensates for procedural losses, for decomposition of
vanillin, and for the relative inhibition of its formation because of the presence of unknown urinary

factors. Indeed, at room temperature the oxidation of VMA by periodate proceeds smoothly in
pure solutions, whereas an elevated temperature (50oC) is required for the oxidation of VMA in
urinary extracts. In occasional urinary samples, the oxidation may be strongly inhibited even at
50oC.

The oxidation of VMA to vanillin is also sensitive to hydrogen ion concentration. In neutral and
acidic solutions, oxidation results in the formation of a yellow pigment; strongly alkaline solutions,
on the other hand, delay the formation of vanillin and cause its decomposition. Optimal
conditions are obtained in (1 to 15 g/100 ml) sodium or potassium carbonate solution at an
approximate pH of 11. The maximum absorption of vanillin occurs at 348 nm. However, at this
wavelength considerable absorbance of the oxidation product (p-hydroxybenzaldehyde) of p-
hydroxymandelic acid, a normal constituent of urine, necessitates measurement of 360 nm, where
the absorbance of vanillin is 80% of its peak value and interference is minimal. It should be
pointed out that the absorbance of vanillin drops sharply from 350 to 380 nm, and it is important
that the wavelength setting remain exactly at 360 nm.
NORMAL VALUES
Normal values range from 1.8 to 7.1 mg of VMA/d, or 1.5 to 7.0 g/mg creatinine.
Results: Urinary vanilmandelic acid in mg/d: ______________________________
Exercise No. 38
SEROTONIN & 5’-HYDROXYINDOLE ACETIC ACID
SEROTONIN AND ITS METABOLITE: 5-HYDROXYINDOLE ACETIC ACID (5-HIAA)
Serotonin (5-hydroxytryptamine, 5-HT), a powerful smooth muscle stimulant and vasocontrictor,

is a derivative of the amino acid tryptophan. This compound is formed predominantly in the
enterochromaffin cells (otherwise known as agentaffin cells, because of their affinity for silver
salts) of the gastrointestinal tract. It is transported in the blood by the platelets and is present in
the brain and other tissues. In recent years, interest in this substance and other related hydroxy
indoles has grown considerably because of the discovery that they are excreted in large amounts
by patients with metastatic carcinoid syndrome (argentaffinoma).
The formation and breakdown of serotonin is depicted in Fig. 13-32. The essential amino acid
tryptophan is hydroxylated to form 5-hydroxytryptophan (5-HTP). Approximately 1 to 3% of
dietary tryptophan is normally metabolized by this pathway, but as much as 60% of this amino
acid is converted to 5-HTP in carcinoid tumors. The 5-hydroxytryptophan is decarboxylated to
serotonin (5-hydroxytryptamine). While the enzymatic decarboxylation is most active in carcinoid
tumors, it may also take place in the liver, kidney, lung and brain.
Serotonin is pharmacologically the most active indole amine; however, its biological activity is
apparently lost when it is bound to tissues or platelets. It may rapidly undergo oxidative
deamination in a tumor or in the blood after release from a tumor. The oxidative deamination of

serotonin by the enzyme monoamine oxidase (MAO) leads to the formation of 5hydroxyindole
acetic acid (5-HIAA), which is quantitatively the most significant metabolite of the 5-hydroxyindole
pathway. The majority of the 5-HIAA is excreted in the free form, although a small amount may
be conjugated as the O-sulfate ester before excretion.

TOXICOLOGY
Exercise No. 39
CARBON MONOXIDE
Hemoglobin and its derivatives have characteristic absorption bands in the visible region that can
be utilized to detect carboxyhemoglobin and to measure the quantity present. Oxygenated
hemoglobin and carboxyhemoglobin have similar double bands in alkaline solution. The
absorption maxima for oxygenated hemoglobin are 576 to 578 and 540 to 542 nm; for
carboxyhemoglobin they are 568 to 572 and 538 to 540 nm. Deoxygenated hemoglobin has a
single broad band at 555 nm.
If a weekly alkaline dilution of blood is treated with sodium hydrosulfite, oxygenated hemoglobin
(and any methemoglobin present) is converted to deoxygenated hemoglobin.
Carboxyhemoglobin is unaffected by such treatment.
This is the basis for several methods for the determination of percent saturation of hemoglobin
by carbon monoxide. The method to be described works satisfactorily with fresh, whole blood,
but is not satisfactory with postmortem blood or specimens containing denatured hemoglobin.
PRINCIPLE
A dilute hemolysate of blood is treated with sodium hydrosulfite, which reduces methemoglobin
and oxyhemoglobin but does not affect carboxyhemoglobin. The absorbance of this solution is
measured at 541 and 555 nm, the absorbance ratio A 541/A555 is calculated, and the %
carboxyhemoglobin is determined from the standard curve.
Objectives: 1. To perform correctly a conventional method of measuring blood carbon

monoxide.
2. To calculate accurately CO levels in blood in % conventional units.
REAGENTS
1. NH4OH, 0.12 mol/l. Dilute 15.9 ml of concentrated NH4OH to 1.0 liter with deionized water.
This solution is stable.

2. Sodium hydrosulfite (sodium dithionite), AR. Preweigh 10 mg portions of sodium dithionite
into individual small test tubes. Stopper the test tubes or cover with Parafilm.
3. Carbon monoxide. Lecture bottle (Matheson Gas Products, Div. of Will Ross, Inc., East
Rutherford, N.J. 07073).
4. Oxygen, CP.
SPECIAL APPARATUS
A narrow band pass (<2 nm) spectrophotometer with 1.00 cm cuvettes is required, although the
use of a recording spectrophotometer with the same specifications is desirable. The procedure
listed below can be performed on a Beckman DB Recoding Spectrophotometer or other similar
instrument.
It is imperative that the spectrophotometer be checked regularly for wavelength and
spectrophotometric accuracy with appropriate calibrating filters (e.g., NBS Reference Material
930) and with liquid photometric standards (e.g., NBS Reference Material 931).
PROCEDURE
1. Add 100.0 l of whole heparinized blood to 25 ml of 0.12 molar NH4OH. Mix the solution and
allow it to stand for 2 min.
2. Transfer 3.0 ml of NH4OH (blank) and 3.0 ml of the hemolysate (test) respectively into 1.0 cm
cuvettes. (Analyze the sample in triplicate).
3. Add 10 mg of sodium dithionate to each of the cuvettes. Cover the cuvettes with Parafilm
and invert gently 10 times.
4. Exactly 5 min after the addition of dithionate to the sample, read the absorbance at 541 and
555 nm against the NH4OH blank. (If a number of samples are analyzed, space the addition
of the reducing agent so that each can be read exactly 5 min).
5. Calculate the ratio of the absorbance at 541 nm to that at 555 nm, and determine the %
carboxyhemoglobin from the calibration chart.
Note: For confirmation and for the purpose of record, the sample without and with dithionite
(steps 1 and 3, respectively) may be scanned between 450 and 600 nm.
PREPARATION OF THE STANDARD CURVE

Caution—Use fume hood when working with carbon monoxide gas.

1. Collect 20 ml of heparinized blood from a healthy person who does not smoke.
2. Transfer a 4.0 ml portion of the fresh, heparinized blood sample into each of two 125 ml
separatory funnels. Treat one sample with pure oxygen and the other with pure carbon
monoxide for 15 min while the funnels are gently rotated. After the addition of the gases,
close the separatory funnels and rotate them gently for an additional 15 min. Analyze the
fully saturated samples immediately, in triplicate, according to the procedure given above.
Use these results for the establishment of the 0 and 100% carboxyhemoglobin calibration
points. These samples may not be used to establish the intermediate calibration points. Plot
the ratio of the absorbance at 541 nm to that of 555 nm for the 0% and for the 100%
carboxyhemoglobin samples and draw a line between the two points.
3. Fill the funnel containing the 100% carboxyhemoglobin sample with nitrogen gas and rotate
it for 5 min. Treatment with nitrogen removes the physically dissolved CO from the sample,
but a small amount of CO will also dissociate from hemoglobin. Determine the exact
carboxyhemoglobin content of this sample by the method described, using the standard curve
just prepared. Prepare intermediate standards by mixing appropriate proportions of the
nitrogen-treated sample with the oxygen-treated sample.
4. Analyze each of the diluted blood samples from step 3 in triplicate, according to the procedure
given above.
5. Plot the calculated concentrations against the absorbance ratios obtained. These points
should fall on the line drawn for the fully saturated samples, since the curve is linear over the
entire range.
Results: % CO-Hb: ________________________

Exercise No. 40
VOLATILE POISONS
These are substances that distilled with steam when the materials containing them are heated
with water and tartaric or dilute sulfuric acid. They include the following: Phosphorus, HCN,
Carbolic acid, Cresols, Thymol, Carbon disulfide, Alcohols, Chloroform, Chloral hydrate,
Formaldehyde, and Acetone among others.
Objectives: 1. To perform correctly spot tests for the detection of organic volatile poisons in
various body fluids.
2. To devise an effective strategy of determining the presence of volatile poisons
in unknown analysis given a time limit.
40.1. Phosphorus:
1. Scherer’s Preliminary tests.

Principle – yellow P is volatile on gentle warming and the vapor blackens AgNO3.
Procedure – place material in a small flask. Cut a v-shaped slit in the cork and place the
latter loosely in the mouth of the flask so that the 2 strips of filter paper hang free.
Moisten one strip with AgNO3, and the other with lead acetate solution.
Warm gently from the water bath and allow to stand for some time protected from light.
Results: Blackening of AgNO3 shows P.

Blackening of AgNO3 and PbAC2- P and H2S present.
No blackening of either – no phosphorus.
2. Confirmatory tests. Evaporate distilled water with Br2 or Cl2 water.
a. Ammonium molybdate test
Reagents – few gtts. conc. HNO3 AND NH4 molybdate.
Result – yellow ppt. of NH4 phosphomolybdate.
Procedure: Acidify the solution with a few drops of concentrated nitric acid. Add
an equal volume of ammonium molybdate solution and warm to about 40 oC.
Phosphoric acid precipitates yellow ammonium phosphomolybdate.
b. Ammonium magnesium phosphate test.
Reagent – magnesia mixture (MgCl2, NH4OH, NH4Cl)
Procedure: Add magnesium mixture to the second portion of distillate. Phosphoric
acid gives a white crystalline precipitate with the microscope. It should consist of
well-formed crystals or at least be crystalline. These crystals are transparent,
acicular prism.

40.2. Hydrocyanic Acid:
1. Schonbein-Pagenstecher test.
Principle – ozonizing action of HCN. A positive test shows its presence but it is not conclusive
for it is also given by O3, HNO3 and Cl2.
Reagents – tartaric acid and guaiac copper
Procedure: Acidify or distillate or material with tartaric acid and put in a small flask. Then
suspend in the flask a strip of “Guaiac-copper” paper without letting it touch the liquid. Gently
warm the contents of the flask upon the water bath. Neither HCN acid nor KCN is present
unless the paper is turned blue or bluish green.
Result – on heating, the paper that is exposed to the fumes becomes blue or bluish green.
This is due to O3 produced.
2. Prussian blue test.

Reagents – KOH, FeSO4 and FeCl3.
Procedure: Add to the solution a little potassium hydroxide solution and 1-2 drops of freshly
prepared ferrous sulfate solution and 1 drop of ferric chloride solution. Shake well and warm
gently. Finally, acidify with dilute hydrochloric acid. If much HCN acid is present, a precipitate
of Prussian blue will appear at once, but if the quantity is small the colloidal solution will have
merely all blue, blue green or green-blue color. After a long time (10-12 hours) a flocculent
precipitate or Prussian blue will settle to the bottom of the test tube.
Result- Prussian blue due to production of ferric ferrocyanide.

3. Sulphocyanate test.
Reagents – HCN, KOH, (NH4)2S, FeCl3
Procedure: Add to a portion of the distillate a few drops of KOH solution, and then a little
ammonium sulfide solution. Evaporate the mixture to dryness in a porcelain dish upon the
water bath. Dissolve the residue in a little water and acidify with dilute HCl. Warm and filter
paper to remove sulfur. Usually it is necessary to pour the filtrate 2-3 drops of dilute ferric
chloride solution.
Result – blood red due to ferric sulphocyanate.

4. AgNO3 test.

Reagents – HNO3 and AgNO3.
Procedure: Acidify a portion of the distillate with dilute nitric acid and add silver nitrate
solution in excess.
Result – white curdy ppt. of AgCN.
40.3. Phenol, Lysol, Cresol, Creosote, Creolin, & Pyrogallol
Tests for Phenol or carbolic acid
1. Millon’s test Reagent

– Millon’s
Procedure: Millon’s reagent heated with a solution containing only a trace of carbolic acid,
produces a red color.
Result – red color (given by aromatic compounds)

2. Bromine water test
Reagent – excess bromine water
Procedure: Add excess of bromine water.
Result - yellowish white crystalline ppt. of tribromo-phenylhypobromite.
3. Ferric chloride test

Reagent – very dilute FeCl3 ggt. by ggt., HCl and H2SO4
Procedure: Add ferric chloride solution drop by drop. A blue-violet color to the aqueous
solution is imparted which with the addition of dilute HCl or H2SO4 is changed to yellow.
This in turn disappears upon addition of alcohol. (Distinction from salicylic acid). 4.
Melzer’s benzaldehyde test
Reagent – conc. H2SO4 – 1 to 2 gtts. Benzaldehyde KOH

Procedure: Add 2 ml. of conc. H2SO4 to 1 cc of the solution to be tested, then 1 or 2 drops of
benzaldehyde and heat. The mixture, at first yellowish brown, will become dark red. At the
same time a red resinous substance will appear unless the solution is too dilute. When cold

add 10 ml. of water and enough KOH solution to give a distinct alkaline reaction. If carbolic
acid is present a violet-blue color will appear.
40.4. Chloroform:
1. Hoffmann’s Phenylisocyanide test

Procedure: Add 1 or 2 drops of aniline to the chloroform solution and then few ml. of aqueous
or alcoholic potassium hydroxide solution.
Result – penetrating repulsive odor of phenylisocyanide.
2. Fujiwara’s pyridine tests

Procedure: Mix 2 ml. of pyridine with 3 ml. of 10% NaOH solution, heat to boiling and add 1
ml. of the liquid to be tested. Even a trace of chloroform will produce a bright, blue-red color.
3. Cyanide test
Procedure: Seal the liquid to be tested for chloroform in a glass tube (pressure tube) with a
little solid NH4Cl and alcoholic potassium hydroxide solution. Heat for several hours in a
boiling water bath. Cool the tube, remove the solution and test for hydrocyanic acid by the
Prussian blue-red.
40.5. Ether
1. Dichromate test
Few mm K2Cr2O7 + 4 gtts H2SO4 = Chromic acid
Add H2O and then CHCl3
Result – greenish color of CHCl3 due to ether (chromium chloride forms)
40.6. Chloral Hydrate

The following are tests to differentiate chloral hydrate from chloroform:
1. With Nessler’s solution

Add a few drops of Nessler’s reagent to an aqueous chloral hydrate solution and shake. It will
produce a yellowish red precipitate. The color of which change after a while to a dirty
yellowish green.
2. With sodium thiosulfate
Boil a few ml. of chloral hydrate solution with 0.2-0.3 of solid sodium thiosulfate. This will give
a turbid liquid of brick-red color. A few drops of KOH solution will remove the turbidity and
change the color to brownish red.

40.7. Iodoform:
1. Lusgarten’s test
Gently warm a few drops of alcoholic iodoform solution in a test tube with a little sodium
phenolate solution. If iodoform is present, a red substance shall be dissolved with a few drops
of dilute alcohol.
Note: prepare sodium phenolate solution by mixing 20 grams of phenol with 40 grams of
sodium hydroxide and 70 grams of H2O.
2. Resorcinol test
Dissolve about 0.1 gram of resorcinol in 2 ml. of H2O, add a few drops of NaOH solution, and
finally the liquid containing CHI3. This mixture heated to boiling will develop even in very
dilute solution a yellowish red color attended by beautiful yellowish green fluorescence.
(Chloral, bromal, bromoform, and chloroform also give this test.)
40.8. Aniline
1. Phenylisocyanide
Procedure: Heat a portion of distillate with a few drops of CHCl3 and KOH solution.
Reagent – few gtts. CHCl3 and KOH
Result – repulsive odor or phenylisocyande
2. Hypochlorite test
Procedure: Add a few drops of aqueous Ca or Na hypochlorate solution drop by drop to a
portion of the distillate, a violet-blue color gradually changing to a dirty red, will appear if
aniline is present. Addition of a little dilute aqueous phenol solution containing some
ammonia will produce a blue color.
Result – violet-blue – dirty red. If phenol with NH3 are added blue.
3. Bromine water test Reagent – bromine water

Result – flesh colored precipitate
4. Chromic acid test
Reagent – extent with ether and evaporate
Rub the residue with 4-5 gtts. of conc. H2SO4. Add 1 gtt K2Cr2O7 – 1 or 2 drops of H2O produces
at once a deep blue color.
40.9. Ethyl Alcohol
1. Lieben’s iodoform test

Warm the unknown then add.
Reagent – iodo potassium iodide solution (I2+KOH) – aqueous.
Result – lemon yellow precipitate of iodoform

4 KOI + C2H5OH = KOH + CHO CI3 + H2O + KI
CI3CHO + KOH = CHI3 = KCOOK
Instead of iodo potassium iodide solution you may use a small crystal of iodine and enough
KOH to give solution a distinct yellow to brownish color.
2. Berthelot’s test
Reagents – shake the liquid containing ethyl alcohol with a few gtts. Benzoyl chloride + excess
10% NaOH.
Result – odor of ethyl benzoate (aromatic fruity odor)

C2H5O2H + C6H5COCl + NaOH => C6H5COO2H5 + NaCl = H2O
3. Chromic acid test

Reagents: warm the liquid containing ethyl alcohol with dilute HCl + 1-2 gtts. K2Cr2O7.
Result – color changes from red to green and also note odor of acetaldehyde.
4. Ethyl acetate test

Reagents – equal amount of unknown + H2SO4. Add anhydrous acetate. Heat. (use no
acetate).

Result – note the odor of acetate (ethyl)
5. Vitali’s test
Reagent – distillate a piece of solid KOH + gtts CS2
Let stand until most of the CS2 has evaporated. Add 1 gtt NH4 molybdate solution + excess
dilute H2SO4.
Result – red due to potassium xanthogenate
40.10. Methyl Alcohol (wood alcohol):
Methanol (methyl or wood alcohol) is a widely used solvent in paints, varnishes and paint
removers. It is used alone as an antifreeze fluid and with ethanol and soap as a solid canned fuel.
Poisonings are usually due to accidental ingestion by children or by alcoholics. In some areas,
methanol may be a contaminant in “moonshine”.
Several test depend upon first oxidizing methyl alcohol to formaldehyde and detecting the latter
by means of a color change.
1. Cu-Oxidation test
Dilute the solution, if necessary, until the total alcoholic strength does not exceed 10%.
Immerse in cold H2O the test tube containing 3 ml. of liquid and insert 3-4 times a Cu spiral
heated to redness, reheating each time.
Filter, expel by boiling color of acetic aldehyde, cool and add 1 drop of 0.5% aqueous
resorcinol solution.
Add this mixture from a pipette to form an upper layer on 2 ml. of conc. H 2SO4 and gently
rotate test tube for three minutes. If a rose-red ring does not appear at the contact surface
of the 2 liquids, less than 2% methyl alcohol is present.
2. The most convenient and reliable method for methanol determination is gas chromatography.
Such a method has already been described. Methanol can also be measured by a variety of
other methods, most of which
involve measuring the color
intensity after oxidation of methanol to
formaldehyde, followed
by the development of a color
by reacting formaldehyde with
chromotropic acid (CTA):

These methods work well since chromotropic acid is specific for formaldehyde and, hence, for
methanol after oxidation. The microdiffusion method referred to earlier is useful for the
determination of methanol, and it also utilizes CTA for color development.
The CTA colorimetric procedure for methanol has two major drawbacks. First, methanol is not
quantitatively oxidized to formaldehyde. It is readily apparent that after formation of
formaldehyde by the oxidation reaction just noted, the formaldehyde itself can be oxidized to
formic acid and further to carbon dioxide as follows:
CH2O + MnO4 - → CO2 + H2O
This means that before a quantitative procedure can be devised, conditions must be chosen such
that constant proportion of methanol are oxidized. Thus, the method is empirical and the set
conditions must be established and adhered to rigidly before quantitative results can be achieved.
Second, the presence of reducing substances other than methanol will affect the system so that
the procedure can no longer be applied quantitatively. The most common interference in cases
of methanol poisoning is ethanol. It is not generally appreciated that the presence of ethanol
invalidates a methanol procedure based on oxidation followed by CTA color development, if the
calibration curve has been set up using pure methanol standards.
The procedure of Hindberg and Wieth obviates both drawbacks. First, the procedure must be
carried out identically for standards and unknowns. Second, an excess of ethanol is added to
both standards and unknowns. This results in a constant “interference”of a magnitude much
greater than would never be encountered in practice.
A third, but minor, drawback of any CTA procedure for determining methanol is the use of
concentrated sulfuric acid for development of the final color. The dehydrating effect of
concentrated sulfuric acid can produce formaldehyde from appropriate organic compounds and
there will be false high results. We have encountered this interference occasionally in patients
with severe acidosis. Apparently, some substances may appear in a trichloroacetic acid filtrate,
such as glycolic acid, which reacts as follows:
This type of interference can be detected by running a blank for comparison. A portion of filtrate
is carried through the procedure except that the oxidation step is omitted. Any color developed
in the unoxidized specimen is due to formaldehyde contaminating the original specimen, or to
glycolic acid. The following is convenient, qualitative screening test for methanol.

REAGENTS
All chemicals used should be analytical reagent grade.
1. Trichloroactic acid, 20 g/100 ml. Dissolve 20 g of trichloroacetic acid in water and dilute to
100 ml.
2. Potassium permanganate, 3 g/100 ml. Add 3.0 g of potassium permanganate to a solution of

15 ml of 85% phosphoric acid and dilute to 100 ml with water.
3. Sodium bisulfite.
4. Chromotropic acid (1,8-dihydroxynaphthalene-3,6-disulfonic acid).
5. Sulfuric acid, concentrated.
PROCEDURE
1. To 2.0 ml of blood, serum, or urine, add 4.0 ml of the trichloroacetic acid solution. Do not use
heparin EDTA as an anticoagulant for blood specimens. Shake the mixture thoroughly, and
then centrifuge.
2. Into each of two tubes labeled “sample” and “sample blank”, pipet 1.0 ml of supernatant. Add
2 drops of the potassium permanganate solution to the sample tube only. Wait exactly 2 min.
3. Add approximately 10 mg of sodium bisulfite to both tubes to decolorize (reduce) the excess
permanganate. Mix thoroughly. If some permanganate color remains, add more sodium
bisulfite, but avoid a large excess.
4. Add approximately 2 mg of chromotropic acid to both tubes and mix the solution by swirling.
5. Carefully underlay this solution with 3.0 ml of concentrated sulfuric acid by inclining test tube
and flowing the acid down the side of the inclined tube. A purple ring at the interference may
be considered a positive test for methanol.
6. Shake the tube to diffuse the purple color. The color is fully developed after about 20 minutes.
COMMENTS

This method is very sensitive and will detect about 10 mg methanol/100 ml sample. An aqueous
control should always be processed in parallel with each batch of samples for comparison with
the specimen. Formalin, heparin, methenamine, and EDTA also give positive tests. If the “sample
blank” is also positive, one of the interferences discussed above may be present.
INTERPRETATION
Methanol poisoning is considerably more dangerous than than due to ethanol. Methyl alcohol is
metabolized in man to formaldehyde and formic acid. The accumulation of formic, and other
acids, severely reduces the alkali reserve, resulting in a metabolic acidosis). In addition, necrosis
of the pancreas and serum amylase elevations have been demonstrated. Therefore, in addition
to blood methanol levels, plasma carbon dioxide content, serum amylase determinations, and
electrolyte studies are useful laboratory tests for determining the severity of the poisoning and
following the progress of treatment.
Metabolites of methyl alcohol can damage the optic nerve, resulting in either temporary or
permanent blindness. The mechanism of this effect is not well understood, nor is it a constant
finding; nevertheless, prompt treatment of these cases may not only be lifesaving but may also
preserve the eyesight.
As little as 2 teaspoonful (10 ml) of methanol are considered toxic; fatal results have been reported
with dosages between 2 and 8 ounces. A blood level greater than 80 mg /100 ml is dangerous
to life.
Treatment is twofold. First, the acidosis is treated, generally with sodium bicarbonate, both
intravenously and orally. Second, it has also been proposed that in severe cases, ethyl alcohol be
administered to saturate the alcohol dehydrogenase enzyme system. Since ethly alcohol is the
preferred substrate for this enzyme, this prevents the conversion of methanol to its toxic
metabolites.
40.11. Acetone
1. Lieben’s Iodoform test Reagents:

Iodine + KOH
Result – Iodoform (note odor and crystals). Acetone differs from alcohol in giving CHI3 when
NH4OH is substituted for KOH NaOH. Acetaldehyde resembles acetone in giving CHI 3 in the
cold and under same conditions as above.
2. Legal’s test
Reagent – a few gtts. of sodium nitroprusside + KOH

Result – a reddish yellow color – yellow HAc (in excess) purplish red. Heat to violet. This test
is given by acetone but not by alcohol. Red color caused by aldehyde fades upon addition of
HAc and changes to green with heat.
3. Benzoldt’s test
Reagent – hot saturated solution of nitrobenzaldehyde (cooled) + K + KOH
Procedure: Prepare a hot saturated solution of nitrobenzaldehyde solution and allow it to

cool. Add this solution to liquid containing acetone and also some sodium hydroxide solution.
At first it is yellow then green and then blue due to indigestion. If indigestion is present in
traces only, shake mixture with CHCl3 and this solvent will dissolve the blue color and become
blue.
Result – yellow-green-blue ppt. of indigotine.
40.12. Carbon Disulfide
1. Lead acetate test

Reagent – PbAc2 + KOH and heat (distinction between CS2 & H2S-no reaction between PbAc2
and CS2).
Result – with PbAc2 – no effect; with KOH – black ppt (PbS)
2. Sulphocyanate test
Heat an aqueous solution of CS2 for a few minutes with concentrated ammonium hydroxide
solution and alcohol. Ammonium sulphocyanate is formed together with (NH4)2S.
Concentrate this solution upon the the H2O both to about 1 cc and acidify with dilute HCl.
Add a drop of ferric chloride solution.
3. Xanthocyanate test
Shake a few cc of distillate for several minutes with 3 or 4 times its volume of saturated solution
of KOH in absolute alcohol. Faintly acidify the solution with acetic acid and add 1 or 2 drops
of CuSO4 solution. If CS2 is present, a brownish black precipitate of Cupric xanthogenate will
appear. This will soon change to a yellow flocculent precipitate of Cuprous Xanthogenate.
40.13. Formaldehyde

1. Phloroglucinol test
a. Acid medium
Gradually heat to boiling the solution to be tested for HCHO with a mixture of equal parts
of HCl and H2O and sprinkle with phloroglucinol-yellow red flecks.
b. Alkaline medium
Mix 2 ml. of a 0.1% phloroglucinol solution with 1 ml. of KOH solution and add the liquid
to be tested for formaldehyde. The appearance of a distinct red color shows the presence
of formaldehyde. This color test is given only by more dilute formaldehyde solution,
stronger solution giving no color.
To detect formaldehyde in milk, add to 10 ml. Milk, 1-2 ml. of a 0.1% phloroglucinol
solution and a few drops of KOH solution.
2. Recorcinol test
Reagents – 5% resorcinol (aqueous) + 40% NaOH. Equal volume of this solution and the
solution to be tested by heating to boiling and keeping at that temperature for about ½
minutes.
Result – red color

3. Hener’s test
Procedure: Mix in a large test tube 5 ml. of the liquid to be tested for formaldehyde with 2
ml. fresh unboiled milk and 7 ml. HCl, 0.2 cc of 10% FeCl3 solution and boil gently for about
1 minute. In presence of formaldehyde-violet color will appear.
40.14. Thymol
1. With concentrated H2SO4

Result – in the cold-faintly yellowish solution when heated-a beautiful rose-red color
2. With glacial acetic acid
Reagents – glacial HAc (1 ml.) and 6 gtts. concentrated H2SO4
Result – blue-green zone at the point of contact the mixture is fluorescent

3. Piria’s test
Reagents - H2SO4 + unknown + 10 vol H2O + white Pb + ferric chloride solution
Results – beautiful violet-red color (unlike phenol it does not give Millon’s test).
Exercise No.41
NON-VOLATILE ORGANIC POISONS

Objectives: 1. To perform correctly spot tests for the detection of organic nonvolatile poisons
in various body fluids.
2. To devise an effective strategy of determining the presence of nonvolatile
poisons in unknown analysis given a time limit.
41.1. Picric acid
1. Isopurpuric acid test. Gently heat (50-60 degC) an aqueous solution of picric acid with a few
drops of saturated aqueous potassium cyanide solution (1:2). The solution will become deep
red owing to the formation of potassium isopurpurate. Even one milligram of picric acid
dissolved in 5 ml. of water (1:5000) gives a deep red color.
2. Picramic acid test
a. With dextrose – heat an aqueous picric acid solution with 2-3 drops of saturated aqueous
sodium hydroxide and dextrose solution. The solution becomes dark red. Avoid excess
of sodium hydroxide solution otherwise a red color dye solely to the action of the alkali
upon dextrose will appear.
b. With ammonium sulfide – the same red color appear when a basic acid solution is warmed
with a few drops of sodium hydroxide and ammonium sulfide solution.
3. Dyeing test – dissolve the substance containing picric acid in hot water put thread of wool,
silk and cotton in the solution. In a few hours (12-24) remove the threads and thoroughly
rinse in pure water. If picric acid is present, the wool and silk will be dyed yellow but not the
cotton. In other words picric acid is not fast upon vegetable fibers, like cotton. Picric acid
dilute 1:100,000 will still produce a yellow color upon wool.
41.2. Acetanilide
Ether or chloroform will completely extract acetanilide from an aqueous acid solution.
1. Indophenol test – this test is due to the presence of a free, primary, aromatic amino group
(NH2). Therefore acetanilide must first be hydrolyzed.
Procedure: Boil acetanilide in a small test tube with about 4 ml. of fuming hydrochloric acid
and evaporate the solution to about 10 drops. Cool and add 2-4 ml. of saturated, aqueous
carbolic acid solution, drop with good shaking. This will produce a more or less dirty red violet
color. The color will become deeper, if the mixture is well shaken for a few minutes. Then
carefully add ammonium hydroxide solution as an upper layer. The latter will become a
beautiful indigo-blue whereas the under layer will retain a red color. This blue color is only

characteristic of acetanilide and other aniline derivatives, when preceded by the dirty redviolet
color since a mixture of an aqueous phenol solution and hypochlorite solution also gives a
blue color with ammonia phenacetin also gives the endophenyl test.
2. Phenyl-isocyanide test. Boil acetanilide for a few minutes with about 5 ml. of alcoholic or
aqueous potassium hydroxide solution. Cool, and add 2 drops of chloroform and heat again.
The offensive odor of phenylisocyanide will be developed. Potassium hydroxide hydrolizes
acetanilide, forming potassium acetate and aniline. And the latter will chloroform gives
phenyl-isocyanide. Phenacetin and other derivatives of phenetidine do not give the phenyl-
isocyanide test.
3. Calcium Hypochlorite test. Boil acetanilide for a few minutes with alcoholic potassium
hydroxide solution. Dilute with water and extract aniline with ether. Remove the other extract,
evaporate upon a watch glass, and test the residue for the aniline by means of calcium
hypochlorite solution by the phenyl-isocyanide test.
4. Examination for acetanilide in urine. To study the behavior of acetanilide in the human
organism, take the material at night, twice in the course of three hours 0.3 gram of this
compound is given at a single dose. Examine in the manner described below the urine pass
in the next twelve hours. An “acetanilide urine”, boil for a few minutes with concentrated
hydrochloric acid, usually gives the indophenol tests. But the test is more certain, if pamino-
phenol is first isolated. Boil a considerable quantity of urine (300-500 ml) for a few minutes
with about 10 ml. of concentrated and repeatedly extract the cold urine with large quantity of
ether. This hydrochloric acid under a reflux. Then add excess of sodium carbonate till or
evaporate the other. The residue usually contains p-amino-phenol as a reddish or brownish
oil. An aqueous solution of this substance will give the indophenol test.
41.3. Phenacetin
Acetophenetedium, acetphenetidine, crystallizes in shiningly leaflets without color, odor or taste

and melts at 134-155o. It is soluble in 1400 parts of cold water, in 70 parts of boiling water 16
parts of alcohol, and freely soluble in ether it is closely related to acetanilide but is not direct
derivative of it. Liquefies with chloral and antipyrine. Dose, 0.3 gram; maximum dose, 1 gram in
powder or capsules.
Phenacetin can be easily and completely extracted by ether or chloroform from an acid, aqueous
solution.
1. Oxidation test. Boil phenacetin for a few minutes with about 3 ml. of concentrated
hydrochloric acid. Dilute the water in about 10 ml. and filter when cold. Addition to the filtrate

of few drops of chromic acid solution will produce a ruby-red color. In this test acetanilide
gives a yellow color gradually turning green. Strong chlorine water may be substituted for
chromic acid.
2. Indophenol test. Treated in this manner describe for acetanilide phenacetin gives a very
good indophenol test.
3. Differentiation test. Phenacetin differs from acetanilide in not giving the phenylisocyanide
test when warmed with chloroform in the presence of potassium hydroxide solution.
42.4. Salicylic Acid
Aspirin is responsible for more cases of accidental poisonings in children than any other
substances. This extremely useful analgesic is so widely used and readily available (and carelessly
handled) that children frequently ingest a toxic quantity by eating the flavored tablets like candy
or mimicking adults. Toxic doses of salicylates initially produce a stimulation of the central
nervous system. This may be reflected by hyperventilation, flushing, and fever. Unfortunately, an
unrecognized case of salicylate poisoning may be thought to be a case of infection and further
aspiring given in a vain attempt to control the fever. Central nervous system stimulation is
followed by depression.
A complex disturbance of acid-base balance results from severe hyperventilation. Initially a

respiratory alkalosis occurs, but this may be followed, especially in infants, by a metabolic acidosis.
The net may be a decrease in the blood pH.
Salicylic acid, or o-oxy-benzoic acid, is a fairly widely spread in the plant kingdom in the free state,
a salicylic aldehyde and as methyl ether, and finally the glucide salicine. Salicylic acid is used as a
preservative for keeping wine, lager beer, cider, jams, etc. Death occurs for about 1 ounce taken
in 4 days. Death results from paralysis of respiration.
1. Ferric-chloride test. Addition of a few drops of ferric chloride solution to an aqueous or

alcoholic solution of salicylic acid produces a blue-violet color. If the solution is very dilute,
the color is more of red-violet. By this test salicylic acid is shown to be a derivative of phenol.
Excess of the reagent affects to the delicacy of test. Addition of hydrochloric acid changes the
violet color to yellow. Presence of organic acid, such as citric lactic, and tartaric

acid, also interferes more or less with this test. This test is not given, if caustic alkalies or
alkaline carbonates are present.
2. Millon’s test. If an aqueous salicylic acid solution is warmed with Millon’s reagent, a deep
red color will appear.
3. Bromine water test. In strong excess this reagent produces a yellowish white crytalline
precipitate of tribomo-phenyl hypobromide even with very dilute salicylic acid solutions. This
precipitate should be examined microscopically and its melting point determined, after it has
been filtered and dried upon a porous plate or in a vacuum-decicator. It melts at 131132 o
with evolution of gas.
4. Methyl Ester test. If salicylic acid is warmed, best in a water bath, with methyl alcohol and
concentrated sulfuric acid, the characteristic odor of methyl salicylate can be recognized.
DETERMINATION OF SALICYLATES IN BIOLOGICAL FLUIDS
5. The procedure to be described for determining salicylate in urine, serum, or other specimen
is based on the formation of a violet complex between ferric ion and phenols.
REAGENTS
1. Color reagent. Dissolve 40 g of mercuric chloride, AR, in 850 ml of water by heating. Cool the
solution and add 120 ml of 1 molar HCl and 40 g of ferric nitrate (Fe(NO 3)3 9 H2O). When all
the ferric nitrate has dissolved, dilute the solution to 1 liter with water. It is stable indefinitely.
2. Salicylate standard, stock. Dissolve 580.0 mg of sodium salicylate (500 mg salicylate acid) in
water and dilute to 250 ml. Add a few drops of chloroform as a preservative. This solution
contains 2.0 mg salicylic acid/ml. Store in refrigerator; it is stable for about 6 months.
3. Working standard. Dilute 25.0 ml of stock salicylate solution to 100.0 ml with water. Add a
few drops of chloroform as a preservative. This solution contains 0.5 mg of salicylic acid.
4. Prepare a series of standards as follows:
A B C D E
ml Working standard 0.2 0.4 0.6 0.8 1.0 ml Water 0.8 0.6 0.4 0.2 0.0

mg Salicylate/100 ml 10.0 20.0 30.0 40.0 50.0
Run these standards as described above (step 1 to 3), reading the absorbances at 540 nm
against the reagent blank, and plot a calibration curve. Beer’s law is followed over this
range.
5. If the unknown absorbance is greater than 0.7, repeat the analysis using a smaller portion of
specimen diluted to 1.0 ml with water.
PROCEDURE FOR SALICYLATE DETERMINATION IN URINE
1. Follow the procedure as outlined under serum. If the urine contains more than 50 mg/100 ml
salicylic acid, make an appropriate dilution of urine with distilled water and repeat the test.
2. After reading the absorbance of the unknown, obtain a sample blank reading by setting the
instrument with water and reading the absorbance of a solution prepared by mixing 1.0 ml of
urine or diluted urine with 5.0 ml of color reagent and 0.1 ml of syrupy phosphoric acid (sp.
gr. 1.75), using the same cuvets as before. Urine solutions may not require centrifuging.
If is necessary to clarify the solutions, centrifuge both unknown and urine sample blank.
CALCULATION
Urine salicylic acid (mg/100 ml) = (mg/100 ml in diluted unknown – mg/100 ml in diluted sample
blank) x dilution factor.
INTERPRETATION
Blank values for serum, cerebrospinal fluid, and plasma are less than 1.1 mg/100 ml as salicylic
acid. For blood, the blank is less than 2.0 mg/100 ml and for urine it is less than 4.5 mg/100 ml.
recoveries of added salicylate are quantitative and the following substances in the indicated
concentrations do not interfere: phoshate (100 mg/100 ml), bilirubin (20 mg/100 ml), phenol (25
mg/100 ml), heparin (10,000 U), glucose (1000 mg/100 ml), and urea (1000 mg/100 ml).
Acetoacetic acid forms a pink color with ferric iron, and at a level of 50 mg/100 ml gives a value
of 1 mg/100 ml as salicylate. The procedure can easily be adapted to micro- or ultamicroscale, a
useful feature of pediatric cases.
Therapeutic levels of salicylic acid rarely rise above 20 mg/100 ml in blood or serum. Above 30
mg/100 ml, toxic symptoms such as headache, tinnitus, flushing, and hyperventilation may be
seen. Serum electrolytes should be followed and any imbalance corrected. Lethal salicylate levels
are usually greater than 60 mg/100 ml.

41.5. Aspirin
Aspirin or aceto-salicylic acid is formed by the action of acetic anhydride of acetyl chloride upon
salicylic acid. Aspirin is a white, inodorous crystalline powder having a sweet, acid taste and
melting at about 137o. It is soluble in 100 parts of water, 4.5 parts of alcohol, 10 parts of ethyl
and 26 parts of chloroform. An aqueous aspirin solution has an acid reaction. Used in five to
fifteen grains or more doses, as an analgesic, anodine, and antipyretic, etc. It is incompatible with
alkalies; it should be taken dry, preferably in table form. It is much used to relieve headaches,
neuralgias, neurities, rheumatis and pleuresy. Used as an analgesic and antipyretic.
1. Heat 0.2-0.5 gram of aspirin for a few minutes with a little sodium hydroxide solution, cool, best
by setting test tube in ice water, and sulfuric acid. After sometime filter of the salicylic acid
that crystallizes out and apply the test already described for its recognition.
The second component of the hydrolysis of aspirin, that is acetic acid, can usually be
recognizes by its odor in the filtrate with some alcohol and concentrated sulfuric acid. The
odor of acetic ether will be developed.
To detect salicylic acid and saliciluric acid in urine, test a portion with ferric chloride. If the
result is not convincing, proceed according to the method employed in detecting salicylic acid
in urine.
41.6. Veronal
Veronal or barbital is the ethyl barbituric acid, diethyl-malonyl-urea. It crystallizes from hot water
in large colorless spear-shaped crystal melting at 191o and is soluble in 146-147 parts of water at
20o and in 15 parts at 100o. Veronal is also freely soluble in hot alcohol and acetone. It dissolves
with difficulty in cold ether, benzene and chloroform.
1. Veronal may be sublimed almost without decomposition when heated in a test tube with a
production of well-formed crystals. The aqueous solution of this veronal crystals react faintly
acid to litmus paper.
2. Veronal is soluble in solutions of potassium or sodium hydroxide, ammonia and sodium

carbonate. If these solutions are as nearly saturated as possible, dilute hydrochloric acid will
precipitate veronal unchanged.
3. The melting point of pure veronal is 187-188o. If the substance of being veronal is mixed with
the same quantity of pure veronal the melting point should not change.

4. With Millon’s reagent, or a soluble of yellow mercuric oxide in 2 ml. of nitric acid. An aqueous
solution of veronal gives a white, gelatinous precipitate soluble in a large excess of Millon’s
reagent.
41.7. Antipyrine
Phenyldimethylpyrazolon is soluble in less than its own weight of water and in 13 its volume of
alcohol. It has a highly bitter taste and melting at 113 o.
1. Ferric chloride test. Add 1-2 drops of dilute ferric chloride solution to an aqueous antipyrine
solution. It will produce a deep red color that can be seen even in a dilution of 1:1000000.
Dilute sulfuric acid changes the red to a pale yellow color.
2. Tannic acid test. Tannic acid solution produces a white precipitate, when added to an
aqueous antipyrine solution. Obviously this test is not characteristic of antipyrine.
3. Fuming Nitric acid test. Add 1-2 drops of fuming nitric acid to an antipyrine solution. The
solution will turn green. Then heat to boiling and add to another drop fuming nitric acid. The
green color will change to red. This test is distinctly given by one cc of an aqueous antipyridine
solution (1:200).
4. Nitrous antipyrine test. Add a few drops of potassium or sodium nitrate solution to an
aqueous antipyrine solution and then dilute sulfuric acid. A green or blue color will appear.
A few drops of acetic acid may be substituted for sulfuric acid but the solution must be heated.
With concentrated antipyrine solutions green crystals of nitroso-antipyrine, will aspartate after
sometime.
41.8. Caffeine
Trimethylxantine, is a freely basic alkaloid. It crystallizes in white shining needles. It is soluble in

80 parts of water, giving a colorless solution with a neutral reaction and a bitter taste. It is quite
easily soluble in hot water (1:2).
Ether will extract more caffeine from an aqueous alkaline solution than an aqueous tartaric acid
solution. Since caffeine dissolve with some difficulty in ether, but more easily in chloroform, the
latter solvent is usually employed after the solution has been made alkaline with ammonia.
After distillation of the solvent, caffeine appears in concentric clusters of long shining needles. In
an analysis by the Stas-Otto method, caffeine will appear in all three extracts. Chloroform,

benzene and also amyl alcohol extract caffeine completely from an acid solution, whereas
petroleum ether will extract it neither from acid nor alkaline solution. Caffeine in dilute sulfuric
acid solution (1:1000) is precipitated only by such general alkaloidal reagents as phosphomolybdic
acid, phosphotungstic acid and potassium bismuthous iodide.
1. Amalic acid test. Gradually evaporated caffeine to dryness in a small dish upon the water
bath with a few ml. of strong chlorine water, taking about 10 times the quantity of the latter.
Red to red-brown residue will remain. If a very little ammonium hydroxide solution is at once
added, a purple violet color will appear. This test may be made by covering the dish containing
the residue with a glass plate moistened with a drop of strong ammonia.
Amalic acid is tetramethyl-alloxanthine, this forms colorless crystals that easily redden in
the air, is difficulty soluble in water and insoluble in absolute alcohol. In contact with
ammonia amalic acid takes on a purple-red color and a blue color when moistened with
potassium or sodium hydroxide solution.
This test is not confined to caffeine alone but it is also given by theophyline and theobromine.
But caffeine may be separated from theophyline and theobromine by the fact that benzene
will extract it from alkaline solution and leave the other 2 bases behind. If the solution is then
acidified, chloroform will extract theophyline and theobromine. Theophyline further differs
from caffeine and theobromine in giving a red color with the diazonium reagent.
2. Diazonium reagent. This reagent should be kept as two separate solution namely:
a) Solution I – containing 0.5 gram of sulfanilic acid and 5 grams of hydrochloric acid in
100 grams of water.
b) Solution II – a 0.5%, solution of sodium nitrite. The reagent is prepared by mixing 10

ml. of Solution I with a drop of Solution II.
3. Tannic acid. This reagent, added to an aqueous solution of caffeine, causes a heavy white
precipitate soluble in an excess of the acid. This test is not characteristic of caffeine.
41.9. Nicotine
A liquid alkaloid obtained from tobacco has been used for suicide and murder. It is very deadly
poison, death occurring in some instances after a few minutes.
Detection:
In suspected nicotine poisoning, the material that should be selected for chemical examination is
urine, blood, stomach and intestine with their contents, liver and lungs.

Ether or low-boiling petroleum ether will extract it from an aqueous alkaline solution. Evaporation
of the solvent leaves the alkaloid as an oily liquid having the odor of tobacco and a strong alkaline
reaction. The following special tests serve for the identification of nicotine.
1. Crystallization test. Evaporate nicotine dissolved in dilute hydrochloric acid upon a watch
glass. A yellow varnish will remain. Microscopic examination will show it to be entirely
amorphous. If kept for a long time in a dessicator over sulfuric acid, it will become distinctly
crystalline.
2. Roussi’s test. Dissolve a trace of nicotine in ether, using a dry test tube. Add to this solution
about the same volume of ether containing iodine. Stopper and set the test tube aside. The
mixture will become turbid and deposit a brownish red resin, gradually becoming crystalline.
After some time, ruby red needles having a dark blue reflex will crystallize. These are
“Roussin’s crystals”. If nicotine is old or resinous, as a rule it will not give these crystals.
3. Melzer’s test. If the alcoholic solution of a drop of nicotine is heated with about 2 ml. of
epichlorhydrine. Epichlorhydrine, obtained by the action of potassium hydroxide (1 mol) upon
a dichlorine. This is a colorless liquid insoluble in water and freely soluble in alcohol and ether.
It has an odor like chloroform and a burning (sweetish test) the mixture after more or less
time, depending upon concentration, will become distinctly red. The limit of delicacy is 0.25
mg of nicotine. Under the same conditions nicotine gives no color.
4. Schindelmeiser’s test. If 5-10 mg of non-resinous nicotine is treated first with a drop of

formaldehyde solution (about 30%) free formic acid and then with a drop of concentrated
nitric acid, the mixture takes on a rose-red color. If nicotine and formaldehyde are in contact
for several hours, the solid residue treated with a drop of concentrated nitric acid gives an
even finer color, that is, a rose to dark to dark red color.
5. Physiological test. If 1 ml. of nicotine hydrochloride is injected to a frog in a sitting position,

the frog will draw up its hind legs over the back so that the heels approach it even touching
each other when the action is stronger or the legs even cross over the back. This typical
posture of the hind legs comes to an end in about 30 minutes and after that time they will
relax. As a result of these and larger doses of nicotine, the muscles of the animal acquire a
characteristic conditions of stiffness.
This effect upon the frog just described is entirely absent in the case of nicotine.
41.10. Strychnine

Strychnine occurs together with brucine, combined with malic and caffetannic acid, in many
strychnos species.
Poisoning may result from swallowing a vermin killer containing meal or flour with strychnine and
perhaps arsenic also. Animals killed with strychnine may cause poisoning when eaten. The drug
is used for suicide and murder. It has been mistaken for stantonine, for salicine etc. Brucine may
be physiologically considered a dilute Strychnine.
Detection:
Potassium and sodium hydroxide, ammonia and alkali carbonates precipitate the free strychnine
base from aqueous solutions of its salts as a white crystalline solid. They extract strychnine from
an alkaline solution and deposit the alkaloid upon evaporation in fine crystalline needles.
Chloroform takes up the alkaloid more freely, since strychnine is considerably more soluble in this
solvent than in other. Even very dilute solutions of strychnine salts give precipitated with most of
the alkaloidal reagents.
1. Sulfuric Acid-Dichromate test. Dissolve a trace of strychnine in 2-3 drops of concentrated

sulfuric acid upon a watch glass. Add a particle of potassium dichromate and hold it firmly in
one place upon the glass. Intense blue or blue-violet streaks will come from the potassium
dichromate, if the watch glass is tilted up and down. If the entire mixture is stirred, the sulfuric
acid will have a beautiful evanescent blue or blue-violet color.
2. Wharton’s test. Dissolve the substance to be tested in a dry condition in chloroform. Put
this solution in a small test tube and evaporate the chloroform by setting the tube in a larger
one containing boiling hot water. When the water is dry or nearly so, add a few drops of
mixture of equal parts of strong sulfuric acid and water and dissolve by shaking. Now
introduce bromine vapor carefully and move the tube to the to and from so that the solution
takes up bromine. Replace the tube to expel excess of bromine vapor. If the strychnine is
present, carmine-red color will appear in a few minutes increasing the intensity as bromine
evaporates. This color fades after a time. Instead of bromine vapor, a solution of a drop of
bromine into cc of chloroform may be used. The quantity of strychnine present is small, only
a little bromine should be added to the solution.
41.11. Atropine
The belladonna plant, a tropabelladonna contains alkaloids in its various parts. Belladonna berries
are sometime eaten by mistake. In fusion of leaves and extract have also been taken for other
substances. Persons were reported poisoned by application by the plaster. Hyoscyamus has been
eaten for turning by mistake. The seeds have likewise been accidentally mixed with celery seeds
and used in cooking.

Detection:
Ether, benzene or chloroform will extract atropine from an aqueous solution rendered alkaline
with sodium hydroxide. Upon evaporation of ether solution atropine usually appears a
noncrystalline varnish.
1. Vitali’s test. Evaporate upon the water bath in a porcelain dish atropine or an atropine salt
with a few drops of fuming nitric acid. Moisten the yellowish residue when cold with a few
drops of a solution of potassium hydroxide in absolute alcohol (about 1:10). An evanescent
violet color at once changing to a fine red will appear.
2. Guglielmo’s Odor test. Heat a little atropine in a small dry test tube over low flame until a
white vapor appears. At the same time agreeable odor, recalling the perfume of black-thorn
blossoms can be detected. Then add about 1 ml. of concentrated sulfuric acid and heat until
the acid begins to darken. Without cooling, dilute at once with 2 ml. of water added from a
small graduated cylinder. During foaming this odor will be still stronger.
3. Color reactions. Atropine differs from most of the alkaloids in not giving, at least in the cold,
any striking colors that are characteristic with reagents usually employed for this purpose such
as concentrated sulfuric and nitric acid, and the reagents of Froehde, Erdmann, Marquis
Mecke.
a) Perhydrol-sulfuric acid (Schaer’s reagent) test. A few drops of perhydrol sulfuric acid
are added to a particle of the alkaloid, after 0.5 minute an intense leaf-green color
beginning at the margin, appears becoming olive-green after a few minutes and finally
discolored or brown-green in color.
b) Para-dimethyl-amino-benzaldehyde-sulfuric acid test. Add a drop of the reagent to a

small quantity of atropine and warm gently, best upon an asbestos plate. A red color will
appear, becoming in a time an intense cherry-red to violet and remaining unchanged for
a day.
4. Alkaline reaction test. An aqueous solution atropine turn the red litmus paper blue and also
reacts with phenol-phthalein. Place a trace of the alkaloid upon a strip of phenolphthalein
paper, and add a drop of absolute alcohol, and allow it to evaporate color will appear. Then
add a drop of water. The paper will at once turn red.

5. Gerrad’s test. Add about 2 ml. of a 1% solution of a mercuric chloride in 50% alcohol to 1
mg of atropine. Warm very gently and a yellow precipitate of mercuric oxide, becoming red
upon boiling will appear.
6. Physiological test. To make this test dissolve the residue from the ether extract of the
aqueous alkaline solution of 4-5 drops of extremely dilute sulfuric acid and introduce a drop
of this solution into the conjunctival sac of 1 eye of cat or dog, comparing the width of two
pupils.
41.12. Cocaine
Cocaine has a two-fold action. It acts upon the central and peripheral nervous system. In small
doses it excites the spinal cord and brain. In large doses it may produce convulsions and then
paralysis. The peripheral action is manifested by numbing sensation.
Ether, chloroform or benzene will extract cocaine from an alkaline aqueous solution.
1. Precipitation test. If 1-2 drops of potassium hydroxide solution are added to an aqueous
solution of a cocaine salt not too dilute, it will become milkly. First oil drop and later fine
crystalline needle of the free base separate.
In applying this test to the residue from the ether extract of aqueous alkaline solution, dissolve
it first of dilute hydrochloric acid and add potassium hydroxide solution drop by drop in
excess, cooling well in setting in ice. Special care has been taken to have alkaloid pure enough
so that after filtration, washing and drying, its melting point can be determined.
2. Potassium permanganate test. Add saturated potassium permanganate solution drop by

drop to a concentrated aqueous solution of cocaine salt. A violet crystalline precipitate of
cocaine permanganate will appear. In applying this test to the residue from the ether solution
of II, B, dissolve first in a few drops of dilute hydrochloric acid, to evaporate the solution upon
the water bath, and test the solution of the dry residue in water in potassium hydroxide
solution (1:100).
3. Chromic acid test. Add 5% of chromic acid solution, or potassium dichromate solution of
responding concentration, to a solution of cocaine salt not too dilute. Each drop will produce
a precipitate that will immediately disappear upon shaking the solution. Then add to the clear
solution about 1 ml. of concentrated hydrochloric acid. This will produce a more or less
crystalline orange precipitate.

When the quantity of cocaine for examination is not too small do test 1-3. A positive result
admits without doubt the presence of the substance. They are applicable therefore for the
identification of cocaine in pharmaceutical examination rather than for the purposes of
forensic chemistry.
4. Detection of benzoyl group. This test requires at least 0.2 gm of cocaine. Warm the cocaine
for a few minutes in a small test tube with 2 ml. of concentrated sulfuric acid in boiling water.
Cool and dilute with water, adding it drop by drop keeping the mixture cold. A white
crystalline precipitate of benzoic acid will appear. Filter, wash with a little ice water and dry
these precipitate. Benzoic acid may be identified by sublimation, or by determining its melting
point (120), provided the quantity is sufficient.
5. Iodic acid test. Add a few drops of concentrated sulfuric acid to a trace of cocaine and then
a small particle of potassium iodate free from iodine, or iodic acid. No color appears in the
cold bath, if a mixture is heated in a porcelain dish over a small flame until vapors of sulfuric
acid comes off, and just a little longer there appears in succession brown, olive-green, blue
and violet shades of color which issue from the particle of iodate and immediately disappears.
Finally vapors of iodine are also given off.
6. Physiological test. Dissolve the given material (the residue from the ether extract from the
alkaline solution) in a few drops of dilute hydrochloric acid and evaporate a solution to dryness
upon the water bath. Dissolve the residue in a little pure water and place a drop of the solution
upon the tongue. If cocaine is present, anesthesia results.
41.13. Quinine
Quinine occurs together with cinchonine and other alkaloids as salts of quinic and quinotannic
acid, especially in the barks of various species of cinchona.

Ether, benzene and chloroform extract quinine from aqueous alkaline solution. Upon evaporation,
ether deposits the alkaloid as a resinous, amorphous varnish in which its presence may be
recognized by the following test.
1. Fluorescent test. Dilute sulfuric acid dissolves the residue from the ether solution and
produces a fine blue fluorescence if the quinine is present.
2. Thallieoquin test. Dissolve the residue from the ether solution in a little dilute acetic acid
and add 5-10 drops of saturated chlorine water. The colorless solution has a faint blue
fluorescence and will at once give a fine green color upon the addition of ammonia in excess,
the quinine is present. Larger quantities of quinine gives a green precipitate, thallieoquin.
Thallieoquin is soluble in alcohol and chloroform but insoluble in ether.
3. Herepathite test. Mix 30 drops of acetic acid, to 20 drops of absolute alcohol, and 1 drop of
dilute sulfuric acid (20%). Add 20 drops of this mixture to 0.01 gm of quinine and heat to
boiling. Finally add 1 drop of alcoholic solution of iodine (1:10). At once, or sometime not
until the solution has stood for sometime, green leaflets having a metallic luster separate. This
product so called Herapathite, an iodine compound of quinine having a constant composition.
4. Abensour’s Erythroquinine test. Add 1 drop of each of half saturated bromine water,
potassium ferrocyanide solution (1:10), and ammonia to 10 ml. of a faintly acid, best with
acetic acid, solution of quinine. The mixture if shaken gradually turns red. This test is given
especially well if the mixture is at once shaken with chloroform. This solvent takes up the color
and has the appearance of a chloroform solution of iodine.
41.14. Codeine
Codeine occurs to the extent of 0.2 to 0.8% in almost all kinds of opium. It is methyl ethyl of the
mono acid phenol morphine.
Codeine can be extracted from the aqueous solution by other benzene or chloroform. These
alkaloids appear in the ether, benzene or chloroform. These alkaloids appear in the ether residue
usually as a non crystalline varnish in which may be detected by the following test:
1. Sulfuric acid test. Concentrated sulfuric acid dissolves pure codeine without color. After
several days standing in the cold or at once after gently heating, the solution becomes reddish
to brownish violet. If the solution of codeine in concentrated sulfuric acid is heated upon a
watch glass to about 150, that is until white vapors appear, and cooled, it will give a deep color
with a drop of concentrated nitric acid. This test like that of Pellagri is due to the formation
of apomorphine.

2. Nitric acid test. Concentrated nitric acid dissolves codeine with a yellow color.
3. Froehde’s test. This reagent dissolves codeine with a yellowish color that soon changes to
green and finally to deep blue. Gentle warming of the solution over a very small flame hastens
this change of color.
4. Marqui’s test. Concentrated sulfuric acid containing formalin dissolves codeine with a
reddish violet color. This soon changes to blue violet which persist for quite a long time.
The spectrum of this solution shows absorption of orange and yellow.
5. Mixed test. Concentrated sulfuric acid containing selenious acid dissolved codeine with blue
color quickly changing to emerald green and finally becoming permanent olive green.
Gentle heat will produce a still blue color.
Exercise No. 42
METALS (INORGANIC POISONS)
All metals are toxic if a sufficient quantity is absorbed. Generally they are not encountered in their
toxic form in the elemental or free state, but rather in the form of salts. The degree of toxicity of
a given metal is dependent on the solubility of the salt; the greater the solubility, the more likely
it is that it will be absorbed and the greater will its toxicity. For example, barium chloride is soluble
and extremely toxic, but barium sulfate is insoluble enough to be used as a radiopaque medium
for the gastrointestinal tract.
In general, metals can be detected after burning away the organic material in the specimen and
measuring the metallic ions in the inorganic residue by some standard procedure. The
combustion process may be either a wet digestion with strong, oxidizing acids, or a dry ashing
procedure in a furnace.
Some metals and their salts are volatile at high temperatures (e.g., mercury) and special
precautions must be taken to avoid loss during the ashing step. Some metal ions, such as sodium,
potassium, calcium, magnesium, and iron, are commonly analyzed in clinical laboratories and thus
will not be considered here. The so-called trace metals are present in biological material in only
minute amounts, even after ingestion of toxic amounts. For this reason most metal
determinations require analytical techniques used in trace analysis. These are difficult and require
considerable experience if they are to be carried out with validity. Instrumental analysis,
particularly atomic absorption spectrophotometry, makes it possible to conduct trace metal
analyses more easily.
Objectives: 1. To perform correctly spot tests for the detection of metals in various body
fluids.

2. To devise an effective strategy of determining the presence of metallic poisons
in unknown analysis given a time limit.
42.1. ARSENIC AND RELATED METALS
Arsenic, despite its reputation, is not a common poison. It is still a favorite homicidal poison, but
homicidal poisonings are rare. Since arsenic is ingredient in some herbicides and insecticides,
accidental poisonings, both acute and chronic, may still be encountered on occasion.
Clinically, the symptom of both acute and chronic arsenic poisonings can easily be confused with
a variety of other conditions. It is not uncommon, therefore, for a clinician to request that the
presence of arsenic be ruled out as an aid in the differential diagnosis. The specimen of choice in
this case is urine, even if 2 or 3 weeks have elapsed after ingestion of the poison. In long-term
chronic cases, analysis of hair and nails may be informative, but this subject to difficulties in
interpretation.
In the older literature, arsenic is frequently described as a “protoplasmic poison”. This term is as
good as any for describing the mode of action of the metal. Arsenic combines readily with
proteins because of its great affinity for sulfhydryl groups. This results in the precipitation of
proteins, producing gastrointestinal irritations and irreversible inhibition of important enzyme
systems, which are important toxic effects of arsenic. The great affinity of arsenic for tissue
proteins is also responsible for the rapid removal of arsenic from the blood. Blood, therefore, is
not good specimen except in cases in which a large overdose of this substance has been ingested.
DETECTION OF ARSENIC
The test to be described is commonly referred to as the Reinsch test. It depends on the fact that
metallic copper in the presence of acid will reduce arsenic to the elemental form. The arsenic is
deposited on the copper as a visible, dark film:
The oxidized forms of antimony, bismuth, mercury and selenium can also be reduced by metallic
copper under these conditions. Thus, the same test constitutes an exclusion test for these metals
as well. The test was applied by Gettler in a systematic way to biological material, and its
modification by Rieders will be described.
REAGENTS
1. Hydrochloric acid, concentrated, AR.
2. Copper spiral. Wind bright, clean copper wire around a 3 mm glass rod about eight to ten
times to make a tight spiral. A 1.0 cm square copper foil may also be used.

PROCEDURE
To 100 ml of urine in a shallow dish, add 10 ml of concentrated HCl. Add a copper spiral and
gently boil the solution until the volume is reduced to about 20 ml. Remove the copper, rinse
gently with distilled water, examine, and note any color change.
INTERPRETATION
If the copper is still bright, arsenic (25 g/l or more), mercury (50 g/100 ml or more) and selenium
(50 g/100 ml or more) have been ruled out. In the presence of arsenic or selenium, the surface
of the copper will be gray to black; in the presence of mercury, the film will be light gray to silvery
and will become shiny on rubbing. Some sulfur compounds, antimony, bismuth, or tellurium, also
give gray to black deposits.
In the case of a positive test, the nature of the deposit on the copper must be verified by further
tests. Arsenic can be quantitated after wet digestion of another specimen, by an excellent
colorimetric method. Recently, the technique of atomic absorption spectroscopy has been used
in a practical determination of arsenic in biological material. The principle of atomic absorption
spectroscopy is discussed in Chapter 3.
Normal arsenic levels in the urine are less than 50 g/l. In cases of chronic poisoning, arsenic
levels in urine will rise to 100 g/l; in acute poisoning, 1.0 mg/l or more may be present.
Since arsenic is readily bound by sulfhydryl groups in protein, considerable arsenic is bound by
keratin and subsequently deposited in hair and nails. This phenomenon has led to the analysis of
hair and nails in an effort to determine whether a previous exposure to arsenic has occurred.
Interpretation of these analyses is difficult because of the problem of differentiating between
surface contamination of the hair and endogenous arsenic. If such an examination is required, a
minimum of 1.0 g of clean hair (a large handful), clipped close to the scalp, should be submitted
to a toxicological specialist.
42.2. LEAD
Lead is still one of the most serious of the metallic poisons. In adults, inorganic and organic lead
compounds may be encountered in industrial exposures. An increasing awareness of this danger
has promoted the use of prophylactic measures. Education of workers about the hazards of lead
intoxication has also been of help in minimizing industrial poisonings.
Unfortunately, children are particularly sensitive to lead poisoning and the exposure of children
to lead-containing paint and plaster, particularly in lower class housing, has continued despite
regulations, labeling laws, and attempts to educate the public. Severe poisoning in a child can

cause lead encephalopathy, the mortality rate for which is high. Those children who survive
frequently show evidence of permanent central nervous system damage.
The diagnosis of lead poisoning is difficult, and the demonstration of an elevated lead level in
blood or urine constitutes the most positive indication of absorption of a lead compound. Being
a ubiquitous element, lead is normally present in trace amounts in biological material. Analytical
procedures must be extremely sensitive and conducted with great care in order to achieve valid
results. These requirements generally make lead analyses the function of a special laboratory,
particularly one which has experience with trace metal analyses and their special problems. This
can be illustrated by some facts relating to lead analysis. An average normal lead level in blood
is 30 g/100 ml and an amount of 100 g/100 ml represents a toxic level. Thus, 5 ml of the
normal blood specimen contains 1.5 g, and the abnormal sample contains 5 g. It is obvious
that any method used must not only be extremely sensitive, but it must also have an excellent
accuracy and precision in order to discriminate between the ends of the 3.5 g range separating
the normal and toxic lead levels in blood. In addition, all of the glassware and reagents used in
the analysis contain traces of lead. Thus, after careful selection of reagents and cleaning of
glassware, the analyst must still exercise meticulous technique in order to keep blank values of
lead low.
DETECTION OF LEAD OR LEAD POISONING
The actual analysis may follow one of many techniques: colorimetric analysis with
diphenylthiocarbazone, polarography, or atomic absorption spectrophotometry, to name some
of the most reliable ones.
The clinical laboratory performs two very important functions that aid in the diagnosis of lead
poisoning, even if the lead analysis is done by others. First, the specimens to be analyzed must
be collected in a valid way, that is, free of contamination. Second, other diagnostic tests can be
done for screening purposes or for confirmation. These tests are based on the effects of lead on
erythropoiesis. Lead interferes in the biosynthesis of hemoglobin, which results in anemia.
Although the precise mechanism of this interference is still under investigation, one result is a
buildup of precursors of hemoglobin. Two precursors that accumulate in lead poisoning are
aminolevulinic acid and coproporphyrin III, and urinary excretion of these substances increases
markedly. Methods for the detection of these substances and related enzymes are discussed in
Chapter 9.
Elevated urinary porphyrin levels can occur in conditions other than lead poisoning; however, after
several hundred comparisons in the author’s laboratory, it has been noted that in all cases of
proven lead poisoning a corresponding elevation of urinary coproporphyrin levels occurred.
Table 2 shows the correlation of urinary lead and elevated coproporphyrin levels in 140 cases of
lead poisoning.

Table 2. COMPARISON OF URINARY LEAD AND COPROPORPHYRIN LEVELS IN 140 CASES OF
ESTABLISHED LEAD POISONING
Number of Cases (total = 140)

Test for Coproporphyrin
Negative
Urinary Lead Test for Test for A* B
Range Coproporphyri Coproporphyri
g/l n n
Positive Doubtful
0—80 5 3 68 --
(Normal)
80-100 4 0 3 2
100-200 19 3 5 1
200-300 10 0 0 2
300-400 3 0 0 1
400-500 2 0 1 0
500-600 2 0 0 0
600-700 2 0 0 0
700-800 2 0 0 0
800-900 2 0 0 0
* A—Single specimen analyzed for lead

B—First specimen, Pb abnormal; other specimens, Pb normal.
SPECIMEN COLLECTION
A 24 h urine specimen is the specimen of choice. The patient should void directly into a leadfree
container (a borosilicate glass or polyethylene container from which surface lead has been
removed by washing, then rinsing with hot 1 molar nitric acid, and rinsing twice with metal-free
water). A preservative should not be added because it might contaminate the specimen. After
noting the total volume, the entire specimen, or a minimum of 100 ml, is submitted to the
toxicological laboratory for analysis. Catheterized specimens should not be used unless it is
unavoidable. In this case, the catheter should be cleaned (as just noted) to remove surface lead
before sterilization. In some cases we have found that an indwelling catheter through which urine
has been flowing freely for 24 to 48 h is usually free from surface lead. The possibility of
contamination should always be borne in mind when catheterized specimens are submitted for
analysis. In an emergency, it may be necessary to analyze a random urine specimen rather than
24 h urine specimen. In such a case, the specimen must be collected with the same care as just

outlined. Interpretation of the result is subject to the same difficulty as discussed next in
connection with blood specimens.
Blood specimens can be analyzed as readily as urine, but lead levels may fluctuate widely in
different blood specimen for the same patient. We have had the experience of seeing normal
lead levels in occasional blood specimens from lead-poisoned patients in well-documented cases.
For this reason, properly collected 24 h urine specimens are preferable. In very young or acutely
ill patients, blood may be the only practical specimen available. In these cases it may be necessary
to run several specimens before lead poisoning can be ruled out.
If the test is to be performed on blood, an anticoagulant or preservative should not be used unless
the exact lead content of these agents is known so that proper correction can be made. The
needle, syringe, test tube, and stopper should be of lead-free material, cleaned as previously
described. Special tubes for blood-lead collection are commercially available. Since most of the
lead is in the erythrocytes, a serum lead level is of little value.
As with any analysis of trace substance, the sensitivity of the analysis and the expected level of
substance controls the amount of specimen to be collected. For example, if a lead method is used
that is sensitive to 1 g of lead and in which the known reagent blank is also 1 g, and the
expected blood level is within normal limits, or about 30 g/100 ml, then a minimum of 10.0 ml
of blood must be collected. This quantity of specimen would contain 3 g of lead, a level that
can be differentiated from a blank with some degree of validity.
NORMAL VALUES
Normal lead levels range up to 80 g/l of urine or 80 g/100 ml of blood, with an average of 30
g/100 ml. Levels higher than normal indicate elevated absorption of lead compounds; levels
greater than 100 g/100 ml of blood, are usually associated with signs and symptoms of lead
poisoning. Normal blood lead levels in children are 15 to 20 g/100 ml. In this age group, levels
of 40 g/100 ml may represent an abnormal exposure to lead compounds. Some clinicians prefer
urine lead levels to be reported on a per diem basis. In the author’s opinion, it is preferable to
report these levels in g/l together with the total volume of the 24 h specimen. This allows the
clinician to correlate the 24 h excretion of lead with other factors that may be related to an
excessively high or low urinary output.
COLORIMETRIC DETERMINATION OF LEAD
PRINCIPLE
Lead (as Pb2+) forms a red complex with diphenylthiocarbazone (dithizone) that is soluble in a
number of organic solvents. Interference by other metal ions, such as zinc, cobalt, nickel,

cadmium, silver, copper, mercury, stannous tin, bismuth and thallous thallium is eliminated by use
of complexing agents and performance of extractions at controlled pH levels.
Specimens are either digested with nitric acid or ashed in a muffle furnace at 500 oC. The residue,
dissolved in HCl, is transferred to a separatory funnel. Citrate is added to complex the iron, the
pH is made alkaline with NH4OH, and the solution is extracted with a CCl4 solution of dithizone.
Pb, Zn, Cu, Ni, Co, Cd, Hg, and Bi are extracted quantitatively and Ag partially. The dithizone
solution is then shaken with dilute HCl, of pH 2. This step removes Pb, Zn, and Cd from the
dithizone solution, leaving the other metals in the CCl4-dithizone layer. The dilute HCl solution is
now treated with citrate, it is made alkaline with NH 4OH, cyanide is added to complex zinc and
cadmium, and the solution is extracted with dithizone in toluene. Unreacted dithizone is extracted
into the alkaline aqueous phase, leaving the red lead dithizone in the toluene. After separating
and filtering the toluene layer, the color intensity is read in a spectrophotometer at 520 nm. The
absorbance is compared with those of standards carried through the same procedure. It is
essential to run blank determinations and correct the final result for any trace quantities of lead
present in the reagents and glassware.
42.3. THALLIUM
This metal is rarely encountered. Thallium salts are used as rodenticides, usually by professional
exterminations. Cases have been reported of children eating fruit slices for other foods used as
bait and treated with thallium salts, which had been carelessly scattered by exterminators
intending to kill rats. Formerly, thallium salts were used, both internally and externally, as a
depilatory. Fortunately, because of the high toxicity of these substances, this use has been
virtually abandoned.
In many respects, the toxic effects of thallium are similar to those of lead. One characteristic
feature of poisoning by this metal is loss of hair or occasionally loss of nails and the skin of the
feet. In some intoxication in children, loss of hair was the only sign. A lethal dose can be as little
as 0.2 g of a soluble thallium salt. Since this metal is not present in biological material, except in
extreme trace quantities, any amount demonstrated in the urine is significant.
The following procedure is simple and useful for the detection of this substance.
DETECTION OF THALLIUM
PRINCIPLE
Thallium is converted to its oxidized form by the action of bromine water. After destruction of
excess bromine, the thallic ion is complexed with methyl violet to form a blue to violet compound
of unknown structure that is soluble in benzene.

REAGENTS
2. Bromine water. Into a glass-stoppered bottle containing 50 ml of water, add 2.0 ml of liquid
bromine (Caution: Do this in fume hood! Avoid contact with skin!) Stopper the bottle and
shake thoroughly. Some undissolved liquid bromine should remain in the bottom of the
bottle. Store in th fume hood.
3. Sulfosalicylic acid, 20 g/100 ml in water.
4. Methyl violet, AR, 20 g/100 ml in water.
PROCEDURE
1. Into a glass-stoppered tube, place 1.0 ml of urine and 3 drops of concentrated HCl; mix.
2. Add 5 drops of bromine water; mix thoroughly.
3. Add 5 drops of sulfosalicylic acid solution to decolorize the bromine.
4. Add 1 drop of methyl violet solution and mix.
5. Add 1.0 ml of benzene, stopper the tube and shake thoroughly. After separation the layers,
decant or aspirate off the benzene and observe its color, if any.
INTERPRETATION
A colorless benzene layer rules out the presence of 0.8 g or more of thallium. A positive test
imparts a blue to violet color to the benzene. No interference to the test is seen with levels up to
1.0 mg of borate, oxalate, chlorate, nitrate, phosphate, sulfate, chloride, bromide, perchlorate, or
EDTA. The color formation is inhibited by 1.0 mg quantities of nitrate, sulfite, sulfide, thiosulfate,
and thiocyanate; 0.2 mg or more of iodide gives a false positive test. Of all the metals selected,
the only one that gives a false positive is 0.01 mg of Hg2+. Alkyl aryl sulfonate detergents give
false positives, but these also give a color if the bromine and sulfosalicylic acids are omitted. This
test can be made quantitative by measuring the absorption at 610 nm.

Other procedures that can be used for the quantitative determination of thallium in blood, tissues,
and other specimens involve emission spectrography or various colorimetric methods following
digestion of the specimen. Atomic absorption spectroscopy can also be applied for the
determination of this metal.
Exercise No. 43
NONMETALS (INORGANIC POISONS)
The toxic nonmetals are usually encountered as compounds with other elements, or as sodium
and potassium salts. They are infrequently found in the free elemental form. Perhaps a more
descriptive heading for this group would be toxic anions.
Objectives: 1. To perform correctly spot tests for the detection of nonmetal poisons in
various body fluids.
2. To devise an effective strategy of determining the presence of nonmetallic
poisons in unknown analysis given a time limit.
43.1. BORON
Boric acid and borate salts are commonly found in the home laundry or medicine cabinet.
Accidental poisonings occur chiefly in children who ingest these preparations, or in infants treated
with talcum powders containing borates, which may be absorbed through abraded or irritated
skin.
Quantitative analysis of biological material for boron is a difficult problem. Not the least of the
difficulties is that boron-free glassware must be used. The following simple test does not require
special equipment and is convenient for screening purposes.
DETECTION OF BORON
PRINCIPLE
Turmeric or curcuma is a plant native to the East Indies and China. It is used as a condiment (curry
powder) in the tropical East. From the root of the plant is obtained an orange-yellow coloring
matter, which is curcumin or turmeric yellow. By an unknown reaction, this substance combines
with borates to form a characteristic color. This has been used as a qualitative test for boron for
many years, and papers treated with turmeric are readily available.
REAGENTS

2. Turmeric paper (commercially available), AR.
3. Ammonium hydroxide, concentrated, AR.
PROCEDURE
1. Mix 5 drops of urine with 1 drop of concentrated HCl.
2. Place 1 drop of the acidified urine on turmeric paper. Observe the color.
3. Let the paper dry and again observe the color.

4. Hold the paper over concentrated NH4OH and observe the color.
INTERPRETATION
A positive test is indicated by a brownish-red color of the acidified urine on the wet or dry paper.
A green-black or blue color results after exposure to ammonia fumes.
If the wet acidified spot does not change color, the urine contains less than 10 mg borate/l.
If, after drying, the spot still does not change color, less than 5 mg borate/l is present. If, after
exposure to ammonia, no color is produced, less than 3 mg borate/l is present.
Normally, less than 2 mg borate/l is present in urine. After a patient has been exposed to borate,
the urine level increases sharply, but even at levels of 10 mg/l no particular signs and symptoms
of borate poisoning are seen. This test, although not sensitive to normal urinary borate levels,
can detect elevated levels before toxic effects are displayed.
43.2. BROMIDES
Bromides are used in both organic and inorganic forms in medicine, chiefly for the purpose of
sedation. These drugs are sometimes abused or may be taken in overdosage accidentally. The
nonprescription status of drugs containing bromide makes them easily available to the patient
prone to drug abuse.
DETERMINATION OF BROMIDE IN SERUM
PRINCIPLE
The procedure to be described measures free Br—only; thus, the bromine in most of the organic
compounds is not detected. When organic bromides are ingested, however, they are metabolized
eventually to inorganic bromide.
The bromide anion readily displaces chloride from gold trichloride, forming gold tribromide:
AuCl3 + 3Br— → AuBr3 + 3Cl—
The formation of gold tribromide may also be accompanied by the formation of AuBrCl 2 and
AuBr2Cl. The resulting brown color is very stable in acid solution and can be read quantitatively
at 440 nm.

REAGENTS
1. Trichloroacetic acid, 10 g/100 ml aqueous solution.
2. Gold (auric) chloride solution. Wash the contents of a 1.0 g ampule of gold chloride into a
200 ml volumetric flask and dilute to the mark with water. The solution is stable.
3. Trichloroacetic acid (10 g/100 ml)—sodium chloride (0.06 g/100 ml) mixture. Place 0.6 g of
NaCl in a 1 liter volumetric flask and add 500 ml of water. Add 100 g of trichloroactic acid and
dilute to volume with water.
4. Standards.
a. Stock, 10 mg/ml. weigh exactly 1.000 g of NaBr, AR, dissolve in water, and dilute to
100 ml.
b. Dilute standard, 0.5 mg/ml. Pipet 10.0 ml of stock standard into a 200 ml volumetric
flask and dilute to volume with the trichloroacetic acid-NaCl mixture.
PROCEDURE
1. Prepare a 1:10 trichloroacetic acid filtrate of serum.
2. Pipet 5.0 ml of clear filtrate (sample) into one tube and 5.0 ml of 10 g/100 ml trichloroacetic
acid solution (blank) into a second tube.
3. Prepare standards as follows:
a. Pipet 0.5 ml of dilute standard into a labeled tube and add 4.5 ml of 10g/100 ml
trichloroacetic acid-NaCl mixture. Mix well (corresponds to 50 mg NaBr/100 ml).
b. Pipet 2.0 ml of dilute standard into a labeled tube and add 3 ml of 10 g/100 ml
trichloroacetic acid-NaCl mixture. Mix well (corresponds to 100 mg NaBr/100 ml).
4. Add 0.5 ml of 0.5 g/100 ml AuCl3 solution to all tubes. Mix well.
5. Read at 440nm

CALCULATION
A unknown x concentration of standard* = mg NaBr/100 ml

A standard*
INTERPRETATION
Although normal bromide levels in serum are 0.8 to 1.5 mg/100 ml, this method may occasionally
give results up to 5 mg/100 ml even with normal serum. It has been suggested that this is due to
a slight turbidity that may at times develop. Therapeutic levels may be in the order of 100 mg/100
ml, and toxic levels are usually greater than 150 mg/100 ml. With a single overdose of an organic
bromide compound, serum levels of inorganic bromide do not rise above normal levels. After
prolonged therapy with these drugs, serum levels of inorganic bromide ma increase to more than
100 mg/100 ml. At these levels, mental disturbances may be elicited.
43.3. FLUORIDE
This element is accessible to the public in the form of the sodium salt. Sodium fluoride is a
common ingredient in roach and ant poisons and, as such, it is frequently kept around the house
and even in the kitchen. For this reason, accidental poisonings have occurred, especially since the
white crystalline material can be mistaken for ordinary salt or baking powder. In recent years a
blue dye is usually added to these preparations to avoid the type of accident.
The fatal dose of sodium fluoride is 5 to 10 g. Once the compound reaches the stomach, the
acidity of the gastric contents converts the salt to free hydrofluoric acid, which produces a dark
red corrosion of the mucous membrane. For this reason, inorganic fluorides could also be
classified with the corrosives.
A number of organic fluoride compounds are extremely toxic, and one of these, sodium
fluoroacetate (sometimes called “1080”), has been used as a rat poison. The toxicity of this
substance is due to its competition with acetate in the tricarboxylic acid cycle with the eventual
formation of fluorocitric acid. It is estimated that a lethal oral dose in man is about 50 mg.
Despite the marked toxicity of these substances, poisonings of this type have not been common
in the past. This was fortunate for the analyst because the difficulty and length of time needed
for fluoride analysis led him to avoid it, if at all possible. Now, with the aid of microdiffusion in
plastic dishes, this analysis has been greatly simplified. Plastic containers are ideal for collecting
specimens as well as for conducting the analysis, since silica in glassware reacts with fluoride to
form a volatile product, resulting in loss of fluoride.

Rieders has described a screening test using modified polypropylene Conway cells. The method
takes about 1 hour for completion; however, the test to be described, although somewhat better
quantitative results.
DETERMINATION OF FLUORIDE IN BIOLOGICAL SAMPLES
PRINCIPLE
Fluoride is separated from the specimen by diffusing it into solid NaOH, at an elevated
temperature for 20 h. The separated fluoride is then estimated by developing a color with a
cerium or lanthanum complex with alizarin complexone.
REAGENTS
1. Perchloric acid, 70 g/100 g.
2. Silver perchlorate, AR.
3. Sodium hydroxide, 0.50 molar. Dissolve 2.00 g of NaOH in 50 ml of water and dilute to 100
ml with 95% ethanol.
4. Dye solution. Dissolve 10.0 g of Amadac-F (Burdick & Jackson Laboratories, Inc. Muskegon,
MI.) in 60% isoproply alcohol and make up to 100 ml with the same solvent. Amadac-F
contains all the necessary components of the dye reagent.
5. Fluoride standard. Dissolve 0.2210 g of sodium fluoride, AR, in water and make up to 100 ml;
1.0 ml of this solution contains 1.0 mg of fluoride, A 1:100 dilution of this solutions will result
in a useful working standard in which 1.0 ml contains 0.010 mg of fluoride.
PROCEDURE
1. Place 0.10 ml of 0.50 molar NaOH in the center of the inside top of a plastic Petri dish (Millipore
Filter Corp., Bedford, MA., Cat. No. PD 10 047 00). Distribute the solution evenly and evaporate
to dryness with a fan.

2. Transfer 1.0 ml of blood, urine, or gastric contents to the bottom of the plastic Petri dish
diffusion unit. Add about 0.2 g of silver perchlorate to the specimen.
3. Add 2.0 ml of perchloric acid to the specimen and cover immediately with the prepared
receiver top. Mix by gently swirling.
4. Place the unit in an oven, previously heated to 50 oC, and allow it to remain at this temperature
for 20 h.
5. Carefully remove the unit from the oven and allow to cool. Remove the receiver top and add
1.0 ml of distilled water to the NaOH residue in the receiver top.
6. Gently stir with the tip of a dropping pipet, aspirate the solution, and transfer to a 5 ml
volumetric flask. Repeat last part of step 5 and first part of step 6 twice more.
7. Add 1.0 ml of Amadac-F reagent and dilute the solution to 5.00 ml.
8. After 1 h, read the color of 620 nm and compare with a water blank and standard carried
through the entire procedure.
INTERPRETATION
Normal blood fluoride levels range up to 0.050 mg/100 ml. In fatal cases, blood levels may be as
high as 0.2 to 0.3 mg/100 ml. Excretion of fluoride in the urine is about 1.0 mg/d. Even subtoxic
doses of fluoride can result in sharp increases in urine levels, and in severe poisoning the urine
concentration can be several milligrams/100 ml.
Exercise No. 44
CORROSIVES
This group includes those strong mineral acids or fixed alkalies that produce chemical burns on
contact. There are no good tests that can be carried out on blood, serum, or urine by which the
type of acid or alkali can be detected and the ingested quantity estimated.
The only specimen that can be examined profitably is gastric contents. Frequently, this specimen
is not available unless the patient has vomited, since gastric lavage is containdicated in this type
of poisoning. If gastric contents are available, the pH should be measured. Ions such as Na +, K+,
Cl-, SO4-2, and PO4-3 can be demonstrated by methods used in the laboratory for routine analysis.
Obviously, most of the common ions would be present normally in gastric contents. To be of

significance in this type of case, a large excess must be present. Since many compounds in the
group of corrosives can cause major disturbances in acid-base balance, it is advisable to perform
electrolyte studies on blood. Usually the clinician has evidence from lesions in the mouth and
esophagus that a corrosive substance has been ingested.
Exercise No. 45
DRUG TESTING
METHAMPHETAMINE (MET)/TETRAHYDROCANNABINOL (THC)
Introduction
The effects of methamphetamine generally last 2-4 hours, and the drug has a half-life of 9-24
hours in the body. Methamphetamine is excreted in the urine primarily as amphetamine and
oxidized and deaminated derivatives. However, 10-20% of methamphetamine is excreted in an
unchanged form. Thus, the presence of the parent compound in the urine indicates
methamphetamine use. Methamphetamine is generally detectable in the urine for 3-5 days after
ingestion, depending on urine pH level. THC ( 9-tetrahydrocannabinol) is the primary active
ingredient in cannabinoids (marijuana). The peak effect of smoking THC occurs in 20-30 minutes
and has a duration of 90-120 minutes after one cigarette. Elevated levels of the urinary
metabolites of THC are found within hours of exposure and remain detectable for 3-10 days after
smoking. The main metabolite excreted in the urine is 9-THC-COOH. The SD BIOLINE MET/THC
test is a rapid urine screening test that can be performed without the use of an instrument. Cut-
off values of 1,000/ml for methamphetamine and 50 ng/ml for 9-THC-COOH have been
established as a guideline for screening results by the National Institute on Drug Abuse (NIDA).
A sample containing methamphetamine (test band 1) at 1,000 ng/ml or greater and/or a sample
containing 9-THC-COOH (test band 2) at 50 ng/ml or greater is considered positive for
methamphetamine and/or THC. Samples containing lower concentrations of these agents are
considered negative. All presumptive positive results must be confirmed by an approved method.
Test principle
This test relies on competition for antibody binding between drug-protein conjugates and drugs
that may be present in the urine sample. The SD BIOLINE MET/THC test contains a membrane
strip, which has two test band regions that are pre-coated with drug-protein conjugates. A gold
pad containing mouse monoclonal drug antibodies conjugated to colloid gold particles is placed
between the sample pad and the membrane. The antibodies on the test strip allow for selective
identification of methamphetamine and 9-THC-COOH in urine with a high degree of sensitivity.
During testing, a urine specimen migrates upward by capillary action. If a drug is present in the
urine specimen below its cut-off concentration, or if no drug is present, the binding sites of the
respective antibody conjugated gold particles in the test device will not be saturated. The

antibody conjugated gold particles will then be captured by their respective immobilized drug-
protein conjugate, and a colored line will appear in the associated test region. The colored line
will not form in the test region if the drug level is at or above its cut-off concentration because it
will saturate all the binding sites of the drug antibodies. A drugpositive urine specimen WILL NOT
generate a colored line in the respective test region due to drug competition, while a drug-
negative urine specimen WILL generate a line in the respective test band region due to the
absence of drug competition. Regardless of whether a test band appears in either test region (1
or 2), a control band should be present in the control region (C) to confirm the viability of the test.
The control band is immobilized with goat anti-mouse immunoglobulin, and will therefore
capture any monoclonal antibody-gold conjugate that passes through the region, producing a
colored band in the control region (C). this function as a procedural control. The control band
should always appear if the test procedure is performed properly and the test reagents of the
control line are working.
Objectives: 1. To perform correctly a qualitative drug test for the detection of two drug
metabolites in random urine samples.
2. To interpret accurately the test results.
Intended use
The SD BIOLINE MET/THC test is a rapid, one-step lateral flow chromatrographic immunoassay
designed for the simultaneous qualitative detection of methamphetamine and 9-THC-COOH,
respectively. This test is intended for professional, in vitro diagnostic use only. This test provides
only a preliminary analytical test result. A more specific alternative chemical method must be
used in order to obtain a confirmatory result. Gas chromatography/mass spectrophotometry
(GC/MS) has been established as the preferred confirmatory method. Clinical consideration and
professional judgment should be applied to any drug of abuse test result, particularly when
preliminary positive results are indicated.
Materials provided and active ingredients of main components
1. The SD BIOLINE MET/THC test kit contains the following items to perform the assay:
• 25 Test devices with desiccant in individual foil pouches
• 25 Disposable urine droppers
• 1 Instructions for use
2. Active ingredients of main components

• 1 test strip includes:
o Gold Conjugates: Mouse monoclonal anti-MET gold colloid (0.47 l),
mouse

monoclonal-THC gold colloid (0.47 l)
o Test Line 1: Methamphetamine-BSA conjugate (0.23 l) o Test Line 2:
THC-BSA conjugate (1.17 l) o Control Lin: Goat anti-mouse IgG (0.35 l)
• Protective gloves, Timer, Biohazard container
Kit storage and stability

1. This kit should be stored at a temperature between 2oC and 30oC. Do not freeze the test kit
or its components.
2. The test device is sensitive to both humidity and heat.
3. Check the humidity indicator on the desiccant for color change and throw the pouch if the
color indicates saturation (yellow to green).
4. Perform the test immediately after removing the test device from the foil pouch.
5. Do not use the test kit beyond its expiration date.
6. The shelf life of the kit is as indicated on the outer package.
7. Do not use the test kit if the pouch is damaged or the seal is broken.
Warnings
1. For in vitro diagnostic use only. Do not reuse the test device.
2. Avoid cross-contamination of urine samples by using a new urine specimen container and
dropper (disposable specimen droppers or pipette tips) for each urine sample.
3. Urine specimens may be potentially infectious. Decontaminate and dispose of all specimens,
reaction kits and potentially contaminated materials in a biohazard container as if they were
infectious waste.
4. The entire instructions for use must be read carefully before using this test. If test procedures
and precautions are not followed, false results may be obtained.
1. Urine specimens should be collected in a clean glass or plastic container. Fresh urine
specimens do not require any special handling or pretreatment.
2. If specimens are not immediately tested, they should be refrigerated at 2-8oC or frozen.
Specimens should be brought to room temperature (15-30oC) before testing.

3. Specimens containing precipitate may yield inconsiderable test results. Such specimens must
be clarified by centrifuging or allowing to settle prior to assaying.
4. For prolonged storage, specimens may be frozen and stored at a temperature below -20oC.
frozen specimens should be completely thawed, thoroughly mixed and brought to room
temperature (15-30oC) prior to testing.
1. Allow all kit components and specimens to reach a temperature between 15 oC and 30oC prior
to testing.
2. Remove the test device from the foil pouch and place it on a flat, dry surface.
3. Holding the urine dropper above the test device, dispense 3-4 drops of urine into the sample
well (S).
4. As the test begins to work, you will see a purple colored band move across the result window
in the center of the test device.
5. Interpret test results at 5 minutes. Do not interpret test results after 5 minutes.
1. Negative Result: The presence of three colored bands (1, 2 and C) within the result window,
regardless of the order in which the bands appear, indicates a negative result. A negative
result indicates that the drug concentration are below the detectable levels.
2. Methamphetamine-Positive Result: The presence of both the THC test band (2) and the
control band (C) within the result window, regardless of which band appears first, indicates a
methamphetamine-positive result. This result indicates that the concentration of
methamphetamine in the sample is at or above the detectable level (1,000 ng/ml).
3. THC-Positive Result: The presence of both the MET test band (1) and the control band (C)
within the result window, regardless of which band appears first, indicates at THC-positive
result. This result indicates that the concentration of THC metabolites in the sample is at or
above the detectable level (50 ng/ml).
4. Methamphetamine- and THC-Positive Result: The presence of only the control band (C) within
the result window indicates a positive result for both methamphetamine and THC. This result
indicates that the concentrations of methamphetamine and THC metabolites are at or above
their detectable levels (1,000 ng/mld and 50 ng/ml, respectively).
5. Invalid Result: If the control band (C) is not visible within the result window after performing
the test, the result is considered invalid. Instructions may not have been followed correctly or
the test may have deteriorated beyond the expiration date. It is recommended that the
specimen be retested using a new test kit.

Test limitations
1. The SD BIOLINE MET/THC test is designed for use with human urine only.
2. The test provides only a preliminary analytical result. A secondary analytical method must be
used to obtain a confirmatory result. Gas chromatography/mass spectrophotometry (GC/MS)
is the preferred confirmatory method.
3. The SD BIOLINE MET/THC test is a qualitative screening assay and cannot determine the
concentration of methamphetamine or THC metabolites in the urine or the level of
intoxication.
4. It is possible that technical or procedural errors, as well as other interfering substances in the
urine specimen, may cause erroneous results.
5. Adulterants such as bleach and/or alum in urine specimens may yield erroneous test results,
regardless of the analytical method used. If adulterations is suspected, the test should be
repeated with another urine specimen.
6. Certain medications containing opiates derivatives may produce a positive result. Foods and
tea containing poppy products may also produce a positive result.
1. The presence of a colored band in the control region (C) acts as the internal procedural control
included in the test. It confirms sufficient specimen volume and correct procedural technique.
A clear background is required.
2. Control standards are not supplied with this kit. However, good laboratory testing practice
recommends that positive and negative controls be tested to confirm the test procedure and
to verify proper test performance; NIDA recommends that positive quality control specimen
be at or near the cut-off concentrations.
1. Expected Values
This kit can detect methamphetamine and THC metabolites in human urine. A positive result
indicates that the sample probably contains methamphetamine and/or 9-THC-COOH at or
above the concentrations of 1000 ng/ml and 50 ng/ml, respectively.
2. Sensitivity and Specificity

The NIDA has suggested that the screening cut-off for positive sample be 1,000 ng/ml for
methamphetamine and 50 ng/ml for 9-THC-COOH.
The accuracy of the SD BIOLINE MET/THC test was evaluated in comparison to a commercially
available test (immunoassay) at the cut-off of 1,000 ng/ml of methamphetamine and 50 ng/ml
of 9-THC-COOH. The results are summarized in the following tables:
SD BIOLINE MET/THC Sensitivity (95%Cl)
Positive Negative

Immunoassay MET Positive (65) 65 0 100% (94.4-100%)
THC Positive (65) 65 0 100% (94.4-100%)
SD BIOLINE MET/THC Sensitivity (95%Cl)
Positive Negative
Immunoassay MET Positive (117) 0 117 100% (96.8-100%)
THC Positive (116) 0 116 100% (96.8-100%)
3. Reproducibility of the SD BIOLINE MET/THC test has been demonstrated by within-run, between-run and
batch-to-batch studies using in-house reference panels. All values were identical to reference panel
acceptance criteria.
4. Analytical Sensitivity
The SD BIOLINE MET/THC can detect 1000 ng/ml of methamphetamine and 50 ng/ml of 9THC-COOH as its
confirmed minimum detection limits. The SD BIOLINE MET/THC tests is equal to other commercial kits for the
detection of low concentration of methamphetamine and THC metabolites.
5. Analytical Specificity
Due to cross-reactivity resulting from the interaction of efavirenz metabolites with the THC rapid test, it is
possible that anti-retroviral therapy with efavirenz produces urine samples that screen false positive for THC,
despite the absence of THC metabolites.
The following table lists compounds that are positively detected in urine by the SD BIOLINE MET/THC test at
5 minutes.
Compound Concentration (ng/ml) Cross-Reactivity (%)
D-Amphetamine 30,000 <5
D,L-Amphetamine sulfate 100,000 <2
(+) Ephedrine 100,000 <2
(-) Ephedrine 75,000 <2
D-Methamphetamine 1,000 100
p-OH-Methamphetamine 10,000 10
Methylenedioxyamphetamine 100,000 <2
Methylenedioxymethamphetamine 7,000 <15
[THC]
Compound Concentration (ng/ml) Cross-Reactivity (%)

Cannabinol 15,000 <1
8
11-nor- -THC-COOH 250 20

9
11-nor- -THC-COOH 50 100
8
-THC 25,000 <1
9
-THC 15,000 <1
11-hydroxy- 9-THC 10,000 <1
A study was conducted to determine the cross-reactivity of the test with compounds in
urine not associated with methamphetamine and THC. The following compounds
showed no cross-reactivity when tested with the SD BIOLINE MET/THC test at a
concentration of 100 g/ml:
Acetamidophen β-Estradiol Diethylpropion (-)Cotinine Maprotiline Nalidixic acid

Acetophenetidin Estrone-3-sulfate Diflunisal Creatinine Meperidine Nalorphine
(Phenacetin) Ethyl-paminobenzoate Digoxin Deoxycorticosterone Meprobamate Naloxone
N-acetylprocainamide Fenoprofen Diphenylhydramine Dextromethorphan Methadone Naltrexone
Acetylsalicylate Furoxemide Domperidone Papaverine Promethazine Naproxen
Aminopyrine Gentisic acid Doxylamine Penicillin-G D,L-Propanolol Niacinamide
Amitryptyline Glucuronide Ecgonine hydrochloride Pentazocaine Propiomazine Thiamine
Amobarbital Glutethimide Ecgonine methyllester Pentobarbital D- Thioridazine
Amoxapine Guaifenesin Nifedipine Perphenazine Propoxyphene D,L-Thyroxine
Amoxicillin Hippuric acid Norcodein Phencyclidine Promazine Tolbutamide
Apomorphine Hydralazine Tetrahydrocortisone Phenelzine Quinidine Triamterene
Aspartame Hydrochlorothiazide Tetrahydrozoline Phendimetrazine Quinine Trifluoperazine
Atropine Hydrocodone Thebaine Phenobarbital Rantidine Trimethoprim
Benzilic acid Hydrocortisone Norethindrone Phentermine L- Salicylic acid Trimipramine
Benzoic acid Hydromorphone O- Noroxymorphone phenylethylamine β- Secobarbital Tryptamine
Benzoylecgonine Hydroxyhippuric acid D-Norpropoxyphene Phenylethylamide Serotonin D,L-Tyrosine
3-Hydroxytyramine Phenylpropanolamin
Benzphetamine Noscapine Sulfamethazine Uric acid
Ibuprofen Prednisolone
Butabarbital Nylidrin Sulindac Verapamil
Imipramine Prednisone
Chloralhydrate D,L-Octopamine Temazepam Zomepirac
Iproniazid Erythromycin
Chloramphenicol Oxalic acid Tetracycline
(-)Isoproterenol
Chlordiazepoxide Oxazepam Diazepam
Isoxsuprine
Chlorothiazide Oxolinic acid Diclofenac
Ketamine
Chlorpromazine Oxycodone
Ketoprofen
Chlorquine Oxymetazoline
Labetalol
Cholesterol Oxymorphone
Levorphanol
Clomipramine Methaqualone
Lidocaine
Clonidine Methylphenidate
Loperamide
Cocaine hydrochloride Methyprylon Morphine-
Loxapine succinate
Codeine 3-β-Dglucuronide
Cortisone

UNKNOWN ANALYSIS: DETECTION OF POISONS
Course, Year & Section: __________ Subject: ______________ Date: ____________

Names of Member: __________________________________________________________
Substance Test Method Observable Result Interpretation Remarks

Tested

References
Annino, J.S., 1964. Clinical Chemistry: Principles and Procedures. 3rd Ed., Boston: Little, Brown and Company.
Bennett, P.N., Brown, M.J. &Sharma, P. 2012. Clinical Pharmacology. 11th ed., China:Churchill Livingstone Elsevier.
Bishop, M.L., Duben-Von Laufen, J.L., & Fody, E.P., 1985. Clinical Chemistry Principles, Procedures &
Correlations. Philidelphia: Lippincott Company.
Bishop, M.L., Fody, E.P. & Schoeff, L.E. 2010. Clinical Chemistry: Techniques, Principles, Correlations. 6th ed.,
Philadelphia: Walter Kluwer/Lippincott Williams & Wilkins.
Burtis, C.A., Ashwood, E.R., & Burns, D.E. 2012. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics.
5th ed., U.S.A.: Elsevier Saunders.
Calbreath, D.F., 1992. Clinical Chemistry: A Fundamental Textbook. Philadelphia:W.B. Saunders Company.
Dialab Procedures on Measurements of Glucose, Lipid Profile, Total Protein, Albumin, Urea, Uric Acid, and
Creatinine.
Hellmann, M.A., Savage, E.P., Keefe, T.J. 1986. Epidemiology of Accidents in Academic Chemistry Laboratories. J.
Chem. Educ. 63:A267-70; A290-293.
Henry, J.B., 1996. Clinical Diagnosis & Management by Laboratory Methods, 19th Ed. Philadelphia:W.B. Saunders
Company.
Hilado, L.D., 1996. Compiled Notes in Clinical Chemistry. Iloilo City, Philippines.
Hilado, L.D., 1990. Med. Tech. 4B Laboratory Manual in Clinical Chemistry. Iloilo City, Philippines.
Kaplan,A., Jack, R., Opheim, K.E., Toivola, B., & Lyon, A.W., 1995. Clinical Chemistry: Interpretation & Techniques,
4th Ed. Philadelphia:Williams & Wilkins.
Kaplan, L.A., & Pesce, A.J. 2010. Clinical Chemistry: Theory, Analysis, Correlation. 5th ed., U.S.A.: Mosby Elsevier.

McPherson, R.A. & Pincus, M.R. 2012. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd
ed., Singapore: Elsevier W.B. Saunders Company.
Sacher, R.A., & McPherson, R.A., 2000. Widmann’s Clinical Interpretation of Laboratory Tests, 11th Ed. Philadelphia:
F.A. Davis Company.
Tietz, N., 1999. Fundamentals of Clinical Chemistry, 3rd Ed. Philadelphia: W.B. Saunders Company.
Tietz, N.W., 1976. Fundamentals of Clinical Chemistry. Philadelphia: W.B. Saunders Company.
Electronic References
Product inserts from reagent kits obtained from the following:
1) A.L.S. Biochemicals (USA)

2) Dialab Produktion und Vertrieb von chemisch – technischen (Austria)
3) EliTech Clinical Systems (France)
WATER BALANCE & ELECTROLYTES

Total Body Water (42L)
Intracellular Water (28 L) Extracellular Water (14 L)
Interstitial fluid Plasma
(10.5 L) (3.5 L )
Potassium K+ Potassium
(110 mmol/L ) ( mmol/L )
K+
4
Na +
sodium sodium
+
(10 mmol/L ) Na (135 mmol/L )
Diffusion sodium pump
The daily obligatory losses are shown as follows:
Skin 500 mL
Lungs 400 mL

Gut 100 mL
Kidneys 500 mL
Total 1500 mL
These obligatory losses are compensated by water taken from the following sources:
Water from oxidative metabolism 400 mL
Minimum in diet 1100 mL
Total 1500 mL
ECF Osmolality
The ECF osmolality is regulated by the level of sodium and associated anions (e.g., HCO3), glucose,
urea, and proteins.
Of these solutes sodium is the major determinant of plasma osmolality.
The ECF volume is directly dependent on the sodium content and is maintained by :
1) Regulation of renal excretion of sodium or glomerular filtration rate.

About 70% of filtered sodium is reabsorbed by the earlier parts of the renal tubules.
2) Aldosterone via the RAA system.

Aldosterone
= promotes sodium reabsorption
= stimulating the release of potassium and hydrogen.

=Water and sodium loss
Overhydration
= excessive intake of water (polydipsia)
= excessive reabsorption of water such as in cases of SIADH and ectopic ADH

secretion.
DISORDERS OF WATER METABOLISM

WATER DEFICIT
Reduced intake/water deprivation
Infancy; Elderly; & Enfeebled (Hot weather complicates)
Defective thirst
Head injuries; and postneurosurgery
Excess solute intake
Formula feeding to supplement nutrition; NGT with heavy solute
Sweating
Renal loss of water
Primary due to loss of renal concentrating ability in nephropathy;
Secondary due to diabetes mellitus; & Osmotic dieresis
WATER EXCESS
Compulsive water drinking
Increased intake with inappropriate secretion of ADH

Renal abnormality that limits or prevents water excretion
Congestive heart failure (CHF) (underperfusion of kidneys)
Cirrhosis of the liver with ascites
Function of Electrolytes
Maintenance of osmotic pressure and hydration e.g., Na+
Buffering functions e.g., HCO3-
Activators in enzyme reactions e.g., Mg++
Normal neuromuscular excitability e.g., Ca++
Redox reaction (electron transport) e.g., Fe++/Fe+++
Levels of Electrolytes in the Plasma (mmol/L)
Plasma Interstitial Intracellular

Fluid
Cations
Na+ 142 140 10
K+ 5 4.1 150
Ca++ 5 4.1 -
Mg++ 5 3.0 40
Total 155
Anions
Cl- 103 115 15
HCO3- 27 27 10
PO4 3- 2 2 100
SO4 2- 1 1.1 20
Organic Acids 6 3.4 -
Proteins 16 10 60
Total 155
Anion Gap
The anion gap refers to the difference between the sums of the concentrations of the principal
cations (e.g., Na+ and K+) and of the principal anions (e.g., CI -and HCO3-).
It represents the unmeasured net negative charge on plasma proteins.
Methods of calculating Anion Gap. The anion gap (AG) may be measured by any of the following
formula:
1. AG = Na+ - (Cl- + HCO3 -) NV: 7-14 mmol/L

2. AG = (Na+ + K+) - (Cl- + HCO3-) NV: 10-18 mmol/L
ALTERATIONS IN ANION GAP INCREASED

ANION GAP
Chloride Normal; Normal or low bicarbonate (increased anions)
Diabetic ketoacidosis
Uremic acidosis due to sulfates, phosphates and fixed acids; Starvation
Alcoholic ketosis (ethanol metabolites, lactate)
Lactic acidosis due to hypoxia/hypoperfusion
Exogenous poisons (ketones, lactate, salicylates, alcohols)
NORMAL ANION GAP ACIDOSIS
Hyperchloremic acidosis
Diarrhea
RTA
Hyperalimentation
Early renal failure
DECREASED ANION GAP (<6 mEq/L)

Cationic myeloma proteins
Hyperlipidemia with decreased plasma content of water & all electrolytes
Erroneous report
Sodium
= most abundant cation in the extracellular fluid.
= accounts for about 92% of the osmotically active solutes in the plasma. Its amount also determines
the ECF volume.
= 70% of sodium is freely exchangeable while 30% is complexed in the bone.
Regulators of Sodium Level

Diet
Kidney
= renal threshold for sodium is 110-130 mmol/L.
= about 70-80% of filtered sodium is reabsorbed at the proximal tubule RAA
= aldosterone promotes retention of sodium in exchange of secretion of K + & H+ ions. Atrial
natriuretic factor (ANF)
= this cardiac peptide promotes natriuresis (urinary excretion of sodium) and relaxation of the
vascular smooth muscle (vasodilatation)
Reference Ranges
sodium in the extracellular fluid is 135-145 mmol/L in
the intracellular fluid, it is within 4-10 mmol/L
Determination of Sodium
Serum sodium may be measured using emission flame photometry (EFP), atomic absorption
spectrophotometry (AAS), or ion-selective electrodes (ISE).

Colorimetric method called Albanese-Lein
= involves combining sodium with zinc uranyl acetate to produce sodium uranyl acetate
precipitate. Addition of water to the precipitate produces a yellow solution
ABNORMALITIES OF SODIUM AND THE EXTRACELLULAR FLUID (ECF)
HYPONATREMIA
Total-body Na & ECF volume LOW
GIT fluid loss
Burns
“Third compartment” accumulation (ascites, ileus)
Salt-losing renal disorders
Diuretic overuse
Total-body Na & ECF volume NORMAL
Acute water intoxication, usually iatrogenic
Syndrome of inappropriate secretion of ADH (SIADH)
Glucocorticoid deficiency
Severe whole-body K depletion
Total-body Na & ECF INCREASED
Acute renal failure with superimposed water load
CHF
Cirrhosis
Nephrotic syndrome
HYPERNATREMIA
Total-body Na NORMAL & ECF volume LOW
Excessive insensible loss: fever, thyrotoxicosis
Diabetes insipidus
Total-body Na & ECF volume LOW
Gastroenteritis
Osmotic diuresis
Pronounced sweating
Total-body Na INCREASED proportionately more than INCREASED ECF volume
Salt ingestion, deliberate or accidental
Inappropriate IV therapy
HYPEROSMOLALITY, without Na alterations

High blood ethanol (high osmolality but plasma not hypertonic)
Hyperglycemia
Radiographic contrast media
Potassium Potassium
is the major intracellular cation.
It is about 20 times greater in concentration inside the cells than outside.

= about 90% of potassium is free or exchangeable

= only 10 % is bound in the red blood cells, bone and brain tissues.
Normal values of potassium in serum samples are in the range of 3.8-5.0 mmol/L.
= elevated levels of potassium (>7.5 mmol/L)can seriously inhibit the irritability of muscles,
including the heart and may lead to paralysis or cessation of heartbeat
= low serum potassium (<3.0 mmol/L) may increase muscle irritability and cause heartbeat
during systole.
Measurement of Potassium
Like sodium, potassium may be measured by flame photometry, atomic absorption
spectrophotometry and ion-selective electrode.
Colorimetric procedure for potassium is Lockhead and Purcell that uses potassium cobaltinitrite. A
blue sodium potassium cobaltinitrite is produced with addition of phenol reagent.
Sodium and Potassium Assay Notes
=anticoagulants which tend to increase plasma volume e.g., oxalates, lowers sodium levels.
= blood samples taken after physical exercise or muscular activity have lower sodium.
= water used in the assay must be free of electrolyte traces and the thumb must not be used when
mixing the tubes since the skin may contain sodium chloride.
= serum potassium levels are usually higher than those obtained using plasma samples due to
platelets release potassium during the clotting process = among the causes of spuriously high
potassium are:
1. Increased platelet count
2. Prolonged application of tourniquet due to juxtavenular cellular injury production leakage of
potassium
3. Increased muscular activity e.g., repeated or excessive clenching of fist prior to and during
drawing of blood
4. Hemolyzed specimen (greatest error)
5. Contamination with potassium EDTA
ABNORMALITIES OF SERUM and WHOLE-BODY POTASSIUM
HYPERKALEMIA
Inappropriate cellular metabolism
Insulin deficiency; Acidemia; Hypoaldosteronism; & Cell necrosis (burns, crush, hemolysis,
antileukemia therapy)
Decreased renal excretion
Acute renal failure; Chronic interstitial nephritis; Tubular unresponsiveness to aldosterone;
& Hypoaldosteronism
Increased K intake
Inappropriate use of salt substitutes for K+ replacement
Potassium salts of antibiotics

HYPOKALEMIA
Inappropriate cellular metabolism
Alkalemia; Familial periodic paralysis; & Very rapid generation of cells (leukemia, treated
megaloblastic anemia)
Increased excretion
Vomiting and/or diarrhea; Diuretic overuse; Hyperaldosteronism; & Renal tubular acidosis
(RTA)
Decreased K intake
Anorexia nervosa; Diet deficient in vegetables and meats; & Clay eating (binds K and
prevents absorption)
Chloride
Chloride is the major extracellular anion.
It is for the maintenance of electrolytes balance, hydration, and maintenance of osmotic pressure.
Measurement of Chloride
Chloride may be measured by the ISE method

= membrane of the chloride ISE is a composite of silver sulfide and silver chloride
= only bromide can possibly cause interference.
Coulometric-amperometric method with adaptation of this is the Cotlove titrator or chloridometer.
Zall color reaction has been used in some semi-automated chloride analyzers
= reagent contains mercuric thiocyanate and ferric nitrate
= the chloride ions displace the thiocyanate ions to form soluble but undissociated
mercuric chloride
= releasing in the process an equivalent amount of thiocyanate
= this reacts with ferric ions derived from the ferric nitrate to produce reddish brown ferric
thiocyanate
Mercuric nitrate titration method of Schales and Schales

= the mercuric nitrate forms soluble but virtually undissociated mercuric chloride, which
does not affect the indicator diphenylcarbazone
= as soon as all the chloride ions are thus combined, the next drop of mercuric nitrate
added will cause the indicator to change to from colorless or faint pink to violet
= the volume of mercuric nitrate required to produce the endpoint is a measure of the
amount of chloride present
CSF Chloride

= normally higher than that of serum because the protein concentration in cerebrospinal fluid is
low, hence, there are practically no proteinate anions.
= about 115-132 mmol/L
= falls to about serum levels in cases of bacterial meningitis when protein concentration in the
CSF is elevated
Sweat Chloride
= screens for cystic fibrosis
= about 50 mg sweat is needed to test for chloride
= sweat-inducing drug, pilocarpine, is introduced into a limited area of the skin by means of an
electric current flowing between two electrodes attached to a limb (of a child) or the back
(on an infant), technique is called iontophoresis
= normal sweat chloride is about 5-40 mmol/L and most patients with cystic fibrosis have levels
above 60 mmol/L.
HYPERCHLOREMIC ACIDOSES
Renal Tubular Failure

Defective exchange of H+ for Na+ causes increased urinary excretion of
HCO3-
Urine often inappropriately alkaline
Urine: Na+ increased, K+ increased, Cl- decreased
Ca++ excretion and bone demineralization, if prolonged
Gastrointestinal Loss of HCO3-

Na+ & K+ loss in intestinal fluid
Fluid and electrolyte loss  hypovolemia
Increased BUN and creatinine, from dehydration and decreased GFR
Urine Na+ and Cl- very low
Calcium
Calcium is the most abundant cation in the body. It is 90% bound to the skeleton. In the bone. Its
combines with phosphates to form the hydroxyapatite crystals which provide strength to the bone.
Functions of calcium
Structural Bone
Neuromuscular Control of excitability
Release of neurotransmitters
Initiation of muscle contraction
Catalytic Coenzyme for coagulation factors
Signal transduction Intracellular secondary messenger

Calcium Regulation
Parathyroid Hormone (PTH)
= increases blood calcium levels
= secretion is inhibited by hypercalcemia and calcitriol
Target PTH Action Effect
Organ
Rapid release of calcium from Increased plasma Ca
Bone osteoclastic resorption
Increased Ca reabsorption Increased plasma Ca
Decreased Pi reabsorption Decreased plasma Pi

Kidney
Increased 1 -hydroxylation of 25- Increased Ca & Pi
Ohcholecalciferol absorption in the
gut
Decreased bicarbonate reabsorption
Acidosis
Calcitriol or Activated Vitamin D3
= also known as 1.25-didydroxy-cholecalciferol
= it is stimulated by increased PTH and decreased phosphate
Calcitonin
= produced by the C cells of the thyroid gland
= decreases blood calcium level (hypocalcemic hormone)
Measurement of Calcium
Aside from ISE (reference method), atomic absorption spectrophotometry and flame
photometry.
Three Measurement Approaches

1) Precipitation of calcium as an insoluble compound followed by titration or
colorimetric methods
ammonium oxalate (Clark-Collip)
chloranilic acid (Ferro-Ham method)
picrolonic acid
2) Formation of colored complexes calcium and a variety of dyes followed by
colorimetric determination of the complex
Alizarin
O-cresolphthalein complexone (Ca-OCP)
Calcein (Diehl-Ellingboe method)
Ammonium purpurate (Murexide)
Nuclear fast red
3) Removal of calcium from a colored complex by titration with a chelating agent
EDTA (ethylene diamine tetraacetic acid)
EGTA (ethylene glyco-bis(2-aminoethyl ether)- tetraacetic acid)
Dyes
The endpoint is reached by recovery of the original color of the dye or the disappearance
of the fluorescence of the calcium-dye complex.

O-cresolphthalein complexone method
= the dye binds calcium tightly in alkaline solution to form a highly colored complex with an
absorbance at 578 nm
= the reaction mixture contains 8-hydroxyquinoline to bind magnesium and prevent its
interference,
= urea to decrease the turbidity of a lipemic serum and increase color intensity of the
calciumdye complex, and
= ethanol to decrease the absorbance of the blank
Normal serum total calcium (Cat) falls within the range of 8.5 to 10.4 mg/dL (2.13 to 2.60 mmol/L).
Ionized calcium (Ca2+) in plasma, serum or whole blood is within the normal range of 4.68-5.32 mg/dL
(1.17 to 1.33 mmol/L).
Multiply mg/dL by 0.25 to convert to mmol/L
Inorganic Phosphate
= in the body exists only as inorganic phosphate esters
= about 80% of the phosphates are incorporated into the bone together with calcium
= most organic phosphates are present inside the cells as components of molecules e.g., the DNA ,
phospholipids, ATP, etc.
= in contrast, most inorganic phosphates are mostly confined in the extracellular fluid where they act
as buffers
= excreted principally via the urine
= phosphate homeostasis is closely linked with calcium regulation since the same hormones regulate
the levels of the two minerals
PTH, for example, stimulates the kidney to excrete phosphate while conserving calcium
Usually, the relationship between calcium and phosphorus is inverse. Measurement

of Phosphates Fiske-Subbarow method
= protein-free filtrate is prepared using trichloroacetic acid
= conversion of the inorganic phosphate in the sample to the heteromolybdenum blue by a reaction
with ammonium molybdate and the reducing agent, aminonaphthol sulfonic acid (pictol) = the
absorbance of the complex is measured at 700 nm
Daly-Ertingshausen method
= the inorganic phosphates is converted into phosphomolybdate polyacid by a reaction
with ammonium molybdate in sulfuric acid
= precipitation of proteins is prevented using a wetting agent called Tween 80
= OD of the phosphomolybdic acid is measured at 340 nm
Magnesium
= the 4th most abundant cation in the body

= majority of this mineral is stored in the bones in complex with calcium and phosphate
= about 70% of magnesium is free and only 30% is bound to protein = most of the
magnesium in the body is located within the cell.

= an essential activator of several enzymes e.g., phosphateses, kinases, phosphorylases and
enolases.
= also necessary in the oxidative phosphorylation occurring in the mitochondria. =
therapeutic agent and has an anti-convulsant laxative and antacid effects
Measurement of Magnesium
Magnesium may be measured by ISE, AAS, colorimetric or fluorometric analysis.
Calmagite (3-hydroxy-4 [(6-hydroxy-m-toly)azo]-1-naphthalene-O-sulfonic acid)

= in the presence of polyvinylpyrrolidone (used to minimize the effects of serum proteins), a
violet complex forms which absorbs light at 520 nm
= the reagent used contains amphoteric betaine detergent Empigen BB to shift the wavelength
of the blank, strontium chelate to mask the effect of calcium, and triethanolamine to mask
the effects of iron
Methylthymol blue
= complex formed is measured at 510 nm =used
in the DuPont aca analyzer.
Titan yellow
= method is called Dye-Lake method
= magnesium reacts with an alkaline solution of titan yellow in the presence of
polyvinylpyrrolidone to form a red lake colloidal precipitate
Fluorometric analysis include the use of the either hydroxyquinoline or calcein.
Normal values magnesium fall within the range of 1.3 to 2.1 mEq/L or 0.65 to 1.05 mmol/L.
TRACE ELEMENTS
Trace elements are present in the body in very small amounts usually less than 1 microgram
per gram of tissue. They form part of the micronutrients of the body and can be subdivided into four (4)
major groupings based on their physiological function:
1) Essential elements for normal growth, development and maintenance with established
Recommended Daily Allowance (RDA) (e.g. Fe, Zn, I, and Se)
2) Elements with definite role in the body but with no RDA yet established (e.g. Cu, Mn, Cr,
Co, Mo, & F)
3) Elements found consistently in body tissues in ultratrace amounts and not known to have a
definite role or detriment to the body (e.g. Li, Ni, Sn, Si, and V)
4) Elements with no known function in the body and cause pathological changes/toxic if
present (e.g. Al, Be, Cd, Hg, Pb, and As)
Dietary Reference Intakes (DRI) Definitions

(According to the Food and National Board of the National Academy of Sciences in 1997)

Recommended Dietary Allowance (RDA) is the average daily dietary intake level that is
sufficient to meet the nutrient requirements of nearly all (97 to 98%) healthy individuals in a
particular life stage and gender group.
Adequate Intake (AI) is a recommended intake value based on observed or experimentally

determined approximations or estimates of nutrient intake by a group or groups of healthy
individuals, which is assumed to be adequate; it is used when an RDA cannot be determined.
Tolerable Upper Intake Level (UL) is the highest level of daily nutrient intake that is likely
to pose no risk of adverse health effects for almost all individuals in the general population.
As intake increases above the UL, the potential risk of adverse effects increases.
Estimated Average Requirement (EAR) is a daily nutrient intake value that is estimated to
meet the requirements of half of the healthy individuals in alife stage and gender group; used
to assess dietary adequacy and serves as the basis for RDA.
Copper
In the blood, copper is seen in red blood cells or is bound to transport proteins e.g., albumin, and
ceruloplasmin.
Ceruloplasmin is necessary for the absorption of iron to the ferric state, a prerequisite for binding by
transferrin. It has a peroxidase activity.
Copper is important in erythropoiesis (hemoglobin synthesis) and catalytic activity of several enzymes
e.g., cytochrome oxidase and uricase.
Serum copper may be measured by AAS.
To convert to SI, multiply ug/dL by 0.157 to get values in umol/L.
Iron
Total body iron in humans is approximately 3-5 g with about 70% incorporated in the red blood cells,
and about 25% is found in the reticuloendothelial system, incorporated with ferritin and
hemosiderin as stored iron.
Two forms of iron in the body are:

Ferrous iron = found in oxyhemoglobin and reduced hemoglobin
Ferric iron = found in ferritin, hemosiderin, transferrin and methemoglobin
The two other proteins that are involved in the transport of iron are:
Haptoglobin. This binds hemoglobin and services to facilitate disposal of the iron from this
molecule

Hemopexin. This binds heme to avoid to aid its removal from the circulation
Measurement of Iron Ferrozine

method
= where serum proteins are precipitated in an acid solution containing thioglycolic acid that
reduces ferric to ferrous ion, thereby dissociating the iron from its binding to transferrin
= chromogen ferrozine is then added to the supernate to form a highly colored ferrous
complex which is measured at 562 nm.
Normal serum iron concentration falls within 65 to 165 ug/dL (11.6-29.5 umol/L) for men and 45 to
160 ug/fL (8.1-28.6 umol/L) for women.
Higher values are obtained in the morning due to diurnal variations.
Total Iron Binding Capacity (TIBC) and Transferrin Saturation
A known amount of ferric ions, more than sufficient to fully saturate the serum transferrin with iron,
is added to a serum sample. The excess ferric ions, not bound to transferrin, is removed by
addition of a small amount of buffered ion-exchange resin.
The sample is diluted and centrifuged, and an aliqout of the supernate is analyzed for iron content
of the fully saturated transferrin - this value is the TIBC.
The % saturation of transferrin is measured as followed.
Transferrin Saturation = Serum Fe x 100

(% saturation) TIBC
TIBC varies from 260 to 440 ug/dL (46.5-78.8 umol/L). Transferrin saturation ranges from
2050%.
Zinc Protopophyrin/Heme Ratio (ZPP/H)

= excellent screening test for detecting iron deficiency anemia and for monitoring the course of
therapy.
= hemoglobin in a drop of blood is converted to cyanmethemoglobin by treatment with cyanide
containing reagent
= portion of the mixture is placed on a cover slip and introduced into a ProtoFlour
hematofluorometer which measure simultaneously the light absorbed by the film of
cyanmethemoglobin at 424 nm and the fluorescent light emitted at 595 nm by zinc
protoporphyrin.
= the results are displayed as a ratio of umol ZPP/mol heme.
The normal ratio is 30 to 80 umol ZPP/mol heme.
= normal in thalassemia but abnormal in iron deficiency anemia.
= the ratio is elevated in all types of iron deficiency syndromes and chronic exposure to lead.

BIOLOGICAL ROLES OF ESSENTIAL TRACE ELEMENTS & ASSOCIATED ABNORMALITIES
Element Biological Role Comments Deficiency/Abnormality/ Toxicity
Chromium Glucose metabolism Potentiates insulin Glucose intolerance in deficiency
action
Cobalt Component of vitamin No other function Pernicious anemia
B12 known in man
Copper Co-factor for oxidase 90-95% Cu bound to Inherited diseases: Wilson’s & Menke’s
enzymes ceruloplasmin
Fluorine Inhibits dental caries; Usually supplied as Excessive intake cause fluorosis
therapeutically improves supplement to drinking
hydroxyapatite crystal water
quality in bones
Iodine Component of T3 & T4 Concentrated in the Iodine deficiency still occurs in various
thyroid; geographic areas
supplementation by
addition to salt is
common
Iron Component of In plasma, bound to Hypochromic, microcytic anemia
heme enzymes; transferrin, stored as in deficiency
hemoglobin, ferritin
cytochromes
Manganese Required for Component of Deficiency not known in man
glycoprotein and mitochondrial peroxide
proteoglycan syntheses dismutase
Molybdenum Component of sulfite Essential for Deficiency reported in total parenteral
and xanthine oxidases production of uric acid nutrition (TPN) patient; inability to
metabolize methionine
Selenium Component of Antioxidant properties, Deficiency may occur where soil Se is low
glutathione Se and vitamin E act and in long-term TPN patients with
peroxidase & synergistically inadequate supplements
iodinothyronine-
5’deiodinase
Silicon Involved in calcification Role in bone, cartilage, Deficiency: Impairment of normal growth
of bones and connective tissue in animals; silicosis may occur from
is poorly understood industrial exposure
Zinc Co-factor or component Involved in many Deficiency: Growth failure,
of more than 200 metabolic processes; hypogonadism, impaired wound healing;
metalloenzymes protein synthesis;
immunological Genetic disease: acrodermatitis
function; growth & enteropathica-impaired absorption;
development
Toxicity:vomiting, gastrointestinal
irritation
MEASUREMENT METHODS & FOOD SOURCES OF TRACE ELEMENTS

Trace Methods Food Sources
Element

Chromium Graphite Furnace AAS with Brewer’s yeast, mushrooms, molasses, nuts,
Zeeman background correction wine, beer, asparagus, prunes, meats,
cheeses, and whole grains
Copper Graphite furnace AAS with Shellfish, liver, kidney, egg yolk, and some
Zeeman background correction legumes
Fluorine ISE Natural or artificially supplemented drinking
water
Iodine ISE for iodide levels and Marine fish and seaweeds; iodized salt
immunoassay for thyroid products
hormones
Manganese Graphite furnace AAS with Bran flakes and wheat products
Zeeman background correction;
Magnesium nitrate as matrix
modifier
Molybdenum Graphite furnace AAS with Milk, milk products, organ meats, and dried
Zeeman background correction legumes and some cereals
Selenium Graphite furnace AAS with Organ meats, seafoods, cereals and grains,
Zeeman background correction; dairy products, and fruits and vegetables
Nickel nitrate or reduced grown in seleniferous areas
palladium as matrix modifier
Zinc Flame AAS for serum, plasma, Seafoods, meats, milk, and eggs; low in
urine, and for erythrocytes fibrous plants and vegetables
ACID-BASE BALANCE
Blood gas analysis routinely involves analysis of blood gases oxygen and carbon dioxide, and blood
pH.
The preferred sample if arterial blood collected in heparinized tubes.
The normal arterial pH falls within the range of 7.35 – 7.45 (average of pH 7.4). This is equivalent to a
molar hydrogen ion concentration of 4.5 x 10-8M to 3.5 x 10-8M buffer.
The bicarbonate buffer system is illustrated as follows:

H2CO3  H+ + HCO3- Equation 1
H2CO3 and HCO3 act as the conjugate acid-base pair with the latter acting as the base. The Ka (acidity
constant) for the equation can be written as follows:
Ka = [H+][HCO3-]
[H2CO3]
Equation 2
where the brackets represent the molar concentration. The equation tells that the higher the
hydrogen ion concentration, the higher is the acidity constant.

If the hydrogen ion concentration is to be solved then the equation becomes
[H+] = Ka [H2CO3]
[HCO3-]
Equation 3
Since pH = –log [H+], then
pH = -log Ka – log [H2CO3] Equation 4

[HCO3- ]
Or,
pH = [HCO3- ]_____
pKa + log [H2CO3] Equation 5
pH = pKa + log [HCO3- ]

[H2CO3]
This is called the Henderson-Hasselbalch equation.
The pKa is a constant and it depends on the buffer involved.
The pKa is the pH at which the molar concentration of the acid is equal to the molar concentration of
its conjugate base.
It is at this pH where the system exerts its maximum buffering activity. Usually the range of pH at which
a buffer is effective is within pKa + 1 pH unit.
The bicarbonate buffer has a pKa of 6.1 which means that it is effective in maintaining the pH of a
solution within the range of pH 5.1 to pH 7.1.
CAH CAH
CO2 + H2O ----- H2CO3 ------- H+ + HCO3-
Equation 6
In the peripheral tissues

= the bicarbonate diffuses out of the red blood cell, to maintain electrical neutrality, this diffusion of
bicarbonate is accompanied by as shift of chloride into the red blood cells (chloride shift) which
is mediated by a transport protein located in membrane of the red blood cell.
In addition to the respiratory component of the acid-base balance, the levels of bicarbonates in the
blood is also closely regulated by the kidney.

= bicarbonates are readily filtered in the glomerulus but it is absorbed in the proximal tubule
especially when a lot of the base needed e.g., in cases of acidosis.
= this mechanism forms the metabolic component of the acid-base balance.

In summary, acid-base balance is controlled by chemical buffers primarily bicarbonate, the lungs and
the kidney. It can be represented as follows:
pH = [HCO3-] which is a function of the kidney (metabolic component)

[H2CO3] which is a function of the lungs (respiratory component)
Normally, the levels of bicarbonate and carbonic acid are maintained at a ratio of 20:1.
Total CO2
Total CO2 consists of the HCO3 , undissociated H2CO3, dissolved CO2, and carbamino-bound CO2.
The bicarbonate is by far the largest (~95% of the total) and accounts for all but approximately 2 mmol/L
of the CO2 content.
The carbamino fraction is negligible in serum, but is appreciable in whole blood because of the
presence of hemoglobin.
Generally, the CO2 content is measured by automated methods or using a CO 2 electrode.
Routinely, the total CO2 content is assumed to be equal to the sum of the dissolved CO 2 and
bicarbonate.
This can be expressed in mmol/L. The normal value of total CO2 is 23 – 27 mmol/L.
pCO2 or dissolved CO2

= constitutes about 5 – 10% of the total CO2 content usually expressed in mmHg
= the normal value of 35 – 45 mmHg.

Sample Problem.
Given a pCO2 of 44 mmHg and total CO2 of 29 mmol/L. Solve for the pH.
= first, convert pCO2 in mmHg to dissolved CO2 by multiplying the solubility coefficient
of CO2 gas which is a constant (0.03 mmol/L/mmHg) i.e.,
44 mmHg x 0.03 mmol/L/mmHg = 1.32 mmol/L
= then determine the HCO3 concentration by finding the difference between total CO 2 and
dissolved CO2 concentration
29 mmol/l – 1.32 mmol/L = 27.68 mmol/L
= Calculate the pH as follows:

pH = 6.1 + log [HCO3- ] = 6.1 + log [HCO3-] [H2CO3]
[pCO2]
pH = 6.1 + log 27.68 mmol/L

1.32 mmol/L = 7.42
Physiologic Buffers
Bicarbonate Buffer.
Hemoglobin.
= this is a major buffer localized inside the red blood cells.
= in the peripheral tissues, carbon dioxide accumulates as a waste product of metabolism.
= as the pressure of carbon dioxide increases in the plasma, the gas diffuses into the red
blood cells where it reacts with water to form carbonic acid as catalyzed by carbonic
anhydrase (also known as carbonic dehydratase).
= the carbonic acid readily splits into hydrogen ions and bicarbonate.
= the hydrogen ion combines with hemoglobin which then releases the oxygen for the
tissues. Hemoglobin therefore can also act as buffer.
Phosphate Buffer.
This buffer system has a minor role in the blood. Instead, along with plasma proteins containing
especially the amino acid glutamine, it is important for the excretion of hydrogen ions in the
kidney. The conjugate acid-base pair of this buffer is shown as follows:
H2PO4  H+ + HPO42-
The pKa of this reaction is 6.8.
Plasma Proteins.
The amino acids present in the proteins are amphoteric and they can act as buffer.
Transport of Blood Gases

The transport of oxygen and carbon dioxide back and forth the lungs and the peripheral tissues is a
function of hemoglobin in red blood cells. Hemoglobin exists in two forms:

“T” or taut structure (deoxyhemoglobin) which has a low affinity for oxygen. “R”
or relaxed structure (oxyhemoglobin) which has high affinity for oxygen.
HPO
H
PO
In the peripheral tissues where the oxygen tension is very low because oxygen is utilized during
metabolism, the hemoglobin molecule exists in the taut form in order to prevent uptake of the
delivered oxygen.
In contrast, the relaxed form is favored in the lungs where oxygen tension is very high. This allows
uptake of oxygen by the hemoglobin molecule.
The affinity of hemoglobin with oxygen depends on many factors. This can be shown with the oxygen-
hemoglobin dissociation curve.

Decreased
100 PCO2
Temp
2,3-BPG Lungs
Shift to the Right
Increased
% saturation of p Increased
H PCO2
hemoglobin
Temp
50 Shift to the 2,3-BPG
Left
Decreased
Peripheral p
Tissues H
4
Partial pressure
0 of oxygen (pO2) in mmHg
The Oxygen-Hemoglobin Dissociation Curve

= the curve is sigmoid in shape
= exhibits cooperative effects, in that, at lower oxygen tension, the affinity of hemoglobin with
oxygen is very small, however, once the hemoglobin molecule has started binding with oxygen,
its affinity for the succeeding oxygen molecules increases
Comparison of Arterial and Venous Blood

Parameter Arterial Blood Venous Blood
Chloride Higher Lower
Bicarbonate Lower Higher
PCO2 Lower Higher
PO2 Higher Lower
pH Less More
Blood Gas Assay Notes
Arterial blood is required for pO2 measurement.
Venous blood may be taken for pH and pCO2 if it is drawn without stasis (no tourniquet) and without
the patient clenching the fist.
“Arterialized” venous blood may be obtained by heating the hand and forearm in water at 45 oc for 5
minutes and then drawing blood from the dilated veins on the back of the hand.
When the blood sample is left in open air, carbon dioxide diffuses from the blood to the surrounding air,
reducing the pCO2 in the blood thereby increasing the pH.
In contrast, oxygen diffuses from the air into the blood since pO2 in air is greater than that in whole
blood.

Measurements of blood pH, pCO2 and pO2 may be done simultaneously in a blood gas instrument.
The pH is measured by a micro glass electrode.
pCO2 is measured by a Severinghaus electrode while pO2 is measured by the Clark electrode.
Natelson-Van Slyke method is an example of a gasometric method for carbon dioxide.
Disorders of Hydrogen Ion Homeostasis/Acid-Base Disturbances
When the bicarbonate level is primarily defective, the condition is referred to as metabolic in nature.
If the level of carbonic acid is primarily defective, the condition is classified as respiratory in nature.
Metabolic Acidosis.
= bicarbonate is very low resulting in a low pH
= can be compensated for by the lungs by hyperventilation lowers the carbonic acid level restoring
the pH.
= clinical observations include:

Kussmaul respirations; Shock, coma, moderate hypokalemia
Metabolic Alkalosis.
= bicarbonate is very high resulting in high pH
= can be compensated for by the lungs hypoventilation which increases carbon dioxide in the
blood.

Paresthesias; tetany; hypokalemia, & weakness
Respiratory Acidosis.
= seen when the carbonic acid levels are very high.
= can be compensated for by the kidneys by reabsorb a lot of bicarbonates to restore the pH of
the blood.

ACUTE: Air hunger, disorientation
CHRONIC: Hypoventilation, hypoxemia, cyanosis
Respiratory Alkalosis.
= occurs when the level of carbonic acid is very low.
= compensated for by the kidneys by allowing more excretion of bicarbonates in the kidney

ACUTE: Hyperventilation, paresthesias, light-headedness
CHRONIC: Hyperventilation, latent tetany
CLINICAL CHEMISTRY 2
LECTURE
Comprehensive Exam
Multiple Choice
Choose the best answer among the choices.
The normal serum osmolality is approximately within: *

180 – 220 mOsm/kg
135 – 145 mOsm/kg
350 – 370 mOsm/kg
280 – 300 mOsm/kg
PFF of plasma
whole blood
urine
Most of the carbon dioxide present in blood is in the form of: *

carbonic acid dissolved CO2
carbonate bicarbonate ion
Which of the following drugs is used as bronchodilator? *

Phenytoin
Clozapine
Theophylline
Amikacin
O-cresolphthalein (OCP) forms a colored complex with *

calcium
magnesium
chloride copper
Which substance has the least contribution to serum osmolality? *

glucose sodium nonprotein
nitrogenous albumin
Which organ does not directly participate in the regulation of plasma osmolality? * liver
adrenals
heart
kidney
Interpret the following blood gas results: pH = 7.26, dissolved CO2 = 2.0 mmol/L (NV:
pCO2 = 35 – 45 mmHg), HCO3- = 30 mmol/L (NV: HCO3 = 21 – 28 mmol/L). *
Metabolic alkalosis
Metabolic acidosis
Reactions in renal tubular cells which contribute to acid-base balance include all, except *
Ammonia production from glutamine
Exchange for Na+ in tubular filtrate for H+ in extracellular fluid Bicarbonate production form
the carbonic anhydrase reaction
Reabsorption of H2O due to stimulation by antidiuretic hormone
What is the recommended name for diphenylhydantoin? *

Carbamazepine
Phenytoi n
Nalorphine
Primidone
The best specimen to collect for acute poisoning due to abused drugs is *
gastric lavage
whole blood
serum
urine
The dissociation constant of carbonic acid in plasma at 37°C is *

7.4
7.2
6.8
6.1
Calcium concentration in the serum is regulated by: *

thyroxine parathyroid hormone
insulin vitamin C & D
The following laboratory results were obtained on arterial blood: Sodium = 136 mEq/L pH = 7.32
Potassium = 4.4 mEq/L PCO2 = 79 mmHg Chloride = 92 mEq/L Bicarbonate = 40 mEq/L. These results
are most compatible with: *
Respiratory alkalosis Respiratory
acidosis Metabolic alkalosis
Metabolic acidosis
With a serum osmolality of 345 mOsm/L and a normal BUN and glucose, you would expect: *
Hyperglycermia Hyponatremia
Hypoproteinemia
Hypernatremia
Which of the following is an antiarrhythmic drug with a metabolite with the same function? *
Nortriptyline
Procainamide
Quinidine
Digoxin
What is the pCO2 if the dissolved CO2 is 1.8 mmol/L? *

60 mmHg
72 mmHg
35 mmHg
24 mmHg
Which of the following is consistent with uncompensated metabolic acidosis? *

pH 7.30 HCO3- 16 mmol/L pCO2 28 mmHg pH 7.34 HCO3- 18
mmol/L pCO2 32 mmHg pH 7.45 HCO3- 22 mmol/L pCO2 40
mmHg pH 7.25 HCO3- 15 mmol/L pCO2 35 mmHg
Determine the anion gap given the serum electrolyte data: Na = 132 mmol/L, CI = 90 mmol/L, HCO3
= 22 mmol/L. * Cannot be determined
2 mmol/L
10 mmol/L
62 mmol/L
2
The T3 resin uptake test is a measure of: *
circulating T3
b und T3 total thyroxine-
o
binding globulin binding capacity of thyroxine-binding globulin

The Zinc protoporphyrin/ Heme ratio (ZPP/H) is an index for effectiveness of therapy for *
iron deficiency anemia
megaloblastic anemia iron toxicity
lead and arsenic poisoning

Liver tissue
Heart tissue
Kidney tissue
Brain tissue
Which serum component is able to alter the free drug level in plasma? *
albumin
urea
creatinine calcium
Reinsch test can detect poisoning from * organophosphates

arsenic
alcohol
carbon monoxide
The metal that forms a complex with sulfonated bathophenanthroline 2,4,6- tripyridylS-triazine is *
magnesium
iron copper calcium
The transport protein associated with bilirubin in the blood is *

albumin
gamma globulin
beta-globulin alpha 1-globulin
Increased trough levels of acetaminophen in the serum are often associated with toxic effects to what
organ? *
heart kidney
liver
pancreas
Which of the following is a cause of metabolic alkalosis? * excessive vomiting
late stage of salicylate poisoning renal failure
uncontrolled diabetes mellitus
Which of the following will shift the oxyhemoglobin dissociation curve to the left? *
Metabolic Acidosis
Bicarbonate infusion
Pernicious Anemia Dengue Fever
An epileptic patient receiving phenytoin develops acute glomerulonephritis (AGN).

What change, if any, would be expected in the patient’s circulating drug level? * decrease in free
drug increase in free drug no change in circulating drug level increase in protein-bound drug
The Trinder reaction is widely used for the colorimetric determination of *

salicylate
phenothiazine
acetaminophen
barbital
In a patient who is suspected of having pheochromocytoma, measurement of which of the following

would be most useful? *
HVA
5’-HIAA
VMA
Δ9-THC
Fasting serum phosphate concentration is controlled primarily by the : *

Small intestine
Parathyroid glands
Pancreas
Skeleton
An increase in aldosterone is usually accompanied by: *

Hyperkalemia
Increased water loss
Hyponatremia
Hypokalemia
Neuromuscular irritability depends in part on the extracellular fluid concentration of *

Complexed calcium Ionized calcium
Protein-bound calcium
Total calcium
Blood gases are drawn with the following results:pH = 7.58 PCO2 = 55 mmHg HCO3-
= 18 mM. What do these data indicate? *
dual problem of acidosis respiratory acidosis,
uncompensated metabolic alkalosis, partially compensated
instrumental
The error
major intracellular anion is *
chloride
bicarbonate
potassium sodium
What abused drug is acidic among these? *

NAPA
Phenobarbital
Marijuana
Acetazolamide
Post-hepatic jaundice may be due to * neoplasm of the liver

Crigler-Najjar syndrome viral hepatitis
Alcoholic cirrhosis
The hormones referred to as 18-carbon steroids with unsaturated A ring are *

glucocorticoids
estrogens
mineralocorticoids
androgens
The bicarbonate ion concentration may be calculated from the total CO2 and PCO2 blood levels by
using which of the following formulas? *
0.03 X (PCO2 - total CO2)
0.03 X (total CO2 - PO2)
total CO2 – (0.03 x PCO 2
(total CO2 + 0.03) x PCO)2
Which of the following is not associated with spirometry? *

Friedewald equation
Henderson -Hasselbalch equation
alveolar air equation Henry’s law
What is the confirmatory method for measuring drugs of abuse? *

EMIT TLC HPLC
GC-MS
Which of the following conditions will cause an increased anion gap? *

Lactate acidosis
Renal failure
All of
these
Diabetes mellitus
The major anion in the extracellular fluid originated from the: * liver diet gastric
lumen kidneys
In what form must a drug be in order to elicit a pharmacologic response? *

unbound
bound to albumin
bound to globulins bound to fatty acids
Calmagite and titan yellow are specifically used to determine the concentration of serum *
chloride calcium phosphorus
magnesium
The most likely explanation for a urease BUN result of zero is: *
Instrument was not properly zeroed
Patient is in renal crisis
Sample was collected in improper anticoagulant
Incubation temperature was too low
Aminoglycosides & other antibiotics are particularly toxic to what organ? *

heart liver
spleen kidney

= 16 mM. What do these data indicate? *
respiratory acidosis, uncompensated dual problem of
acidosis
metabolic alkalosis, partially compensated error in one of the
blood gas measurements
Which reagent would not dissolve indirect bilirubin? * dimethylsulfoxide

(DMSO) caffeine-benzoate methanol water
Which of the following is an example of a long-acting barbiturate? *

Pentobarbital
Phenobarbital Amobarbital
Secobarbital
Which body compartment has the highest fraction of water? *

Hollow organs
Interstitial
Intracellular
Plasma
Which of the following compounds has a sedative effect and used to treat anxiety? *
Cannabinoids
Amphetamine
Benzodiazepine
Opiates
What is the active metabolite of the antiarrhythmic drug procainamide? *

PEMA
Pronestyl
NAPA
Disopyramide
The highest levels of gonadotropins occur * at the midpoint

of the menstrual cycle several days prior to ovulation
during follicular phase of the menstrual cycle during the luteal phase
of the menstrual cycle
When is a blood sample for determination of the trough level of a drug appropriately drawn? *
two hours after drug administration during the distribution
phase of a drug shortly before drug administration during
the absorption phase of the drug

= 25 mM. What do these data indicate? * respiratory
acidosis, uncompensated metabolic alkalosis, partially
compensated dual problem of acidosis
error in one of the blood gas measurements
To obtain the anion gap, the levels of which of the following is not needed? * chloride and
bicarbonate sodium and potassium plasma proteins major cations
The
iontophoresis for cystic fibrosis determines the levels of sweat *

chloride and potassium sodium and chloride
chloride and bicarbonate sodium and
potassium
The extracellular concentration of potassium is *

100 mmol/L
50 mmol/L
135 mmol/L
5 mmol/L
The Fiske-Subbarow method for inorganic phosphorus is based upon the reaction of inorganic
phosphorus with: *
Potassium oxalate
O-cresolphthalein
Ammonium molybdate
Mercuric nitrate
If the ratio of bicarbonate to carbonic acid is 30:1, what would the blood pH be? *
decreased increased normal
stable
Which test is not a colorimetric method for the quantitation of an electrolyte? *

Albanese -Le n
Lockhead -iPurcell
Fiske-Subbarrow
Daly-Ertingshausen
The indicator used in the Schales and Schales method of chloride measurement is *
permanganate phenolsulfonphthalein
periodate diphenylcarbazone
The prostatic ACP is characterized by the following, except: *

It is associated with prostatic cancer
It is tartrate-stable
It is not affected by Cu++ ions
It is formol-stable

= 38 mMWhat do these data indicate? *
error in one of the blood gas measurements metabolic
alkalosis, partially compensated respiratory acidosis,
uncompensated dual problem of acidosis
One international unit of enzyme activity is the amount of enzyme that, under specified reaction
conditions of substrate concentration, pH, and temperature, causes utilization of substrate at the rate
of: *
1 nanomole/min
1 millimole/min
1 mole/min
1 micromole/min
Which of the following is an example of a phenothiazine drug? *

Cyclosporine Theophylline Phenytoin
Chlorpromazine
The formula for calculating serum osmolality that incorporates a correction for the water content of
plasma is: *
Na + [(2xGlucose)/20] x (BUN/3) 2 Na + Glucose/20
+ (BUN/3)
2 Na + Glucose/3 +(BUN/20)
2 Na x (Glucose/20) x (BUN/3)
In the colon, bilirubin is converted to: * bilirubin diglucuronide
conjugated bilirubin bilirubin-albumin complex

urobilinogen
Select the polypeptide chain combination designated LD3. *

M2H2
H4
MH3
M4
The hormone responsible for retention of water in the kidney tubules is: *
ADH
Both
Aldosterone
Neither
Which of the following statements can be associated with the enzymatic assay of ammonia? *
reaction catalyzed by glutamate dehydrogenase NAD required as a
cofactor
ammonium ion isolated from specimen before the enzymatic step increase in absorbance
monitored at 340 nm
The urinary excretion product measured as an indicator of epinephrine production is: *

homovanillic acid dopamine
dihydroxyphenylalanine (DOPA) vanillylmandelic acid
Which of the following is used to treat manic depression? *

calcium potassium
lithium
chloride
In the Evelyn-Malloy method for the determination of serum bilirubin concentration, quantitation is
obtained by measuring the: *
Purple azobilirubin Green
azobilirubin Red azobilirubin
Blue azobilirubin
The reference method for the quantitation of ionized calcium in blood is *

AAS GC-MS
EFP
ISE
Serum “anion gap” may be decreased in patients with: *

Renal tubular acidosis
Lactic acidosis
Metabolic acidosis due to diarrhea
Diabetic alkalosis
Which of the following mechanisms is responsible for metabolic acidosis? *

Hyperaldosteronism Accumulation of volatile acids
Excessive retention of dissolved CO2
Bicarbonate deficiency
The cofactor needed in the catalyzed reaction by hexokinase is *

copper calcium manganese
magnesium
In the sweat test, the sweating stimulant is introduced to the skin by application of: * filter paper moistened
with pilocarpine nitrate
an electric current
copper electrodes
filter paper moistened in deionized water
The major androgen produced in the adrenal cortex is *

androstenedione testosterone
dehydroepiandrosterone
dihydroxytestosterone
Which of the following is NOT true? * Chloride concentration in normal CSF is

greater than that in serum.
Normal chloride concentration in CSF ranges from 115-132 mmol/L.

Chloride concentration in normal CSF is less than that in serum.
Normal blood chloride does not reflect always a normal CSF chloride
Total iron-binding capacity measures the serum iron transporting capacity of: *
Ceruloplasmin Hemoglobin
ferritin transferrin
Which two conditions can “physiologically” elevate serum alkaline phosphatase? *

healing fractures, pregnancy viral hepatitis, infectious
mononucleiosis rickets, hyperparathyroidism obstructive
jaundice, biliary cirrhosis
The main determinant of plasma colloid osmotic pressure is: * Protein

Glucose
Electrolyte
Urea
Matching Type
Enzyme and its functions
digestive cleavage of C-C, C-O & C-N bonds *

Oxidoreductase
Transferase Ligase Lyase Hydrolase hydrolytic removal of phosphoryl
group from proteins *
Isomerase lactic dehydrogenase
chymotrypsin protein kinase protein
phosphatase
changes in structural arrangement & geometry *

protein phosphatase chymotrypsin lactic dehydrogenase protein kinase
Isomerase cleavage of C-C, C-O & C-N leaving double bonds *
Hydrolase
Oxidoreductase Lyase
Transferase Ligase
phosphorylation of proteins with ATP *

lactic dehydrogenase protein kinase
chymotrypsin protein phosphatase
Isomerase
protein hydrolysis at seryl residues *

protein kinase lactic dehydrogenase Isomerase protein phosphatase
chymotrypsin exchange of glycosyl, methyl or phosphoryl groups *
Transferase
Oxidoreductase Lyase
Hydrolase Ligase joining of two molecules with ATP hydrolysis
*

Ligase
Transferase
Oxidoreductase
Hydrolase Lyase
conversion of lactate to pyruvate *

chymotrypsin protein kinase Isomerase protein phosphatase
lactic dehydrogenase removal of H+/electron and O
radicals *
Lyase
Oxidoreductase Transferase Hydrolase
Ligase
Matching Type
Enzymes and their principal clinical applications.
Acid phosphatase & PSA *

brain disorders acute pancreatitis cardiac disorders
parenchymal liver diseases bone and obstructive
liver diseases prostatic cancer
Amylase & lipase *

bone and obstructive liver diseases cardiac disorders
acute pancreatitis prostatic cancer brain
disorders parenchymal liver diseases
AST/ALT ratio, OCP *

parenchymal liver diseases acute pancreatitis
cardiac disorders bone and obstructive liver
diseases prostatic cancer brain disorders
LD1&2, CK-MB & AST * parenchymal liver

diseases prostatic cancer cardiac disorders bone
and obstructive liver diseases brain disorders acute
pancreatitis
Alkaline phosphatase *
prostatic cancer bone and obstructive liver diseases
acute pancreatitis cardiac disorders parenchymal
liver diseases brain disorders
Matching Type
Match the acid-base disorder with their characteristic parameters: metabolic alkalosis *
pH < 7.4; decreased bicarbonate; decreased PCO2 pH > 7.4;

increased bicarbonate; increased PCO2 pH > 7.4; decreased
bicarbonate; decreased PCO2 pH < 7.4; increased bicarbonate;
increased PCO2 respiratory acidosis *
pH > 7.4; increased bicarbonate; increased PCO2 pH < 7.4;
decreased bicarbonate; decreased PCO2 pH < 7.4; increased
bicarbonate; increased PCO2 pH > 7.4; decreased bicarbonate;

decreased PCO2 respiratory alkalosis * pH > 7.4; increased
bicarbonate; increased PCO2 pH < 7.4; increased bicarbonate;
increased PCO2 pH > 7.4; decreased bicarbonate; decreased PCO2
pH < 7.4; decreased bicarbonate; decreased PCO2 metabolic
acidosis * pH < 7.4; decreased bicarbonate; decreased PCO2 pH >
7.4; increased bicarbonate; increased PCO2 pH > 7.4; decreased
bicarbonate; decreased PCO2 pH < 7.4; increased bicarbonate;
increased PCO2
UNIT 1
Heart tissue
Brain tissue
Liver tissue
Kidney tissue
All of the following methods have been used for the measurement of of serum bilirubin EXCEPT: *
Bilirubinometer
BSP excretion
Jendrassik-Grof
Bilirubin oxidase

Arterial blood
Any of these
Metabolic acidosis
Metabolic alkalosis

insoluble in water
The buffering capacity of blood is maintained by a reversible exchange process between bicarbonate
and: *
Sodium
Potassium
Calcium
Chloride

Holoenzyme
Apoenzyme
Prosthetic group
Isoenzyme
pH, PCO2, and PO2
HCO3, PCO2, and PO2
alpha 1-globulin
beta-globulin
albumin
ligandin

M4
M2H2
MH3
H4

AST/ALT
LD1/LD5 ratio
In the bilirubin oxidase method, bilirubin is oxidized to a colorless compound which is: *

urobilinogen
biliverdin
bilierythrin
What is an enzyme cofactor?
Inorganic ions
Organic molecules
All of the choices
One international unit of enzyme activity is the amount of enzyme that, under specified reaction
conditions of substrate concentration, pH, and temperature, causes utilization of substrate at the
rate of: *
1 mole/min
millimole/min
1 micromole/min
1 nanomole/min

Which two physiological conditions are characterized by elevated serum alkaline phosphatase? *
Transferrin
Ceruloplasmin
Albumin
Immunoglobulin
A physician suspects his patient has pancreatitis. Which test(s) would be most indicative of this
disease? *
Creatinine
LD isoenzymes
B-hydroxybutyrate
amylase
A person suspected of having dual case of alkalosis would have which of the following laboratory
findings? *

Uroerythrin
Urochrome
Urobilin
Stercobilin
Which of the following enzymes catalyzes the conversion of starch to glucose and maltose? *

Amylase (AMS)
The determination of total amount and ionized amount may be performed in serum and plasma: *

Chloride
Sodium
Iron
Calcium
ACP
CK
AST
LDH
Lipoprotein
Bilirubin
Hematoxylin
Bence Jones protein
Vitamin
Vitamin B12
Vitamin C
Vitamin D

respiration rate
ammonia formation
blood PCO2
Colon
Liver
Spleen
Small intestine
urobilinogen
bilirubin products
urobilins
mesobilirubins


transport molecules
all of these
If the total bilirubin is 4.3 mg/dL and the conjugated bilirubin is 3.1 mg/dL, the unconjugated bilirubin
is: *
A. 20.52 umol/L
B. 58.14 umol/L
C. 37.62 umol/L
D. 34.20 umol/L
Which of the following is a characteristic shared by lactate dehydrogenase, malate dehydrogenase,

isocitrate dehydrogenase and hydroxybutyrate dehydrogenase? *

Jaffe reaction
In the Jendrassik – Grof method for the determination of serum bilirubin concentration, quantitation
is obtained by measuring the green color of: *
azobilirubin
diazobilirubin
urobilinogen
Which of the following enzyme pairs cannot be used in the diagnosis of liver disorders? *
ALP & LAP

GGT & 5’-NT
LDH & AST
ACP & ALS


Lipid profile
Zinc protoporphyrin or free erythrocyte protoporphyrin measurement are useful to assess blood
concentrations of: *
Lead
Mercury
Arsenic
Beryllium

ACP & ALP

AST & ALT
AMS & LPS
CK & LDH
Sodium
Iron
Magnesium
Potassium
alpha1-globulin
gamma-globulin
beta-globulin
albumin

Liver
Bone
Intestine
Placenta
Creatinine Clearance Tes

Hippuric Acid Test
CK
LD
AST
ALT
What was the name given to enzymes by Louis Pasteur that he based from a process he himself
discovered? *
enzyme
ferment
catalyst
ligand
Pasteur
Kuhne
Buchner
Harden
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both elevated in which
of the following disease? *
muscular dystrophy
viral hepatitis
diazobilirubin
urobilinogen
Gilbert syndrome
Viral hepatitis
Rotor syndrome
In which year Edward Buchner discovered that yeast extract can cause the fermentation of sugar to
alcohol? *

1888
1896
1900
1907
Delta- bilirubin
Oxidized bilirubin
Beta - bilirubin
If the total bilibirubin is 3.1 mg/dL and the conjugated bilirubin is 2.0 mg/dL., unconjugated bilirubin
is *
0.5 mg/dL
1.1 mg/dL
2.2 mg/dL
5.1 mg/dL
manganese
copper
calcium
magnesium
Which of the following chemical determinations may be of help in establishing the presence of
seminal fluid? *

Acid phosphatase
Sodium
Bicarbonate
Carbon dioxide
Phosphate

Reye’s syndrome
renal failure
hepatic cirrhosis
diabetes mellitus
intrinsic factor
gastrin
secretin
folic acid
Vitamin A
Vitamin C
Niacin
Thiamine
Metal ions
Both a and b
None of the above
What metal is the most affected by even the slightest hemolysis? *
Calcium
Chloride
Potassium
Sodium
dilute HCl
caffeine-benzoate
sodium hydroxide
hemolytic jaundice
neonatal jaundice

Total lactic dehydrogenase (LD) activity, confirmed to fraction 4 and 5 , is most likely to be associated
with: *
Hemolytic anemia
Which of the following serum constituents is greatly affected if a blood specimen is left standing at
room temperature for 8 hours before processing? *
Glucose
TAG
Bilirubin
Metabolic acidosis
Metabolic alkalosis
Reitman and Frankel

Isaacson – Jensey
Biuret
Jendrassik – Grof
Holoenzyme

Prosthetic group
Apoenzyme
None of these
A scanning of a CK isoenzyme fractionation revealed two peaks: a slow cathodic peak (CK-MM) and
an intermediate peak (CK-MB). A possible interpretation for this pattern is: *
Brain tumor
Muscular dystrophy
Viral hepatitis
Jaffe
Diazo
Zimmerman
Lowry
In early 19the century, which scientist studied fermentation of sugar to alcohol using a cell-free
extract? *
Edward Buchner
Louis Pasteur
Alfred Joseph
Willy Kuhne
1926
1928
1924
1907
ALP & LAP

GGT & 5’-NT
LDH & AST
ACP & ALS
A critically ill patient becomes comatose. The physician believes the coma is due to hepatic failure.
The assay most helpful in this diagnosis is: *
Ammonia
ALT
AST
GCT

Phospholipids
Bilirubin
Cholesterol
Triglycerides
In which of the following disease states is unconjugated bilirubin NOT a major serum component? *
Gallstones
HDN
Neonatal jaundice
Which suffix is added to the name of the substrate or to a word or to a phrase describing the activity
of enzyme, to name an enzyme? *
-Ise
-Ase
–Ic
-Ace
porphobilinogen
stercobilinogen
urobilin
protoporphyrin
Manganese
Magnesium
Calcium
Iron
bacterial oxidases
In the determination of lactate dehydrogenase at 340nm, using pyruvate as the substrate, one
actually measures the: *

Decrease in NADH
Increase in NADH
Apoprotein
Apoenzyme
Coenzyme
Prosthetic group
heme
bilirubin
urobilinogen
the reaction between bile and 2,4- dichloronitrobenzene to a yellow color

the liberation of oxygen by bile to oxidize orthotolidine to a blue -purple color
iodide
calcium
manganese
iron
storage of iron

Alcoholic cirrhosis

Pancreatic tumor
Gallstones
Which of the following enzymes are used in the diagnosis of pancreatic inflammation? *

methanoic acid
caffeine-benzoate
cetrimide
Type I glycogen storage disease is due to a deficiency in glucose-6-phoshatase in the liver. This
condition is called: *
Gaucher’s disease
Hers disease
Pompe’s disease
In bilirubin determinations, the purpose of adding a concentrated caffeine solution or methyl alcohol
is to: *

Precipitate protein
The metal deficient in hypochromic microcytic anemia that forms a complex with TPTZ (sulfonated
bathophenanthroline 2,4,6- tripyridyl-S-triazine) is: *
copper
magnesium
iron
cobalt

buffer base
base excess
Which of the following parameters using ISE does not require a glass membrane (oxides of Si, Al
and Na) ? *
Sodium
Chloride
pH
Calcium
An emphysema patient suffering from fluid accumulation in the alveolar spaces is likely to be in what
state? *
Metabolic acidosis
Metabolic alkalosis
Proteins
RNA
Both protein & RNA
Diabetes mellitus
Kidney function
Sodium
Chloride
Potassium
Calcium
An electrophoretic separation of lactate dehydrogenase isoenzymes that demonstrates an elevation

in LD-1 and LD-2 in a “flipped” pattern is consistent with: *
Viral hepatitis
Pancreatitis
Renal failure

A serum sample was assayed for bilirubin at 10AM and the result was 12 mg/dL. The same sample
was retested at 3PM. The result now is 8 mg/dL/. The most likely explanation for this discrepancy
is: *

Siggaard-Andersen
Gibbs – Donnan
Natelson
Acetaminophen is particularly toxic to the:
Liver
Kidney
Heart
Spleen
Which ion is affected by the use of the chelating agent sequestrene? *
Calcium
Iodine
Bicarbonate
Bromide
A coenzyme or metal ion that is very tightly or even covalently bound to the apoenzyme is called? *
Holoenzyme
Prosthetic group
Apoprotein
None of these
Aldolase
Phosphofructokinase
Pyruvate kinase

Glycine
Taurine
50%
75%
95%
99%
A complete catalytically active enzyme together with its bound enzyme or metal ions is called? *
Complex enzyme
Apoenzyme
Prosthetic group
Zymogen
The sensitive enzymatic indicator for intravascular hemolysis and acute myocardial infarction is: *

Triglyceride
Amylase

increased
decreased
balanced
ALP
GGT
LDH
ACP
Iodide
Chromium
Zinc

Magnesium
15:1
25:1
20:1
30:
rice
wheat
meat
yeast
UNIT 2
Intestinal cancer
Pheochromocytoma
Diabetes mellitus
None of these
Thyroid gland
PTG
Osseous tissues
Hypothalamus
Which of these hormones are especially related in the delicate balance of production and utilization
of glucose in the body? *

ADH acts on the: *
Pancreas

Adrenal cortex
Adrenal medulla
Kidneys

Albumin
CRP
cortisol
aldosterone
testosterone
corticosterone
Pineal gland
Hypothalamus
Pituitary gland
Thyroid gland
Normal
Unknown
Decreased
Increased
Significantly lower
Higher
Normal
Persistent hypoglycemia is seen in which of the following conditions?1. insulinoma 2. acromegaly 3.
hyperthyroidism 4. Cushing’s disease *
1 and 2
1 only
1 and 3
1,2,and 3
Somatotropin
ADH
LH
ACTH

Glycerophosphate

None of the above
Adrenal glands
Pituitary gland
Pancreas
Kidneys
GC
ELISA
RIA
HPLC
Increased
Normal
Slightly decreased
Markedly decreased
acetate
cholesterol
sitosterol
triglycerides
albumin
TBG
transcortin
SHBG
Ehrlich’s reagent
m-dinitrobenzene
Acetic anhydride

hyperglycemia: *
Insulin
Glucagon
Parathyroid hormone
Thyroid hormone
Pituitary gland
Osseous tissues
Adrenal cortex
Muscle tissues

17-ketosteroids
Tryptophan
5’-HIAA
Which of the parameters is the gold standard for thyroid function testing? 1. T4 2. T3 3. Resin T3
Uptake 4. TSH *
1,2 and 3
1,2,3 and 4
1 and 2
4 only

PFF of plasma
urine
whole blood
Dwarfism

Cushing’s disease
Thyrotoxicosis
Pregnancy
Which of the following methods would yield reliable quantification of ethanol in the presence of other
alcohols? *

T3
T4
TSH
calcitonin
8 am
12 noon
12 midnight
4 pm

Calcitonin
Somatotropin
Insulin
Thyroxine
thyroxine
aldosterone
insulin
parathyroid hormone
Decreased
None of the above
Increased
Normal

Growth hormone
Insulin
Sex hormones
Thyroid hormone
Reinsch’s test is used to screen urine for toxic concentrations of all of the following except *
mercury
cyanide
bismuth
arsenic
GC
RIA
ELISA
HPLC
Because of infertility problems, a physician would like to determine when a woman ovulates. The
physician orders serial assays of plasma progesterone. From these assays the physician can tell
when ovulation occurs because *

Kober
Porter-Silber
Pisano
Zimmermann
All of these
TRH stimulation
TSH
T3U
Cardiac muscles
Adrenals
Renal tubules
Mammary gland


12-hour urine
24-hour urine
pooled urine
random urine
Calcitonin
ACTH
T3
Thyroxine
The predominant estrogen in post-menopausal women is *
estrone
progesterone
estriol
estradiol

8 PM
12 AM
12 PM
8 AM

Bound to globulin
Bound to albumin

Free
Bound to prealbumin

A patient shows the following data: T4 concentration of 8 ug/dL and T3 uptake of 30%. What is the
condition? *
Hypothyroidism
Hyperthyroidism
Euthyroidism
Persistent hypoglycemia is seen in which of the following conditions? 1. insulinoma 2. galactosemia
3. Hypothyroidism 4. Addison’s disease *
1 and 2
1 and 3
1,2,and 3
1,2,3,and 4
16-epiestriol
estrone
estriol
estradiol
estradiol
progesterone
estriol
estrone
Zimmerman
Porter-Silber
Murphy-Pattee
Kober

Steroid hormones
Peptide hormone

Graves disease
Adenohypophysis
Pancreas
Adrenal medulla
Adrenal cortex
lead
methanol
carbon monoxide
ethylene glycol
ethanol
ethanol
methanol
lead
carbon monoxide
ethylene glycol
ethylene glycol
methanol
carbon monoxide
ethanol
lead
lead
ethylene glycol
ethanol
carbon monoxide
methanol
methanol
ethanol
ethylene glycol
lead
carbon monoxide

organophosphate
arsenic
Iron
mercury
cyanide
mercury
organophosphate
arsenic
Iron
cyanide
arsenic
cyanide
mercury
Iron
organophosphate
mercury
arsenic
cyanide
Iron
organophosphate
Iron
cyanide
mercury
arsenic
organophosphate
prostatic CA *

Bence-Jones protein
alpha-feto protein
colon CA *

Bence-Jones protein
alpha-feto protein
liver CA *

Bence-Jones protein
alpha-feto protein

Bence-Jones protein
alpha-feto protein
multiple myeloma *

Bence-Jones protein
alpha-feto protein
Calcitonin *
Pancreatic, GIT
Ovarian, Breast
Breast only
Medullary thyroid
CA-125 *
Breast only
Medullary thyroid
Pancreatic, GIT
Ovarian, Breast
CA-27, CA-29 *
Medullary thyroid
Breast only
Pancreatic, GIT
Ovarian, Breast
CA-19-9, CA 19-5, CA 242, CA-50 *
Breast only

Ovarian, Breast
Medullary thyroid
Pancreatic, GIT
CA-15-3, CA549, MCA *
Breast only
Medullary thyroid
Pancreatic, GIT
Ovarian, Breast
RB 1 *
lymphoma, leukemia
Wilm’s tumor
breast only
p53 *
Wilm’s tumor
lymphoma, leukemia
breast only
c-abl/bcr *

breast only
Wilm’s tumor
lymphoma, leukemia
K-ras, bcl-2 *

breast only
lymphoma, leukemia
Wilm’s tumor

Wilm’s tumor
lymphoma, leukemia
breast only
hCG *
Neurohypophysis
Placenta
Adenohypophysis
Liver
Thymus
Ovaries
Thyroid gland
Somatomedins *
Adenohypophysis
Liver
Placenta
Thyroid gland
Neurohypophysis
Thymus
Ovaries
Vasopressin *
Thymus
Liver
Ovaries
Adenohypophysis
Thyroid gland
Neurohypophysis
Placenta
T3 & T4 *
Liver
Ovaries
Thymus
Thyroid gland
Placenta
Neurohypophysis
Adenohypophysis
Estradiol *
Liver
Placenta
Thyroid gland
Ovaries

Thymus
Adenohypophysis
Neurohypophysis
Liver cirrhosis *
euthyroidism
hypothyroidism
hyperthyroidism
Decrease RT3U *
euthyroidism
hyperthyroidism
hypothyroidism
Iodine deficiency *
hyperthyroidism
euthyroidism
hypothyroidism
Renal failure *
euthyroidism
hyperthyroidism
hypothyroidism
Starvation *
hypothyroidism
euthyroidism
hyperthyroidism
Increased TBG *
hypothyroidism
euthyroidism
hyperthyroidism
Increased RT3U *
hypothyroidism
hyperthyroidism
euthyroidism
Decreased TBG *
hypothyroidism
hyperthyroidism
euthyroidism
Thyroid cancer *
euthyroidism

hypothyroidism
hyperthyroidism
Cystic fibrosis *
euthyroidism
hypothyroidism
hyperthyroidismts
THC *
Tranquilizers
Opiates
Sedative-hypnotics
Hallucinogens
Benzoylecgonine *
Sedative-hypnotics
Tranquilizers
Opiates
Phencyclidine *
Sedative-hypnotics
Hallucinogens
Tranquilizers
Opiates
Naloxone *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens
Amobarbital *
Hallucinogens
Opiates
Sedative-hypnotics
Tranquilizers
Heroin *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens

Methadone *
Opiates
Sedative-hypnotics
Tranquilizers
Hallucinogens
Phenobarbital *
Sedative-hypnotics
Opiates
Tranquilizers
Hallucinogens
Cocaine *
Opiates
Hallucinogens
Tranquilizers
Sedative-hypnotics
Oxazepam *
Sedative-hypnotics
Hallucinogens
Opiates
Tranquilizers

LONG QUIZ 1
DIRECTION: Write all answers on a whole sheet of paper. You are not allowed to change your answer.
I.) FILL IN THE BLANKS. Identify the missing term per statement given the starting letter.
1. The functional units of the liver is the a ACINUS
2. The liver and gall bladder are located in the a ABDOMINAL cavity.
3. The BILE serves to emulsify fats into fat droplets called micelles.
4. The hepatic sinusoids conduct BLOOD within the liver.
5. The gallstones are usually made of CHOLESTEROL
6. Bile salts and bile acids are synthesized from CHOLESTEROL
7. The normal yellow color of plasma or serum is due to BILIRUBIN
8. ICTERUS is another term for jaundice.
9. The VERY-LOW DENSITY LIPOPROTEINS/VLDL transports liver-produced triglycerides.
10. The VITAMIN K dependent coagulation factors are produced in the liver.
II. MATCHING TYPE. Match column A with column B. Letters only.

Column A Column B
Terms used in Liver Function Testing
1. The parenchymal cells of the liver R A. portal triad
2. Functional and structural units of the liver L B. excretory function
3. Collective term for OCT, LDH, AST & ALT G C. hepatic blood vessels
4. Compounds produced from amino acid & bile acid P D. HDL, IDL & LDL
5. Collective term for hepatic artery, portal vein & sinusoids C E. dual wavelength method by Allen
6. Collective term for CRP, AFP, fibrinogen & orosomucoid I F. albumin
7. Metabolic pathway involving citrulline, ornithine & arginine J G. hepatic parenchymal enzymes
8. Catabolic products of nitrogenous bases of genes S H. diazotized sulfanilate
9. Collective term for the portal vein, hepatic artery & bile duct A I. globulins
10. Collective term for LAP, ALP, 5’NT & GGT M J. urea cycle
11. Transport protein of fatty acids, calcium & bilirubin F K. bilirubin oxidation products
12. Collective term for bilicyanin, bilierythrin & biliverdin K L. liver lobules
13. Liver function that is tested involving Kupffer cells B M. hepatobiliary enzymes
14. Bilirubin fraction that includes alpha- & delta-bilirubin T N. liver biopsy
15. Confirmatory test for NAFLD & autoimmune hepatitis N O. B2
16. Transport protein for cholesterol in the blood D P. bile salt
17. Chromogen used in Evelyn-Malloy and Jendrassik-Grof methods H Q. urobilinogens
18. Collective term for the reductive products of bilirubin Q R. hepatocytes
19. Metabolic pathway used to detoxify ammonia J S. urates & orotic acid
20. Means of removing interference due to icteric serum samples E T. B1
U. Beta-oxidation pathway
V. V. None of the above
Column A Column B
Blood levels of substances in specific liver disorders
21. Iron in hemochromatosis A 31. Copper in Wilson’s disease A A. Increased
22. AAT in SERPIN1 mutation B 32. Ammonia in cirrhosis A B. Decreased
23. B2 in Criggler-Najjar syndrome C 33. UDPGT in Criggler-Najjar dse B C. Normal
24. Polar bilirubin in Rotor syndrome A 34. Albumin in liver cirrhosis B

25. AST levels after a heavy meal C 35. De Ritis ratio in iatrogenic hepatitis B
26. ALT levels in viral hepatitis A 36. ALP levels in poor bone fracture healing B
27. α- bilirubin in pernicious anemia A 37. Total bilirubin in viral hepatitis A
28. Prothrombin levels in liver cirrhosis B 38. Urea in severe liver disease B
29. Conjugated bilirubin in Gilbert’s dse C 39. Cholesterol in NAFLD A
30. ALP after meals in type B patient A 40. Prothrombin time in chronic hepatitis A
Match column A with column B. You are not allowed to change your answer. Letters only.
Column A Column B
CAUSES OF HYPERBILIRUBINEMIAS
41. hereditary spherocytosis B A. elevated direct bilirubin
42. acute quartan malaria B B. elevated indirect bilirubin
43. hypoalbuminemia B C. elevated direct and indirect bilirubin
44. deficiency in conjugating enzyme B
45. erythroblastosis fetalis B
46. idiopathic cholangitis A
47. cholelithiasis A
48. decreased hepatic uptake of bilirubin B
49. viral hepatitis A
50. use of chlorpromazine drug A
TYPES OF BILIRUBIN
51. prompt bilirubin B A. B1
52. free bilirubin A B. B2
53. beta-bilirubin B
54. cholestatic bilirubin B
55. hemobilirubin A
56. alpha-bilirubin A
57. direct bilirubin B
58. membrane with lipid affinity A
59. bilirubin diglucuronide B
60. indirect bilirubin A
LIVER FUNCTION TESTS

61. Lipid profiling B A. Carbohydrate function test
62. Shank-Hoagland test C B. Lipid function test
63. Cephalin-cholesterol flocculation test C C. Protein function test
64. Prothrombin time C D. Excretory function test
65. Indocyanin green test D
66. Fecal urobilinogen assessment D
67. Icterus index D
68. Jendrassik-Grof method D
69. A/G ratio determination C
70. Galactose tolerance test A
III. ENUMERATION. Give three (3) answers for each of the following:
A. Congenital hepatic diseases
Congenital hepatic diseases are the 1st column in the table below
GENETIC DISEASES OF THE LIVER
Disorder Gene Protein Product

Alpha1-antitrypsin SERPIN1 Alpha1-antitrypsin
deficiency (Pi)
Criggler-Najjar UGTA1 UDP-glucuronyl transferase 1
syndrome
Dubin-Johnson COMOAT Canalicular multispecific
syndrome organic transporter
Gilbert’s syndrome UGTA1 UDP-glucuronyl transferase 1
(promoter)
Hemochromatosis HFE HFE protein (cell membrane
protein regulating iron
metabolism)
Wilson’s disease ATP7B ATPase, Cu(2+)-transporting
beta polypeptide
B. Specific tests for liver protein metabolism
TP, A/G ratio is a WRONG answer) it should be specific like with Biuret Kjeldahl, Lowry, etc with the
discoverers name in the test OR mentions a specific protein like CRP test, IgG test, albumin test, etc
are also correct; other possible answers:
Cephalin cholesterol flocculation test
Thymol turbidity test (Shank & Hoagland; MacLagan)
Hippuric acid test
C. Examples of Gene – Liver disorder combination
GENETIC DISEASES OF THE LIVER
Disorder Gene Protein Product

Alpha1-antitrypsin SERPIN1 Alpha1-antitrypsin
deficiency (Pi)
Criggler-Najjar UGTA1 UDP-glucuronyl transferase 1
syndrome
Dubin-Johnson COMOAT Canalicular multispecific
syndrome organic transporter
Gilbert’s syndrome UGTA1 UDP-glucuronyl transferase 1
(promoter)
Hemochromatosis HFE HFE protein (cell membrane
protein regulating iron
metabolism)

Wilson’s disease ATP7B ATPase, Cu(2+)-transporting
beta polypeptide
Answers SHOULD BE PAIR OF TERMS like COMOAT - Dubin-Johnson, HFE-Hemochromatosis,
etc
D. Tests for the presence of bilirubin in urine samples
The following are BLOOD tests and we are looking for URINE TESTS
Blood Tests for Bilirubin
Evelyn- Malloy
Jendrassik and Grof
Anino, Ducci & Watson
Stoner & Wiseberg
Modified Michaelson
Thamhauser & Anderson
Alkaline methanolysis
Icterus index
Possible answers are ICTOSTIX, ICTOTEST, Foam test, Harrison Spot test, Gmelin test etc.
E. Analytes decreased in serum in severe liver disease
Platelet is NOT an analyte or substance, if answered it will be

WRONG & platelets are affected EARLY in the liver disease.
Possible ANSWERS are ALBUMIN, CHOLESTEROL & BUN/Urea!
LABORATORY FINDINGS IN PROGRESSION OF CHRONIC

HEPATITIS TO CIRRHOSIS
Laboratory Change Early, Mid, or Late Finding
Parameter
Platelet Decrease Early
Count
Prothrombi Increase Early
n Time
AST/ALT >1 Early-Mid
ratio
Albumin Decrease Early-Mid
Globulins Increase Early-Mid
AFP Increase Early-Mid

ALP Increase Mid
Cholesterol Decrease Late
BUN/Urea Decrease Late
Ammonia Increase Late
F. Substances elevated in blood during severe liver disease
• Enzymes like ALP, AST, & ALT; Globulins & tumor markers like AFP.
G. Common causes of acute hepatitis (vary your answers)

Possible ANSWERS are VIRUSES, ALCOHOLISM, TOXINS, DRUG USE & ANTIBODIES!
ANSWERS must vary. Give only 1 point to similar answers like HAV, HBV & HCV (all of
these are viruses).
TYPICAL LABORATORY FINDINGS IN ACUTE HEPATITIS OF

VARIOUS CAUSES
Viral Alcoho Ischemic/ Drug- Autoim

lic Toxic induce mune
Feature d
(iatrog
enic)
Peak AST 300- 100-300 1000- 300-800 300-1000
800 10,000
Peak ALT 400- 50-125 800-6000 400- 400-1200
1200 1200
AST/ALT <1 >1, usu. >1 for 1-2 <1 <1
Ratio (De >2 days, then
Ritis ratio) <1
Duration ALT 4-5 4-5 10-12 days 1-3 2-6
weeks weeks weeks months
Peak ALP <3 URL <3 URL <1.5 URL >3 URL >3 URL
Prothrombin Normal N to sl. Increased N to sl. N to
Time to increas increase increased
slightly ed d
increas
ed
Other Viral None None None Autoimm
serologi une
es markers:
low
albumin,
increased
globulins

H. Pairs of terms made up of oxidized products from bilirubin & their color
Pairs of terms made up of oxidized products from bilirubin & their color
Possible ANSWERS are paired terms as follows:
Biliverdin - green
Bilicyanin - blue
Bilierythrin - red
I. Specific lab tests performed to assess the excretory function of the liver
ANSWERS should be SPECIFIC!
STOOL EXAM is WRONG!!! What specific stool test? Urobilinogen, Stercobilinogen ( stool color
assessment).
Indocyanin green (ICG) dye excretion test & BromSulphonPhthalein (BSP) dye excretion test.
J. Reagents that accelerate coupling of B1 with diazotized sulfanilic acid
Alcohol (methanol), Dimethylsulfoxide (DMSO), caffeine-benzoate, & Cetrimide
IV. MULTIPLE CHOICE QUESTIONS.On the prescribed answer sheet, choose the letter that corresponds to
the correct answer. You are not allowed to use pencil, make alterations and change your answer.
1. In the liver, bilirubin is converted to:
A. Urobilinogen C. bilirubin-albumin complex
B. Urobilin D. bilirubin diglucuronide
2. In the Jendrassik – Grof method for the determination of serum bilirubin concentration, quantitation is
obtained by measuring the green color of:
A. azobilirubin C. diazobilirubin
B. bilirubin glucuronide D. urobilinogen
3. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both elevated in which of the
following disease?
A. muscular dystrophy C. obstructive disease of the liver
B. liver parenchymal disease D. infectious mononucleosis
4. Which of the following serum constituents is greatly affected if a blood specimen is left standing at room
temperature for 8 hours before processing?
A. Cholesterol C. Creatinine
B. Triglyceride D. Bilirubin
5. The most sensitive enzymatic indicator for liver damage from ethanol intake (alcoholic cirrhosis) is:
A. alanine aminotransferase (ALT)
B. aspartate aminotransferase (AST)
C. gamma-glutamyl transferase (GGT)
D. alkaline phosphatase (ALP)
6. Which two physiological conditions are characterized by elevated serum alkaline phosphatase?
A. rickets, hyperthyroidism
B. obstructive jaundice, biliary cirrhosis
C. growth, third trimester of pregnancy
D. viral hepatitis, myocardial infarction

7. Given the following results:
Alkaline phosphatase Marked increase
Alanine aminotransferase Slight increase
Aspartate aminotrnaferase Slight increase
Gamma – glutamyl transferase Marked increase
This is most consistent with:
A. Acute hepatitis C. Chronic hepatitis
B. Osteitis fibrosa D. Obstructive jaundice
8. Kernicterus is an abnormal accumulation of bilirubin in:

A. Heart tissue C. Liver tissue
B. Brain tissue D. Kidney tissue
9. In the total bilirubin assay, bilirubin reacts with diazotized sulfanilic acid to form:
A. Diazo bilirubin C. Azobilirubin
B. Biliverdin D. Bilirubin glucuronide
10. In the Malloy and Evelyn method for the determination of bilirubin, the reagent that is reacted with
bilirubin to form a purple azobilirubin is:
A. Dilute sulfuric acid C. sulfobromophthalein
B. Diazonium sulfate D. Diazotized sulfanilic acid
11. Bile acid concentrations are useful to assess:
A. Diabetes mellitus C. Intestinal malabsorption
B. Hepatobiliary disease D. Kidney function
12. Which of the following enzymes are used in the diagnosis of obstructive jaundice?
A. Amylase (AMS) and lipase (LPS)
B. Aspartate aminotransferase (AST) and leucine aminopeptidase (LAP)
C. 5’-nucleotidase (5’NT) and gamma-glutamyl transferase (GGT)
D. Aspartate aminotransferase (AST) and lactate dehydrogenase (LD)
13. In which of the following disease states is unconjugated bilirubin NOT a major serum component?
A. Bile duct atresia C. Neonatal jaundice
B. Hemolysis D. Erythroblastosis fetalis
14. If the total bilirubin is 4.3 mg/dL and the conjugated bilirubin is 2.1 mg/dL, the unconjugated bilirubin is:
A. 20.52 umol/L C. 37.62 umol/L
B. 58.14 umol/L D. 34.20 umol/L
15. The principle of the tablet test for bilirubin in urine or feces is:
A. the reaction between bile and 2,4- dichloronitrobenzene to a yellow color
B. the liberation of oxygen by bile to oxidize orthotolidine to a blue-purple color
C. chemical coupling of bile with a diazonium salt to form a brown color
D. chemical coupling of bilirubin with a diazonium salt to form a purple color
16.A serum sample was assayed for bilirubin at 10AM and the result was 12 mg/dL. The same sample was
retested at 3PM. The result now is 8 mg/dL/. The most likely explanation for this discrepancy is:
A. The reagent has deteriorated
B. The sample was exposed to light
C. A calculation error in the first assay
D. The sample was not refrigerated
17. What substance gives feces its normal color:
A. Uroerythrin C. Urobilin
B. Urochrome D. Stercobilin
18. A breakdown product of hemoglobin is:
A. Lipoprotein C. Hematoxylin
B. Bilirubin D. Bence Jones protein
19. Serial bilirubin determinations are charted below with the best explanation for the results due to:
Day Collected Assayed Results

1 7 Am 8 AM 14.0 mg/dL
2 7 Am 6 PM 9.0 mg/dL
3 6 AM 8 AM 15.0 mg/dL
A. Sample hemolysis and hemoglobin deterioration
B. Sample exposure to light
C. Sample left in warm location
D. Reagent deterioration
20. The following bilirubin results are obtained on a patient:

Day 1: 4.3 mg/dL Day 4: 2.2 mg/dL
Given that the controls were within range each day, what is a probable explanation for the result on day
4:
A. No explanation necessary
B. Serum, not plasma, was used for testing
C. Specimen had prolonged exposure to light
D. Specimen was hemolyzed
21. Urobilinogen is formed in the:
A. Kidney C. Liver
B. Spleen D. Intestine
22. In bilirubin determinations, the purpose of adding a concentrated caffeine solution or methyl alcohol is to:
A. Allow indirect bilirubin to react with color reagent
B. Dissolve conjugated bilirubin
C. Precipitate protein
D. Prevent any change in pH
23. If the total bilibirubin is 3.1 mg/dL and the conjugated bilirubin is 2.0 mg/dL., unconjugated bilirubin is
A. 0.5 mg/dL C. 2.2 mg/dL
B. 1.1 mg/dL D. 5.1 mg/dL
24. The most widely used methods for bilirubin measurement are those based on the :
A. Jaffe reaction C. 8-hydroxyquinoline reation
B. Schales and Schales method D. Jendrassik Grof method
25. A critically ill patient becomes comatose. The physician believes the coma is due to hepatic failure. The
assay most helpful in this diagnosis is:
A. Ammonia C. AST
B. ALT D. GCT
26. In which of the following conditions does decreased activity of glucuronyl transferase result in increased
unconjugated bilirubin and kernicterus in neonates?
A. Gilbert’s disease C. Dubin-Johnson syndrome
B. Rotor’s syndrome D. Crigler-Najjar syndrome
27. A 21-year-old man with nausea, vomiting and jaundice has the following laboratory findings:
Total serum bilirubin level 8.5 mg/dL (normal, 0-1.0)
Conjugated serum bilirubin level 6.1 mg/dL (normal, 0-0.5)
Urine urobilinogen Increased
Fecal urobilinogen Decreased
These can best be explained as representing:
A. Unconjugated hyperbilirubinemia, probably due to hemolysis.
B. Unconjugated hyperbilirubinemia, probably due to toxic liver damage.
C. Conjugated hyperbilirubinemia, probably due to biliary tract disease.
D. Conjugated hyperbilirubinemia, probably due to hepatocellular obstruction.
28. Which of the following tests has no clinical application in assessing liver function?

A. Triglyceride C. Gamma-glutamyl transferase
B. Amylase D. Lactate dehydrogenase isoenzymes
29. Urobilinogen is synthesized in the:

A. Common bile duct C. Hepatocytes
B. Intestinal lumen D. Renal tubules
30. Which enzyme ratio is the best indicator of acute or chronic hepatitis:
A. AST/ALT ratio C. Amylase/lipase ratio
B. CK-MB/total CK ratio D. LD1/LD5 ratio
31. When measuring serum bilirubin, the purpose of adding caffeine-sodium benzoate or methanol to the
reaction mixture is to:
A. accelerate the reaction of conjugated bilirubin
B. Accelerate the reaction of uncojugated bilirubin
C. Destroy excess diazo reagent
D. Shift the wavelength absorbed by azobilirubin
32. The diazo-bilirubin color appearing after addition of methanol represents:

A. Indirect bilirubin C. Free bilirubin
B. Direct bilirubin D. Total bilirubin
33. Which of these patterns of serum results is consistent with obstructive liver disease:
Total Bilirubin Conjugated Bilirubin Total ALP
A. Increase Increase Increase
B. Increase Normal Increase
C. Normal Increase Decrease
D. Increase Normal Decrease
34. Formation of bile acids is the major mechanism for eliminating:

A. Phospholipids C. Cholesterol
B. Bilirubin D. Triglycerides
35. To quantitate serum bilirubin levels, it is necessary that bilirubin couples with diazotized sulfanilic acid
to form:
A. Verdobilirubin C. Biliverdin
B. Azobilibinogen D. Azobilirubin
36. The quantitation of both conjugated ad unconjugated bilirubin may be helpful in differentiating between
several types of jaundice. A condition characterized by an elevation of total bilirubin primarily because
of an increase in the conjugated bilirubin fraction is:
A. Hemolytic jaundice C. Neonatal jaundice
B. Crigler-Najjar syndrome D. Obstructive jaundice
37. Which assay below will most likely require a sample blank to reduce sample interference:
A. Bilirubin determination using a hemolyzed serum
B. Triglyceride determination using the Hantzsch reaction
C. Cholesterol determination using CHOD-PAP
D. Albumin determination by turbidity
38. Acetaminophen is particularly toxic to the:

A. Liver C. Heart

B. Kidney D. Spleen
39. Type I glycogen storage disease is due to a deficiency in glucose-6-phoshatase in the liver. This
condition is called:
A. Gaucher’s disease C. Hers disease
B. von Gierke’s disease D. Pompe’s disease
40. Cholesterol is utilized in the synthesis of:

1. Bile acids 3. Bile salts
2. Steroids 4. Bilirubin
A. 1 & 3 only C. 1, 2 & 3 only E. 4 only
B. 2 & 4 only D. 1, 2, 3 & 4
41. The term  bilirubin refers to:

A. Water-soluble bilirubin C. Bilirubin tightly bound to albumin
B. Free unconjugated bilirubin D. Direct-reacting bilirubin
42. Which of the following is not a function of the liver?

A. 25-hydroxylation of vitamin D C. storage of iron
B. production of bile acids D. production of insulin
43. Elevated blood levels of ammonia occur in all of the following disorders except
A. Reye’s syndrome C. chronic liver failure
B. renal failure D. diabetes mellitus
44. The transport protein associated with bilirubin in the blood is

A. alpha 1-globulin C. gamma globulin
B. beta-globulin D. albumin
45. Which reagent would not dissolve indirect bilirubin?
A. dimethylsulfoxide (DMSO) C. caffeine-benzoate
B. methanol D. cetrimide E. water
46. Post-hepatic jaundice may be due to

A. cirrhosis C. hepatitis
B. Crigler-Najjar syndrome D. neoplasm of the common bile duct
47. The immediate precursor of bilirubin is

a. mesobilirubinogen b. delta bilirubin c. biliverdin d. urobilinogen
48. This compound is partially reabsorbed from the intestine through the portal circulation is
a. biliverdin b. urobilin c. urobilinogen d. bilirubin
49. The type of bilirubin which is covalently linked to albumin is

a. alpha b. beta c. delta d. gamma
50. Caffeine-benzoate reagent is used in this bilirubin test

a. Evelyn-Malloy b.Jendrassik-Grof c. Zimmermann d. Icterus index
51. Post-hepatic jaundice is caused by all of these except

a. cholelithiasis b. pancreatic CA c. hemoglobinopathies d. biliary atresia
52. The conversion factor to use to convert bilirubin in mg% to micromoles/L is

a. 0.357 b. 17.1 c. 0.059 d. 88.4

53. Dubin-Johnson syndrome is characterized by all of the following except
a. impaired excretion of bilirubin b. normal hepatic uptake of bilirubin
c. inability to form conjugated bilirubin d. increased bilirubin in urine and CSF
54. The use of methanol or caffeine benzoate in bilirubin determination will measure
a. B1 b. B2 c. delta bilirubin d. total bilirubin
55. Which of the following is not a function of the liver?

A. 25-hydroxylation of vitamin D C. storage of iron
B. production of bile acids D. production of insulin
56. Post-hepatic jaundice may be due to

A. cirrhosis C. hepatitis
B. Crigler-Najjar syndrome D. neoplasm of the common bile duct
57. What is the immediate precursor of bilirubin?

A. mesobilirubinogen C. urobilinogen
B. biliverdin D. urochrome
58. What enzyme system is responsible in the conjugation of bilirubin?

A. leucine aminopeptidase C. glucose-6-phosphate dehydrogenase
B. uridine diphosphate glucuronyltransferase D. carbamoyl phosphate synthetase
59. Oxidation of urobilinogen by microorganisms will produce

A. porphobilinogen C. urobilin
B. stercobilinogen D. protoporphyrin
60. Which of the following functions as a transport protein for bilirubin in the blood?
A. alpha1-globulin C. beta-globulin
B. gamma-globulin D. albumin
61. Which bilirubin fraction is unconjugated and weakly bound to albumin?

A. alpha C. beta
B. delta D. gamma
62. All of the following describe the direct bilirubin except

A. insoluble in water C. conjugated in the liver
B. conjugated with glucuronic acid D. excreted in jaundiced patients’ urine
63. All of the following methods have been used for the measurement of of serum bilirubin except
A. Bilirubinometer C. Jendrassik-Grof
B. BSP excretion D. Bilirubin oxidase
64. Which method uses sulfanilic acid, HCl and sodium nitrite?
A. Jaffe C. Zimmerman
B. Diazo D. Lowry
65. Indirect-reacting bilirubin may be quantified by reacting it initially in which reagent?

A. dilute HCl C. dilute sulfuric acid
B. caffeine-benzoate D. sodium hydroxide
66. In the bilirubin oxidase method, bilirubin is oxidized to a colorless compound

A. diazotized sulfanilic acid C. biliverdin

B. urobilinogen D. bilierythrin
67. Mesobilirubinogen, urobilinogen and stercobilinogen are collectively known as

A. urobilinogen C. urobilins
B. bilirubin products D. mesobilirubins
68. Increased total bilirubin due to increased direct bilirubin suggests

A. hemolytic jaundice C. neonatal jaundice
B. Crigler-Najjar syndrome D. obstructive jaundice
69. Complete obstruction of the common bile duct would yield all of the following results except
A. negative urine urobilinogen C. negative fecal urobilinogen and urobilin
B. negative urine bilirubin D. passage of acholic stools
70. Post-hepatic jaundice is caused by all of these except
A. cholelithiasis C. pancreatic CA
B. hemoglobinopathies D. biliary atresia
71. The use of methanol or caffeine benzoate in bilirubin determination will measure
A. B1 B. B2 C. delta bilirubin D. total bilirubin
72. Inside the liver, bilirubin is conjugated with:

A. Uronic acid C. Uridine disphosphate gluconate
B. Ligandin D. Glucuronic acid
73. Which of the following substances is NOT increased in liver disease?

A. Lactate dehydrogenase C. Alanine aminotransferase
B. Albumin D. Bilirubin
74. In which method is diazotized sulfanilic acid used?

A. Evelyn-Malloy method
B. Neither Evelyn-Malloy nor Jendrassik-Grof
C. Both Evelyn-malloy and Jendrassik-Grof
D. Jendrassick-Grof method
75. The conjugation of bilirubin occurs in the:

A. Spleen C. Gall bladder
B. Small intestine D. Liver
76. The polarity and water solubility of bile acids are increased upon conjugated with:
A. Glycine C. Neither glycine not taurine
B. Either glycine or taurine D. Taurine
77. Which of these is NOT a liver function test:

A. Thymol turbidity test C. Immunoglobulin electrophoresis
B. ICG test D. PAH clearance
78. Increased levels of indirect bilirubin only is seen in:

A. Bile duct obstruction C. Hyperadrenalism
B. Hemolytic disease D. Hepatitis
79. It is one of the best tests for bilirubin:

80. A. Reitman and Frankel C. Biuret
81. B. Isaacson – Jensey D. Jendrassik – Grof
82. One of the following is used to assess liver function. Which one is it?
A. Creatinine Clearance Test C. Para-aminohippurate test
B. Inulin Clearance test D. Hippuric Acid Test

GOOD LUCK!!!
LONG QUIZ 2
I) Match column A with column B regarding the electrolytes and their associated properties, method
of testing, and terminologies. Letters only. There are more-than-one answer occasionally as
indicated by a number after the phrase.
Column A Column B
1. Contributes 92% to ECF osmolality A A. Sodium
2. Most abundant cation in the human body D B. Potassium
3. Mostly affected by even the slightest of blood laking B C. Chloride
4. Produced daily in the stomach C D. Calcium
5. Has renal threshold of 110 mmol/L A E. Phosphorus
6. Counterion in the sodium pump process B F. Magnesium
7. Has bromide as its common interferent C G. Copper
8. Has calcium as its interfering species (2) F&H H. Iron
9. Effectively removed from solution with hydroxyquinoline F
10. Measured in sweat together with chloride to screen for cystic fibrosis A
11. Affected by the use of the chelating agent sequestrene B
12. Counterion in the chloride shift phenomenon C
13. Known as an ECF buffering agent E
14. Forms hydroxyapatite crystals with calcium ions E
15. Ninety percent of this metal is transported by ceruloplasmin G
16. Total amount in the body is 3.0-5.0 grams H
17. Regulated by the aldosterone and ANP (2) A & B
18. Regulated by the PTH, calcitriol, and calcitonin (2) D & E
19. Exhibits diurnal variation H
20. Measured in serum using the AAS method (6) A,B,D,F,G,H
21. Measured in serum using the ISE method (5) A,B,C,D,F
22. Serum levels are affected by patient diaphoresis (3) A,B,C
23. Known to act as activators of enzymes (5) C,D,F,G,H
24. Can be measured using tetraphenylboron B
25. Can be commonly measured using dye-binding methods (3) D,F,H
26. Has diagnostic importance in meningitis C
27. Can be measured using the Zall color reaction C
28. Can be precipitated with (NH4)2C2O4 and titrated with permanganate D
29. Can be reacted with ammonium molybdate E
30. Deficiency can be assessed by ZPP/H ratio determination H
31. Determination of total amount and ionized amount may be performed in serum D
32. Can bind calmagite, methylthymol blue and xylidyl blue F
33. Can bind OCP, alizarin and EGTA D
34. Affects largely blood pH whenever its concentration varies in blood C
35. Can be measured using Albanese-Lein method A

36. Can be measured using the Lockhead-Purcell method B
37. Can be measured using the Fiske-Subarrow method E
38. Can be measured using the Clark-Collip method D
39. Can be measured using the Schales & Schales method C
40. Can be measured using the Terosite method H
II) What does each of the following acronyms/symbol stand for? (2 points each) WRONG SPELLING
IS VERY WRONG with no point!
1. ZPP/H zinc protoporphyrin/heme ratio 6. ANF atrial natriuretic factor
2. EGTA ethylene glyco- bis (1-aminoethyl ether) tetraacetic acid 7. Pi - inorganic phosphorus/phosphate
3. OCP ortho-cresolphthalein 8. ACE angiotensin-converting enzyme
4. SIADH syndrome of inappropriate secretion of antidiuretic hormone 9. TIBC total iron binding capacity
5. Cat -total calcium 10. TPTZ sulfonated bathophenanthroline 2,4,6-
tripyridyl-S-triazine
III) Write all answers on a separate whole sheet of pad paper. You are not allowed to change your
answers.
A) Write the Henderson-Hasselbalch equation correctly on the main blood buffer system and do the
following: (20 points)
1. Encircle the metabolic component
2. Underline twice the respiratory component
3. Give the value for the pKa
4. Give the normal range for the blood pH 7.35-7.45 NOT 7.4 ; range is asked for
5. Give the normal ratio of the acid with the base 1:20 NOT 20:1
pH = 6.1 (pKa) + log Bicarbonate or HCO3 - ion or Conjugate Base

Carbonic acid or H2CO3 or Acid
__________________________
A1 to A5 is 2 points each if followed correctly. The formula is 10 points. All in all is 20 points.
B) Draw the normal Hb-O2 Dissociation Curve with correct labels of the x and y axes. (10 points) Enumerate
5 factors that would cause a shift to the left and enumerate also 5 factors that would cause a shift to the
right BELOW the illustrated graph. (10 points)
10 points for the S-shaped curve with correct x and y parameters. If 50% y axis DOES NOT fall on
40 mmHg of x axis DEDUCT 5 points from 10!
SHIFT TO THE LEFT : Decreased PCO2 , Temperature, 2,3-BPG and Increased pH & PO2

SHIFT TO THE RIGHT : Increased PCO2 , Temperature, 2,3-BPG and Decreased pH & PO2
Count the factors to be 5 for shift to the left and to the right.
C) Solve for the pH of the blood of a patient with the following data: (10 points)
ANSWER: 7.29 ; with shown process as follows: pH = 6.1 + log 31 ÷ 2; without process 2
points only
TCO2 = 33 mM
PCO2 = 2 mM
Tell the type of acid-base disorder and the expected Hb-O2 dissociation curve that the patient would
have and explain why. (10 points)
ANSWERS: Acid-base disorder is COMPENSATED RESPIRATORY ACIDOSIS (5 points) if resp.
Acidosis only 2 points; Expected curve of Hb-oxygen dissociation is SHIFT TO THE RIGHT due
to HIGH CO2 therefore HIGH Carbonic acid (H2CO3) with DECREASED pH. (5 points) if no
reasoning 2 points only
D) Tabulate the differences between a heparinized blood exposed to air for 3 hours and a heparinized blood
with cap for 1 hour in terms of their PO2, PCO2, and pH. Do not anymore explain why. (15 points)
Construction of correct table with headings/labels = 3 points
Correct descriptions 2 points each = 6 x2 = 12 points; all in all = 15
pH PCO2 PO2
Capped tube with LOW HIGH LOW
blood
Uncapped tube with HIGH LOW HIGH
blood
E) What are the four (4) parameters that the TCO2 is comprising of? (10 points)
Bicarbonate ions, Carbonic acid (PCO2), dissolved CO2 gas, & carbamino compound (CO2
bound to proteins).
2 points each x 4 = 8. ADD 2 if all the 4 are correct. If only 3 are given, 6 points only & no
+2.
F) Indicate in the table (copy & answer) whether the parameters are low, normal or high in the given
conditions: (30 points) You take it by three columns, if one of the 3 columns is wrong - NO
POINT. All 3 columns should be correct. Five conditions x 6 = 30 points.
CONDITION PCO2 Bicarbonate pH

Dual case of Alkalosis LOW HIGH HIGH
Metabolic Acidosis NORMAL LOW LOW
uncompensated
Respiratory Alkalosis LOW LOW HIGH

Partially compensated
Metabolic Alkalosis HIGH HIGH HIGH

Instrumental error NORMAL NORMAL LOW

Many
possible

Answers
G) Provide a correct explanation of the effects on the specified electrolytes for each of the following clinical
conditions: (25 points) 5 points each for exemplary answers; impartial correctness or students
have coherent reasoning somehow- give 2 points; any excess and lack of substance in answers
give NO POINT.
1. ECF principal ions in Water Deficit
ECF principal ions are Na and Cl ; will be INCREASED due to pure water loss.
2. ICF principal ions in Starvation
ICF principal ions are K & Bicarbonate; both are diet-dependent & will be DECREASED.
3. Principal cations in Acute renal failure
Principal cations are Na & K; Na is LOW because reabsorption is absent; K is low 7 wasted.
4. Principal anions in Pulmonary emphysema
Principal anions are Cl & Bicarbonate; Bicarb is HIGH due to more CO2 where it comes
from; Chloride is LOW due to chloride shift phenomenon
5. All principal ions in Diarrhea
Principal ions are Na, K, Cl & Bicarb; all are LOW because of osmotic loss wherein loss is
not only water but with osmotic substances like the ions.
IV-A). CLINICAL SIGNIFICANCE. Tell whether each of the given parameters alongside a medical condition
from nos. 1- 30 is A) increased, B) decreased, or C) normal. For nos. 31- 40, tell whether the relationship
between the given pairs of parameters is A) Direct or B) Inverse. For nos. 41-50, decide on whether the
parameter is A) higher or B) lower based on the given pair of clinical conditions following the parameter.
Letters only.
1. pH in hypercapnia B 31. [H+] & Ph B

2. PO2 in respiratory distress B 32. PO2 & PCO2 B
3. PCO2 in left-shifted Hb-O2 dissociation curve B 33. Ka & pH B
+
4. Chloride in hyperventilation A 34. pKa & [H ] B
5. Bicarbonate ions in respiratory alkalosis B/C 35. Chloride & bicarbonate B
6. H2CO3 concentration in pulmonary embolism B 36. CO2 level & pH B
7. TCO2 in asthmatic attacks A 37. H2CO3 & PO2 B
8. [H+] in metabolic acidosis A 38. Chloride & PO2 A
9. 2,3-BPG level resulting in a left-shifted Hb-O2 dissociation curve B 39.% Hb saturation & PO2 level A
10. pH level of blood left exposed to air for one hour A 40. pH & PO2 A
11. PO2 in patients with emphysema due to chain smoking B
12. PCO2 resulting in dual case of acidosis A
13. Chloride in a patient with lung edema B
14. Bicarbonate ions in uncompensated respiratory acidosis C
15. H2CO3 concentration in IDA A
16. TCO2 in partially compensated metabolic alkalosis A
17. [H+] in a dual case of alkalosis B
18. PO2 in a blood sample placed in a capped tube B
19. PCO2 in renal disorder characterized by decreased excretion of HCO 3- A/C
20. Chloride level in metabolic acidosis A

21. Bicarbonate ions in compensated respiratory alkalosis B
22. H2CO3 concentration in salicylic acid poisoning A
23. TCO2 in a patient with blood PCO2 = 1.2mM & HCO3- level of 25Mm C
24. [H+] in respiratory acidosis A
25. PO2 in euphoric states A
26. PCO2 in dual case of alkalosis B
27. Chloride in any disease with decreased CO2 A

28. Bicarbonate ions in any disease with increased oxygen tension B
29. H2CO3 concentration in any disease with decreasing Ph A/B
+
30.[H ] in metabolic alkalosis B
Parameters Clinical Condition

B 41. oxygen is ________ in a right-shifted compared with a left-shifted Hb-O2 dissociation curve
B 42. chloride is _______ in metabolic alkalosis compared with metabolic acidosis
A 43. chloride is _______ in respiratory alkalosis compared with respiratory acidosis
B 44. PCO2 is _______ in normal lung function compared with lungs with clogged bronchi
A/B 45. PCO2 is _______ in a patient with a blood pH of 7.25 compared with one with pH of 7.45
A 46. PO2 is _______ in a blood exposed to air compared with a capped tube
B 47. PO2 is _______ in compensated respiratory acidosis compared with uncompensated one
A 48. [H+] is ______ in a solution with pKa of 4.5 compared with a pKa of 6.5
B 49. [H+] is ______ in a solution with pH 6.0 compared with pH of 5.0
A 50. [H+] is ______ in a solution with a Ka value that increased after adding it with salt.
IV-B). MULTIPLE CHOICE. Choose the letter that corresponds to the correct answer. Letters only.
51. The specific protein for the transport of copper present in plasma is:
A. Transferrin C. Albumin
B.Ceruloplasmin D. Immunoglobulin
52. The bicarbonate and carbonic acid ratio is calculated from an equation by:
A. Siggaard-Andersen C. Natelson
B. Gibbs – Donnan D. Henderson – Hasselbalch
53. Zinc protoporphyrin or free erythrocyte protoporphyrin measurement are useful to assess blood
concentrations of:
A. Lead C. Arsenic
B. Mercury D. Beryllium
54. Specimens for blood gas determination should be drawn into a syringe containing:
A. No preservative C. EDTA
B. Heparin D. Oxalate
55. Valinomycin enhances the selectivity of the electrode used to quantitate:

A. Sodium C. Potassium
B. Chloride D. Calcium
56. Which of the following parameters using ISE does not require a glass membrane (oxides of Si, Al and Na)
?
A. Sodium C. pH
B. Chloride D. Calcium
57. Which vitamin is required for the normal absorption of dietary calcium:
A. Vitamin A C. Vitamin C
B. Vitamin B12 D. Vitamin D
58. When determining blood pH, CO2 and O2 concentrations, the best sample is:
A. Arterialized capillary blood from finger C. Plasma using heparin
B. Arterial blood D. Any of these
59. The normal serum osmolality is approximately within:

A. 280 – 300 mOsm/kg C. 350 – 370 mOsm/kg

B. 180 – 220 mOsm/kg D. 135 – 145 mOsm/kg
60. The metal deficient in hypochromic microcytic anemia that forms a complex with TPTZ (sulfonated
bathophenanthroline 2,4,6- tripyridyl-S-triazine) is
A. copper C. iron
B. magnesium D. cobalt
61.Which analyte cannot be measured using AAS?

A. iodide C. manganese
B. calcium D. iron
62.The cofactor needed in the catalyzed reaction by hexokinase is

A. manganese C. calcium
B. copper D. magnesium
63. Reactions in renal tubular cells which contribute to acid-base balance include all, except
a. Ammonia production from glutamine
b. Bicarbonate production form the carbonic anhydrase reaction
c. Exchange for Na+ in tubular filtrate for H+ in extracellular fluid
d. Reabsorption of H2O due to stimulation by antidiuretic hormone
64.In ketoacidosis, the blood pH would most likely be affected in what way?
A. unchanged from normal C. decreased
B. increased D. balanced
65.The sum of carbonic acid and bicarbonate in plasma is referred to as

A. standard bicarbonate C. buffer base
B. total carbon dioxide D. base excess
66.If a blood gas specimen is left exposed to air, which of the following changes
occur?
a. PO2 and pH increase; PCO2 decreases
b. PO2 and pH decrease; PCO2 increases
c. PO2 increases; pH and PCO2 decrease
d. PO2 decreases; PCO2 and pH increase
67. How would blood gas parameters change if a sealed specimen is left at room
temperature for 2 or more hours?
a. PO2 decreases, pH decreases, PCO2 increases
b. PO2 increases, pH increases; PCO2 increases
c. PO2 decreases, pH decreases; PCO2 decreases
d. PO2 increases, pH decreases; PCO2 increases
68. The bicarbonate ion concentration may be calculated from the total CO2 and
PCO2 blood levels by using which of the following formulas?
A. 0.03 X (PCO2 - total CO2) C. (total CO2 + 0.03) x PCO2
B. 0.03 X (total CO2 - PO2) D. total CO2 – (0.03 x PCO2)
69. Blood gases are drawn with the following results:

pH = 7.49 PCO2 = 59 mmHg HCO3- = 38 mM
What do these data indicate?
A. metabolic alkalosis, partially compensated
B. respiratory acidosis, uncompensated
C. dual problem of acidosis

D. error in one of the blood gas measurements

pH = 7.29 PCO2 = 50 mmHg HCO3- = 25 mM

pH = 7.25 PCO2 = 56 mmHg HCO3- = 16 mM

pH = 7.58 PCO2 = 55 mmHg HCO3- = 18 mM
73. The following laboratory results were obtained on arterial blood:

Sodium 136 mEq/L
PH 7.32
Potassium 4.4 mEq/L
PCO2 79 mmHg
Chloride 92 mEq/L
Bicarbonate 40mEq/L
These results are most compatible with:
A. Respiratory alkalosis C. Metabolic alkalosis
B. Respiratory acidosis D. Metabolic acidosis
74. The buffering capacity of blood is maintained by a reversible exchange process between bicarbonate and:
A. Sodium C. Calcium
B. Potassium D. Chloride
75. At blood pH 7.40 what is the ratio between bicarbonate and carbonic acid?
A. 15:1 C. 25:1
B. 20:1 D. 30:1
76. The bicarbonate and carbonic acid ratio is calculated from an equation by:
A. Siggaard-Andersen C. Natelson
B. Gibbs – Donnan D. Henderson – Hasselbalch
77. Acidosis and alkalosis are best defined as fluctuations in blood pH and CO 2 content due to changes in:
A. Bohr effect C. Bicarbonate buffer
B. O2 content D. Carbonic anhydrase
78. A common cause of respiratory alkalosis is:

A. Vomiting C. Asthma
B. Starvation D. Hyperventilation
79. Metabolic acidosis is describe as a (n):

A. Increase in CO2 content and PCO2 with a decreased pH
B. Decrease in CO2 content with an increased pH
C. Increase in CO2 with an increased pH
D. Decrease in CO2 content and PCO2 with a decreased pH
80. Respiratory acidosis is described as an a (n):

A. Increase in CO2 content and PCO2 with a decreased pH
B. Decrease in CO2 content with an increased pH
C. Increase in CO2 content with an increased pH
D. Decrease in CO2 content and PCO2 with decreased pH
81. Normally the bicarbonate concentration is about 24 mEq/L and the carbonic acid concentration is about
1.2:pK = 6.1. Using the equation pH = pK + log [salt]/[acid], calculate the pH.
A. 7.28 C. 7.40
B. 7.38 D. 7.42
82. The normal range for the pH of arterial blood measured at 37oC is:
A. 7.28-7.34 C. 7.35-7.45
B. 7.33-7.37 D. 7.45-7.50
83. Unless blood gas measurement are made immediately after sampling, in vitro glycolysis of the blood causes
a:
A. Rise in pH and PCO2 C. Rise in pH and a fall in PO2
B. Fall in pH and a rise in PO2 D. Fall in pH and a rise in PCO2
84. Hydrogen ion concentration (pH) in blood is usually determined by means of which of the following
electrodes?
A. Silver C. Platinum
B. Glass D. Platinum-lactate
85. A person suspected of having metabolic alkalosis would have which of the following laboratory findings?
A. CO2 content and PCO2 elevated, pH decreased
B. CO2 content decreased and pH elevated
C. CO2 content PCO2, and pH decreased
D. CO2 cotent and pH elevated
86. If the pKa is 6.1, the CO2 content is 25 mmol/L, the salt equals the total CO 2 content minus the carbonic
acid, the carbonic acid equals 0.03 x PCO2 and PCO2 is 40mm Hg, it may be concluded that:
A. pH=6.1 +log [(40-0.03)/(0.03)] C. pH=6.1 + log[)25-1.2)/(1.2)]
B. pH=6.1 + log [(25-0.03)/(0.03)] D. pH=6.1 + log [(1.2)/(1.2-25)]
87. A patient is admitted to the emergency room in a state of metabolic alkalosis. Which of the following would
be consistent with this diagnosis?
A. high PCO2, increased HCO3 C. high TCO2, decreased H2CO3
B, low TCO2, increased HCO3 D. low TCO2, decreased H2CO3
88. In respiratory acidosis, a compensatory mechanism is the increased in:

A. respiration rate C. blood PCO2
B. ammonia formation D. plasma bicarbonate concentration

89. A blood gas sample was sent to the lab on ice, and a bubble was present in the syringe. The blood had
been exposed to room air for at least 30 minutes. The following change in blood gases occurred:
A. CO2 content increased/PCO2 decreased
B. CO2 content and PO2 increased/pH increased
C. CO2 content and PCO2 decreased/pH decreased
D. PO2 increased/HCO3 – decreased
90. The following blood gas results were obtained:

pH 7.18
PO2 86 mm Hg
PCO2 60 mm Hg
O2 saturation 92%
HCO2 21 mEq/L
TCO2 23 mEq/L
Base
The excessresults are -8.0
patient’s mEq/L with which of the following?
compatible
A. Fever C. Emphysema
B. Uremia D. Dehydration
91. An emphysema patient suffering from fluid accumulation in the alveolar spaces is likely to be in what state?
A. Respiratory acidosis C. Metabolic acidosis
B. Respiratory alkalosis D. Metabolic alkalosis
92. The expected blood gas results for a patient in chronic renal failure would match:
A. Metabolic acidosis C. Metabolic alkalosis
B. Respiratory acidosis D. Respiratory alkalosis
93. Severe diarrhea causes:

A. Metabolic acidosis C. Respiratory acidosis
B. Metabolic alkalosis D. Respiratory alkalosis
94. Absorption of vitamin B12 requires the presence of:

A. Intrinsic factor C. secretin
B. gastrin D. folic acid
95. Most automated blood gas analyzers directly measure:

A. pH, , and % O2 saturation C.. HCO3, PCO2, and PO2
B. pH, PCO2, and PO2 D. pH, PO2and % O2 saturaturation
For nos. 96-100 & 101-105, match the two columns regarding acid-base disorders versus lab results:
pH, pCO2, HCO3- Levels Interpretation
96. decreased, decreased, decreased A A. metabolic acidosis, compensated
97. decreased, increased, decreased B B. respiratory and metabolic acidosis
98. increased, decreased, decreased D C. instrumental error
99. increased, increased, decreased C D. respiratory alkalosis, compensated
100. decreased, increased, increased E E. none of the above
pH, pCO2, HCO3- Levels Interpretation

101. decreased, decreased, increased E A. metabolic acidosis, uncompensated
102. increased, increased, increasedB B. metabolic alkalosis, compensated
103. increased , decreased, increased D C. metabolic acidosis, uncompensated
104. decreased, normal, decreased A D. dual case of alkalosis

105. decreased, normal, normal E E. instrumental error
For nos. 106 – 125, match analytes/parameters with suitable reference method for quantitation.
Column A (Parameters) Column B (Methods)
106. copper A A. AAS
107. bromide B B. ISE
108. chloride B/C C. Amperometric
109. manganese A D. Colorimetric
110. magnesium A/B E. None of these
111. iron A
112. iodide B
113. sodium B
114. ammonium B
115. phosphate D
116. pH B
117. PCO2 B
118. PO2 C
119. bicarbonate B
120. potassium B
121. molybdenum A
122. selenium A
123. cobalt A
124. fluoride B
125. zinc A
For nos. 126-135, match the electrolyte/BGA-related tests and reference values. Letters only.
Column A Column B
126. Plasma osmolality B A. 600-900 mOsm/kg HOH
127. AG C B. 285-310 mOsm/kg HOH
128. Urine osmolality A C. 12-18 mEq/L
129. Bicarbonate D D. 22-27 mmol/L
130. % Hb-O2saturation E E. 95-100% of PO2
-----------------------------------------------------------------------------------------------------------------
131. PO2 A A. 85-100 mmHg

132. PCO2 B B. 1.14-1.26 mM
133. Blood [H+] D C. 24-29 mM
134. Blood pH E D. 44-36 nM
135. TCO2 C E. 7.36-7.44
GOOD LUCK!
LONG QUIZ 3

DIRECTION. Choose the letter that correspond to the correct answer for numbers for all multiple choice question.
For matching type questions, match column A with column B and choose the letter that corresponds to the correct
answer. A letter can be used as answer more than once. Write all answers on a sheet of paper. You are not
allowed to change your answer.
1. What is a coenzyme?
a. Inorganic ions b. Organic molecules c. Organic molecules containing metals d. Both b and c
2. A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme is called?
a. Holoenzyme b. Prosthetic group c. Apoenzyme d. None of these
3. A complete catalytically active enzyme together with its bound coenzyme is called?
a. Holoenzyme b. Prosthetic group c. Apoenzyme d. None of these
4. The protein part of holoenzyme is called?
a. Apoprotein b. Apoenzyme c. Coenzyme d. Both a and b
5. Which suffix is added to the name of the substrate or to a word or to a phrase describing the activity of
enzyme, to name an enzyme?
a. -Ise b. -Ase c. –Ic d. -Ace
6. Which enzyme transfers phosphate groups?
a. Glucose oxidase b. Hexokinase c. Transferase d. Nuclease
7. The site where enzyme catalyzed reaction takes place is called?

a. Active site b. Catalytic site c. Activity site d. Functional site
8. The molecule that is bound and acted upon by the enzyme is called?
a. Biomolecule b. Substance c. Reactant d. Substrate
9. Who proposed the existence of proteolytic enzymes as proteins?

a. Kuhne b. Hayden c. Sumner d. Northrop
10. What will happen to the enzyme-catalyzed reaction if temperature is increased?

a. Rate of reaction decrease c. Rate of reaction increases
b. Reaction equilibrium shifts forward d. Reaction equilibrium shifts backward
11. What will happen to reaction if more enzymes are added?

a. Rate of reaction decrease c. Rate of reaction increase
12. How metal ions participate in catalysis?
a. By causing reduction and oxidation reactions between enzyme and substrate
b. By causing ionic interactions between enzyme and substrate
c. All of the above
13. What is Vmax?
a. Maximum rate of reaction
b. Rate of reaction increase with increase in enzyme concentration
c. Both a and b
d. None of the above
14. What is Km in Michaelis-Menten equation?
a. Michaelis-Menten constant
b. Substrate
c. Enzyme
d. All of the above
15. Which enzymes are said to follow Michaelis-Menten kinetics?
a. Enzymes which show parabolic dependence of rate of reaction and substrate
b. Enzymes which show circular dependence of rate of reaction and substrate
c. Enzymes which show hyperbolic dependence of rate of reaction and substrate

16. Double-reciprocal plot is also called?

a. Michaelis plot c. Line plot
b. Lineweaver-Burk plot d. Inverse plot
17. Which scientist proposed lock and key model in 1894?
a. Daniel Koshland b . Emil Fischer c. Einstein d. Michael
18. What is induced fit?

a. when enzyme change shape due to absence of substrate
b. when enzyme do not change shape due to absence of substrate
c. when enzyme change shape due to presence of substrate
d. when enzyme do not change shape due to presence of substrate
19. Who postulated induced fit in year1958?
a. Daniel Koshland b. Emil Fischer c. Linus Pauling d. Einstein
20. A purely competitive enzyme inhibitor has which of the following kinetic effects?
A. increases Km without affecting Vmax C. decreases Km without affecting Vmax
B. increases Vmax without affecting Km D. decreases Vmax without affecting Km
E. decreases both Vmax and Km
21. Enzymes as classic catalysts accomplish which of the following energy effects?
A. raise the energy of activation C. lower the energy of activation
B. raise the energy level of the products D. lower the energy levels of the reactants
E. decrease the free energy of the reaction
22. Synthesis of an enzyme promoted by the substrate on which it acts, is characterized by the term
A. activation B. derepression C. gratuity D. induction E.
constitutivity
23. Which statement about the active site is incorrect?

A. It is composed of linearly arranged amino acid chain.
B. It is relatively small compared to the total bulk of the enzyme.
C. It does not generally form covalent interaction with substrates.
D. It is three-dimensional in quality.
E. none of these
24. Which statement about most enzymes is incorrect?
A. They increase the rapidity of the reactions they catalyze.
B. They are specific for the substrate as well as the reaction catalyzed.
C. They are large polypeptides with high molecular weight.
D. They are most active near neutral pH.
E. They are not affected by changes in temperature.
25. Michaelis & Menten did not make which of the following assumptions concerning analyses of enzyme action?
A. The initial reaction of velocity should be measured since most of the substrate has not been converted to product.
B. Maximal velocity is reached when the concentration of ES complex is equal to the total number of enzymes.
C. The formation of the ES complex does not appreciably decrease the [S].
D. For analysis of enzyme kinetics, the total [E] studied at each [S] is fixed.
E. Plotting the reciprocal of [V] and [S] will produce an ideal linear curve.
26. Which enzymes are used to diagnose liver diseases?
A. ACP & ALP C. AMS & LPS
B. AST & ALT D. CK & LDH

27. Which enzyme cannot be used to detect acute myocardial infarction (AMI)?
A. ACP C. AST
B. CK D. LDH
28. Which of the following enzyme pairs cannot be used in the diagnosis of liver disorders?
A. ALP & LAP C. LDH & AST
B. GGT & 5’-NT D. ACP & ALS
29. Using the choices in no. 28, which pair has clinical utility for AMI detection?
Answer: C
30. Which fraction is expected to be elevated in alcoholic cirrhosis of the liver?
A. ALP C. LDH
B. GGT D. ACP
31. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both elevated in which of
the following disease?
C. muscular dystrophy C. pulmonary emboli
D. viral hepatitis D. infectious mononucleosis
32.Which two physiologic conditions can greatly elevate blood alkaline phosphatase?
E. rickets, hyperparathyroidism
F. obstructive jaundice, biliary cirrhosis
G. growth, third trimester of pregnancy
H. viral hepatitis, infectious mononucleiosis
33. A physician suspects his patient has pancreatitis. Which test(s) would be most indicative of this disease?
A. Creatine kinase C. AST/ALT
B. LD isoenzymes D. Amylase
34.Which of the following chemical determinations may be of help in establishing the presence of seminal fluid?
A. Lactic dehydrogenase C. Acid phosphatase
B.Isocitrate dehydrogenase D. Alkaline phosphatase
35. The most sensitive enzymatic indicator for liver damage from ethanol intake is
E. alanine aminotransferase (ALT)
F. aspartate aminotransferase (AST)
G. gamma-glutamyl transferase (GGT)
H. alkaline phosphatase (ALP)
36. A serum sample drawn in the emergency room from a 42-year-old man yielded the following laboratory
results:
CK 385 Units (Normal = 15-160)
AST 73 Units (Normal = 0-48)
CK-MB 106 Units (Normal = 2-12)
Which of the following conditions might account for these values?
A. Myocardial infarction C. Pulmonary infarction
B. Cerebrovascular accident D. Early acute hepatitis

37. In competitive inhibition of an enzyme reaction the
A. Inhibitor binds to the enzyme at the same site as the substrate
B. Inhibitor often has a chemical structure different from that of the substrate
C. Activity of the reaction can be decreased by increasing the concentration of the substrate
D. Activity of the reaction can be increased by decreasing the temperature
38. The presence increased CK-MB activity on a CK electrophoresis pattern is most likely found in a patient
suffering from A. Acute muscular stress following strenuous exercise
B.Malignant liver disease
C. Myocardial infarction
D. Severe head injury
39. Which of the following enzymes catalyzes the conversion of starch to glucose and maltose?
A. Malate dehydrogenase (MD) C. Creatine kinase (CK)
B. Amylase (AMS) D. Isocitric dehydrogenase (ICD)
40. Which of the following enzymes are used in the diagnosis of acute pancreatitis?
E. Amylase (AMS) and trypsin
F. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
G. 5’-nucleotidase (5’N) and gamma-glutamyl transferase (GGT)
H. Aspartate aminotransferase (AST) and lactate dehydrogenase (LD)
41. The specific activity of an enzyme would be reported in which of the following units of measure:
a. Micromoles per minute c. Milligrams per micromole
b. Millimoles per liter d. Units of activity per milligram of protein
e. Units of activity per minute
42. The Km value & Vmax in competitive inhibition are C
43. The Km value & Vmax in noncompetitive inhibition are D
44. The Km value & Vmax in uncompetitive inhibition are E
Choices for nos. 42-44.
A. increased and decreased respectively.

B. decreased and increased respectively.
C. increased and unchanged respectively.
D. unchanged and decreased respectively.
E. both decreased.
45. The functions of many enzymes, membrane transporters, and other proteins can be quickly activated or
deactivated by phosphorylation of specific amino acid residues catalyzes by enzymes called:
a. Cyclases b. Kinases c. Phosphatases d. Proteases e. Zymogens
46.The chemotherapy drug fluorouracil undergoes a series of chemical changes in vivo that results in a covalent
complex such that it is bound to both thymidylate synthase and methylene-tetrahydrofolate. The inhibition of
deoxythymidilate formation and subsequent blockage of cell division is due to which of the following:
a. Allosteric inhibition c. Competitive inhibition
b. Irreversible inhibition d. Noncovalent inhibition
e. Noncatalytic inhibition
47.The Lineweaver-Burk plot is used to graphically determine K m and Vmax for an enzyme that obeys classic
Michaelis-Menten Kinetics. When V is the reaction velocity at substrate concentration S, the Y axis
experimental data in the Lineweaver- Burk plot are expressed as:
a. V b. 1/V c. S d. 1/S e. V/Km
48.In the Lineweaver-Burk plot, the Vmax of an enzyme is:

a. Reciprocal of the absolute value of the intercept of the curve with the x axis
b. Reciprocal of the absolute value of the intercept of the curve with the y axis
c. Absolute value of the intercept of the curve with the x axis

d. Slope of the curve
e. Point of inflection of the curve
49.A noncompetitive inhibitor of an enzyme does which of the following:
a. Decreases Vmax c. Increase Vmax
b. Decrease Km and decreases Vmax d. Increases Km and increases Vmax
e. Increases Km with no or little change in Vmax
50.Digestive enzymes such a pepsin, trypsin, and chymotrypsin are synthesized as inactive precursors. The
preproteins of the active enzymes are termed :
a.Kinases b. Induces c. Isozymes d. Phosphatases e. Zymogens
51. Competitive inhibitors typically resemble the:

a. Enzyme b. Product(s) c. Substrate(s) d. Transition state
52-54. Choose the letters that corresponds to the enzymes used to detect hepatobiliary diseases:
a. GGT b. ALT c. ALP d. ALS e. LAP
52. A
53. C
54. E
55-57. Using the choices in nos. 52-54, choose the letters that corresponds to the enzymes used to detect
hepatic parenchymal disorders.
a. SDH b. LDH c. CPK d. AMS e. ALT
55. A
56. B
57. E
58-60. Choose the letters that corresponds to the diagnostic enzymes for acute myocardial infarction:
a. CK-MB b. ALT c. Troponin T d. AST e. HBD
58. A
59. D
60. E
61-62. Choose the letters that corresponds to the diagnostic enzymes for prostatic cancer:
a. LPS b. PSA c. ALP d. ACP e. ACE
61. B
62. D
63. Which enzyme is used to detect insecticide poisoning?

a. Creatine kinase
b. Acetylcholinesterase
c. Alkaline phosphatase
d. Lactate dehydrogenase
64. The graph that plots substrate concentration versus reaction rate is called
a. Michaelis-Menten plot c. Double reciprocal plot
b. X-Y plot d. Scatter plot
65. Which of the following is not the property of enzyme:

a. to be converted in the course of reaction
b. specificity towards a substrate or a group of substrates

c. alters the rate of reaction a thousand or a million-fold
d. protein with which a substrate can attach reversibly
66. On which organism or sample was the term enzyme literally associated?
a. Rice b. wheat c. meat d. yeast
67. In early 19the century, which scientist studied fermentation of sugar to alcohol using a cell-free extract?
a. Edward Buchner b. Louis Pasteur c. Alfred Joseph d. Willy Kuhne
68. What was the name given to enzymes by Louis Pasteur that he based from a process he himself
discovered?
a. Enzyme b. ferment c. catalyst d. ligand
69. In which year Edward Buchner discovered that yeast extract can cause the fermentation of sugar to
alcohol?
a. 1888 b. 1896 c. 1900 d. 1907
70. Who gave the word “enzymes” to catalytic molecules?
a. Pasteur b. Kuhne c. Buchner d. Harden
71. In which year James Sumner isolated and crystallized urease?
a. 1926 b. 1928 c. 1924 d. 1907
72. All enzymes are made up of which biomolecules (or, biomolecules)?
a. Proteins b. RNA c. Both a and b d. Neither a nor b
73. What is an inorganic cofactor?
a. Metal ions c. Vitamin-derived molecules
b. Both a and b d. None of the above
74. What is a coenzyme?
a. Inorganic ions c. Organic molecules
b. Organic molecules containing metals d. Both b and c
75. Enzymes that differ in structure and origin but same reaction catalyzed are called:
a. Holoenzyme c. Prosthetic group
b. Apoenzyme d. Isoenzyme
76. A complete catalytically active enzyme together with its bound enzyme or metal ions is called?
a. Complex enzyme c. Prosthetic group
b. Apoenzyme d. Zymogen
77. The nonprotein part of holoenzyme is called?
a. Apolipoprotein c. Apoenzyme
b. Prosthetic group d. Both a and b
78. Which suffix is added to the name of the substrate or to a word or to a phrase describing inactivity?
a. -ise b. –ogen c. –pro d. -ase
79. In how many classes enzyme is divided by the IUBMB Enzyme Commission?
a. 4 b. 5 c. 2 d. 6
80. The site where enzyme catalyzed reaction takes place is called?
a. Active site b. Allosteric site c. Activity site d. Functional site
81. The molecule that is bound and acted upon by the enzyme but suppresses its function is termed:
a. Biomolecule b. Substance c. Inactivator d. Reactant e. Substrate
82. The action of a biocatalyst affects:
a. Rate of reaction c. Reaction equilibrium
b. pH of the reaction d. Active site structure
83. What will happen to reaction if temperature is increased?
84. What will happen to reaction if enzyme is added?
85. The enzyme that does not require a carbohydrate substrate is:
a. Catalase b . AMS c. Hexokinase d. Cellulase
86. The step which decides the rate of reaction is called?

a. Acceleration step c. Reaction inhibiting step
b. Rate limiting step d. Reaction enhancing step
87. Is it possible to have several steps with equal activation energy?
a. Yes
b. No
88. How metal ions participate in catalysis?

a. By causing reduction and oxidation reactions between enzyme and substrate
b. By causing ionic interactions between enzyme and substrate
c. Both a & b
d. Neither a nor b
89. What are the most common point group for protease enzymes?
a. Serine b. tryptophan c. tyrosine d. aspartate
90. The enzyme chymotrypsin has this number of point groups in its active site:
a. 1 b. 2 c. 3 d. 4
91. What is enzyme kinetics?
a. Studying the mechanism of rate of reaction of enzyme
b. Looking into the factors affecting enzyme activity
c. Both a and b
92. What is Vmax?
a. Maximum rate of reaction
b. Rate of reaction increase with increase in enzyme concentration
c. Associated with Michaelis constant
d. Amount of substrate converted to products per enzyme
93. Who gave the general theory of enzyme action and saturable plot between [S] and V in 1913?
a. Leonor Michaelis and Maud Menten
b. Alfred Michaelis
c. Maud Michaelis
d. Lineweaver and Burk
94. In an enzyme catalyzed reaction, enzyme exists in which form?
a. Free form
b. Combined form
c. ES form
d. All of the above
95. What does Km stand for in Michaelis-Menten equation?
a. Michaelis-Menten constant
b. Substrate concentration
c. Enzyme reaction constant
d. Competitive inhibition constant
96. Which enzymes are said to follow Michaelis-Menten kinetics?
a. Enzymes which show parabolic dependence of rate of reaction and substrate
b. Enzymes which show circular dependence of rate of reaction and substrate
c. Enzymes which show hyperbolic dependence of rate of reaction and substrate
d. Enzymes which show sigmoidal curve of rate of reaction and substrate
97. Double-reciprocal plot is also called?
a. Michaelis plot
b. Line plot
c. Lineweaver-Burk plot
d. Inverse plot
98. What is k1?
a. The rate constant of the enzyme-substrate complex formation
b. The rate constant equivalent to ½ Vmax
c. The rate constant of the separation of substrate from the enzyme

d. The rate constant of the conversion of substrate to new substances
99. What is the relationship between Km and enzyme affinity for substrate?
a. Direct
b. Inversely proportional
c. Indirect
d. Depends on parameters
100. A purely competitive enzyme inhibitor has which of the following kinetic effects?
A. increases Km without affecting Vmax C. decreases Km without affecting Vmax
B. increases Vmax without affecting Km D. decreases Vmax without affecting Km
E. decreases both Vmax and Km
101. Which statement about the active site is incorrect?

A. It is composed of linearly arranged amino acid chain.
B. It is relatively small compared to the total bulk of the enzyme.
C. It does not generally form covalent interaction with substrates.
D. It is three-dimensional in quality.
E. none of these
102. Which statement about most enzymes is incorrect?
A. They increase the rapidity of the reactions they catalyze.
B. They are specific for the substrate as well as the reaction catalyzed.
C. They are large polypeptides with high molecular weight.
D. They are most active near neutral pH.
E. They are not affected by changes in temperature.
103. The official name of an enzyme is accompanied by an EC followed by 4 numbers separated by dots.
What does EC stand for?
A. Enzyme Committee C. Enzyme Commission
B. Enzyme Company D. Enzyme Constitution
104. Which anticoagulant cannot be used for plasma collection for enzyme assays because it is regarded as
an enzyme poison?
A. heparin B. EDTA C. fluoride D. citrate
105. In enzyme analysis of serum substances, buffers actually

A. stabilize electrolytes C. act as a carrier for ions
B. maintain acidic pH D. affect protein configuration
106. A physician suspects his patient has acute hepatitis. Which test would be least indicative?
A. GOT C. ALT
B. LD isoenzymes D. Amylase
107. In competitive inhibition of an enzyme reaction the
A. Inhibitor binds to the enzyme at the same site as the substrate
B. Inhibitor often has a chemical structure different from that of the substrate
C. Activity of the reaction can be decreased by increasing the concentration of the substrate
D. Activity of the reaction can be increased by decreasing the temperature
109. The specific activity of an enzyme would be reported in which of the following units of measure:

A. Micromoles per minute C. Milligrams per micromole
B. Millimoles per liter D. Units of activity per milligram of protein
E. Units of activity per minute
110. The inactive form of an enzyme is called the
A. Apoenzyme C. Holoenzyme
B. Coenzyme D. Proenzyme
111. Aminotransferase enzymes (AST and ALT) catalyze the
A. Exchange of phosphate groups with sulfur-containing acids
B. Exchange of amino and keto groups between alpha-amino and alpha-keto acids
C. Hydrolysis of amino acids to ammonia and carbonate
D. Reversible transfer of hydrogen from amino acids to coenzymes
112. The apoenzyme-coenzyme complex is also regarded as
A. metalloenzyme C. proenzyme
B. holoenzyme D. zymogen
113. Enzyme activity can be measured via the following EXCEPT
A. decrease in substrate concentration C. appearance of products due to catalysis
B. extent of utilization of coenzyme D. increase in temperature due to catalysis
114. Biologic catalysts are characterized by all of the following EXCEPT
A. can either be organic or inorganic C. not consumed or altered in the reaction
B. decrease the energy of activation D. relatively high stereospecificity
115. Which of the following are surely derived from vitamin?
A. Activator C. Prosthetic group
B. Coenzymes D. Cofactors
116. The site where catalysis and conformational changes occur in an enzyme is the
A. active site C. cleavage site
B. allosteric site D. blocking site
117. The amount of enzyme that will convert 1 mole of substrate converted per second per liter of sample:
A. IU/L C. Katal unit
B. Turnover number D. Catalytic activity per mg. protein
118. The amount of enzyme that will convert 1 micromole of substrate converted per minute per liter of
sample:
A. IU/L C. Katal unit
B. Turnover number D. Catalytic activity per mg. protein
119. The prostatic ACP is characterized by the following, except:

A. It is tartrate-stable C. It is formol-stable
B. It is not affected by Cu++ ions D. It is associated with prostatic cancer

120. Select the polypeptide chain combination designated LD 5.
A. M4 C. M2H2
B. MH3 D. H4
121. A physician suspects his patient has pancreatitis. Which test(s) would be most indicative of this disease?
A.Creatinine C. B-hydroxybutyrate
B.LD isoenzymes D. amylase
122. One international unit of enzyme activity is the amount of enzyme that, under specified reaction conditions
of substrate concentration, pH, and temperature, causes utilization of substrate at the rate of:
A.1 mole/min C. 1 micromole/min
B.1 millimole/min D. 1 nanomole/min
123. Which of the following chemical determinations may be of help in establishing the presence of seminal
fluid?
A. Lactic dehydrogenase (LD) C. Acid phosphatase
B. Isocitrate dehydrogenase (ICD) D. Alkaline phosphatase
124. Increased total lactic dehydrogenase (LD) activity, confirmed to fraction 4 and 5 , is most likely to be
associated with:
A. Pulmonary infarction C. Myocardial infarction
B. Hemolytic anemia D. Acute viral hepatitis
125. Regan isoenzyme has the same properties as alkaline phosphatase that originates in the :
A. Skeleton C. intestine
B. Kidney D. placenta
Alkaline phosphatase Slight increase
Alanine aminotransferase Marked increase
Aspartate aminotransferase Marked increase
Gamma-glutamyl transferase Slight increase
A. acute parenchymal liver disease C. obstructive jaundice
B. chronic hepatitis D. liver hemangioma
Alkaline phosphatase Marked increase
Alanine aminotransferase Slight increase
Aspartate aminotransferase Slight increase
Gamma – glutamyl transferase Marked increase
A. Acute hepatitis C. Chronic hepatitis
B. Osteitis fibrosa D. Hepatobiliary disease
128. The presence increased CK-MB activity on a CK electrophoresis pattern is most likely found in a patient
suffering from:
A. Acute muscular stress following strenuous exercise C. Malignant liver disease
B. Myocardial infarction D. Severe head injury
129. A serum sample drawn in the emergency room from a 42-year-old man yielded the following laboratory
results:
CK 185 Units (Normal = 15-160)
AST 123 Units (Normal = 0-48)
CK-MB 6 Units (Normal = 2-12)
Which of the following conditions might account for these values?
A. Crush injury to the thigh C. Pulmonary infarction
B. Cerebrovascular accident D. Early acute hepatitis
130. When myocardial infarction occurs, the first enzyme to become elevated is:
A. CK C. AST

B. LD D. ALT
131. In the determination of lactate dehydrogenase at 340nm, using pyruvate as the substrate, one actually
measures the:
A. Decrease in pyruvate C. Increase in NADH
B. Decrease in NADH D. Increase in NADH
133. A scanning of a CK isoenzyme fractionation revealed two peaks: a slow cathodic peak (CK-MM) and an
intermediate peak (CK-MB). A possible interpretation for this pattern is:
A. Brain tumor C. Myocardial infarction
B. Muscular dystrophy D. Viral hepatitis
134. Which of the following enzymes are used in the diagnosis of acute pancreatitis?
A. Amylase (AMS) and lipase (LPS)
B. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
C. 5’-nucleotidase (5’N) and gamma-glutamyl transferase (GGT)
D.Aspartate aminotransferase (AST) and lactate dehydrogenase (LD)
135. Which of the following is a glycolytic enzyme that catalyzes the cleavage of fructose-1, 6-diphosphate to
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate?
A. Aldolase C. Pyruvate kinase
B. Phosphofructokinase D. Glucose-6-phosphate dehydrogenase
136. The greatest activities of serum AST and ALT are seen in:
A. Acute hepatitis C. Metastatic hepatic carcinoma
B. Primary biliary cirrhosis D. Alcoholic cirrhosis
137. An electrophoretic separation of lactate dehydrogenase isoenzymes that demonstrates an elevation in LD-
1 and LD-2 in a “flipped” pattern is consistent with:
A. Myocardial infarction C. Pancreatitis
B. Viral hepatitis D. Renal failure
138. Which of the following is a characteristic shared by lactate dehydrogenase, malate dehydrogenase,
isocitrate dehydrogenase and hydroxybutyrate dehydrogenase?
A. They are liver enzymes. C. They catalyze redox reactions.
B. They are cardiac enzymes. D. They are class III enzymes.
139. The most heat labile fraction of alkaline phosphatase is obtained from:
A. Liver C. Intestine
B. Bone D. Placenta
140. The aldehyde transport coenzyme is derived from:
A. Vitamin A C. Niacin
B. Vitamin C D. Thiamine
Column A Column B
Enzymes and their assay methods
141. LDH B A. Michel; Ellman
142. CHS A B. Wroblewski-LaDue; Wacker
143. LPS C C. Cherry-Crandall; Shihabi-Bishop
144. AST; ALT E D. Oliver-Rosalki-Hess; Tanzer-Gilvarg
145. CK D E. Reitman-Frankel; Babson
-----------------------------------------------------------------------------------------------------------------------
Coenzymes and their transported molecules
146. NAD+; FAD C A. amino
147. Biocytin E B. acyl
148. PLP A C. electrons

149. CoASH B D. hydroxyl
150. Ascorbate D E. carboxyl
LONG QUIZ 4
I) TUMOR MARKERS. For nos. 1-40, match column A with columns B. For nos. 41- 55, match column A with
columns B & then column A with column C. Write your answers on a whole sheet of paper. Letters only.
You are not allowed to change your answers. (70 points)
Column A Column B
Chemical Nature of Different Classes of Tumor Markers
A ___ 01. PSA A. enzyme
B ___ 02. calcitonin B. hormone
C ___ 03. AFP C. oncofetal antigen
D ___ 04. CA 125 D. carbohydrate marker
E ___ 05. C-peptide E. nonenzyme protein
A ___ 06. thymidine kinase
B ___ 07. PRL
C ___ 08. CEA
D ___ 09. episialin
E ___ 10. IgG
A ___ 11. aryl sulfatase B
B ___ 12. ACTH
C ___ 13. squamous cell antigen
D ___ 14. DU-PAN-2
E ___ 15. 2-microglobulin
A ___ 16. CK-BB
B ___ 17. hCG
C ___ 18. Tennessee antigen
D ___ 19. CA-19
E ___ 20. pregnancy specific antigen
Tumor Markers and associated Diseases

C ___ 21. multiple myeloma A. CEA
E ___ 22. hydatidiform mole B. AFP
B ___ 23. liver CA C. BJP
A ___ 24. intestinal CA D. PSA
D ___ 25. prostatic CA E. hCG
Protein Markers of Tumor Types

___ 26. A ___ 26. Aldolase, Alcohol Dehydrogenase, Hexokinase A. Liver only
___ 27. B ___ 27. ACTH, Bombesin, Neurophysins B. Small-cell lung cancers
___ 28. I ___ 28. CA-125 C. Embyonal, choriocarcinomas
___ 29. D ___ 29. PSA, CK-BB D. Prostate
___ 30. E ___ 30. LDH, tdt, thymidine kinase E. Leukemias
___ 31. J ___ 31. CA-15-3, CA549, MCA F. Brain, lung, colon, GIT, breast
___ 32. G ___ 32. Alkaline Phosphatase G. Ovarian, Lung, Bone, Liver
___ 33. L ___ 33. CA-19-9, CA 19-5, CA 242, CA-50 H. Breast only
___ 34. C ___ 34. hCG I. Ovarian, endometrial
___ 35. H ___ 35. CA-27, CA-29 J. Ovarian, Breast
___ 36. K ___ 36. Aryl sulfatase B K. Colon, Breast
___ 37. P ___ 37. Catecholamine metabolites L. Pancreatic, GIT
___ 38. M ___ 38. Calcitonin M. Medullary thyroid
___ 39. N ___ 39. Tennessee antigen N. Colon, GIT, gall bladder
___ 40. O ___ 40. Amylase O. Pancreas only
Column A Column B Column C

Oncogenes & Tumor-suppressor Genes
Genes Types Cancer types
AH ___ ___ 41. N-ras A. Oncogene A. breast only
BJ ___ ___ 42. VHL B. Tumor suppressor B. T- & B-lymphoma,
lung
AI ___ ___ 43. K-ras, bcl-2 C. Combination of A&B C. bladder, melanoma,
glioblastoma
BE ___ ___ 44. DCC, APC D. Chronic myeloid
leukemia (CML)
AF ___ ___ 45. N-myc E. colorectal
BA ___ ___ 46. P16 E-cadheria, BRCA2, RB1 F. neuroendocrine
AL ___ ___ 47. c-erb B-2 G. meningioma,

neurofibromatosis 2
BN ___ ___ 48. p53 H. AML, neuroblastoma
BK ___ ___ 49. WT 1 I. lymphoma, leukemia
AB ___ ___ 50. c-myc J. kidneys
BC ___ ___ 51. cdk a2 K. Wilm’s tumor
AD ___ ___ 52. c-abl/bcr L. breast, ovarian, GIT
BG ___ ___ 53. NF 2 M. retinoblastoma,
osteosarcoma
B ___ ___ 54. RB 1 N. breast, liver,
M ___ ___ 55. p16 bladder,sarcomas
BC
II) ESSAY. Briefly discuss your answers to the following questions. All answers should be in sentence form.
Scoring depends on the number of correct answers/examples that you give per question. Each item requires 3
answers/examples. A point will be given if only 1 example/answer is correct, 3 points for 2 correct
answers/examples and 5 points if all 3 answers/examples are correct. (25 points)
1) What are the potential uses of tumor markers in the clinical setting? Cite three.
• Screening in general population
• Differential diagnosis in symptomatic patients
• Clinical staging of cancer

• Estimating tumor volume
• Prognostic indicator for disease progression
• Evaluating the success of treatment
• Detecting the recurrence of cancer
• Monitoring responses to therapy
• Radioimmunolocalization of tumor masses
• Determining direction for immunotherapy
2) Give three tumor markers and their discoverers.
Year Author Marker

1846 H. Bence-Jones Bence-Jones protein
1928 W.H. Brown Ectopic hormone syndrome
1930 B. Zondek hCG
1932 H. Cushing ACTH
1949 K. Oh-Uti Deletions of blood group antigens
1959 C. Market Isoenzymes
1963 G.I. Abelev AFP
1965 P. Gold and S. Freeman CEA
1969 R. Heubner and G. Todaro Oncogenes
1975 H. Kohler and G. Milstein Monoclonal antibodies
1980 G. Cooper, R. Weinberg, and M. Bishop Oncogene probes and transfection
1985 H. Harris, R. Sager, and A. Knudson Suppressor gene
3) Discuss three means of measuring tumor markers and provide examples.

Possible answers are as follows:
Enzyme markers - spectrophotometric assays, activity assays, colorimetric, UV Hormones, oncofetal
antigens & carb markers - immunoassay techniques like RIA, EMIT, IRMA, ELISA, etc.
Genetic markers like oncogenes and tumor-suppressor genes - PCR, fluorometric
4) What are the three mechanisms of how exposure to carcinogens lead to cancer?
Exposure to such an agent may cause cancer either by producing
1) direct genotoxic effects on deoxyribonucleic acid (DNA) (e.g., as with radiation) or 2) by increasing cell
proliferation (e.g., by a hormone), or 3) both 1 and 2 (e.g., through the use of tobacco).
Any other answers depends on the discretion of the teacher.
5) Cite three types of carcinogen with specific examples.

Carcinogens may be 1) physical (e.g., radiation), 2) chemical (e.g., a polycyclic hydrocarbon), or 3)
biological (e.g., a virus).

LONG QUIZ 5
Southwestern University Phinma - College of Medical Technology

LONG EXAM 5 on Endocrinology
I) IDENTIFICATION. Identify what each of the following describes or defines: (10 points)
1. The study of endocrine glands and their secretions Endocrinology
2. The chemical regulators that are produced by ductless glands Hormones
3. The medium which transports hormones to their target organs Blood
4. The specific membrane proteins that bind hormones Receptors
5. The gland that regulates adenohypophyseal secretions Hypothalamus
6. The target tissues of the hormone somatotropin all organs
7. The largest endocrine gland in the body Skin
8. The other name for the posterior lobe of pituitary gland Neurohypophysis
9. The term used for the constantly maintained internal equilibrium of the body Homeostasis
10. The only hormone produced by the adenohypophysis that exhibits a positive feedback
mechanism
Prolactin
II) MCQ: Select the best answer for each of the following questions. Shade the box that corresponds
to the correct answer. Final answers on the answer sheet. NO ERASURES / ALTERATIONS
ALLOWED.
1. TSH is produced by the:
A. Hypothalamus B. Pituitary gland C. Adrenal cortex D. Thyroid
2. The major control mechanism for serum calcium level is played by

A. estrogen C. parathyroid hormone
B. growth hormone D. thyroid hormone
3. Persistent hypoglycemia is seen in which of the following conditions?

1. insulinoma
2. galactosemia A. 1,2,3,and 4 B. 1 and 3
3. Hyperthyroidism
4. Addison’s disease C. 1 and 2 D. 1,2,and 3
4. One of the following is NOT secreted by the anterior pituitary gland. Which one is it?
A. LH C. HGH
B. VMA D. ACTH
5. Increased urinary catecholamine metabolites with intermittent hypertension may indicate which of
the
following conditions:
A. idiopathic hypertension C. arteriosclerosis
B. adrenal medullary disease D. Hyperthyroidism
6. The major thyroid hormone is

A. Somatotropin B. Calcitonin C. Insulin D. Thyroxine
7. Plasma renin activity is most useful in the differential diagnosis of:

A. Hypertension B. Diabetes mellitus C. Lead Poisoning D. Alcoholism
8. Assessments of primary & secondary hypothyroidism include the following EXCEPT:

A. T 3 C. TSH
B. T 4 D. Calcitonin
9. Hyperthyroidism has been associated with the following, EXCEPT BONUS

A. Decreased cholesterol C. Increased T 32
B. Increased sugar level D. Increased T 4
10. The reference method for pituitary secretions is the

A. RIA technique B. Spectrophotometry C. HPLC D. Dynamic testing
11. The secretion of anterior pituitary hormones is regulated by:

A. Hypothalamus C. Aldosterone
B. Serotonin D. ACTH
12. Which of the following is caused by oversecretion of the adrenal cortex?

A. Pagets disease C. Pheochromocytoma
B. Cushing’s disease D. Grave’s disease
13. The use of iodized salt serves to provide iodine for the synthesis of:
A. Sex hormones B. Insulin C. Thyroid hormone D. Growth hormone
14. Diurnal variations is important to consider when collection blood for the assay of:
A. Catecholamines C. Cortisol
B. Creatinine D. Thyroid hormones
15. The estrogen designated as E 2 whose urine concentration is the greatest is:
A. Estriol C. Estrone
B. Estradiol 17β D. Progesterone
16. What is the hormone which controls the reabsorption of sodium in the kidneys:
A. Aldosterone C. Estrogen
B. ADH D. Growth hormone
17. Catecholamines are secreted by the:

A. Kidney B. Pituitary C. Adrenal cortex D. Adrenal medulla
18. Which of the following is a metabolite of epinephrine?
A. 17-ketosteroids C. Vanillylmandelic acid
B. Follicle stimulating hormone D. thyroid stimulating hormone
19. Patient has a TSH result markedly elevated. This maybe due to:
A. Hypothyroidism due to pituitary disorders
B. Primary aldosteronism C. Graves disease D. Hyperthyroidism
20. Patient’s value is normal T 3 , T 4 , what do you think is the value of TSH?
A. Increased C. Normal
B. Decreased D. None of the above

21. In which of the following are the thyroid hormones classified?
A. Amino acid derivatives
B. Steroid hormones
C. Fatty acid derivatives
D. Peptide hormone
22. When is the best time to get plasma cortisol samples:3

A. 8 AM C. 12 PM
B. 8 PM D. 12 AM
23. Why is it important to time samples for cortisol level:

A. Actually, there is no timing
B. The best period to get is from 10 PM to 2 AM
C. Because maximal secretion of cortisol has a diurnal pattern
D. None of the above
24. The relationship of a cholesterol and thyroid hormone is:

A. Directly proportional C. Inversely proportional
B. Not related D. none of the above
25. Diabetes insipidus is associated with deficiency in:

A. ACTH C. HCG
B. TSH D. ADH
26. TRH is produced by the:

A. Hypothalamus C. Adrenal cortex
B. Pituitary gland D. Thyroid
27. Calcium concentration in the serum is regulated by:

A. Insulin C. thyroxine
B. parathyroid hormone D. vitamin C
28. Which of the following steroids is an adrenal cortical hormone?

A. Angiotensinogen C. progesterone
B. Corticosterone D. HCG
29. Which of these hormones are especially related in the delicate balance of production and
utilization of glucose in the body?
A. Growth hormone and cortisol
B. Epinephrine and thyroid hormones
C. Insulin and glucagon
D. Glucagon and thyroxine
30. Which of the reactions measures 17-ketosteroids?

A. Porter-Silber C. Murphy-Pattee
B. Zimmerman D. Kober
31. The net effect of calcitonin on the metabolism of calcium and calcium and phosphorus is:
A. Decreased calcium, increased phosphorus
B. Decreased calcium and phosphorus
C. Increased calcium and phosphorus

D. Increased calcium, decreased phosphorus
32. Patients with Cushing’s syndrome exhibit:

A. decreased plasma 17-hydroxysteroid concentration
B. decreased urinary 17-hydroxysteroid excretion
C. serum cortisol concentrations greater than 15 mg/dL
D. decreased cortisol secretory rate
33. Fasting serum phosphate concentration is controlled primarily by the :

A. Pancreas C. Parathyroid glands
B. Skeleton D. Small intestine
34. A patient has the following test results:

Decreased serum calcium levels
Increased serum phosphorous levels
Decreased levels of parathyroid hormone
This patient most likely has:

A. hyperparathyroidism C.nephrosis
B. hypoparathyroidism D. steatorrhea
35. The Porter-Silber reaction is used in the assay of:

A. Urinary estrogen C. Testosterone
B. Glucocorticoids D. Epinephrine
36. ACTH is produced in:

A. Pancreas C. Adenohypophysis
B. Adrenal cortex D. Adrenal medulla
37. This hormone is produced by the posterior pituitary gland:

A. Vasopressin C. Prolactin
B. Both A & D D. ADH
38. Serotonin is secreted by the:

A. Kidney B. Liver C. Adrenal cortex D. Small intestine
39. Dark-light cycle is an important function of:

B. Melatonin D. Thyroid hormones
40. Glucagon is produced by:

A. B-cell of the pancreas C. Alpha cell of the pancreas
B. Pituitary gland D. Adrenal Cortex
41. ACTH acts on the:

A. Pancreas
B. Adrenal cortex
C. Posterior pituitary gland
D. Adrenal medulla

42. The Zimmerman determination of 17-ketosteroid is based on reaction with:
A. Acetic anhydride C. Meta-dinitrobenzene
B. Ehrlich’s reagent D. Potassium ferricyanide
43. Which of these hormones lowers the blood glucose level?

A. Cortisol C. Glucagon
B. Epinephrine D. Insulin
44. At low or absent levels, which one of the following hormones has the ability to produce
hyperglycemia:
A. Glucagon C. Thyroid hormone
B. Insulin D. Parathyroid hormone
45. Which of these have receptors for ADH?

A. DCT C. Adrenals
B. Thyroid D. Mammary gland
46. One of the following is NOT secreted by the adenohypophysis. Which one is it?
A. LH C. hCG
B. Somatotropin D. ACTH
47. The main thyroid hormone is

C. T 3 D. ACTH C. Insulin D. Calcitonin
48. Elevated serotonin metabolites may indicate which of the following?

A. Idiopathic hypertension B. Pheochromocytoma C. Diabetes mellitus D. Intestinal cancer
49. Hypothyroid test is shown in:

A. Normal T 3
B. Normal Free T 4 level
C. Decrease T 3 , increase T 4
D. Decrease resin T 3 T 4
50. The androgen whose urine concentration is the greatest is:

B. Estradiol D. Testosterone
51. What is the hormone which controls the natriuresis in the Kidney:
A. Aldosterone C. ANF
52. Erythropoietin is secreted by the:

A. Kidneys B. Pituitary C. Adrenal cortex D. Adrenal medulla
53. Which of the following is a metabolite of serotonin:

B. 5’-HIAA D. Tryptophan

54. Which is an advantage of RIA assay over other tests for hormones?
A. Lengthy analysis
B. Any volume for sample
C. Measures interferences as well
D. High specificity
55. Patient has a TRH result that is markedly elevated. This maybe due to:
A. Primary hypothyroidism
B. Primary aldosteronism
C. Tertiary thyroid hypofunction
D. Secondary hypothyroidism
56. Patient’s T 3 , & T 4 levels are low, what do you think is the value of TSH?
B. Decreased D. None of the above
57. In which of the following are the eicosanoids classified?

A. Amino acid derivatives
B. Steroid hormones
C. Fatty acid derivatives
D. Peptide hormone
58. A deficiency of somatotropin will result in

A. Grave’s disease C. Cretinism
B. Dwarfism D. Hashimoto’s thyroiditis
59. The tumor marker associated with ovarian carcinoma is:

A. Parathormone C. Calcitonin
B. HCG D. ACTH
60. When is the best time to get plasma testosterone samples:

A. 6 AM C. 12 PM
B. 8 PM D. 12 AM
61. Which of these hormones are especially related in the delicate balance of production and
utilization of glucose in the body?
A. Growth hormone and cortisol
B. Epinephrine and thyroid hormones
C. Insulin and glucagon
D. Glucagon and thyroxine
62. Which of the reactions measures 17-OHcorticoteroids?

A. Porter-Silber C. Murphy-Pattee
B. Zimmerman D. Kober
63. The net effect of calcitonin on the metabolism of calcium and phosphorus is:
E. Decreased calcium, increased phosphorus
F. Decreased calcium and phosphorus
G. Increased calcium and phosphorus
H. Increased calcium, decreased phosphorus

64. The Kober reaction is used in the assay of:
A. Urinary estrogen C. Testosterone
B. Glucocorticoids D. Epinephrine
65. The estrogen whose urine concentration is elevated in gestation is:

B. Estradiol D. Progesterone
66. What is the hormone which controls the reabsorption of water in the Kidney:
A. Aldosterone C. Estrogen
67. ACTH is produced in:

A. Pancreas
B. Adrenal cortex
C. Adenohypophysis
D. Adrenal medulla
68.This hormone is produced by the posterior pituitary gland:

A. Vasopressin C. Prolactin
B. Both A & D D. ADH
69. Catecholamines are secreted by the:

A. Kidney
B. Pituitary
C. Adrenal cortex
D. Adrenal medulla
70. The menstrual cycle is important to consider when collection blood for the assay of:
B. Gonadotropins D. Thyroid hormones
71. GHRH is produced in:

A. Hypothalamus
B. Pituitary gland
C. Alpha cell of the pancreas
D. Adrenal cortex
72. ACTH acts on the:

A. Pancreas
B. Zona fasciculata
C. Posterior pituitary gland
D. Adrenal gland
73. The Zimmerman determination of 17-ketosteroid is based on reaction with:

A. Acetic anhydride C. Meta-dinitrobenzene
B. Ehrlich’s reagent D. Potassium ferricyanide
74. Which of these hormones lowers the blood glucose level?
A. Cortisol C. Glucagon

B. Epinephrine D. Insulin
75. At low or absent levels, which one of the following hormones has the ability to produce
hyperglycemia:
A. Glucagon C. Thyroid hormone
B. Insulin D. Parathyroid hormone
76. The major control mechanism for serum calcium level is played by
A. estrogen C. parathyroid hormone
B. growth hormone D. thyroid hormone
77. Persistent hypoglycemia is seen in which of the following conditions?

1. insulinoma 3. hypothyroidism
2. galactosemia 4. Addison’s disease
A. 1,2,3,and 4
C. 1 and 2
B. 1 and 3
D. 1,2,and 3
78.Normal thyroid function is termed

A. hypothyroidism C. thyrotoxicosis
B. hyperthyroidism D. Euthyroidism
79. Which of these have receptors for ADH?

A.DCT C. Adrenals
B. Thyroid D. Mammary gland
80. One of the following is secreted by the neurohypophysis. Which one is it?
A. LH C. HCG
B. Oxytocin D. ACTH
81. Increase values of Thyroxine (T 4 -RIA) is usually associated with the following,
EXCEPT:
A. Hyperthyroidism C. Nephrosis
B. Acute thyroiditis D. Graves’ disease of the thyroid
82. Elevated serotonin metabolites may indicate which of the following?

A. Idiopathic hypertension8
B. Pheochromocytoma
C. Diabetes mellitus
D. Intestinal cancer
83. Plasma renin activity is most useful in the differential diagnosis of:
A.Hypertension C. Lead Poisoning
B. Diabetes D. Alcoholism
84. Following are thyroid function test, EXCEPT:
A. Free thyroxine T 4 C. Thyroglobulin
B. Free Triodothyronine D. Testosterone
85. Estrogen and progesterone receptor assays are useful in assessing prognosis in

which of the following:
A. Cancer of the uterus C. Breast cancer
B. Liver cirrhosis D. Cushing Syndrome
86. One of the following is NOT associated with hyperthyroidism;

A. Decreased calcium C. Increased protein-bound iodine
B. Increased T 3 D. Decreased Cholesterol
87. One of the following is not thyroid function test:

A. T 3 C. TSH
B. T 4 D. ACTH
88. Hyperthyroidism has been associated with the following, EXCEPT BONUS
A. Decreased cholesterol C. Increased T 3
B. Increased sugar level D. Increased T 4
89. Which hormone is responsible for calorigenesis and thermogenesis?

A. T4 and T3
B. Estrogens
C. Both
D. Neither
90. The secretion of anterior pituitary hormones is regulated by:

A. Hypothalamus C. Aldosterone
B. Serotonin D. ACTH
91. Which of the following is caused by versecretion of the thyroid gland?

A. Paget’s disease C. Pheochromocytoma
B. Addison’s disease D. Grave’s disease
92. The major nonspecific binding protein for hormones is:
A. Albumin
B. TBPA (thyroid-binding prealbumin)
C. TBG (thyroid binding globulin)
D. CRP
93. T 3 uptakes is actually a measurement of:

A. Thyrotoxicosis B. Cushing’s disease C. Pregnancy D. Dwarfism
94. The major androgen produced in adrenals is:

A. Estriol C. DHEA
B. Estradiol D. Testosterone
95. Which of the following is a metabolite of epinephrine:
B. 5’-HIAA D. Tryptophan
96. Patient has a TSH result that is markedly elevated. This maybe due to:
A. Primary hypothyroidism
B. Primary aldosteronism
C. Graves disease
D. Primary hyperthyroidism

97. Patient’s T 3 , & T 4 levels are normal, what do you think is the value of TSH?
B.Decreased D. None of the above
98. In which of the following is melatonin hormone classified?

A. Amino acid derivatives B. Steroid hormones C.Fatty acid derivatives D.Peptide hormone
99. The deficiency of the thyroid hormone in children is known as

A. Grave’s disease C. Cretinism
B. Myxedema D. Hashimoto’s thyroiditis
100. What hormone is responsible for circadian rhythm:

A. ACTH
B. GH
C. Cortisol
D. Melatonin
III.) HORMONE TARGETS & ORIGINS. Match column A with columns B & C. Letters
only. You are not allowed to change your answers. (80 points)
IIIA)
Column A
1. TRH Origins: K. Hypothalamus Targets: I. Anterior pituitary lobe
2. ACTH G. Adenohypophysis A. Thyroid gland
3. Vasopressin Neurohypophysis C. Renal collecting ducts
4. T3 & T4 Thyroid gland E. All organs and tissues
5. PTH PTG Kidneys & Bones
6. Insulin Pancreas All organs and tissues
7. Aldosterone Adrenal cortex PCT of kidneys
8. Estradiol Ovaries Female gonads/accessory organs
9. Catecholamines Adrenal medulla Heart, blood vessels, liver
10. ANF Heart PCT of Kidneys
11. CRH Hypothalamus Anterior Pituitary Lobe
12. Testosterone Testes Male gonads/accessory organs
13. Thymosin Thymus Lymphoid tissues
14. Somatomedins Liver All organs & tissues
15. CCK-PZ Small intestine Common bile duct
16. EPO Kidneys Bone Marrow
17. Vitamin D3 Kidneys Small Intestine
18. hCG Placenta Female gonads/accessory organs
19. MSH Adenohypophysis Skin
20. Calcitonin Thyroid gland Kidneys and Bones
Match column A with column B. Should there be three columns, match the first column with both the
second and third columns. A letter may be used more than once.
IIIB). CHEMICAL NATURE OF HORMONES.
Column A Column B Column C
21. Amines C & B A. derived from fatty acid A. prostaglandins, leukotrienes
22. Steroids B & E B. derived from cholesterol B. melatonin, thyroxine

23. Eicosanoids A&A C. derived from amino acids C. ADH, insulin, glucagon
24. Glycoproteins E&D D. composed solely of amino acids D. FSH. LH. hCG, GH
25. Polypeptides D & C E. composed of amino acid and sugars E. androgen, estrogen, progestin
IIIC). HORMONE GROUPING.

26. lipophilic A A. Group I hormones
27. requires intracellular receptors A B. Group II hormones
28. generally orally given if treated as drug A
29. requires secondary messengers B
30. transported in blood not bound to proteins B
31. rapid initiation of chemical reaction A
32. plasma half-life of hours to days A
33. examples are catecholamines and insulin B
34. examples are androgens and thyroxine A
35. receptor-hormone complex formation is enough to produce cellular changes A
IIID) HORMONES & THEIR DESCRIPTIONS

T 36. CRH A. tryptophan-derived and induces sleep
P 37. TRH B. hypothalamic secretion also referred to as somatostatin
I 38. GnRH C. responsible for the maturation of ovarian follicles
S 39. PIF D. increases the blood levels of prolactin
D 40. PRF E. hyperglycemic glycogenolytic factor from the pancreas
R 41. GHRH F. major hormone derived from proopiomelanocortin gene
B 42. GHIH G. principal hormone in the maintenance of corpus luteum
K 43. MIF H. derived from mammotrophs
O 44. ADH I. stimulates the production of pituitary FSH & LH
N 45. Oxytocin J. stimulates the release of thyroid hormones
A 46. Melatonin K. decreases the level of the blood MSH
L 47. Serotonin L. source of urinary 5’-hydroxyindole acetic acid
E 48. Glucagon M. another name is somatotropin
H 49. PRL N. peptide hormone for milk ejection and uterine contraction
M 50. GH O. directly regulates the production of aquaporins in collecting ducts
C 51. FSH P. stimulates production of thyroid-stimulating hormne
J 52. TSH Q. cardiac peptide that decreases blood sodium levels
F 53. ACTH R. otherwise known as somatocrinin
G 54. LH S. believed as a dopamine-like inhibitor of prolactin
Q 55. ANF T. stimulates the production of beta-endorphins
LONG QUIZ 6
I. IDENTIFICATION. Identify what each of the following defines or describes: (15 points)
1. gland 1. a bilobed organ responsible in increasing the basal metabolic rate

2. thyroid follicles 2. the fundamental structural and functional unit of the thyroid gland

3. colloid 3. the proteinaceous fluid in the lumen bounded by the thyroid follicles
4. calcitonin 4. the hypocalcemic hormone produced by the thyroid gland
5. thyroglobulin 5. regarded as the prohormone of thyroid hormones T 3 and T4
6. tyrosine 6. the amino acid precursor of thyroid hormones T 3 and T4
7. reverse T3 7. the inactive type of triiodothyronine
8. 1.23-3 nM) 80- 8. the reference range of T3 in the plasma
200 ng/dl
9. 72-163 nM) 5.5 - 9. the reference range of T4 in the plasma
12.5 ug/dl
10. deiodinases 10. the enzymes responsible in the peripheral conversion of T 4 to T3
11. t4 or thyroxine 11. another name for 3,5,3’,5’-tetraiodothyronine
12. 10:1 NOT 1:10 12. the normal T4 to T3 ratio
13. euthyroid 13. the state characterized by normal blood thyroid hormone levels
14. TRH or 14. the hypothalamic hormone that regulates thyroid hormone synthesis
thyrotropin
releasing hormone
15. negative 15. the type of feedback exhibited by the thyroid gland to the pituitary
feedback
II.) MULTIPLE CHOICE. On a whole sheet of paper, write the letter that corresponds to the correct answer for
each of the following items. You are not allowed to change your answer.
1. A patient shows the following data: T4 concentration of 8 ug/dL and T3 uptake of 30%. What is the
condition?
A. Hyperthyroidism B. Euthyroidism C. Hypothyroidism D. Euthyroidism with high TBG
2. Which secretion is involved in calcium homeostasis?

A. estrogen B. VIP C. androgen D. thyroid hormone
3. Which is an advantage of T4 assay over PBI (protein bound iodine) test?

A. Quicker analysis
B. Lesser volume for sample
C. Prevent contamination of extraneous iodine
D. Simpler
4. Cretinism in infants is caused by

A. hypothyroidism B. hyperthyroidism C. Thyrotoxicosis D. euthyroidism
5. Which of the parameters are decreased in hypothyroidism?

1. T4 2. T3 3. Resin T3 Uptake 4. TSH
A. 1 and 2 C. 1,2,3 and 4
B. 1,2 and 3 D. 3 and 4
6. Majority of the thyroid hormones in blood are bound with:
A. Thyroxine –binding albumin B. Glycerophosphate C. Phenolphthalein phosphate D. Disodium
phenyl phosphate
7. Majority of plasma thyroxine ( T4) is:

A. Bound to globulin B.Bound to prealbumin C. Free D. bound to albumin
8. Which of these antibodies are associated with auto-immune thyroiditis?

A. Neither microsomal nor thyroglobulin antibodies

B. Microsomal antibodies
C. Thyroglobulin antibodies
D. Both microsomal and thyroglobulin antibodies
9. The gold standard for the assessment of thyroid function is the measurement of
A. Both T3 and T4
B. TSH
C. TRH stimulation
D. RT3U
10. The metabolically active thyroid hormone is
A. T3 C. rT3
B. T4 D. Thyroxine
11. The methods for determining thyroid hormones are as follows, EXCEPT:
A. Ash method protein bound iodine C. Radioactive tracer method
B. Chromatography D. Colorimetric method
12. Increase values of Thyroxine (T4-RIA) is usually associated with the following, EXCEPT:
A. Hyperthyroidism C. Nephrosis
B. Acute thyroiditis D. Graves’ disease
13. The following are thyroid function tests, EXCEPT:

A. Free thyroxine B. Free Triiodothyronine C. Thyroglobulin D. Testosterone
14. One of the following is NOT associated with hyperthyroidism;

A. Decreased calcium B. Increased T3 C. Increased PBI D. Decreased Cholesterol
15. Thyroid function tests exclude:

A. T3 B. TSH C. T4 D. ACTH
16. Hyperthyroidism has been associated with the following, EXCEPT

A. Increased cholesterol B. Increased T3 C. Increased sugar level D. Increased T4
17. Assay of T3 & T4 levels, and TSH maybe done by this method, EXCEPT:
A. Spectrophotometer B. Radioimmunoassay C. Flame photometer D. Gamma
counter
18. Hypothyroid test is shown in:

A. Normal T3 C. Decrease T3, increase T4
B. Normal Free T4 level D. Decrease resin T3 & T4
19. Test for T3 and T4 can be determined by

A. RIA technique B. EIA technique C. Both D. Neither
20. Methods for determining T3 and T4 exclude

A. EIA B. Spectrophotometer C. RIA D. Hemagglutination test
21. Patient has a TSH result markedly elevated. This maybe due to:
A. Hypothyroidism due to pituitary disorders
B. Primary hypothyroidism
C. Graves disease
D. Hyperthyroidism

22. Determine the free thyroxine index (FT 4I) using the given data:
Total T4 = 10 microgram/dL
Resin T3 uptake of patient = 27%
Resin T3 uptake of serum pool = 30%
A. 2.7 C. 9.0
B. 11.0 D. 3.0
23. The use of iodized salt serves to provide iodine for the synthesis of:
A. Sex hormones B. Insulin C. Thyroid hormone D. Growth hormone
24. Normal T3 in serum and normal T4 is interpreted as:
A. Euthyroidism B. Thyrotoxicosis C. Hyperthyroidism D. Hypothyrodism
25. What is the primary purpose of thyroidal radioiodine uptake test?
A. Diagnosis of hypothyroidism
B. Diagnosis of hyperthyroidism
C. Differentiate hypothyroidism from euthyroidism
D. Differentiate the causes of hyperthyroidism
26. In hyperthyroidism, resin T3 uptake is:
A. Markedly decreased B. Increase C. Normal D. Slightly decreased
27. The radioactive count in RT3U test in hypothyroidism is expectedly:
A. Significantly lower B. Normal C. Slightly below than normal D. Higher
28. In which of the following are the thyroid hormones classified?
A. Amino acid derivatives B. Steroid hormones C. Fatty acid derivatives D. Peptide

hormone

29. Presence of a very high titer for long acting thyroid stimulator antibodies is highly suggestive of what
disorder:
A. Pernicious anemia B. Multinodular goiter C. Hashimoto’s thyroiditis D. Grave’s disease
30. The major function of thyroid gland is:

A. Production of thyroid hormones
B. Increase the BMR and Calcium in blood
C. Thermogenesis & calorigenesis
D. Negative feedbacking to pituitary gland secretion
31. The parent compound of steroid hormones is

A. acetate B. cholesterol C. sitosterol D. triglycerides
32. In the chemical tests for urinary steroids, the first step is hydrolysis using an
A. enzyme B. acid C. both A & B D. neither A nor B
33. The organic reagent used to hydrolyze steroid hormones in the sample is
A. acid B. enzyme C. salt D. alkali
34. The reference method for the assay of steroid hormones is

A. GC B. RIA C. ELISA D. HPLC
35. The method used to quantitate ketogenic steroids is

A. Porter-Silber B. Pisano C. Zimmermann D. Kober
36. Which adrenal tissue produces secretions which react with DNPH-sulfuric acid?
A. glomerular cells C. reticular cells
B. fascicular cells D. chromaffin cells
37. The major transport protein of cortisol which binds 90% of circulating cortisol is
A. SHBG B. albumin C. transcortin D. TBG
38. The best time to collect a blood sample for testosterone measurement is
A. 12 midnight B. 8 am C. 12 noon D. 4 pm
39. The hormones referred to as 18-carbon steroids with unsaturated A ring are
A. mineralocorticoids B. glucocorticoids C. estrogens D. androgens
40. The major androgen produced in the adrenal cortex is

A. testosterone C. androstenedione
B. dehydroepiandrosterone D. dihydroxytestosterone
41. In secondary hypocortisolism, plasma ACTH is

A. decreased B. increased C. normal D. out of range
42. The 17-ketosteroids include all of the following except

A. androsterone C. DHEA
B. androstenedione D. testosterone
43. Dexamethasone suppression test is used to

A. assess estrogen production by adrenals C. establish the cause of hypocortisolism

B. assess androgen production by adrenals D. establish the cause of hypercortisolism
44. The following are true regarding the Zimmerman reaction except
A. the reaction requires an acidic medium C. dinitrobenzene is the chromogen
B. red-purple product is measured at 520nm D. used to assess androgen production in adrenals
45. The Kober method is performed to

A. assess estrogen production by adrenals C. establish the cause of hypocortisolism
B. assess androgen production by adrenals D. establish the cause of hypercortisolism
46. Transcortin is able to bind all of the following hormones except

A. cortisol B. progesterone C. aldosterone D. estradiol
47. The predominant hormone in the menstrual luteal phase is

A. estrogen B. progesterone C. testosterone D. cortisol
48. Which hormone-function dyad is incorrect?

A. estrogen – stimulates uterine endometrium to proliferate or thicken and negative feedbacking
to the pituitary gland
B. FSH – stimulates meiosis in ovaries; release of the ovum
C. Progesterone – maintains levels of estrogen
D. LH – development of the ovum into a corpus luteum
49. The predominant estrogen in a gestating woman is

A. estrone B. estradiol C. estriol D. progesterone
50. The predominant estrogen in post-menopausal women is

A. estrone B. estradiol C. estriol D. progesterone
51. Pituitary secretion of ACTH is inhibited by levels of

A. cortisol B. progesterone C. estradiol D. corticosterone
52. In the Porter-Silber reaction, the 17,21-dihydroxy-20-ketone side chain reacts with
A. dinitrobenzene C. sodium metaperiodate
B. quinone imine D. phenylhydrazine
53. The most potent estrogen which is considered the true ovarian hormone is
A. estriol B. estradiol C. estrone D. 16-epiestriol
54. All of the following glands secrete steroid hormones except

A. ovaries B. pituitary gland C. placenta D. adrenal cortex
55. The placenta secretes numerous hormones both protein and steroid. Which of the
following hormones is not secreted by the placenta?
A. hCG B. progesterone C. estrogen D. LH

56. Because of infertility problems, a physician would like to determine when a woman ovulates. The
physician orders serial assays of plasma progesterone. From these assays the physician can tell when
ovulation occurs because
A. after ovulation, progesterone rapidly increases

B. right before ovulation progesterone spikes
C. after ovulation progesterone rapidly decreases
D. right before ovulation progesterone rapidly increases
57. Which of the following is the major mineralocorticoid?

A. aldosterone B. corticosterone C. cortisol D. testosterone
58. The adrenal medulla secretes which of the following in greatest quantity:
A. epinephrine B. norepinephrine C. HVA D. VMA
59. In a patient who is suspected of having pheochromocytoma, measurement of which of the following would
be most useful?
A. VMA B. 5’-HIAA C. HVA D. 9-THC
60. The highest levels of gonadotropins occur

A. during follicular phase of the menstrual cycle
B. during the luteal phase of the menstrual cycle
C. at the midpoint of the menstrual cycle
D. several days prior to ovulation
61. The reference method for the assay of catecholamines is

A. GC B. RIA C. ELISA D. HPLC
62. In a patient who is suspected of overproducing serotonin, the measurement of which of the following would
be most useful?
63. In a patient who is suspected of overproducing dopamine, measurement of which of the following would
be most useful?
64. In the Zimmerman reaction, the chromogen is

A. dinitrobenzene C. sodium metaperiodate
B. quinone imine D. phenylhydrazine
65. The determination of estrogens by the Kober reaction requires which clinical sample?
A. random urine B. 12-hour urine C. 24-hour urine D. pooled urine
III.) MATCHING TYPE. Match column A with column B. A letter may be used more than once. Place all answers
to the matching type test on the same answer sheet. You are not allowed to change your answer.
A.) SPECIAL THYROID TESTS I

B1. RAIU A. evaluates response to anti-thyroid drug therapy
A2. T3 suppression test B. differentiate causes of hyperthyroidism
D3. Needle biopsy C. diagnosis of hypothyroidism in borderline cases
E4. Thyroid antibodies D. remove thyroid mass to check for malignancy
C5. TSH measurement E. measured to assess presence of anti-thyroid antibodies

B.) SPECIAL THYROID TESTS II
D6. Thyroid-binding proteins A. thyroid scan and RAIU
E7. Thyroid stimulating antibodies B. PBI, BEI, CPB & RIA
A8. Radioactive thyroid tests C. RT3U & % uptake of labeled T3
C9. Tests for free T3 and free T4 D. TBG, TBPA, & TBA
B10. Tests for total T3 and T4 E. LATS & TBI
C.) REFERENCE RANGE

C11. Total T3 by RIA A. 5.5-12.5 µg/dl
A12. Total T4 by RIA B. 25-35%
E13. RT3U C. 80-200 ng/dl
B14. % T3 uptake D. 1:10
F15. T4 to T3 ratio E. 0.85-1.15
F. none of the above
D.) TERMS ON THYROID FUNCTION

D16. Murphy-Pattee method A. TBG
I17. Conjugating compounds for T4 B. sulfates
E18. Prohormone for thyroid hormones C. RIA
G19. Metabolically inactive triiodothyronine D. CPB assay
H20. Gold standard for thyroid function E. thyroglobulin
A21. Major thyroid hormone transport protein F. equilibrium dialysis
B22. Conjugating compound for T3 G. reverse T3
F23. Gold standard for free T3 and T4 assay H. TSH measurement
J24. Radioactive elements used in thyroid scan I. glucuronate
C25. Most sensitive method for TSH assay J. radioiodine and pertechnitate
E.) TBG LEVELS

A26. intake of anabolic steroids A. low TBG level
A27. advanced liver disease B. high TBG level
A28. advanced renal disease
B29. pregnancy
B30. increased estrogen level
F.) THYROID HORMONE LEVELS & DISEASE
B31. increased RT3U A. hypothyroidism

A32. decreased TBG B. hyperthyroidism
A33. liver cirrhosis C. euthyroidism
A34. renal failure
A35. starvation
A36. cystic fibrosis
B37. thyroid cancer
A38. decrease RT3U
A39. increased TBG
A40. iodine deficiency
IV) FILL IN THE TABLE. Write I if the hormone is increased and write D if the hormone is decreased
corresponding to the column headings and row labels. (20 points) COPY & ANSWER

HYPOTHYROIDISM HYPERTHYROIDISM
primary secondary tertiary primary secondary tertiary
TSH
I D D D I I
TRH
I I D D D I
T3 and T4
D D D I I I
V). FILL IN THE BLANKS. Fill in the blanks under A with the organ producing the hormone. Supply blanks under B with
the function of the hormones. (10 points)
A B
1. T3 ___thyroid gland__________ __increase BMR, calorigenesis, thermogenesis, prod. of thyroid hormones__
2. T4 ___thyroid gland__________ __same as no. 1____________________________
3. TSH ___pituitary gland_________ __stimulate thyroid to prod. secretion__________
4. TRH ___hypothalamus_________ __stim. release of TSH from pitui. gland_________
5. Calcitonin ___thyroid gland__________ __decrease plasma Ca levels__________________
VI). SEQUENCE OF THYROID PROCESSES. Assign a number before each step of the processes A (from 1 to 5) and B
(from 1 to 7) to come up with the correct sequence of each of the said processes occurring in the thyroid gland. Sample
answers are : A: 43521; B 7243165
A) Thyroid Hormone Synthesis……………………………34251 (score is either 5 or 0) 3

B) Release of Thyroid Hormones from the Follicle…….6427531 (score is either 7 or 0)
A) Thyroid Hormone Synthesis

___ Incorporation of iodine with the tyrosyl residues of the thyroglobulin
___ Coupling of iodinated precursors to produce T 3 and T4
___ Oxidation of iodide to iodine via peroxidase action
___ Cleavage of thyroid hormones via protease action to release them
___ Iodide absorption by the thyroid gland from the plasma
B) Release of Thyroid Hormones from the Follicle

___ Lysosomal fusion
___ Formation of vesicles on apical membrane (endocytosis of thyroglobulin)
___ Activation of cAMP
___ Digestion of thyroglobulin to release thyroid hormones
___ Colloid droplets undergo phagocytosis
___ Phosphorylation of cellular proteins that mediate cellular processes
___ TSH binds to the basal side receptor of the follicular cell

LONG QUIZ 7
I) MATCHING TYPE: On one whole sheet of paper, match column A with column B from nos.1-90
regarding drugs of abuse, therapeutics, drug metabolites and antidotes. Letters only. You are not
Column A Column B
I) Drugs of abuse and their descriptions.
1. Only acidic group of drugs of abuse D. A. morphine

2. Tertiary amines with benzene rings J. B. amobarbital
3. Condensation product of urea and malonic acid N. C. phenytoin
4. Urinary metabolite of cocaine K. D. barbiturates
5. Popularly known as “shabu” S. E. met-enkephalin
6. Metabolite of marijuana I. F. diazepam
7. Also known as “hashish” causing red eyes R. G. MAO
8. Naturally produced opiate peptide in the brain E. H. phenobarbital
9. Opiate that is a powerful analgesic A. I. Δ9-tetrahydrocannabinol
10. Opiate that is used to treat cough O. J. opiates
11. Opiate that is the source of morphine & N-acetylmorphine Q. K. benzoylecgonine
12. Commercially available as Valium F. L. pentobarbital
13. Long-acting barbiturate H. M. LSD
14. Intermediate-acting barbiturate B. N. barbituric acid
15. Short-acting barbiturate L. O. codeine
16. Snorted drug derived from the Coca plant; known as “crack” T. P. phencyclidine
17. Enzyme inhibited by amphetamines G. Q. heroin
18. Also known as “angel dust” P. R. marijuana
19. Most commonly abused hallucinogen in the U.S. M. S. methamphetamine
20. Anticonvulsant structurally similar to phenobarbital; Dilantin C. T. cocaine
II) Drugs of abuse and their classes.
21. Codeine D. A. Sedative-hypnotics

22. Methaquaalone C. B. Dopaminergic stimulants
23. Methamphetamine B. C. Hallucinogens
24. Thiopental A. D. Opiates
25. Diazepam E. E. Tranquilizers
26. LSD C.
27. Propoxyphene D.
28. Methylphenidate B.
29. Morphine D.
30. Pentobarbital A.
31. Oxazepam E.
32. Benzoylecgonine B.
33. THC C.
34. Phenobarbital A.
35. Naloxone D.
36. Phencyclidine C.
37. Cocaine B.
38. Methadone D.
39. Amobarbital A.
40. Heroin D.

Column A Column B
THERAPEUTICS Part 1
41. cyclophosphamide, cyclosporin D. A. cardiotropic drug
42. phenobarbital, phenytoin B. B. anticonvulsant drug
43. chlorpromazine G. C. antiasthmatic drug
44. aspirin, acetaminophen E. D. immunosuppressive drug
45. theophylline, Ventolin C. E. antiphlogistic drug
46. methotrexate H. F. manic depressive drug
47. digitoxin, quinidine A. G. antipsychotic drug
48. lithium, doxepin F. H. chemotherapeutic drug
49. Haldol G.
50. Leucovorin H.
DRUGS & THEIR METABOLITES
51. Digitoxin E. A. salicylic acid and acetic acid

52. Procainamide H. B. PEMA
53. Primidone B. C. N-acetylmorphine
54. Ethosuximide I. D. benzoylecgonine
55. Theophylline J. E. digoxin
56. Aspirin A. F. THC
57. Heroin C. G. hydroxyquinidine
58. Cocaine D. H. NAPA
59. Marijuana F. I. desmethylmethosuximide
60. Quinidine G. J. 3-methylxanthine
THERAPEUTICS Part 2
61. cyclophosphamide D. A. cardiotropic drug

62. phenobarbital B. B. anticonvulsant drug
63. chlorpromazine G. C. antiasthmatic drug
64. aspirin E. D. immunosuppressive drug
65. theophylline C. E. antiphlogistic drug
66. methotrexate H. F. manic depressive drug
67. digitoxin A. G. antipsychotic drug
68. lithium carbonate F. H. chemotherapeutic drug
69. haloperidol G.
70. Leucovorin H.
71. cyclosporine D.
72. phenytoin B.
73. acetaminophen E.
74. ibuprofen E.
75. Ventolin C.
76. quinidine A.
77. doxepin F.
78. indiral A.
79. anti-histamine C.

80. Tylenol E.
DRUGS/TOXINS & ANTIDOTES
81. Acetaminophen J. A. Deferoxime

82. Aluminum A. B. Charcoal hemoperfusion
83. Arsenic C. C. Dimercaprol
84. Benzodiazepines G. D. Digibind
85. Cyanide F. E. Naloxone
86. Digoxin D. F. Amyl nitrite
87. Opioids E. G. Flumazenil
88. Organophosphorus H. H. Pralidoxime, atropine
89. Salicylates/TCAD I. I. Bicarbonate
90. Theophylline/Barbiturates B. J. N-acetylcysteine
II) MCQs: On the same answer sheet, write the letter that corresponds to the correct answer. You are not
1. The Trinder reaction is widely used for the colorimetric determination of

A. acetaminophen B. phenothiazine C. salicylate D. barbital
2. Reinsch test can detect poisoning from

A. carbon monoxide B. arsenic C. alcohol D. organophosphates
3. The confirmatory method for identification of drugs in body fluids is

A. AAS B. GC-MS C. HPLC D. FPIA
4. Which of the following serum components is able to alter the free drug level in plasma?
A. creatinine B. urea C. albumin D. calcium
5. All of the following statements are true regarding drug distribution patterns except
A. Drug metabolism is slower in newborn than adults.
B. Drug metabolism is more rapid for 6-year old children than for adults.
C. Renal clearance of drugs is faster in newborn than adults.
D. Drug metabolism often changes during pubescence.
6. When is a blood sample for the determination of the trough level of a drug appropriately drawn?
A. during the absorption phase of the drug
B. during the distribution phase of a drug
C. shortly before drug administration
D. two hours after drug administration
7. Free drug levels can generally be determined by analyzing what body fluid?
A. whole blood B. plasma ultrafiltrate C. urine D. PFF of plasma
8. An epileptic patient receiving phenytoin develops acute glomerulonephritis (AGN). What change, if any,
would be expected in the patient’s circulating drug level?
A. decrease in free drug C. increase in protein-bound drug

B. increase in free drug D. no change in circulating drug level
9. In what form must a drug be in order to elicit a pharmacologic response?

A. free C. bound to globulins
B. bound to albumin D. bound to fatty acids
10. What are the approximate half-life periods for a serum drug concentration to reach 97-99% of the steady-
state?
A. 1-3 B. 2-4 C. 5-7 D. 7-9
11. The first-pass effect after oral administration occurs in the

A. liver B. kidney C. colon D. intestine
12. The study of what the body does to the drug is

A. first-order kinetics C. pharmacokinetics
B. zero-order kinetics D. pharmacodynamics
13. The route of drug administration with 100% bioavailability is via

A. SQ B. IM C. IV D. intranasal
14. The study of what the drug does to the body is
A. first-order kinetics C. pharmacokinetics
B. zero-order kinetics D. pharmacodynamics
15. The route of drug administration with the least bioavailability is via
A. SQ B. IM C. rectal D. intranasal
16. The study of drug action or effects is
A. pharmacognosy C. pharmacokinetics
B. pharmacology D. pharmacodynamics
17. The painless route of drug administration is via
A. SQ B. IM C. IV D. intranasal
18. Which of the following drugs is used as bronchodilator?

A. Theophylline B. Phenytoin C. Amikacin D. Clozapine
19. What is the active metabolite of the antiarrhythmic drug procainamide?

A. Pronestyl B. Disopyramide C. PEMA D. NAPA
20. What is the recommended name for diphenylhydantoin?

A. Phenytoin B. Nalorphine C. Primidone D. Carbamazepine
21. Which of the following is an example of a phenothiazine drug?

A. Cyclosporine B. Theophylline C. Phenytoin D. Chlorpromazine
22. Which of the following is used to treat manic depression?

A. potassium B. lithium C. calcium D. chloride
23. What is the major active metabolite of the anticonvulsant drug primidone?
A. Phenytoin B. Acetazolamide C. NAPA D. PEMA
24. Which of the following is the most commonly encountered xanthine that could potentially interfere with the
determination of theophylline?
A. Nicotine B. Caffeine C. Amphetamine D. Procainamide
25. Which of the following drugs is used as an anti-cancer?

A. Methotrexate B. Amiodarone C. Tacrolimus D. Paroxetine
26. Acetaminophen is particularly toxic to what organ?

A. heart B. kidney C. spleen D. liver
27. Anticoagulated whole blood is the preferred specimen in determining exposure to what compound?
A. methanol C. acetaminophen
B. mercury D. carbon monoxide
28. Free erythrocyte protoporphyrin levels are useful as a screening method for exposure to which of the
following metals?
A. zinc B. lead C. iron D. mercury
29. The identification of the urinary metabolite ecgonine would be useful in determining exposure to which of
the following drugs?
A. Codeine B. Cocaine C. Amphetamine D. Propoxyphene
30. 9 THC is the principal active component of what drug?

A. Benzodiazepine B. Marijuana C. Morphine D. Codeine
31. Reinsch’s test is used to screen urine for toxic concentrations of all of the following except
A. bismuth B. arsenic C. mercury D. cyanide
32. Which of the following methods would yield reliable quantification of ethanol in the presence of other
alcohols?
A. reaction with permanganate and a chromotropic acid
B. Conway diffusion followed by dichromate reaction
C. Alcohol dehydrogenase reaction
D. Gas liquid chromatography
33. Levels of 8-9% Hb-CO saturation of whole blood are commonly found in which of the following situations?
A. fatal CO poisoning C. acute CO poisoning
B. nonsmoking residents of rural areas D. cigarette smokers
34. Bioavailability of a drug refers to the:

A. Availability of therapeutic administration.
B. Availability of the protein-bound fraction of the drug.
C. Drug transformation.
D. The fraction of the drug absorbed into the systemic circulation.
35. The drugs secobarbital & amobarbital are classified as:

A. Basic drugs C. Acidic drugs
B. Neutral drugs D. Unclassified
concentrations of:
A. Lead C. Arsenic
37. The most widely employed screening technique for drug abuse is:
A. High-performance liquid chromatography C. Thin layer chromatography
B. Gas-liquid chromatography D. UV spectrophotometry
38. Lithium therapy is widely used in the treatment of:

A. Hypertension C. Aggression
B. Hyperactivity D. Manic-depression
39. A drug that relaxes the smooth muscles of the bronchial passages is:
A. Acetaminophen C. Phenytoin
B. Lithium D. Albuterol
40. A cardiac glycoside that is used in the treatment of congenital heart failure and arrhythmias by increasing
the force and velocity of myocardial contraction is:
A. Digoxin C. Lithium
B. Acetaminophen D. Phenytoin
For nos. 41-50, match the poison with their distinctive properties.
41. wood alcohol; produces metabolic acidosis B. A. ethanol

42. basophilic stippling in RBCs; produce hypochromic anemia D. B. methanol
43. presence of CaOx crystals in urine; anti-freeze C. C. ethylene glycol
44. the most common toxic substance A. D. lead
45. measurement of carboxyHb; cherry red face E. E. carbon monoxide
--------------------------------------------------------------------------------
46. assay of CHS & AcCHS; affects parasympathetic nerves E. A. arsenic

47. garlic odor of breath; positive Reinsch test A. B. mercury
48. may result to hemosiderosis D. C. cyanide
49. measurement of HiCN; breath with odor of bitter almonds C. D. Iron
50. dimercaprol or penicillamine can be used as treatment B. E. organophosphate
51. Which of the following drugs is used as bronchodilator?

A. Leukotriene B. Phenytoin C. Amikacin D. Clozapine
52. What is the active metabolite of the primidone?

A. Pronestyl B. Disopyramide C. PEMA D. NAPA
53. Which of the following compounds has antitussive property?

A. Amphetamine B. Codeine C. Cannabinoids D. Benzodiazepine
54. What is the brand name for haloperidol?

A. Phenytoin B. Nalorphine C. Primidone D. Haldol
55. Which of the following is an example of antiepileptic drug?

A. Cyclosporine B. Carbamazepine C. Quinidine D. Chlorpromazine
56. Which of the following is used to treat manic depression?
A. La B. Li2CO3 C. Lu D. Le
57. Methylxanthine is the metabolite of which of the following drugs:

A. Amitriptyline B. Desipramine C. Imipramine D. Theophylline
58. Which of the following can be used to treat arrhythmias?

A. Phenytoin B. Acetazolamide C. NAPA D. Phenobarbital
59. Which of the following is the most commonly encountered xanthine that could potentially interfere with the
determination of theophylline?
A. Nicotine B. Caffeine C. Amphetamine D. Procainamide

60. Which of the following drugs is used as analgesic?
A. Methotrexate B. Amiodarone C. Morphine D. Paroxetine
61. Which of the following is an antiarrhythmic drug with a metabolite with the same function?
A. Quinidine B. Digoxin C. Procainamide D. Nortriptyline
62. Increased trough levels of aminoglycosides in the serum are often associated with toxic effects to what
organ?
A. heart B. kidney C. pancreas D. liver
63. Which of the following is an example of a intermediate -acting barbiturate?

A. Phenobarbital B. Amobarbital C. Secobarbital D. Pentobarbital
64. Acetaminophen is particularly toxic to what organ?

A. heart B. kidney C. spleen D. liver
65. Anticoagulated whole blood is the preferred specimen in determining exposure to what compound?
A. methanol C. acetaminophen
B. mercury D. carbon monoxide
66. Free erythrocyte protoporphyrin levels are useful as a screening method for exposure to which of the
following metals?
A. zinc B. lead C. iron D. mercury
67. Of the following specimens, which of the following would be appropriate for determining exposure to lead?
A. EDTA plasma B. serum C. whole blood D. CSF
68. The identification of the urinary metabolite ecgonine would be useful in determining exposure to which of
the following drugs?
A. Codeine B. Cocaine C. Amphetamine D. Propoxyphene
69. Heroin is synthesized from what drug?

A. Diazepam B. Morphine C. Ecgonine D. Chlorpromazine
70. 9 THC is the principal active component of what drug?

A. Benzodiazepine B. Marijuana C. Morphine D. Codeine
71. After absorption, codeine is metabolized to what compound?

A. Phencyclidine B. Morphine C. Methadone D. Propoxyphene
72. Reinsch’s test is used to screen urine for toxic concentrations of all of the following except
A. bismuth B. arsenic C. mercury D. cyanide
73. Which of the following tests would be particularly useful in determining exposure to isopropanol?
A. serum osmolality and urine acetone C. urine acetone and urine osmolality
B. urine osmolality and serum osmolality D. serum sodium and serum acetone
74. Which of the following methods would yield reliable quantification of ethanol in the presence of other
alcohols?
A. reaction with permanganate and a chromotropic acid
B. Conway diffusion followed by dichromate reaction
C. Alcohol dehydrogenase reaction
D. Gas liquid chromatography

75. Levels of 8-9% Hb-CO saturation of whole blood are commonly found in which of the following situations?
A. fatal CO poisoning C. acute CO poisoning
B. nonsmoking residents of rural areas D. cigarette smokers
76. A urine screening test for porphobilinogen is positive. The MOST likely disease state is:
A. lead poisoning C. lithium poisoning
B. arsenic poisoning D. Mercury poisoning
77. Bioavailability of a drug refers to the:

A. Availability of therapeutic administration.
B. Availability of the protein-bound fraction of the drug.
C. Drug transformation.
D. The fraction of the drug absorbed into the systemic circulation.
78. The cyclic antidepressants are classified as:

A. Basic drugs C. Acidic drugs
B. Neutral drugs D. Unclassified
concentrations of:
A. Lead C. Arsenic
80. Gas chromatography with the nitrogen/phosphorus detector is the most commonly used technique for the
analysis of:
A. Digoxin C. Ethyl alcohol
B. Acetysalicylic acid D. Cyclic antidepressants
81. The most widely employed screening technique for drug abuse is:
A. High-performance liquid chromatography C. Thin layer chromatography
B. Gas-liquid chromatography D. UV spectrophotometry
82. THC is a metabolite of:

A. Cocaine C. Opiate
B. Marijuana D. Phencyclidine
83. Lithium therapy is widely used in the treatment of:
A. Hypertension C. Aggression
B. Hyperactivity D. Manic-depression
84. The anticonvulsant used to control tonic-clonic (grand mal) seizures is:
85. A drug that relaxes the smooth muscles of the bronchial passages is:
A. Acetaminophen C. Phenytoin
B. Lithium D. Theophylline
86. A cardiac glycoside that is used in the treatment of congenital heart failure and arrhythmias by increasing
the force and velocity of myocardial contraction is:
87. A carbonate salt used to control manic-depressive disorders is:

88. Which of the following elevates carboxyhemoglobin?
A. Nitrite poisoning C. sulfa drug toxicity
B. exposure to carbon monoxide D. sickle cell anemia
89. The reason carbon monoxide is so toxic is because it:

A. is a protoplasmic poison
B. combines with cytochrome oxidase
C. has 200 times the affinity of oxygen for hemoglobin binding sites
D. sensitizes the myocardium
90. When the clinical response does not agree with total drug concentration, free drug levels may be of clinical
use in all of the following cases, EXCEPT:
A. uremia C. ingestion of ther drugs
B. hypoalbuminemia D. patient noncompliance
91. Testing for the diagnosis of lead poisoning should include:

A. ion-exchanged analysis of urine for porphobilinogen
B. analysis of morning urine for delta-aminolevulinic acid
C. analysis of feces for porphyrin
D. ion-exchange analysis of feces for protoporphyrin
92. The main reason for suboptimal drug levels in therapeutic monitoring is:
A. Renal failure
B. Liver failure
C. Improper dosage prescribed
D. Patient noncompliance with dosage regimen.
93. About 90% of phenytoin is excreted in the urine as:
A. Phenobarbital C. Phenobarbital
B. Para-hydroxyphenyl phenylhydantoin D. N-acetylprocainamide
94. Nortriptyline is a metabolite of:

A. Amitriptyline C. Butriptyline
B. Protripyline D. Norbutriptyline
95. Cocaine is metabolized to:

A. Carbamazepine C. Hydrocodone
B. Codeine D. Benzoylecgonine
96. If a drug has a half-life of 7 hours, how many doses given at 7-hour intervals does it usually take to achieve
a steady state or plateau level?
A. One C. Five
B. Three D. Eight
97. An analgesic that alleviates pain without causing loss of consciousness is:
98. Thin-layer chromatography is of particular use in the identification of:

A. Lipids C. Inorganic ions
B. Drugs D. Enzyme inhibitors

99. A blood sample was received from the emergency room with a request for narcotic analysis. The request
slip indicated that the patient (a known heroin addict) responded to naloxone administration. The serum was
negative for morphine by EIA. What other tests should be performed in assessing suspected opiate abuse?
A. Heroin C. Alcohol
B. Salicylate D. Methaquaalone
100. After adding 8 drops of 10% ferric chloride to 5 ml of urine, the mixture develops a stable deep red purple
color. This is presumptive evidence of:
A. Lead C. Salicylates
B. Bromide D. Quinidine
101. The most common substance of abuse is

A. Acetone C. Distilled water
B. Ethanol D. Methyl alcohol
102. Four children are admitted with malaise, anorexia and abdominal pain. Further evaluations reveal mild
anemia, erythrocyte basophilic stippling and profound pica habits. Poisoning by which heavy metal is most
likely responsible?
A. Arsenic c. Mercury
B. Iron D. Lead
103. The most toxic among the alcohols is

A. Acetone C. Isopropyl alcohol
B. Ethanol D. Methyl alcohol
104. Production of alcoholic beverages by fermentation is probably among the first chemical processes
employed by humans. Alcohol concentration is commonly described as the proof number. Nineteenth-century
fur traders lacking testing equipment could determine proof of spirit. They would pour a small amount of black
gunpowder onto a stump, add some of the alcohol, and set it afire. If the alcohol were at least 50% (v/v), the
gunpowder would ignite after the liquid burned off and the preparation was described as 100 proof. Suppose that
a certain whiskey were 45% (v/v). The alcohol proof number would be:
A. 10 B. 20 C. 22.5 D. 45 E. 90
105. The most toxic among the arsenicals is the gaseous

A. tryparsamide C. arsenic sulfate
B. arsine D. organic arsenic
GOOD LUCK !!!

LONG QUIZ 8
1. What is the most important role of a DTL in solving societal problems?

The most important role of DTL in solving societal problems is to regulate the number of illegal
drug users to secure the safety and minimize the health concerns in the society brought about
by the usage of these prohibited drugs.
2. Why do people subject themselves to drug testing? Give three answers.

If they have been ordered by the court. If they are employees of public and private offices for
the company’s work rules and if they are secondary/tertiary students to prevent present and
future drug use.
3. Name three (3) governmental agencies that help in ensuring the smooth operation of DTL in
the Philippines.
Department of Health through Bureau of Health Facilities and Services, Department of Social
Welfare and Development and Philippine Drug Enforcement Agency.
4. Give two (2) differences between a screening lab and a confirmatory lab.
Screening lab provides presumptive test that determines + result and the type of drug used
while that of confirmatory is conclusive and more specific and confirms a + screening test.
5. Cite three (3) clinical sample-drug combinations that are commonly encountered in DTL.
MDMA or Methylenedioxymethamphetamine(ecstasy), Methamphetamine Hydrochloride
(Shabu) and Cannabis/ Synthetic Cannabis (Marijuana/Synthetic Marijuana).
6. What is the RA 9165 all about?

Mandates the prohibition of the usage of illegal drugs, includes the monitoring of the
integration of all drug rehabilitation, intervention, aftercare, follow-up programs as well as the
penalties of those involved in the usage and operation of drug establishments.
7. According to your group, what is the most menacing drug in the Philippines? Explain
For us, methamphetamine hydrochloride/shabu is the most menacing drug in the Philippines
because according to our research, it is the most used/prevalent one followed by marijuana.
Most of Filipino drug addicts were caught with “shabu’’ which has the capacity to cause
unsound mind and can contribute to insanity and mental disorders and destroying their lives as
a whole.
8. Cite three functions of the NRL for Toxicology Assay.

It provides laboratory reference for confirmatory testing, maintains quality assurance program
and trains laboratory personnel.
9. Name three individuals who can become a DTL Analyst.

Registered Medical Technologists, Chemical Engineers and Pharmacists.
10. What are the training requirements for the Authorized Specimen Collector? Give three.
Qualification training, Initial Proficiency Demonstration and Error Correction Training.
11. Describe the best collection device for urine specimen in drug testing.
Multi-Drug Integrated Split Specimen Test Cup is a urine collection device with a built-in
qualitative test panel for the simultaneous detection of multiple drugs and metabolites in
human urine without specimen handling. It is intended for professional in vitro diagnostic only.
12. What are 3 out of the many functions of the Analyst of DTL?
Supervises the operations including collection and encoding of results; interprets, records and
releases results and ensures that all legal requirements are compiled.
13. In what two (2) ways do you think can the government solve the drug-related problems in the
Philippines based on the RA 9165?
It can be solved by pursuing intensive campaign against the use of illegal drugs through the
implementation of anti- drug abuse programs and enforcing penalties to those found guilty of
using it.
14. If you are chosen to be part of random drug testing, are you amenable to it? Reason out why
or why not.
As a righteous citizen of the country who understands well the purpose of having random drug
testing, I am willing to do it to follow my nation’s rules and regulations. As long as it is for the
betterment of the country, I am willing to support it.
15. What do you think is the worst drug-related problem that this country have and why?
I guess, it’s the Philippines’ War on Drugs the previous year. I can say that it’s worst because it
doesn’t follow proper due process which involves the killing of the innocent ones.
16. How can you help solve the drug-related problems in our country?
I would educate the people regarding the risks and health complications that these drugs will
cause them and giving them knowledge on how they could recover from it; institutions they
would go to and practices they can follow.
17. Give three characteristics of the prescribed Collection Site for urine in a DTL.
A restroom with toilet for the employee to have privacy while providing the urine specimen; a
source of water (if practical, external) for washing hands and a suitable, clean surface as a
work area.
18. Cite three testing reasons for Oral Fluid according to DTL.
Three testing reasons for oral fluid are for pre-employment, random testing and reasonable
suspicion/cause.
19. How is the integrity of the urine for drug testing ensured?

Procedures and protocols are strictly followed from the collection of the specimen, transport
and analysis in accredited testing laboratory and other precautions such as eliminating water
sources and prohibiting personal belongings in the collection room.
20. Compare and contrast a COC from an MFR.

Chain of Custody references a paper trail/ a course of action of documenting the manage of
storage of a specimen from the moment a donor provides the specimen to the final destination
of the specimen and review and reporting of the final test result while Memorandum for
Records contain information that is usually not recorded in writing during a process. Both are
essential in maintaining the integrity of the patient specimen in DTL as well as integrity of
evidence

MLS 045 – Clinical Chemistry 2
ANSWERS to Modules
2 to 8 INSTRUCTOR’S GUIDE BS Medical Technology / Third Year
Session # 2
Module 2
You have an experience of encountering a lot of parts or aspects of the course and relate them with one another.
It is now time to test your ability to organize the following activities within the scope of the course by suggesting
where to put them in the illustration above: (NOTE: You can look for the definition of the terms below.)
MEASUREMENT/DETECTION: 2
ELECTROLYTES AND TRACE METALS: 19, 27, 30,
BLOOD GASES: 22, 20
PRODUCTS AND BY-PRODUCTS OF METABOLISM: 8, 12, 21
ENZYMES: 3, 5, 10, 24, 28, 17
VITAMINS: 9
TUMOR MARKERS: 29, 23, 15
HORMONES: 11, 18, 12
XENOBIOTICS (DRUGS AND POISONS): 26, 1, 6, 7, 13, 14, 25, 4

1. Pharmacokinetics 16. LSD
2. Instrumentation 17. Lineweaver-Burk plot
3. Szasz method 18. Zona fasciculata
4. Vaccines 19. TPTZ
5. De Ritis ratio 20. Hamburger phenomenon
6. LD50 21. Reye syndrome
7. Chromatographic separation 22. Bohr effects
8. Allen correction method 23. Tartrate inhibition
9. Avidin-binding method 24. LD-flipped pattern
10. Wroblewski-LaDue method 25. Pharmacodynamics
11. Clonidine Inhibition Test 26. Hashish
12. OGTT 27. Gibson & Cooke iontophoresis
13. Biotransformation 28. Turnover number
14. Bioconcentration 29. tdt
15. Philadelphia chromosome 30. Glucose tolerance factor
Module 3
Check for Understanding
I. Classify the different liver function tests
1. Excretory Function Tests

• Test for urobilinogen and other bilirubin pigments in the urine and feces
• Icterus index
• Dye excretion tests
• Serum bilirubin
2. Metabolic Function Tests
• Carbohydrate Metabolism (Galactose tolerance test and Oral Glucose Tolerance Test)
• Protein Metabolism (TP, A/G ratio, Cephalin cholesterol flocculation test, and Hippuric Acid Test)
• Lipid metabolism (Lipid profile)
3. Enzyme Tests
• Alanine aminotransferase
• Aspartate aminotransferase
• De Ritis ratio (AST/ALT)
• Lactate dehydrogenase
• Isocitrate dehydrogenase
• Ornithine Carbamoyl transferase
• Sorbitol dehydrogenas3
• Alkaline phosphatase
• Gamma-Glutamyl transferase
• 5’-Nucleotidase
• Leucine aminopeptidase
Study the illustration showing the normal bilirubin biosynthesis and excretion starting from hemoglobin to
urobilinogen for 5 minutes.

After 5 minutes, answer the following questions regarding the bilirubin synthesis:
1. What is the starting material for bilirubin formation?

Answer: Hemoglobin/Erythrocyte/Heme/Intravascular hemolysis
2. What is the parent compound for bilirubin?
Answer: Heme
3. Where is bilirubin produced?
Answer: Reticuloendothelial system (Spleen and bone marrow)
4. What structural changes happen from hemoglobin to bilirubin production?
Answer: rupture of alpha methene bridge and the addition of hydrogen through biliverdin reductase
5. What happens to the bilirubin in the blood? In the liver? In the intestine?
Answer: Bilirubin binds to albumin and is transported to the liver. In the liver, bilirubin is conjugated
with the help of your UDPGT and moves to your intestine. In the intestine, conjugated bilirubin is
broken down into urobilinogen.
II. What are the 2 most common methods in bilirubin determination? Describe the principle of how
bilirubin is measured in each method.
1. Evelyn-Malloy Method: the diazotized product has a red to reddish-purple color in an acid pH. It has an
absorption maximum at 560 nm. Methanol is used as dissociating agent to measure the total bilirubin.
2. Jendrassik-Grof Method: Sodium acetate (buffer) and caffeine sodium benzoate (coupling accelerator)
are used. The diazotization is terminated by the addition of ascorbic acid. With the use of strong alkaline
tartrate solution, the pink azobilirubin is then converted to a blue azobilirubin.

Module 4
Check for Understanding
I. List the major electrolytes and give their normal value

ELECTROLYTE NORMAL VALUE
Sodium ECF: 135-145 mmol/L

ICF: 4-10 mmol/L
Potassium Serum: 3.8-5.0 mmol/L
Calcium Serum Total Calcium: 8.5-10.4 mg/dL

Ionized Calcium: 4.68-5.32 mg/dL
Magnesium Serum: 1.3-2.1 mEq/L
Chloride ions Sweat Chloride: 115-132 mmol/L
Inorganic phosphate Serum: 3.0-4.5 mg/dL
II. Enumerate the tests used to detect these electrolytes
ELECTROLYTE TEST/S
Flame Emission Photometry

Sodium Atomic Absorption Spectrophotometry
Ion-Selective Electrode
Colorimetric Method (Albanese-Lein)
Potassium Flame Emission Photometry
Atomic Absorption Spectrophotometry
Ion-Selective Electrode
Colorimetric Method (Lockhead and Purcell)
Calcium O-cresolphthalein Complexone Method
Ferro-Ham Method
Clark-Collip Method
Diehl-Ellingboe Method
Ethylenediaminetetracetic acid
Chloride Ion-Selective Electrode
Zall Color Reaction
Schales and Schales
Magnesium Ion-Selective Electrode

Atomic Absorption Spectrophotometry
Colorimetric Method
Fluorometric Analysis
Inorganic phosphate Fiske-Subbarow Method
Daly-Ertingshausen Method
III. List down the disease/s associated with below and above normal levels of electrolytes in the body

ELECTROLYTE BELOW NORMAL ABOVE NORMAL
Sodium Syndrome of Inappropriate Secretion of ADH Diabetes insipidus

Glucocorticoid deficiency Gastroenteritis
Nephrotic Syndrome Thyrotoxicosis
Cirrhosis
Potassium Leukemia Hypoaldosteronism
Renal Tubular Acidosis Chronic Interstitial Nephritis
Hyperaldosteronism Acute Renal Failure
IV. Choose the letter of the correct answer and write it on the blank provided before the number.
A 1. Which hormone promotes increased sodium retention?

A. Aldosterone C. Both
B. Antidiuretic hormone D. Neither
D 2. Which of the following indicate/s pseudohyponatremia?

A. Hyperglycemia C. Hyperproteinemia
B. Hyperlipidemia D. All of the above
A 3. Which of the following cause/s hyperkalemia?

A. Addison’s disease C. Both
B. Cushing syndrome D. Neither
C 4. What is the major extracellular anion?

A. Sodium C. Chloride
B. Potassium D. Calcium
D 5. What analyte is important in coagulation, complement fixation and myocardial contraction?

A. Sodium C. Chloride
B. Potassium D. Calcium
B 6. Phosphorus has a/an _____________ relationship with Calcium.

A. Direct C. Both
B. Inverse D. None of the above
C 7. Regulators of Calcium and Phosphorus levels is/are:

A. Vitamin D C. Both
B. Parathyroid hormone D. Neither
A 8. Which of the following is the most abundant form of calcium?

A. Free Calcium C. Protein bound
B. Complexed Calcium
B 9. What element is essential for the insulin-mediated entry of glucose into cells?
A. Calcium C. Bicarbonate
B. Phosphate D. Magnesium
C 10. This ion diffuses out of the cell in exchange for chloride to maintain pH balance.
A. Calcium
B. Phosphate
C. Bicarbonate
D. Phosphorus

Module 5
Match column A with column B regarding the electrolytes and their associated properties, method of
testing, and terminologies. Write your answer before the number. Letters only. There are more-than-
one answer occasionally as indicated by a number after the phrase.
Column A Column B
41. Contributes 92% to ECF osmolality - A A. Sodium
42. Most abundant cation in the human body - D B. Potassium
43. Mostly affected by even the slightest of blood laking - B C. Chloride
44. Produced daily in the stomach -C D. Calcium
45. Has renal threshold of 110 mmol/L - A E. Phosphorus
46. Counterion in the sodium pump process - B F. Magnesium
47. Has bromide as its common interferent -C G. Copper
48. Has calcium as its interfering species (2) – F, H H. Iron
49. Effectively removed from solution with hydroxyquinoline - F
50. Measured in sweat together with chloride to screen for cystic fibrosis- A
Part 3A: Check for Understanding
I. Write the Henderson-Hasselbach equation correctly on the main blood buffer system and
do the following: (20 points)
6. Encircle the metabolic component
7. Underline twice the respiratory component
8. Give the value for the pKa
9. Give the normal range for the blood pH
10. Give the normal ratio of the acid with the base
pH = pKa + log (HCO₃¯) (HCO₃

(H₂CO₃) ¯)
pH = 6.1 + log
(H₂CO₃)
pH: 7.35-7.45
Acid:Base: 1:20

Part 3B: Check for Understanding
I. Interpret the given BGA results
1. pH: 7.32
pCO₂: 28 mmHg
HCO₃¯: 13 mmol/L
DUAL CASE OF ACIDOSIS
2. pH: 7.46
pCO₂: 26 mmHg
HCO₃¯: 19 mmol/L
PARTIALLY COMPONSATED RESPIRATORY ALKALOSIS
3. pH: 7.10
HCO₃¯: 20 mmol/L
pCO₂: 70 mmHg
DUAL CASE OF ACIDOSIS
4. pH: 7.48
HCO₃¯: 24 mmol/L
pCO₂: 32 mmHg
NON-COMPENSATED RESPIRATORY ALKALOSIS
II. Indicate in the table whether the parameters are low, normal or high in the given conditions.
CONDITION PCO2 Bicarbonate pH

Dual case of Alkalosis LOW HIGH HIGH
Metabolic Acidosis NORMAL LOW LOW

uncompensated
Respiratory Alkalosis LOW HIGH HIGH

Metabolic Alkalosis HIGH HIGH HIGH

Instrumental error NORMAL NORMAL HIGH
Module 6
Part 3A: Check for Understanding
I. Give three trace elements essential for growth and development

1. Iron
2. Zinc
3. Iodine
4. Selenium

II. Give four trace elements known to be toxic if present
1. Aluminum
2. Beryllium
3. Cadmium
4. Mercury
5. Lead
6. Arsenic
You have to be familiar also with the common electrolyte patterns in the clinical setting. You are given a table
with eleven (11) commonly encountered electrolyte patterns involving the four (4) principal ions found in the
body namely: sodium ion, potassium ion, bicarbonate ion, and chloride ion. You need to come up with
explanation for each of the said patterns except for one which will be made as an example. The electrolyte
pattern involving severe diarrhea wherein all the principal ions are decreased in blood is due to the uncontrolled
huge losses of the ions during the egestion of watery and electrolyte-heavy stools by the patient with the
diarrhea. The electrolyte balancing mechanisms cannot act as it usually does because the diarrhea is severe
in that the body fluid loss is abrupt and in large amounts. The body can only respond to subtle changes in
electrolyte concentrations and hydration state.
Now, it is your turn to reason out why the remaining ten (10) electrolyte patterns happen given the conditions
opposite each. This portion is part of the assessment of how you retain, retrieve, and reuse stored knowledge
in new learning situations. Your answers should be placed in a whole sheet of paper to be turned in the next
face-to-face meeting.
Clinical Condition Na+ K+ HCO3- Cl-

Dehydration High Normal Normal or High
Low
Diarrhea Low Low Low Low
Congestive Heart Failure Normal or Normal Normal Low

Low
Malabsorption Syndrome Low Low Normal or Normal
Low
Pyloric Obstruction Low Low High Low
Acute Renal Failure Low High Low High
Pulmonary Emphysema Normal Normal High Low
Diabetic Acidosis Low Normal or Low Low

High
Excessive Sweating Low Normal Normal Low
Starvation Normal Low Low Normal
Ammonium chloride administration Low Low Low High
Part 3B: Check for Understanding

Provide a correct explanation of the effects on the specified electrolytes for each of the following clinical
conditions:
1. ECF principal ions in Water Deficit
Less water will make the ECF ions appear elevated
2. ICF principal ions in Starvation

Sodium appears normal because of renal reabsorption. Chloride concentration is dependent on
hydrochloric acid production in the stomach, chloride only decreases when there is loss of HCl
via vomiting. There’s no renal threshold for potassium and it is diet dependent.
Bicarbonate: Glucose (C₆H₁₂O₆) + O₂ → CO₂ + H₂O + Energy
3. Principal cations in Acute Renal Failure

In Renal failure, sodium is excreted while potassium is retained. Hyperchloremia is due to
decreased production of bicarbonate in the cell and failure of the kidney to reabsorb
bicarbonate.
4. Principal anions in Pulmonary Emphysema

Oxygen will be decreased with resultant hypercapnia. Increased in CO₂ will result in high
bicarbonate levels. Chloride will enter the cell for electrical neutrality. Sodium and potassium will
only be affected once there’s a gastrointestinal or kidney involvement.
5. All principal ions in Diarrhea

All the principal ions are decreased in blood due to the uncontrolled huge losses of the ions during
the egestion of watery and electrolyte-heavy stools by the patient with the diarrhea.
Module 7
Part 3: Check for Understanding
I. You are now given some questions to answer correctly in order to assess the extent of your learning. They
are as follows:
◆ What are the types of pure enzyme inhibition studies?
1. COMPETITIVE INHIBITION
2. NON-COMPETITIVE INHIBITION
3. UNCOMPETITIVE INHIBITION
◆ Why are enzyme inhibition studies performed in the laboratory?
It is where the scientist will find out whether the enzyme is an inhibitor or activator.
◆ What is the most important application of enzyme inhibition studies?
Discovery of drugs (Application in pharmacology)
◆ How can one differentiate competitive and non-competitive inhibition?
Enzyme in competitive inhibition binds at the catalytic site while enzyme in non-competitive
inhibition binds other than the catalytic site.
◆ Which theory on substrate-enzyme interaction is more acceptable to scientists? Reason out why.

Koshland’s Induced Fit model. This model proposes that the initial interaction between enzyme
and substrate is relatively weak, but that these weak interactions rapidly induce conformational
changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds
to be altered. After binding takes place, one or more mechanisms of catalysis generates transition-
state complexes and reaction products.
◆ Compare and contrast between a coenzyme and a prosthetic group.
Coenzyme and Prosthetic group are both non-protein component. Prosthetic group attachment to
apoenzyme is covalent (Strong). While coenzyme is non-covalently bound to apoenzyme (Weak).
◆ Differentiate a metalloenzyme from metal-activated enzyme.
Metalloenzymes are enzymes that contain a tightly bound metal ion while metal-activated enzymes
are enzymes that are not covalently bound to an apoenzyme.
◆ How does the body control enzyme activity?
Enzyme activity is controlled by gene regulation, compartmentalization and via the presence of an
inhibitor or an activator.
◆ What are the means with which enzyme speed up chemical reactions?
Lowering the activation energy by creating an environment in which the transition state is
stabilized. Lowering the energy of the transition state, but without distorting the substrate, by
creating an environment with the opposite charge distribution to that of the transition state.
Providing an alternative pathway. Reducing the reaction entropy change by bringing substrates
together in the correct orientation to react.
◆ Degradative methods requires the presence of enzymes. Explain why.
Enzymes are effective in catabolizing (breakdown) substances like the nutrients. These nutrients
are broken down into digestible forms. Out of the 6 classes of enzymes, 5 classes are degradative.
II. Enumerate the following as delimited by the descriptions given:
1 - 8 - General Properties of Enzymes
Almost are proteins (ribozymes are exceptions), thus have the physicochemical properties of proteins
Heat labile
Water-soluble
Can be removed by salting out process
Made of 16% nitrogen
Deficiency or lack will lead to inborn errors of metabolism
Suffix –ase; prefix pro- & suffix –ogen are inactive forms
9 - 13 - Properties of coenzymes
Transfer a functional group

Essential for enzyme activity
Heat stable low MW organic substance
Combines loosely with apoenzyme
Can be separated from apoenzyme via dialysis
Can bind to two or more substances
Derivatives of vitamin B complex substances
Involved in oxidoreductases (e- transport) and non-electron transport mechanisms
14 - 18 - Mechanisms of controlling enzyme activity
Enzyme gene regulation.

Enzyme compartmentalization.
Enzymes can be regulated in the presence of inhibitors and activators.
Enzymes can be regulated through post-translational modification.

III. Discuss your answers to the following questions
1. Discuss the application of competitive enzyme inhibition in the clinical setting by citing two (2) examples.
DRUG ENZYME CLINICAL USE
Allopurinol Xanthine Oxidase prevention of Gout
Methotrexate Dihydrofolate reductase Anti-cancer
2. Explain the relationship between Km and enzyme affinity.

The relationship between Km and enzyme affinity is inversely proportional. A low Km value of an enzyme
reflects the high affinity of enzyme for the substrate, whereas a high Km value of an enzyme reflects the
low affinity of enzyme for the substrate.
Module 8
Part 3: Check for Understanding
I. You are now given some questions to answer correctly in order to assess the extent of your learning. They
are as follows:
◆ Cite three (3) enzymes that can be used to diagnose cardiac disorders.
Creatine kinase, LDH, HBD, AST
◆ What five (5) enzymes that can be used to diagnose liver disorders?
Parenchymal liver disease: AST, ALT, LD, OCT, SDH; Hepatobiliary disease: LAP, ALP, 5’-NT,
GGT
◆ What is the reaction principle in AST assay? CK assay? CHS assay? AMS assay?
AST:
Aspartate + alpha-ketoglutarate (AST, Pyridoxal phosphate) Glutamate + Oxaloacetate
CK:
CK
Creatine-Phosphate + ADP  Creatine + ATP
Hexokinase, Mg++
ATP + Glucose --------------→ Glucose-6-Phosphate + ADP
G6PD
Glucose-6-Phosphate +NADP → Phosphogluconolactone + NADPH
CHS:
Acetylcholine (Cholinesterase) Acetic acid + Choline
AMS:
Amylase
Starch → maltose + glucose
Maltase
Matlose → glucose
Glucose oxidase
Glucose → gluconic acid + H2O2
◆ Name two (2) enzymes that are indicative of prostatic cancer.

Acid phosphatase, Prostate-specific antigen
◆ Cite ALP isoenzymes that migrate electrophoretically together with the placental type.
Regan isoenzymes, Nagao isoenzymes, Ishihara isoenzyme
◆ What coenzymes facilitate Class 1 enzymes?
NAD, NADP, FAD
◆ Which vitamins give rise to coenzymes that transport organic functional groups?
Folic acid, Biotin, Pantothenic acid, Ascorbic acid, Thiamine, Riboflavin, Pyridoxamine, Niacin
◆ Give five (5) properties of coenzymes that are not found in enzymes.
Heat stable Can pass through dialysis membrane
Low molecular weight Derived from water soluble vitamins
Non-protein
◆ How is enzyme activity measured?
Enzyme activity can be measure by the amount of substrate converted to products, catalytic action
per milligram of protein and the amount of substrate converted by a single enzyme.
◆ How is the conventional unit of enzyme activity expressed in IU/L converted to microkatal/L?
International unit/Liter (IU/L) x 0.0067 = microkatal/L (µkat/L)
IU/L x 6.7 = millikatal/L
IU/L x 6,667 = katal/L
◆ Enumerate enzymes that can be separated via chemical inhibition? Heat inhibition?
Chemical inhibition: ACP, ALP, LD
Heat inhibition: ALP bone isoenzymes
II. MATCHING TYPE. Match column A with column B. You can use a lettered choice more than once.
Enzymes and their principal clinical applications

1. Amylase & lipase - C A. cardiac disorders
2. AST/ALT ratio - B B. liver diseases
3. LD1&2, CK-MB & AST - A C. acute pancreatitis
4. Acid phosphatase - E D. bone and obstructive liver diseases
5. Alkaline phosphatase - D E. prostatic cancer
F. brain disorders
III. Directions: Encircle the letter that corresponds to the correct answer for each of the following items.
1.) Which of the following conditions would a NORMAL level of creatine kinase be found?
A. Acute myocardial infarct
B. Hepatitis
C. Progressive muscular dystrophy
D. Intramuscular injection

Aminotransferase enzymes (AST and ALT) catalyze the
A. Exchange of phosphate groups with sulfur-containing acids
B. Exchange of amino and keto groups between alpha-amino and alpha-keto acids
C. Hydrolysis of amino acids to ammonia and carbonate
D. Reversible transfer of hydrogen from amino acids to coenzymes
2.) The enzymes that exists chiefly in skeletal muscle, heart and brain; is grossly elevated in
active muscular dystrophy; and rises early in myocardial infarction is
A. Lipase C. Lactate dehydrogenase
B. Transaminase D. Creatine kinase
3.) The enzymes present in almost all tissues that may be separated by electrophoresis into
five
components is
A. Lipase C. Creatine kinase
B. Transaminase D. Lactate dehydrogenase
4.) Measurement of the serum acid phosphatase is used to detect neoplastic disease of
the
A. Liver C. Ovary
B. Lung D. Prostate

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CONTENTS:

Sir M (LEC VID) NOTES

On which organism or sample was the term enzyme literally associated? *

Who gave the word “enzymes” to catalytic molecules? *

Which enzymes are used to diagnose liver diseases? *

Kernicterus is an abnormal accumulation of bilirubin in: *

Which vitamin is required for the normal absorption of dietary calcium: *

It has bromide as its common interferent in its analysis *

Which of these is NOT a liver function test: *

Respiratory acidosis is described as an a (n): *

An electrophoretic separation of lactate dehydrogenase isoenzymes that demonstrates

In the determination of lactate dehydrogenase at 340nm, using pyruvate as the

The aldehyde transport coenzyme is derived from: *

It is one of the best tests for bilirubin: *

Which method uses sulfanilic acid, HCl and sodium nitrite? *

The transport material associated with bilirubin in the liver is? *

Select the polypeptide chain combination designated LD 5. *

A scanning of a CK isoenzyme fractionation revealed two peaks: a slow cathodic peak

What metal is the most affected by even the slightest hemolysis?

The sum of carbonic acid and bicarbonate in plasma is referred to as? *

What substance gives feces its normal color: *

In respiratory acidosis, a compensatory mechanism is the increase in: *

Formation of bile acids is the major mechanism for eliminating: *

Indirect-reacting bilirubin may be quantified by reacting it initially in which reagent? *

Zinc protoporphyrin or free erythrocyte protoporphyrin measurement are useful to

A breakdown product of hemoglobin is: *

In which year James Sumner isolated and crystallized urease? *

What is an inorganic cofactor? *

Increased levels of indirect bilirubin only is seen in: *

Which of the following is a characteristic shared by lactate dehydrogenase, malate

Post-hepatic jaundice may be due to: *

An emphysema patient suffering from fluid accumulation in the alveolar spaces is

What is the immediate precursor of biliverdin? *

In ketoacidosis, the blood pH would most likely be affected in what way? *

The protein part of holoenzyme is called? *

The most heat labile fraction of alkaline phosphatase is obtained from: *

Type I glycogen storage disease is due to a deficiency in glucose-6-phoshatase in the

Which fraction is expected to be elevated in alcoholic cirrhosis of the liver? *

In bilirubin determinations, the purpose of adding a concentrated caffeine solution or

Which suffix is added to the name of the substrate or to a word or to a phrase

Valinomycin enhances the selectivity of the electrode used to quantitate: *

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are both

Metabolic alkalosis is described as a (n): *

Which enzyme CANNOT be used to detect acute myocardial infarction (AMI)? *

The term delta bilirubin refers to: *

What is an enzyme cofactor? *

Oxidation of urobilinogen by microorganisms will produce: *

Which enzyme ratio is the best indicator of acute or chronic hepatitis: *

What enzyme system is responsible in the oxidation of bilirubin? *

The buffering capacity of blood is maintained by a reversible exchange process

Absorption of vitamin B12 requires the presence of: *

Which two physiological conditions are characterized by elevated serum alkaline

Which of the following enzymes are used in the diagnosis of pancreatic

Which reagent would not dissolve indirect bilirubin? *

The counterion in the chloride shift phenomenon is: *

Which ion is affected by the use of the chelating agent sequestrene?

The cofactor needed in the catalyzed reaction by hexokinase is: *

Most automated blood gas analyzers directly measure: *

Acetaminophen is particularly toxic to the: *

Which of the following is not a function of the liver? *

All enzymes are made up of which biomolecules (or, biomolecules)? *

Increased total bilirubin due to increased direct bilirubin suggests: *

Which of the following chemical determinations may be of help in establishing the

All of the following describe the direct bilirubin EXCEPT: *