CC LAB-MODULE 1
regulator molecules binds in the allosteric site;
ENZYMES DISCUSSIONS
ENZYMOLOGY
ENZYMES
- Primary Structure Protein
 A specific biologic protein that catalyzes
biochemical reaction without altering
the equilibrium point of the reaction or
consumes or changes in composition.
 They are measured in terms of their
inactivity and not in terms of their
absolute values
 These are catalysts that can be used in
the monitoring and diagnosis of disease
and their remarkable properties makes
them sensitive indicators of pathologic
change.
 COMPONENTS: Active Site and
Allosteric site
NOTE: Catalytic activity of enzymes depends on
the integrity of its structure. When active and
allosteric site is filled no further binding can
occur until an active site discharges its contents.
Active Site- water-free cavity/ substrate
interact w/ particular charge amino acid
residues; substrate interacts
Allosteric Site- cavity other than active site &
TERMS ASSOCIATED WITH ENZYMES
 Substrates
 Cofactors
 Isoenzyme
 Apoenzyme
 Holoenzyme
 Proenzyme
SUBSTRATES
 Substances that acts upon enzymes,
and they are specific for each of their
particular enzymes
e.g. triglycerides-substrate is lipase;
starch-substrate is amylase
COFACTORS
 Non-protein substances added in the
enzymes substrate complex to manifest
the enzyme activity.
ISOENZYMES
 Enzymes with similar enzymatic activity
but differ in their physical, biochemical,
and immunologic characteristics.
 Helps in understanding of organ specific
patterns of metabolism.
 Usually tissue specific or organ specific
e.g. lactate dehydrogenase & creatine
kinase (CK) – exist due to prescence of
multiple genes.
APOENZYMES
 Protein portions of enzymes subject to
denaturation in which enzyme loses its
activity.
  Minimal denaturation may be reverse
by removal of denaturing reagent.
 Affected by extreme pH temperature
and chemical addition.
HOLOENZYMES
 Active substance formed by
combination of a coenzyme and Apo
enzyme
 Apoenzyme +Prosthetic Group
PROENZYMES OR ZYMOGEN
 Inactive enzyme precursor
e.g. coagulation factors & digestive
enzyme
ENZYME CLASSIFICATION NOMENCLATURE
 Enzymes are classified according to
their biochemical functions, indicating
substrate and class of reaction
catalyzed, and are designated by
individual identification numbers.
 Consists of 4 numbers separated by
periods. (e.g. 1.1.1.27)
Example: EC 2.7.3.2.
EC- Enzyme Classification
1st number- Class
2nd number- Sub-Class
3rd number- Sub-subclass (Nitrogenous group or
acceptor)
4th number- Serial number/Enzyme number
within sub-subclass
Classification of Enzymes- “OTHLIL”
1. Oxidoreductases
 Catalyze the removal or addition of
electrons
 Redox reaction
e.g. Dehydrogenases: Cytochrome
oxidase, LDH, MDH, Isocitrate
dehydrogenase, G-6-PD
A+B→A+B
2. Transferase
 Catalyze the reaction of a chemical
group other than hydrogen from one
substance to another
 Transfer of a chemical group other than
hydrogen from 1 substrate to another
e.g. Kinases, Transaminases,
Aminotransferases: CK, GGT, AST, ALT,
OCT
A–X+B→A+B–X
3. Hydrolases
 Catalyze hydrolysis or splitting of a
bond by the addition of water
e.g. Esterases: ACP, ALP, CHS, LPS
Peptidases: Trypsin, Pepsin, LAP
Glycosidases:AMS, Galactosidases
A - B + H2O → OH – B - H
 4. Lyases
 Catalyze removal of groups from
substrates without hydrolysis. The
product contains double bonds.
e.g. Aldolase and Decarboxylase:
Glutamate decarboxylase, Pyruvate
decarboxylase, Tryptophan
decarboxylase
ATP → cAMP + PP¡
ENZYME THEORY
1) EMIL FISHERS / LOCK AND KEY THEORY
 Is based on the premise that the shape
of the key (substrate) must fit into the
lock (enzyme)
2) KOCHLAND’S/ INDUCED FIT THEORY
 Is based on the substrate binding to the
active site of the enzyme.

5. Isomerases
 Catalyze the intramolecular
arrangement of the substrate
compound
e.g. Glucose phosphate isomerqase,
and Ribose phosphate isomerase
A→B
ENZYME KINETICS
6. Ligases
 A chemical reaction may occur
 Catalyze the joining of two substrate
spontaneously if the free energy or
molecules, couples with breaking of the
available kinetic energy is higher for the
pyrophosphate bond in the ATP or
substrate than the product
similar compound.
e.g. Synthases
 Enzyme catalyze physiologic reactions
AB + C → A - C + B
by lowering the activation energy level
ENZYME CLASSIFICATION NOMENCLATURE that the substrate must reach for the
reaction to occur
E + S → ES → E + P

 CATALYTIC MECHANISM
o Absolute Specificity
o Group Specificity
o Bond Specificity
o Steriosometric Specificity

Binding process transforms the substrates

molecule to its activated process transforms the
substrates molecule to its activated state. –
E.C. nomenclatures with red color are the 8
natawa ako parang riddle. -
enzymes to be discussed.
CATALYTIC MECHANISM
ABSOLUTE SPECIFICITY
 Enzyme combines with only 1 substrate
and catalyzes only 1 reaction
GROUP SPECIFICITY
 Enzymes combines with all substrate in
a chemical group
BOND SPECIFICITY
 Enzymes reacting with specific chemical
bonds
STERIOISOMETRIC SPECIFICITY
 Enzymes that combines with only 1
optical isomer
ENZYME REACTION
 Zero-order reaction – the reaction rate
depends only on enzyme
concentration; independent on
substrate concentration
 First-order reaction – the reaction rate
is directly proportional to substrate
concentration; independent on enzyme
concentration
ENZYME ACTIVITY
Enzymes are measured in terms of:
1. Change in the substrate concentration
2. Change in the product concentration
3. Change in coenzyme concentration
GENERAL METHODS OF MEASURING
ENZYMATIC REACTION
1) FIXED-TIME- the reactants are
combined; the reaction proceeds for
designated time; the reaction is
stopped and measurement is made;
2) CONTINOUS MONITORING/KINETIC
ASSAY- multiple measurements of
changed in absorbance are made during
reaction; preferred method.
FACTORS AFFECTING ENZYMATIC REACTIONS
1) ENZYME CONCENTRATION
 The higher the enzyme concentration,
the faster is the reaction, because more
enzyme is present to bind with the
substrate
2) SUBSTRATE CONCENTRATION
 With the amount of enzyme exceeding
the amount of a substance, the reaction
rate steadily increases as more
substrate is added.
 However, when the substrate
concentration reaches as maximal
value, higher concentration of substrate
no longer result in increased rate of
reaction (saturation kinetics.)
UNITS FOR EXPRESSING ENZYMATIC ACTIVITY:
1) INTERNATIONAL UNIT (IU or U)
 1 micromole of substrate/minute
2) KATAL UNIT (KU)
 1 mole of substrate/second
  Enzymes are quantitated based on
their activity rather than absolute
values.
 The units used to report enzyme
levels are activity units.
 The definition for activity unit must
consider change in pH,
temperature, substrate, etc.,
A. COMPETITIVE INHIBITOR
 Physically binds to the active site of
an enzyme.
 Reversible (Substrate > Inhibitor)
 Substrate concentration ↑ than the
concentration of inhibitor will still
proceed, called REVERSIBLE.

3) COFACTORS
 Non-protein entities that must bind
to particular enzymes before a
reaction occurs.(no cofactor, no
reaction)
A. COENZYMES- Organic compound;
 essential to achieve absolute
enzymatic activity and increasing its
concentration will increase the
velocity of an enzymatic reaction
B. NON-COMPETETIVE INHIBITOR
E.g. Nicotinamide Adenine
 It does not compete with the
Dinucleotide (NAD), Nicotinamide
substrate but look for areas other
Adenine Dinucleotide Phosphate
than the active site.
(NADP)
 Binds to the allosteric site (cofactor
site)
B. ACTIVATORS- Inorganic Ions;
 Irreversible
 alters spatial configuration of the
enzymes for proper substrate
E.g. Calcium, Zinc, Chloride,
Magnesium, Potassium
--sabi ni ma’am organic ions, sabi ni
rodriguez notes inorganic. --

C. METALLOENZYMES- Inorganic ion

attached to a molecule
E.g. Catalase and Cytochrome
oxidase

C. UNCOMPETITIVE INHIBITOR
4) INHIBITORS
 The inhibitor binds to the enzyme-
 Enzymatic reactions may not
substrate (ES) complex.
progress if an inhibitor interferes
 Increasing the substrate
with the reaction; (interferes with
concentration results in more
the enzymatic reactions)
 enzyme substrate complex to which
the inhibitor binds thereby
increases the inhibition
↑Substrate concentration= ES = ↑
Inhibitor/inhibition
5) ISOENZYMES
 These are enzymes (polypeptide chains)
having the same catalytic reactions but
slightly different molecular structures –
various forms occur because of
differences in the amino acid sequence
of enzymes.
 The importance of the total enzyme
activity is enhanced by fractionating the
isoenzymes; (fractionation of
isoenzymes)
6) TEMPERATURE
 Enzymes are active at 25°C, 30°C, or
37°C
 37°C is the optimum temperature of
enzymatic activity
 Increasing temperature = Increasing
rate of chemical reaction (movement of
molecules
 Increasing temperature = Increasing
rate of denaturation (40°C to 50°C)
 Inactivation of Enzymes: 60°C to 65°C)
 Temperature Coefficient (Q10) means
for every 10°C increase in temperature
there will be a two-fold increase in
enzyme activity.
 Repeated freezing and thawing tends to
denature proteins and should be
avoided.
 Low temperature renders enzymes
reversibly inactive-
refrigerated/freezing temperature.
7) HYDROGEN ION CONCENTRATION OR
Ph
 Most physiologic reaction occurs in the
pH range of 7-8
 Extreme pH level may denature an
enzyme or influence its ionic state
resulting in structure change or change
of amino acid residue in the active site
8) STORAGE
 Low temperature
(refrigeration/freezing) render enzymes
reversibly inactive
 Repeated freezing and thawing to
denature proteins and should be
avoided
 Enzymes: -20°C= for longer period of
time
 Substrate and coenzymes: 2-8°C
 LDH(LD4 & 5): room temperature
9) HEMOLYSIS
 Mostly increases enzyme concentration
10) LACTESCENCE OR MILKY SPECIMEN
 Decreases enzyme concentration
NOTES TO REMEMBER: *in enzymes*
 An enzyme accelerates the rate of reaction,
reducing the time required to reach
equilibrium.
 A constant change in absorbance per unit
time occurs only when the rate of the
reaction is zero order.
 A substrate concentration of >99 x Km is
needed to achieve zero-order reaction.
 It is easier to measure small increases in
product than to measure small decreases in
a large amount of substrate.
 Enzymes do not alter the free energy or
direction of a reaction, but it alters the
energy of activation by forming a
metastable intermediate, the ES complex.
 Most enzymes are measured by monitoring
the rate of absorbance change (kinetic
assay) at 340nm as NADH is reduced or
consumed, and it allows direct reporting
either by IU or KU.
 In first-order reaction, the enzymes are
used as reagents to measure a specific
analyte.
 In non-kinetic assay absorbance is made at
10-seconds intervals for 100 seconds.
 Endpoint measurement determines the
concentration of substrate or product at
specific time after addition of the sample
(beside glucose testing using strips.)
 Enzyme activity measurements may not be
accurate if enzymes inhibitors are present,
essential cofactors are not included in the
assay, and improper specimen storage.
SPECIFIC ENZYMES
ALKALINE PHOSPHATASE (ALP) E.C.3.1.3.1
 AKA: Alkaline Orthophosphoric
Monoester Phosphohydrolase/
E.C.3.1.3.1
 A nonspecific enzyme capable of
reacting with many different substrates
 It functions to liberate inorganic
phosphate from an organic phosphate
ester with the contaminant production
of an alcohol
 Major tissue sources: liver (sinusoidal
and bile canalicular membranes), bone
(osteoblasts), placenta and intestinal
 Reference values: 30-90 U/L
 Major isoenzymes:
1) Liver ALP – most anodal
2) Bone ALP
3) Placental ALP
4) Intestinal ALP- least anodal;
most cathodal
 ALP comes in many forms and some of
which are true isoenzymes, encoded in
separated gene loci (chromosome 1)
1) ALPL- found in liver, bone and
kidney
2) ALPI- found in intestine
3) ALPP- found in placenta
 Activator of ALP includes Mg, Co, Mn,
and Zn.
 Inhibitor includes borates, oxalates, and
cyanide ions.
 Carcinoplacental ALP:
1) Region ALP- is found in lung,
breast, ovarian and
gynecological cancers; bone ALP
co-migrator; most heat stable
ALP (65°C for 30 minutes);
inhibited by phenylalanine
reagent
2) Nagao ALP- found in
adenocarcinoma of the
pancreas and bile duct, pleural
cancer; variant of Regan ALP;
inhibited by L-leucine and
phenylalanine. (56°C for 10-
15mins.)
ALKALINE PHOSPHATASE METHODS METHODS SUBSTRATE END PRODUCTS
1. Bodansky
1) ELECTROPHORESIS Beta- glyceroPO4 InorganicPO4 +
2. Shinowara
 (+) Liver ALP – most anodal ← Bone 3. Jones Glycerol
ALP ← Placental ALP ←Intestinal 4. Reinhart
ALP – least anodal (-) 5. King and Phenylphosphate Phenol
Armstrong
2) HEAT FRACTIINATION/STABILITY TEST 6.Bessy, Lowry, P-nitro phenyl P-nitrophenol or
 It is performed at 56°C for 10-15 and Brock PO4 yellow
minutes 7. Bowers and (PNPP) nitrophenoxide
McComb ion
(▲ Stable) Regan ← Placenta ←
8. Huggins and Phenolphthalein Phenolphthalein
Intestine ← Liver ← Bone (▲Labile) Talalay diPO4 red
9. Moss Alpha naphthol Alpha-naphtol
3) CHEMICAL INHIBITION TEST PO4
 Uses different concentrations of 10. Klein, Babson, Buffered Free
phenylalanine, synthetic urea and and Read phenolphthalein phenolphthalein
levamisole solutions PO4
o Phenylalanine- Inhibits Regan,
Placental and Intestinal ALP ALP- CLINICAL SIGNIFICANCE
o L-leucine- inhibits Nagao ALP
o Levamisole- inhibits Liver and  Total ALP levels are increased, major
Bone ALP liver fraction that is most frequently
o 3M urea- Inhibits bone ALP elevated--especially in obstructive
jaundice
4) BOWERS AND MC COMB (SZASZ  Conditions in which ALP is ELEVATED-
MODIFICATION)-PNPP (substrate) o Hepatitis
 Considered as the most specific o Bone cancer
method; IFCC recommended o Osteomalacia
method o Paget’s disease
 It is a continuous-monitoring o Biliary obstruction
technique which requires a pH o Hyperthyroidism
environment of 10.15 and should o Hyperparathyroidism
be read at 405nm o Myocardial infraction
o Pregnancy
 Conditions in which ALP is LOWERED-
o Hypophophatasia
P-NITROPHENYLPHOSPHATE + H2O ←ALP→ P- o Pernicious Anemia
NITROPHENOL + PHOSPHATE ION o Aplastic Anemia
o Achondroplasia
OTHER ALKALINE PHOSPHATASE METHODS
o Cretinism
(4°C) Low temperature = Increased ALP o Wilson’s disease
o Post-menopausal women
 o Cardio pulmonary bypass PROCEDURE, RESULT, & CALCULATION
(blood transfusion)
 Placental ALP- tumor marker in serum  Reagent Preparation and Stability:
in CSF foremost germ cell tumors; CSF o Procedure 1 with reagent start
o Reagents are ready for use,
levels occur placental ALP are diagnostic
value in differentiating whether tumor stable even after opening up to
in pineal body. expiry date. Storage at 2-8°C.
Contamination of reagents
ALP-REFERENCE RANGE must be avoided.
o Procedure with sample start
 ALP (total), (37°C) o Working reagents is stable for 4
weeks at 2-8°C 5 days at 15-
MALES-FEMALES 4-15 Y/O 54-369 U/L
25°C,one must be kept light
MALES 20-50 y/o 53-128 U/L protected.
 Specimen:
>/= 60 y/o 56-119 U/L
o Serum or Heparinized plasma
FEMALES 20-50 y/o 42-98 U/L o Avoid hemolysis
>/=60 y/o 63-141 U/L o Loss of activity within 7 days:
0% at 4°C; 10% at 20-25°C
ALP- EXPERIMENT
 ASSAY:
METHOD, PRINCIPLE, & MATERIALS o Wavelength: Hg 405nm (400-
420nm)
 METHOD USE: Bessey – Lowry - Brock o Optical path: 1 cm
 Alkaline Phosphatase Liquicolor, o Temperature: 25°C, 30°C, 37°C
Orthophosphoric Monoester o Measurement: Read against air
Phosphohydrolase (increasing absorbance)
 PRINCIPLE OF TEST:  Pipette 1 ml from bottle of substrate
into one bottle of buffer. Mix
thoroughly.
 Warm the reagent and the cuvette to
P-NITROPHENYLPHOSPHATE + H2O ←ALP→ the desired temperature. Temperature
PHOSPHATE + P-NITROPHENOL must be kept constant (+/- 0.5°C) for
duration of the test.
 MATERIALS:  Procedure 1 with Reagent start
 BUFFER
 Diethanolamine Buffer (pH 10.35 +/- Pipette into cuvette 25°C, 30°C, 37°C
0.2)-----------1.2 mol/L Sample 20ul
 Magnesium chloride--------------- Buffer 1000ul
 Mix, incubate 1 min. at the desired
0.625mmol/L
temperature
 SUBSTRATE
 P-nitrophenyl phosphate--------------- Substrate 250 ul
50mmol/L
  Mix, Read the absorbance after 1 min. ALP-EXPERIMENT-REFERENCE RANGE:
and the same time start the stopwatch.
Read the absorbance again exactly after Temperature 25°c u/i 30°c u/i 37°c u/i
1, 2, and 3 minutes.
 Procedure 2 with Start Women 40-190 19-232 64-306

Pipette into cuvette 25°C, 30°C, 37°C Men 50-190 61-232 80-306
Sample 20ul
Working reagent 1000ul
(previously prepared) Children up to 15 up to 400 up to 488 up to 644
 Mix, read the absorbance after 1 min. y/o
and at the same time start the Children up to 17 up to 300 up to 366 up to
stopwatch. Read absorbance again y/o 483
exactly 1, 2, and 3 minutes.
 RESULT.CALCULATION:
NOTE: during the reaction-nitrophenol is
 CALCULATION: from the readings,
produced. This substance is poisonous when
calculate the mean absorbance change
inhaled, swallowed or when absorbed through
per minute (A/min.)
the skin. It comes into contact with skin or
 Calculate the alkaline phosphatase
mucous membranes, wash thoroughly with
activity in the sample using the
water and consult a doctor
following factor:
VIDEO: SLIDE NUMBER 31 (ALP VIDEO-5:37)
U//l = ▲ A/min. x 3433 (procedure 1w/ reagent
2:02:00-2:08:17
start)
ACID PHOSPHATASE (ACP) E.C.3.1.3.2
U/l = ▲ A/min. x 2757 (procedure 2 w/ sample
start)  AKA: E.C. 3.1.3.2 or Orthophosphoric-
monoester phosphohydrolase
 Conversion factor from traditional units
 It catalyzes the same reaction made by
(U/I) in SI-units (kat/I)
ALP, except that it is active at pH 5.0
 1U/I = 16.67 x 10-3 ukat/l
 Major tissue sources: prostate (major
 1 ukat/l = 60 U/I4
source), RBC, platelets, liver and bone.
 ACP is classified based on acidic pH it
works at and based on source:
BASED ON PH
o PH 6.0- found in RBC
o PH 5.0- found in prostate,
spleen, and kidney
 BASED ON SOURCE (ISOENZYME)  RBC ACP inhibited by cupric ions and
formaldehyde
o Prostatic ACP- found in
 REFERENCE RANGE:
prostate gland, strongly
inhibited by dextrorotary TOTAL ACP: Male: 2.5-11.7 u/L
tartrate ions. Function is mainly Female: 0.3-9.2 u/L
to preserve spermatozoa and PROSTATIC ACP: Male:0.2-5.0 u/L
protects them from phagocytic Female0.0-0.8 u/L
activity of female genital tract. ACP METHODS
o Non-prostatic ACP (TR-ACP)-
METHODS SUBSTRATES END PRODUCTS
found in other tissues, cannot
be inhibited by tartrate but 1. GUTMAN AND Phenyl PO4 Inorganic PO4
inhibited by formaldehyde and GUTMAN
cupric ions. 2. SHINOWARA PNPP p-nitrophenol
 Unstable in ph 7.0 or > 7.0; temperature
> 37°C 3. BABSON, READ, Alpha napthyl PO4 Alpha-naphtol
 To stabilize enzyme activity, acidify AND PHILIPS
serum specimen in ph below 6.5
 ACP used in forensic clinical chemistry 4. ROY AND Thymolphthalein Free
in investigation of great cases. HILLMAN MonoPO4 thymolpthalein
 ISOENZYME: Things to remember:

ACP ISOENZYMES TISSUE SPECIFICITY  Separate red cell to the serum asap to
prevent the leakage of erythrocyte and
Band 1 Prostate Platelet ACP
Band 2 Granulocyte  Serum activity is decreased within 1-2
Band 3 Plasma ( Platelets, hours if the sample is left at room
Erythrocytes and
temperature without the addition of
Monocytes)
preservatives. Results of increased ph
Band 4 Granulocytes
 Hemolysis should be avoided;
Band 5 Osteoclast
contamination from erythrocyte ACP;
 SUBSTRATES:
Low result of ACP due to improper
o Thymophthalein
anticoagulant
monosphosphate –quantitative
endpoint reaction ACP-CLINICAL SIGNIFICANCE
o A-naphthyl PO4 –continous
monitoring methods  Prostate cancer
 Prostatic ACP is inhibited by 20mM L-  Benign Prostatic Hypertrophy
tartrate ions while 1mM cupric sulfate  Paget Disease
and 2% formaldehyde ions inhibit red  Breast Cancer with bone metastases
cell ACP  Gaucher Disease
 Platelet Damage
  Idiopathic Thrombocytopenia
 Vaginal washings of victim can be used
for seminal fluid examination, for
detection of ACP within 4 days.
OTHER CAUSES OF INCREASE SERUM ACP
 Urinary tract obstruction
 Acute urinary retention
 Extensive prostatic massage
 Prostatic inflammation
 Prostatic manipulations- immunobiopsy
and cystoscopy
INCREASED ACP (METASTATIC BONE
INVOLVEMENT):
 Prostatic carcinoma
 Breast, Lungs, and Thyroid carcinoma
 Gaucher’s Disease
 Niemann Pick Disease
ACP- EXPERIMENT
 METHOD USE: Colorimetric Humazym
M-Test (Babson and Reed)
o 1-naphthyl phosphate is
hydrolyzed by acid phosphatase
to phosphate and 1-naphthol,
which is converted with FRTR-
salt to an azo dye. The increase
of absorbance at 405nm is
proportional to the total acid
phosphatase activity in the
sample.
 REACTION PRINCIPLE:
1-naphthyl phosphate + H2O (ACP)→phosphate
+ 1napthol
1-naphthol + FRTR-salt (ACP)→ azo dye
METHOD, PRINCIPLE, & MATERIALS
 MATERIALS:
 BUFFER: Citrate Buffer (pH 5.2)-----
100mmol/l
 SUBSTRATE:
1-naphthyl phosphate-------19umol/l
FRTR-salt------------------------2umol/l
 STABILISER: Acetic Acid ------0.7mil
 CALIBRATOR: AUTOCAL (for 5 ml
Lyophilised calibrator for HUMAN
Clinical Chemistry reagents)
 CONTROL: Huma Trol N; Huma Trol P;
or SERODUS; SERODUS plus (Lyophilised
control serum for Human clinical
Chemistry reagents
PROCEDURE, RESULT, & CALCULATION
 REAGENT PREPARATION:
o Dissolve the contents of 1
bottle Substrate with exactly
2ml of Buffer. (Mark label with
date of preparation.)
 SPECIMEN: SERUM (No plasma)
 SAMPLE CONSIDERATIONS:
 PROCEDURE, RESULT, & CALCULATION
o Avoid hemolytic and icteric sera
o Separate serum from RBC after
centrifugation
o Analyze immediately (within 1-2
hours)
 SAMPLE PREPARATION:
o Stabilize samples, AUTOCAL,
and Controls by addition of one
drop of stabiliser to 1 ml sample
immediately after
 separation/reconstitution.  REFERENCFE VALUE:
Samples will remain stable for 3
days at 2-8°C. 21 hours at 15- TOTAL ACID PHOSPHATASE
Assay temperature 37°C
25°C (room temperature)
Men up to (U/I) 6.6
 ASSAY:
Women up to (U/I) 5.5
o Wavelength: Hg 405nm
o Optical path: 1cm
o Temperature: 37°C (ACP ASPARTATE AMINOTRANSFERASE (AST)
active @ this temp.) E.C.2.6.1.1
o Measurement: against air ( ↑
 AKA: E.C. 2.6.1.1 OR L-Aspartate 2-
absorbance)
Oxoglutarate Aminotransferase or
 Warm working reagent and cuvettes up
aspartate transaminase. Formerly
to desired temperature (37°C).
known as SGOT (serum glutamic-
Temperature must be kept constant
oxaloacetic transaminase)
(+/- 0.5°C) for the duration of the test.
 Is involved in the transfer of an amino
 SEMI MICRO METHOD
group between aspartate and a-keto
 Pipette into Cuvette
acids with the formation of
SAMPLE 100ul oxaloacetate and glutamate
WORKING REAGENT 1000ul  It has 2 isoenzyme fractions, cytoplasm,
and mitochondrial ASTs – the
 Mix, read the absorbance A1 after 5 cytoplasmic isoenzyme is the
minutes and start the stopwatch at the predominant form in serum
same time.  Major tissue source: cardiac tissue,
 Read the absorbance A2 exactly after 3 liver, and skeletal muscle
minutes at 37°C  Other sources: kidney, pancreas, and
 A2 – a1 = a RBC
 REFERENCE VALUE: 5-37 U/L
 CALCULATION:  ISOENZYYME FRACTIONS:
 Calculate the total acid phosphatase o Cytoplasmic AST- predominant
activity in the sample using the form in serum
following factor. o Mitochondrial AST- 5-10% of
total AST
U/I 37°C  METHODS:
Total Acid 248 o Karmen Method/Couple
Phosphatase (A)
Enzyme assay
(multiply)
o Reitman and Frankei
 CONVERSION:
o 1 U/I = 16.67 X 10-3 UKAT/L
o 1 UKAT = 60 U/I
  Coupled enzyme assay (Karmen  AST is also used in monitoring therapy
Method) pH level 7.5 with wavelength with potentially hepatotoxic drugs, a
340nm result 3x of upper border of normal
o It uses malate dehydrogenase sensation of therapy.
(MD) and monitors the change
in absorbance at 340 nm
ASPARTATE AMONITRANSFERASE-
EXPERIMENT

METHOD, PRINCIPLE, & MATERIALS

Aspartate + a-ketoglutarate ←AST→  METHOD USE: LiquiUV Test (KARMEN

Oxaloacetate + Glutamate METHOD)
o Kinetic method for the
Oxaloacetate + NADH + H4 ←MD→ Malate + determination of AST activity
NAD+ according to the
reccomendations of the Expert
---Karmen method uses the principle AST
Panel of the ifcc (Int4ernational
catalyzes the transamination.
Federation of Clinical
o Reitman and Frankel is also used using Chemistry) without
dinitrophenylhydrazone (color pyridoxalphosphate activation.
developer) which produces blue color at
505nm due to diazonium salt coupled  REACTION PRINCIPLE
with keto acid (+ NaOH – color
intensifier)

AST- CLINICAL SIGNIFICANCE

 Viral Hepatitis (100x)

2-Oxoglutarate + L-aspartate ←GOT→ a L-
 Infectious Mononucleosis (20x)
glutamate + oxaloacetate
 Cirrhosis (4x)
 Skeletal Muscle Disorder (8x) Oxaloacetate + NADH + H+ ←MDH→ L-malate +
 Pulmonary Embolism (3x) NAD
 Acute Pancreatitis
 MATERIALS
 Myocardial Infarction
 BUFFER/ ENZYME REAGENT
 Acute Myocardial Infarction (AMI)- AST
o TRIS Buffer (pH 7.9) 100mmol/l
levels begin to rise 6-8 hours, peak after
o L-aspartate 300mmol/l
24 hours, and normalize within 5 days;
o LDH 1.13kU/I
at the onset of Myocardial infarction
o MDH 0.75kU/I
AST level increased
o Sodium Azide 0.095%
 Evaluation of myocardial infarction,
 SUBSTRATE
hepatocellular disorders and skeletal
o 2-oxoglutarate- 60mmol/l
muscle involvement.
 o NADH - 0.9mmol/l
o Sodium Azide - 0.095%
 SPECIMEN: Serum, Heparinized plasma
or EDTA plasma
o Avoid hemolysis
o Loss of Activity within 3 days at
+4°C: 8%; at 20-25°C: 10%
 Test tubes, cuvettes, parafilm,
micropipettes, pipet tips, centrifuge
 ASSAY
 Wavelength: Hg 365nm; 340nm; or Hg
334nm
 Optical path: 1cm
 Temperature: 25°C, 30°C, 37°C
PROCEDURE, RESULT, & CALCULATION
PROCEDURE
 Procedure with sample start
 Reagent Preparation:
 Pipette 2 ml from bottle of Substrate
into a Buffer. Mix thoroughly
 Stable for 4 weeks at 2-8°C
 Warm the reagents and the cuvettes to
the desired temperature. Temperature
must be kept constant for the duration
of the test
Pipette into 25°C, 30°C 37°C
cuvettes
Sample 200ul 100ul
Working reagent 1000ul 1000ul
 Mix, read the absorbance after 1
minute and at the same time start the
stop watch. Read the absorbance again
exactly 1, 2, and 3 minute.
CALCULATION OF RESULTS
 For a ▲ A/min within 0.06-0.08 (Hg 365
nm) or 0.12-0.16 (Hg 334nm, 340nm)
 Use only measurements from the first 2
minutes for calculation (1minute
incubation, 2 min. measurements).
U/I + ▲ A/min. SAMPLE START
Wavelength 25°C, 30°C 37°C
Hg 334nm 971 1780
340 nm 952 1745
Hg 365 1765 3235
 Conversion factor from traditional
unit (U/I) in SI units (kat/I)
o 1U/I = 16.67 x 10 ^-3 ukat/l
o 1ukat = 60 U/L
 Note: If the absorbance change per
minute or activity exceeds: dilute
0.1 ml of the sample with 0.9ml
physiological saline (0.9%) and
repeat the assay using this dilution.
Multiply the result by 10.
VIDEO: SLIDE NUMBER 49 (AST VIDEO -4:50)
2:44:45-2:49:17
ALANINE AMINO TRANSFERASE (ALT) E.C.2.6.2
 AKA. E.C 2.6.1.2 or L-alanine: 2-
oxoglutarate aminotransferase or
alanine transaminase.. Formerly known
as SGPT (serum glutamic-pyruvic
transaminase)
 It has enzymatic activity similar to AST
 It catalyzes the transfer of an amino
group from alanine to a-ketoglutarate
with the formation of glutamate and
pyruvate
 The highest concentration is in the liver,
more liver-specific than AST
 Major Tissue Source: Liver
 Other resources kidney, pancreas, RBC,
heart, skeletal muscles, lungs.
  Reference value: 6-37 U/L at 37°C
 Coupled Enzymatic reaction: using pH
ALT- INCREASED TRANSFERASES:
7.5; reading at 340nm
1. Toxic hepatitis
2. Acute Myocardial Infraction – AST
3. Wolff-Parkinson White Syndrome
4. Trichinosis – AST
Alanine + a-ketoglutarate ←ALT→ Pyruvate 6 5. Chronic Alcoholism
Glutamate 6. Dermatomyositis – AST
7. Hepatic Cancer
Pyruvate + NADH + H+ ←LD→ Lactate + NAD+
8. Reye’s syndrome
AST/GOT ALT/SGPT 9. Viral Hepatitis
10. Muscular Dystrophy – AST
Major Organ Heart Liver
11. Acute Pancreatic - AST
affected
Substrate Aspartic Alpha Alanine Alpha THINGS-TO-REMEMBER:
Ketoglutaric Acid Ketoglutaric Acid
End products Glutamic Acid + Glutamic Acid +  The highest elevations of
Oxoloacetic Acid Pyruvic Acid
transaminases is seen in acute
Color developer 2.4 DNPH 2.4 DNPH
hepatitis
Color intensifier 0.4N NaOH 0.4NaOH  Severe viral or toxic hepatitis may
Methods Reitman and Reitman and produce elevations of transaminases
Frankel Frankel up to 20x the normal limits.
 Moderate elevation of transaminases
in chronic hepatitis, hepatic cancer,
ALT- CLINICAL SIGNIFICANCE
and infection mononucleosis.
 Hepatocellular disorder (hepatitis and  End stage cirrhosis, AST and ALT level
cirrhosis) are not elevated but as low as massive
 Shock Liver tissue destruction
 Infectious Mononucleosis
ALANINE AMINOTRANSFERASE- EXPERIMENT
 Polymyositis
 Severe Pancreatitis METHOD, PRINCIPLE, & MATERIALS

Most important cause for increase  LiquiUV Test

transaminase activity is liver disease
METHOD:
If liver is affected both AST and ALT is increased
 Kinetic method for the determination of
ALT more liver specific ALT activity according to the
recommendations of the Expert panel
Alcoholic hepatitis, liver cirrhosis, and liver
of the IFCC (International Federation of
neoclasia, ↑ASP
Clinical Chemistry) without pyridoxal
phosphate activation
 REACTION PRINCIPLE RESULTS AND CALCULATIONS:

 For ▲ A/min. within 0.06-0.08 (Hg

365nm) or 0.12-0.16 (Hg
334nm/340nm) use only measurements
from the first 2 minutes
2-Oxoglutarate + L-alanine ←GPT→ L-glutamate  For calculations (1 minute incubation, 2
+ pyruvate minutes measurement)

Pyruvate + NADH + H+ ←LDH→ L-lactate + NAD U/L = ▲ a/min. x Sample start

MATERIALS:
Wavelength 25°C, 30°C 37°C
 BUFFER/ ENZYME REAGENT Hg 334nm 972 1780
o TRIS Buffer (pH7.4) 125mmol/l 340nm 952 1745
o L-alanine 625mmol/l Hg 365nm 1765 3235
 Conversion factor from traditional
o LDH 1.5kU/I
units (U/I) in SI-units (kat/I)
o Sodium azide 0.095%
1 U/I=16.67X10^-3ukat/I 1ukat/I = 60U/I
 SUBSTRATE
o 2- oxaglutarate 75mmol/l VIDEO: SLIDE NUMBER 49 (AST VIDEO -4:50)
o NADH 0.9mmol/l 2:44:45-2:49:17 – same lang ang AST at ALT
o Sodium Azide 0.095% process, watch mo ulet if u want, only the
difference is the substrate because diffuse
 SPECIMEN
 Note: if the absorbance change per
o Serum, heparinized plasma or EDTA
minute (A/min.) or the activity exceed,
plasma
dilute 0.1ml of the sample with 0.9ml
o Avoid Hemolysis
physiological saline (0.09%) and repeat
o Loss of activity within 3 days at 4°C:
the assay using this dilution. Multiple
10% at 20-25°C: 17%
the results by 10.

 Test tubes, cuvettes, micropipette, REFERENCE VALUE:

pipet tips, centrifuge
 Procedure with sample start Temperature 25°C 30°C 37°C IFCC*
Men up to 22 U/I 30 U/I 42 U/I 45 U/I
Pipette into 25°C, 30°C 37°C Women up to 17 U/I 23 U/I 32 U/I 34 U/I
cuvettes
Sample 200ul 100ul
Working reagent 1000ul 1000ul
 Mix, read the absorbance after 1
minute and at the same time, start the
stop watch. Read the absorbance again
exactly after 1, 2, and 3 minutes.
AMYLASE (AMS/AMY) E.C.3.2.1.1
 AKA: E.C. 3.2.1.1 OR alpha 1-4-glucan-4
glucohydrolase
 These enzymes catalyzes the hydrolysis
of 1,4-a-glucosidic linkages in
polysaccharides.
 The enzyme is small enough to pass
through the glomerulus, and is the only
enzyme that is normally found in urine.
 It is the earliest pancreatic marker
 Major Tissue Source acinar cells of the
pancreas and the salivary glands
 Other Tissue Source: adipose tissue,
fallopian tubes, small intestine and
skeletal muscles
 Isoenzymes:
1. Salivary amylase (ptyalin or S-
type) - found in salivary glands
2. Pancreatic amylase (amylopsin
or P-type) – found in pancreas
 S1 – slowest salivary gland (near the
cathode)
o S bands are strongly
inhibited by a protein
isolated from wheat
 P2
 P3 – increased in pancreatitis; most
predominant pancreatic AMS
isoenzyme in AP
REFERENCE RANGE:
 Adult: 23-85 U/L
CLINICAL SIGNIFICANCE:
 In acute pancreatitis (AP), AMs levels
rise 2-12 hours after onset of attack,
peak at 24 hours, and normalize within
3-5 days.
 In AP, Increased AMS blood levels are
accompanied by increased urinary
excretion.
 AMS in urine remains elevated for up to
7 days.
 In acute renal failure in the absence of
AP (acute pancreatitis), increased
plasma AMS is accompanied by
decreased urine AMS
 The magnitude of AMY levels does not
reflect the severity of pancreatic
involvement, but higher levels increases
the probability of acute pancreatitis.
 Biliary tract diseases such as
cholecystitis cause up to 4x increase in
serum P-AMY activity as a result of
Primary or Secondary pancreatic
involvement.
 Hyperamylasemia also occurs in
neoplastic diseases.
 Salivary gland inflammation (parotitis)
due to mumps can also release AMS
into the circulation causing an elevated
serum AMS.
Things-to-remember in amylase detection:
 Heparin may inhibit the activity of
amylase. (using some, but not all,
methods)
 Triglycerides may inhibit serum
amylase activity
 Sample is w/ high activity of
amylase, should be diluted with
sodium chloride to prevent
inactivation.
 Many endogenous inhibitors of
AMS, such as wheat germ are
present in serum.
  The administration of morphine and
other opiates for pain relief blood
sampling will lead to falsely
elevated serum AMS levels.
5-glucose-6-phosphates + 5 NAD ←G-6-PD→
5,6-phosphogluconolactone + 5 NADH
Notes-to- remember:

SUBSTRATE FOR ALL THE METHODS: STARCH  Salivary amylase is inhibited by

1. SACCHAROGENIC
 It is the classic reference method
expressed in Somogyi units
 It measures the amount of reducing
sugars produced by the hydrolysis
of starch by the usual glucose
methods.
wheat germ lectin.
 Enzyme molecules are too large to
pass through the healthy
glomerulus of the kidneys, thus,
urinary excretion is not a major
route for elimination except
amylase; Elimination of amylase is
not in urinary excretion

AMYLASE- EXPERIMENT
2. AMYLOCLASTIC
 It measures amylase activity by METHOD, PRINCIPLE, & MATERIALS
following the decreases in substrate
concentration (degradation of METHOD: Caraway Method, Modified
starch)
MATERIALS/RAGENTS:

3. CHROMOGENIC  Amylase Substrate

 It measures amylase activity by the
increase in color intensity of the
soluble dye-substrate solution
produced in the reaction.
4. COUPLED- ENZYME
 It measures amylase activity by a
continuous-monitoring technique
 Test Sera/N – AB controls
 Color developer
 Deionized water
 Test tubes, cuvettes, paraffin,
micropipette, pipet tips, centrifuge
PROCEDURE, RESULT, & CALCULATION
PROCEDURE:

Reagents Reagent Blanks Sample/Controls

AMS substrate 250ul 250ul
Test Sera/N-AB ---- 10ul
Maltospentose ←AMS→ Maltrotriose + Maltose controls
 Incubate test and control for 5 minutes
Maltotriose + Maltose ←glucosidase→ 5- at 37°C
glucose
Color developer 250ul 250ul
5-glucose + 5 ATP ←hexokinnase→ 5-glucose-6- De-ionized water 2000ul 2000ul
phosphates + 5 ADP
  Invert to mix and read % T/absorbance
at 610/640nm against water blank.
(dH2O)
RESUTLTS/CALCULATION OF AMYLASE
ACTIVITY: (read by: absorbance mode)
 Factor use: 750 ul
MINUS THEN DIVIDED
 Calculate the given readings using the
formula and interpret the value
obtained.
Blank Abs = 0.495
Sample Abs = 0.420
 For Statfax: Use absorbance mode at
600nm against water blank (dH2O)
 NORMAL RANGE: 60-180 U/I
VIDEO: SLIDE NUMBER 67 (AMS/AMY VIDEO
3:42 )3:18:20-3:21:50
LIPASE (LPS/LIP)
 Is an enzyme that hydrolyzes the ester
linkages of fats to produce alcohol and
fatty acid
 It catalyzes partial hydrolysis of dietary
TAG in the intestines to the 2-
monoglyceride intermediate, w/ the
production of long chain fatty acids.
 It is the most specific pancreatic
marker – secreted exclusively in the
pancreas; not affected by renal
disorders.
 Major Source: Pancreas
 Minor Source: Gastric mucosa,
Intestinal mucosa, and adipose tissue
 Hemolysis should be avoided because
haemoglobin inhibits lipase activity
CLINICAL SIGNIFICANCE:
 In acute pancreatitis (AP), LPS levels rise
6hours after onset of attack, peak at 24
hours, remains elevated for 7 days, and
normalize in 8-14 days
 In chronic pancreatitis, acinar cell
degradation occurs resulting in loss of
amylase and lipase production
REFERENCE VALUE:
 0-0.1 U/ml
Things-to-Remember in Lipase degradation:
 It uses olive oil as the substrate
because other esterase can hydrolyze
TAG and synthetic diglycerides
 Addition of colipase (protein secreted
by the pancreas) and bile salts will
make assay more sensitive an specific
for AP detection.
 Hemoglobin inhibits the activity of LPs
leading to falsely low values.
 Triolein (more pure form of TAG) is
used also as a substrate for LPS assay
LIPASE –EXPERIMENT
METHOD, PRINCIPLE, & MATERIALS
METHOD:
1. CHERRY CRANDAL (reference method)
 Principle: Hydrolysis of olive oil after
incubation for 20 hours at 37 °C and
titration of fatty acids using NaOH
 Substrate: 50% olive oil
 End Product: Fatty acids
 MATERIALS:

BUFFER:

Triglycerides (olive oil) + 2 H2O ←LPS→ 2-  Goods Buffer pH8.0

monoglyceride + 2 fatty acids  Taurodesoxysholate 1.0mmol/l
 Desoxycholate 1.0mmol/l
2. TIETZ AND FLEREC
 Calcium ions 1.0mmol/l
3. PEROXIDASE COUPLING –most
 Colipase 2mg/l
commonly used method; does not use
50% olive oil  Sodium Azide 0.095%

METHOD USE: Enzymatic Colorimetric Test for SUBSTRATE/SUBSTRATE REAGENT:

the Quantitative Determination of Lipase  Tartrate buffer pH 4.0
REACTION PRINCIPLE:  Lipase Substrate 0.1mmol/l
 Sodium azide 0.095%
 Lipase catalyses the reaction
 Synthetically produce lipase substrate CALIBRATOR:
is added to micro emulsion w/c is
 AUTOCAL/SERODS
specifically split by lipase in the
presence of colipase and bile salts. MATERIALS:
Combination of Colipase and Bile salts
ensure the specific determination of  Test tubes, cuvettes, parafilm,
pancreatic lipase w/o any reaction due micropipettes, micropipette tips,
to lipolitic enzymes/esterase centrifuge

SPECIMEN: SERUM OR HEPARINIZED PLASMA

STABILITY: 7 days at 20-25°C; 3 weeks at 4-8°C;

1,2-0 dilauryl-rac-glycero-3-glutaric acid – 196-
methylresorufin) ester ←LIPASE/COLIPASE→
1,2-0-dilauryl-rac-glycerin + Glutaric Acid – (6-
methylresorufin) ester
Glutaric Acid –(6-methylresorufin) ester 2
←spontaneous degradation→ Glutaric Acid
+Methylresorufin
 End color red
1 year at -20°C
 Discard contaminated samples
 Reagents are ready for use and stable
until expiration date stated on the label
when stores at 4-8°C.
 Avoid freezing and contamination.
 Mix at room temperature before use.
PROCEDURE, RESULT, & CALCULATION
PROCEDURE:

 Pipetting scheme

Pipette into Blank Sample/Calibrato

 cuvettes r Precipitation may also appear on
Calibrator/Samples --- 20ul storage, especially if stored lower than
the recommended temperature, it does
Dist. Water 20ul ---
not affect the analysis. It just necessary
Buffer 1000ul 1000ul to slightly rotate the vial to resuspend
before the analysis.
 Mix carefully (do not shake, incubate
3. Sodium azide is a preservative so extra
for 5 minutes Start reaction by adding
careful not to swallow. Avoid in contact
substrate
to skin and the mucous membranes.
Substrate 250ul 250ul
VIDEO: SLIDE NUMBER 73 (LPS/LIP
 Mix, incubate for 2 minutes at 37°C,
VIDEO )3:18:20-3:21:50
read absorbance again and start stop
watch. LACTATE DEHYDROGENASE E.C. 1.1.1.27
 After exactly 1 and 2 minutes Read
absorbance again and then calculate  AKA E.C. 1.1.1.27 or L-lactate; NAD
A/min. oxidoreductase
 It is a n enzyme that catalyzes the
RESULT CALCULATION: interconversion of lactic and pyruvic
acid
 it is a zinc-containing enzyme that is
part of the glycolytic pathway and is
found in virtually all cells in the body.
A/min = [ A/min sample or calibrator ] – [ A/min blank ]
 It is a hydrogen-transfer enzyme that
Lipase [ U/ I ] = A/min (sample) x concentration calibrator uses the coenzyme nicotinamide
[ U/I ] dinucleotide (NAD+)
 It is a tetrameric molecule containing
Reference value: < 60 U/I four subunits of two possible forms (H
heart and M muscle)
REFERENCE VALUE:
 In plasma, the majority of LD comes
 </= 60 U/I from breakdown of erythrocytes and
platelets, with varying contributions
IMPORTANT PROCEDURAL NOTES: from other organs
1. As many other clinical chemistry  Tissue Source: heart, RBC’s kidneys (LD-
reagents, specially reagents for 1 & LD-2); lungs, pancreas, spleen (LD-
determination of triglycerides, HDL – 3); skeletal muscles, liver, intestine(LD-4
and LDL – cholesterol contain lipase or & LD-5)
high concentrations of detergents carry  Reference value: 100-225 U/L (Forward
over should be avoided by clearing the reaction) ; 80-280 U/L (Reverse
glassware thoroughly. reaction)
2. SUB is a turbid orange colored micro-
emulsion, if it becomes red discard,
LD ISOENZTME as a percentage of total LD:
LD-1 (HHHH or H4) = 17-27%
LD-2 (HHHM or H3M = 27-37%
LD-3 (HHMM or H2M2) = 18-25%
LD-4 (HMMM or HM3) = 3-8%
LD-5 (MMMM or M4) = 0-5%
 RBCs and cardiac tissues contain high
levels of LD-1
 LD-1 is relatively abundant in cardiac
muscle, whereas LD-5 is more abundant
in skeletal muscle
 LD-1 is not found in the skeletal muscles
and Liver; LD-2 is never found in the
skeletal muscles
 LD-2 is the majority isoenzyme in the
sera of healthy persons
 LD-2 is greater the LD-1 is seen in
healthy sera.
 LD-5 has undetectable level in heart,
RBCs and renal cortex.
 LD-6 represents the alcohol
dehydrogenase enzyme, 6th band in
electrophoresis; elevated in drug
hepatoxicity and obstructive jaundice;
it is responsible for the metabolic
conversion of methanol and ethylene
glycol to toxic compounds; present in
patients with arteriosclerotic failure.
 Another form of LD composed of four C
subunits in spermatozoa and in semen
but has never been detected in serum,
even in individuals with seminoma
CLINICAL SIGNIFICANCE:
 Highest serum levels are seen in
pernicious anemia and hemolytic
disorders. Include megaloblastic
anemia
 In AMI, LD levels begin to rise
within 12-24 hours, peak levels
within 48-72 hours and remains
elevated for 10-14 days.
 Hepatic carcinoma and toxic
hepatitis will have 10-fold
increased.
 Viral hepatitis and cirrhosis would
give LD slightly increased values (2-
3x URL
 LD-1 > LD-2 also known as “flipped
pattern” is seen in myocardial infarction
and hemolytic anemia
 LD-2, Ld-3, and LD-4 = LD cancer
markers (predominantly LD-30; acute
leukemia, germ cell tumors and lung
cancers
 LD-5 is moderately increased in acute
viral hepatitis, and cirrhosis and
markedly increased in hepatic
carcinoma and toxic hepatitis.
INCREASED LDH:
1) Anemias- pernicious, hemolytic,
megaloblastic
2) Myocardial Infarction
3) Leukemia
4) Renal infarction
5) Hepatitis and Hepatic cancer
6) Muscular dystrophy
7) Delirium tremens
8) Malignancy
9) Pneumocystis jerovecii pneumonia
METHOD:
 Lactate is a more specific substrate
compared to pyruvate.
 LD-1 prefers forward reaction, whereas
LD-5 prefers the revers reaction.
  LD is stable at room temperature for 48  Modified method base on the
hours. recommendations of the SCE
1. WACKER METHOD (FORWARD/DIRECT (Scandinavian Committee on Enzyme)
REACTION) – reaction is at pH 8.8
REACTION PRINCIPLE OF THE TEST
Ps. Sinasabi ni ma’am sa recording WRECKER
pero kay rodriguez WACKER desisyon ka na
lang ano gusto mo. – ynor qt-

 It is the most commonly used Pyruvate + NADH + H+ < ----LDH------> Lactate +

method because it produces a NAD+
positive rate (NADH) and not
affected by product inhibition MATERIALS/REAGENTS
 Most widely method;
BUFFER:
 less contamination in NAD and
inhibiting products.  TRIS Buffer (pH 7.35) 62.5mmol/l
 Pyruvate 1.5mmol/l
 Sodium Azide 0.095%
Lactate + NAD ----LD--→ Pyruvate + NADH @
SUBSTRATE:
340nm

2. WORBLEUSKI LA DUE  NADH 0.75mmol/l

(REVERSE/INDIRECT REACTION)-  Sodium Azide 0.095%
reaction is at pH 7.2
MATERIALS:
 It is about 2x faster as the
forward reaction.  Test tubes, cuvettes, parafilm,
 It is the preferred method for micropipettes, pipet tips, centrifuge
dry slide technology
REAGENT PREPARATION
 It uses a less costly cofactor and
it has a smaller specimen  Pipette 2 ml from bottle Substrate into
volume requirement one bottle Buffer. Mix thoroughly, the
working reagent is stable at 2-8°C for 3
weeks and must be protected from
light.
Pyruvate + NADH -----LD---→ Lactate + NAD
SPECIMEN: Serum, Heparinized or EDTA plasma
LACTATE DEHYDROGENASE- EXPERIMENT
 Avoid Hemolysis
METHOD, PRINCIPLE, & MATERIALS  Loss activity within 3 days at 8% at +4°C

LiquiUV Test SPECIMEN CONSIDERATION:

METHOD USE: WORBLEWSKI LAUDE

  Erythrocytes contains LD contains LD 1 U/l = 16.67 x 10-3 ukat/l ; 1 ukat/l = 60U/l
concentration 100-150 time that found
in serum, thus immediate separation is  Factor to convert results to the new
advised. Store at 25°C and examined IFCC recommended method:
within 24 hours after collection. U/l (LDH SCE ) x 0.4796 = U/l ( LDH IFCC)
ASSAY: VIDEO: SLIDE NUMBER 84 (LD VIDEO 4:04)
PART 2 15:51-21:37
 WAVELENGTH: Hg 334nm; 340nm;
Hg365nm CREATININE KINASE E.C. 2.7.3.2
 OPTICAL PATH: 1cm
 TEMPERATURE: 25°C, 30°C, 37°C  AKA E.C.2.7.3.2. or ATP creatine, N-
 MEASUREMENT: against air phosphotransferase
(decreasing absorbance)  it catalyzes the transfer of transfer of a
phosphate group between creatine
PROCEDURE, RESULT, & CALCULATION phosphate and adenosine diphosphate
 It is involves in the storage of high-
PROCEDURE:
energy creatinine PO4 in the muscles
Pipette into cuvettes 25°C, 30°C, 37°C  It is a dimeric molecule with small
molecular sixe, composed of a pair of
Sample 20 ul 10 ul two different monomers called M and B
Working Reagents 1000 ul 1000 ul  It is found in small amounts throughout
 Mix and Read the absorbance after 1 the body, but is found in high
minute and at the same time start the concentrations only in muscle and
stop watch. Read the absorbance again brain, although CK from brain virtually
exactly after 1, 2, and 3 minutes never crosses the blood-brain barrier to
reach plasma.
RESULT & CALCULATION  Major Tissue Sources: brain tissue,
smooth and skeletal muscles and
 Using the absorbance readings,
cardiac muscles
calculate the mean absorbance change
 Reference Values: Male= 15-160 U/L;
per minute (a/min.)
Female 15-130 U/L; CK-MB= <6% of
 Calculate the LDH activity in the sample
total CK
by multiplying A/min. using the
 CK is released due to hypoxia and other
following factors.
muscle injuries this a sensitive but not
Hg 334nm 340nm Hg 365nm
specific for MI
U/l(250C,300C)= 8250 8095 15000
A/min. x  Optimal pH both forward and reverse is
U/l ( 370C) = 16345 16030 29705 9 and 6.7; Magnesium is an obligate
A/min. x activator of CK, however excessive
amount of Magnesium can also act as
 Conversion factor of the traditional inhibitor.
units (U/I) in SI units (ukat/I)
 Other metal ions; manganese, calcium,
zinc and copper.
ISOENZYMES:
 CK-BB or CK-1 – Found primarily in
brain, GI tract muscle and urinary
bladder muscle
 CK-MB or CK-2 - found primarily in
heart and smooth muscles
 CK-MM or CK-3 – found in skeletal
muscles, and heart , found in
smooth muscles in small amounts
 CK-MT – located at chromosome 15
constitutes 15% of total heart CK
activity
 CK-BB- Most Anodal; BRAIN TYPE
 CK-MB- myocardium (20%); HYBRID
TYPE
 CK-MM - least anodal, skeletal and
smooth muscles (major 94-100%);
MUSCLE TYPE
 CK-1 is the most anodal and labile
enzyme; CK-3 is the least anodal
(cathode)
 CK-BB is the dominant isoenzyme
of CK found in brain, intestine, and
smooth muscle
 Serum of adults rarely contains CK-
BB of brain due to its high
molecular size; it may be normally
present in neonatal sera.
 Cardiac tissues contains significant
amount of CK-MB (20%) –
myocardium is the only tissue from
which CK-MB enters the serum in
significant quantities.
 Physically well-trained individuals
tend to have elevated baseline
levels of total CK
 Direct muscle trauma, as seen in
contact sports, surgery, strenuous
exercise, and intramuscular
injections, are common causes of
mild elevations of serum CK ( up to
about 5-6 times reference limits)
 Intramuscular injections are known
to increase CK (<5x URL)
 Bedridden patients may have
decreased CK activity
OTHER FORMS OF CK INCLUDES THE MACRO-
CK IT EXIST IN 2 TYPES
1) MACRO-CK TYPE 1 – complex of CK-
BB and non-pathologic origin but
can interfere with other CK tests
2) MACRO CK TYPE 2 – found in
severely ill with malignancy or liver
disease. Detected by abnormally
migrating bands towards the
cathode.
CLINICAL SIGNIFICANCE:
 It is very sensitive indicator of acute
myocardial infraction (AMI) and
Duchenne disorder.
 Highest elevation of total CK is seen in
Duchenne’s muscular dystrophy (50x
URL)
 CK-MB is found mainly in myocardial
tissue – it is used as a serodiagnostic
test for AMI.
 Following AMI, the CK-MB levels begin
to rise within 4-8 hours, peak at 12-24
hours and normalize within 48-72
hours.
 CK-MB is not elevated in angina
 Injury both cardiac and skeletal muscle
accounts for the majority of CK-MM
elevations.
  Total CKis markedly elevated after TROPONIN T 3-12 hrs. 12-48 hrs 5-14 dys
trauma to skeletal muscle from crush
LDH 12-24 hrs. 24-48 hrs 10-14 dys
injury, convulsions, tatany, surgical
AST
incisions or intra muscular injections. 12-24 hrs. 24-48 hrs 10-14 dys

METHODS:
NOTES-TO REMEMBER:
1) TANZER-GILBARG ASSAY
(FORWARD/DIRECT METHOD) – pH  Adenylate Kinase (AK ) released after red
9.0;340nm cell lysis interferes with CK assay
particularly with hemolysis of > 320 mg/L
 Liver cells and RBC do not contain CK
 To increase both the sensitivity and the
specificity of CK-MB in the diagnosis of
acute AMI, it has been found necessary to
perform serial determinations of MB
Creatine + ATP --CPK--→ Creatine PO4 + ADP fraction (at 3- to 4- hour intervals over a 12-
to 16 hour period) that show a progressive
ADP + phosphoenolpyruvate ←--PK--→ Pyruvate + ATP rise that reaches a peak, followed by a fall
to low levels.
Pruvate + NADH ←- LD-→ Lactate + NAD
 Adenosine monophosphate (AMP) is added
2) OLIVER-ROSALKI to the reverse method to inhibit AK which
(REVERSE/INDIRECT METHOD) may be present in the serum from
hemolysis – AK hydrolyzes ADP
 N-acetylcysteine is added to CK reagent to
activate the enzyme (aside from Mg+2) and
partially reversed the inhibition of oxidized
sulfhydryl groups.
 Imidazole serves as a buffer; urate and
Creatine PO4 + ADP ←- CPK--→ Creatine + ATP cysteine are potent CK inhibitors.
 CK is light and pH sensitive; it is also lost
ATP + glucose ←- *HK--→ ADP + glucose-6-PO4
with excessive storage.
Glucose-6-PO4+NADP ←-G6PD-→ ^-phosphogluconate +  Cleland’s reagent and glutathione – partially
NADPH restore lost activity of CK
 CK mass units assay are more sensitive than
AMI MARKERS: electrophoresis, but electrophoresis is still
the reference method for CK
AMI ONSET PEAKS AT NORMALIZE
MARKER AT
METHOD, PRINCIPLE, & MATERIALS
CK-MB 3-12 hrs. 18-24 hrs 48-72 hrs
MYOGLOBIN 2-3 hrs. 9-12 hrs 24 hrs CK-MB NAC ACTIVATED

TROPONIN I 3-12hrs. 24 hrs 5-10 dys METHOD:

  IMMUNOINHIBITION METHOD  Test tubes, parafilm, cuvettes,
(OLIVER-ROSALKI) micropipettes, pipet tips, centrifuge.

REACTION PRINCIPLE TEST:

PROCEDURE, RESULT, & CALCULATION

PROCEDURE:

Creatine phosphate + ADP <--CK------> Creatine  Reagent Preparation: Reconstitute one

+ ATP
ATP + d-GLUCOSE <---HK---------> ADP + D-
glucose-6-phosphate (G6P)
G6P + NADP <----G6P-DH------> 6-phospho-D-
glucono-lactone + NADPH + H+
MATERIALS/REAGENTS
vial ENZyme with exactly 3ml Buffer
Swirl gently and incubate for 5 minutes
at room temperature prior to use. This
working solution is stable for 5 days at
2-8°C
SPECIMEN: Serum, heparinized plasma or EDTA
plasma

BUFFER:  Loss of activity within 1 day at 2-8°C <

10%
 Imidazole buffer (pH 6.7) 0.10mmol/l
 Glucose 20mmol/l ASSAY:
 Mg. acetate 110mmol/l  WAVELENGTH: Hg 334nm; 340 nm; Hg
 EDTA 2.00mmol/l 365nm
ENZYME: enzyme and antibody reagent  OPTICAL PATH: 1cm
(Lyophilized) 1 VIAL contains  TEMPERATURE: 25°C, 30°C, 37°C
 MEASUREMENT: against air (increasing
 ADP 2.00mmol/l absorbance)
 AMP 2.00mmol/l
 Diadenosine IMPORTANT PROCEDURAL NOTES:
pentaphosphate 10.00mol/l  Prior to determining CKMB activity. It is
recommended to measure total
 HK > 2.50 U/ml Activity. It is recommended to measure
 NADP 2.00mmol/l total CK activated method (CK-NAC
 G6P-DH >1.50U/ML LIQUIUV in order to ensure correct
 N-Acetylcysteine 20mmol/l diagnostic interpretation of he results
 Creatinine Phosphate 30mmol/l  Warm reagents and cuvettes to the
 Polyconal Antibody to CK-M subunit desired temperature. Temperature be
 CK-MB controls kept constant for the duration of the
 CK-MB calibrators test:

MATERIALS: Pipette into cuvettes

 Sample/CAL 40u/l method can be employed. Only control
Working reagent 1000il sera with human CK can be used
 Mix and incubate at the desired
temperature for 10 minutes VIDEO: SLIDE NUMBER 97 (CK VIDEO 4:57)
 Read the absorbance A, Exactly PART 2 43:00-48:35
after 5 minutes read the
absorbance A.

RESULTS AND CALCULATION:

 Calculate the absorbance change per

minute (A/min.) according to A/min. =
(A2-A1):5
 The CK-MB activity is calculated using
the following factors:

CK-MB ACTIVITY (U/I)

SEMI-MICRO
Hg 334nm 8414 x A/min
340nm 8254 x A/min.
Hg 365nm 14858 x A/min.
 Conversion factor units (U/I) in SI units
(kat/I)

1 U/l = 16.67 x 10-3 ukat/l ; 1ukat/l = 60 U/l

REFERENCE RANGE: MYO CARDIAL

INFARCTION (MI)

 The likelihood of myocardial damage is

high if the following 3 criteria are met

25°C 30°C 37°C

1.Total CK
MEN >80U/I >130U/I >195U/I
WOMEN >70U/I >110U/I >170U/I
2. CK-MB >10U/L >16U/L >25U/I
3.CK-MB activity Ranging between 6% and 25%
of the total CK activity

QUALITY CONTROL

 All control sera with CK-MB values

determined by this method by this
CC2 MODULE 2-LABORATORY
ELECTROLYTES
 Electrolytes are ions that carry an
electric charge.
 Classified as anions and cations.
 Anions have a negative (-) charge and
move toward the anode (+).
 Cations migrate toward the cathode (-)
because cations are positive (+) charge.
 Electrolytes are essential component of
numerous processes:
o Volume and osmotic regulation
o Myocardial rhythm and
contractility
o Cofactors in enzyme regulation
o Regulation of adenosine
triphosphatase (Atpase) ION
PUMPS
3 process of sodium regulation:
o ACID-BASE BALANCE
o BLOOD COAGULATION
o NUEROMUSCULAR
EXCITABILITY
o PRODUCTION AND USE OF ATP
FROM GLUCOSE
 Extracellular fluid (ECF) 1/3 total body
water(16 liters)
 Intracellular fluid(ICF) 2/3 of total body
water; fluid inside the cell (24 liters)
 40-75% is the average water content of
the human body (advanced age and
obesity – decrease values
HORMONES AFFECTING SODIUM LEVELS:
 Normal plasma is composed of 93%
water and 7% solutes (glucose lipids,
proteins, NPN, amino acids, and ions)
 Water content of plasma is 12% higher
than that whole bloods
SODIUM (Natrium, Na+)
 it is the major cation in the
extracellular fluid (ECF), plays a
central role in maintaining the
normal distribution of water and
osmotic pressure in the ECF
compartments
 It is also known as “natrium”
 most abundant cation in the ECF
representing 90% of all extracellular
cation.
 Na+ determines the osmolality of
plasma
 Na+ ATP ion pump moves 3 sodium
ions out of cell exchange to 2
potassium ions moving in to cell as
ATP is converted as ADP.
 Regulations for Sodium per plasma
sodium concentrations depend
greatly in the intake and excretion
of water.
1. Intake of water in response to thirst can
be stimulated or suppressed by plasma
osmolality.
2. Excretion of water, it is greatly affected
by AVP- which release in the response
to changes to either volume or
osmolality.
3. Blood volume status, affects Na+
secretion through aldosterone,
angiotensin II and atrial natriuretic
peptide (ANP)
1) ALDOSTERONE
 It promotes absorption of
sodium in the distal tubule.
 It promotes sodium retention
and potassium excretion.
 2) ATRIAL NATRIURETIC FACTOR (ANF) HYPONATREMIA W/ NORMAL RENAL FUNCTION
 it is an endogenous CAUSE Serum Na Urine Na 24-hr
antihypertensive agent Una
secreted from cardiac atria. 1.Overhydration Low Low Low
 It blocks aldosterone and renin 2.Diuretics Low Low High
secretion, and inhibits the 3.SIADH Low High High
action of angiotensin II and
4.Adrenal failure Mildly Elevated Normal
vasopressin.
5.Bartter’s Low Low High
 It causes natriuresis
Syndrome
6.Diabetic Low Normal Normal
Hyperosmolarity
ELECTROLYTE ABNORMALITIES:

HYPONATREMIA:
HYPONATREMIA W/ NORMAL RENAL FUNCTION
 low-serum sodium level; below normal CAUSE Urine Serum K
value Osmolality
1.Overhydration Low Normal or Low
 the most common electrolyte disorder,
2.Diuretics Low Low
 defined as reduced plasma sodium 3.SIADH High Normal or Low
concentration to a value less than 135 4.Adrenal failure Highi High
mmol/L 5.Bartter’s Syndrome Low Low
6.Diabetic normal High
CAUSED BY: Hyperosmolarity
 over hydration, use of diuretics,
syndrome of inappropriate ADH (SIADH) HYPERNATREMIA:
 Aldosterone deficit secondary to
Addison’s disease.  Serum sodium concentration above the
 BARTTER’S SYNDROME: It is a rare upper limit of the reference interval
condition wherein sodium chloride  Caused by loss of water, gain of sodium,
(NaCl) gradients cannot form in the loop or both
of Herle causing the ret4ention of  It usually results from excessive water
chloride ion that is not available for the loss
countercurrent; hyponatremia is not  ELEVATION IN CONDITIONS:
corrected with fluid restriction. dehydration; diabetes insipidus;
 PSEUDOHYPONATREMIA: condition hyperaldosteronism;
characterized by falsely elevated serum hyperadrenocorticism
Sodium level usually cause by the
presence of excess lipids in the serum;
reduction in serum concentration
caused by a systematic error in
measurement
.
ANALYTICAL METHODS:
SPECIMEN: serum, plasma, and urine; whole
blood (depends on analyzer)
PLASMA- Lithium heparin, ammonium heparin,
and Lithium oxalate for anti-coagulant
Haemolysed sample can cause decreased
sodium level
URINE must be 24-hr collection
Sweat can also be used in the analysis
 ION SELECTIVE ELECTRODE (ISE)- most
routinely used method in clinical
laboratories
 ATOMIC ABSORPTION
SPECTROPHOTOMETRY (AAS)
 FLAME EMISSION
SPECTROPHOTOMETRY (FES)/FLAME
EMISSION PHOTOMETRY (FEP)
 CHEMICAL METHODS outdated due to
lack of precision
 COLORIMETRY (Albanese and Lein)
REFERENCE LEVEL:
 SERUM, PLASMA: 136-145mmol/L
 URINE (24-h): 40-220 mmol/day; varies
with diet
 CSF: 136-150 mmol/L
ION SELECTIVE ELECTRODE
 Uses a semipermeable membrane to
develop a potential produced by having
different ion concentrations on either
side of the membrane.
 Two Electrodes are Used:
o One electrode has a constant
potential- making in the
reference electrode
o Another electrode- measuring
electrode
o The difference in potential
between the reference and
measuring electrodes can be
used to calculate the
“concentration” of the ion in
solution.
 Most analyzers use a glass ion-
exchange membrane in its ISE system
for Na+ measurement.
Types Of ISE Measurement
Direct Method Provides an undiluted
-Commonly used- sample to interact with
the ISE membrane
Indirect Method Diluted sample is used
for measurement
No significance in result except if the sample is
hyper lipidemic or hyper proteinemia; excess
protein and lipids present in the serum because
displace water that can cause decreased result
POTASSIUM (Kalium, K+)
 MAJOR INTRACELLULAR CATION- only
2% of the body’s total potassium
circulates in plasma
 Potassium is > inside the cell than extra
cellular fluid, 20x > potassium
concentration inside the cell
o Functions include regulations
of: Neuromuscular excitability;
contraction of the heart;
 intracellular fluid volume;
hydrogen ion concentration
o Potassium concentration has a
major effect in skeletal and
cardiac muscle
 Concentration in RBC is 105 mmol/L
 Reciprocal relationship with H+
 Crump is signs of low potassium level.
 Factors that influence the distribution
of potassium between the cell and
extracellular cell fluid: potassium loss
frequently occurs whenever the
potassium and sodium potassium
ATPase pump is inhibited by conditions
such as: hypoxia, digoxin overdose,
hypomagnesemia and insulin promotes
acute entry of potassium in skeletal
muscle, and insulin promotes also acute
entry of potassium in liver by increasing
sodium potassium ATPase activity
 Cathecolamines such as epinephrine
promotes cellular entry of potassium to
the cell, and beta blockers/propranolol
impairs cellular entry of potassium
 In potassium level determination, avoid
hemolysis. Haemolysed sample will
result in hyperkalemia
 Potassium removes in dialysis; in
hazing, potassium are
decrease/remove.
ELECTROLYTE ABNORMALITIES:
(↑)HYPERKALEMIA seen in
 Dehydration; diabetes insipidus;
hypoadrenalism; acidosis hemolysis
 Is almost always due to impaired renal
excretion.
 3 major diminished renal potassium
excretion: reduced aldosterone or
sldosterone responsiveness, renal
failure, and reduced distal delivery of
sodium.
 Chronic Hyperkalemia
 Severe Hyperkalemia: loss of muscle
excitability
 Plasma K: 6-7 mmol/L alters ECG
 Plasma K: 10mmol/L cardiac arrest
(fatal)
 Acidosis: increased by 0.2-1.7 mmol/L
per unit pH reduction
 Low insulin: increased Digitalis;
heparin, captopril, spironolactone,
cyclosporine
EFFECTS OF HYPERKALEMIA TO CARDIA
MUSCLE
 Hyperkalemia decreases the resting
membrane potential (RMP) of the cell.
 Severe hyperkalemia can ultimately
cause a lack of muscle excitability
(8mmol/L)
(↓)HYPOKALEMIA seen in
 Infusion of insulin to diabetics: alkalosis;
vomiting; over hydration; use of loop
diuretics; syndrome of inappropriate
ADH (SIADH) secretion; Barter’s
syndrome (it is a condition whose
primary cause is the excess excretion of
potassium)
 Arrhythmia and paralysis
 Hyperaldosteronism: K wasting
 Alkalosis: K decrease by 0.4mmol/L per
unit pH increase
 Hypomagnesia: K excretion
SPECIMEN:
 Serum, plasma, and urine (24-h). Whole
blood (depends on analyzers)
 Anti-coagulant for plasma is heparin
  Urine is a 24-hr sample to avoid diurnal
variation.
METHODS OF K+ DETERMINATION
 FLAME EMISSION
SPECTROPHOTOMETRY (FES)
o Violet- end color
 ATOMIC ABSORPTION
SPECTROPHOTOMETRY (AAS)
 ION SELECTIVE ELECTRODE
o Current methos of choice
o Valinomycin membrane is used
to selectively bind K, causing an
impedance change that can be
correlated to K concentration
o KCI is the inner electrolyte
solution
 COLORIMETRY
 Lock head and Purcell
REFERENCE VALUE:
 SERUM: 3.5-5.1 mmol/L
 PLASMA: Males: 3.5-4.5 mmol/L;
Females: 3.4-4.4 mmol/L
 URINE (24-h): 25-125 mmol/day
CHLORIDE (Cl-)
 It is the major extracellular anion
 With sodium, they represent the
majority of the osmotically active
constituent of the plasma.
 FUNCTIONS: Chloride also maintains
osmolality and blood volume and
electric neutrality
 Most of the process Cl shifts secondarily
to the movement of sodium or
bicarbonate and Cl is ingested in the
diet and completely absorbed by
intestinal tract and filtered by
glomerulus—possibly reabsorbed in
conjunction with sodium in the
proximal tubule and the excess Cl is
excreted in urine and sweat; excess
sweating stimulates aldosterone
secretion.
 Aldosterone acts on sweat glands to
conserve sodium and potassium levels.
ELECTROLYTE ABNORMALITIES:
(clinical significance)
 HYPOCHLOREMIA: seen in conditions
salt-losing nephritis; prolonged
vomiting; metabolite alkalosis
 HYPERCHLOREMIA: seen in conditions;
dehydration; renal tubular acidosis;
acute renal failure; metabolic acidosis
associated with prolonged diarrhea;
diabetes insipidus; primary
hyperparathyroidism; salicylate
intoxication
ANALYTICAL MEHTODS:
SPECIMEN: serum, plasma, sweat
 anti-coagulant Lithium heparin
 hemolysis does not cause significant
change in serum or plasma value as a
result of decrease level of intracellular
chloride
 mark hemolysis decrease chloride level
because of dilutional effect
 ISE (ION SELECTIVE ELECTRODE)
 MERCURIMETRIC TITRATION (SCHALES
AND SCHALES METHOD) –it involves
titration Cl with a solution of Mercury
(Hg) forming soluble but nonionized
mercuric chloride. The endpoint is
reached when excess Hg forms a
complex with an indicator, such as
 diphenylcarbazone, producing a violet-
blue color
2Cl + Hg HgCl2 + diphenylcarbazone (indicator)
→ violet blue color
 CUOLORIMETRIC METHOD: mercuric
thiocyanate and ferric nitrate to form a
reddish colored complex with a peak at
480nm
 CUOLOMETRIC-AMPEROMETRIC
TITRATION (COTLOVE
CHLORIDOMETER)- it is a method using
coulometric generation of silver ions
(Ag) which combine with Cl to
quantitate Cl ion concentration. When
all Cl ions are bound to Ag ions, excess
DISTRIBUTION:
free Ag ions are used to indicate the
end point of the reaction. As Ag ions
accumulate, the coulometric generators
and timer are turned off. The elapsed
time is used to calculate the
concentration of the Cl ions in the
sample. The Cotlove chloridometer uses
this principle in chloride analysis.
Ag + Cl → AgCl
REFERENCE VALUE:
 PLASMA/SERUM: 98-107 mmol/L
 URINE (24-h): 11.-250 mmol/Day; varies
with diet
CALCIUM (Ca2+)
 Fifth most common element and the
most prevalent cation in the human
body
FUNCTIONS:
 Important in skeletal mineralization
 Plays vital role in: blood coagulation;
neural transmission, enzyme activity;
maintenance of normal tone;
excitability of skeletal and cardiac
muscle
 It is involved in glandular synthesis and
regulation of exocrine and endocrine
glands
 It preserves the cell membrane’s
integrity and permeability, particularly
in terms of sodium and potassium
 According to ringer, calcium is essential
for myocardial infarction
 99% is part of bones and 1% is in the
blood and ECF
 Free or ionized form (45%);
 Bound to plasma protein (40%);
 Complex form (15%); bicarbonate,
citrate, phosphate, and lactate.
o Ionized calcium is sensitive and
specific marker for calcium
disorders.
o For every 1g/dL serum albumin
decreased, there is 0.8 mg/dL
decrease in total Ca+2—
hypocalcemia can be a
consequence of reduced plasma
albumin
o Ionized calcium concentration
in the blood is 5000 to 10x
higher than cytosol of cardiac or
smooth muscle cells.
FACTORS AFFECTING PLASMA CALCIUM
LEVELS:
1) 1,25 Dihydroxycholecalciferol (1,25-
(OH)2-D3)
 It increases intestinal
absorption of calcium
 It increases reabsorption in the
kidneys.
 It increases mobilization of
calcium from bones
 Enhances the effect of
parathyroid hormone on bone
resorption.
2) Parathyroid Hormone
 It conserves calcium by
increasing reabsorption in the
kidneys
 It increases the level by
mobilizing bone calcium
 It activates the process of bone
resorption; activated osteoclast
breakdown, subsequently a
release of Ca to the ECF.
 It suppresses urinary loss of
calcium
 It stimulates the renal
production conversion of
inactive vitamin D to active
vitamin D3 in the kidneys
 PTH Secretion in blood is
stimulated by the decrease in
ionized calcium. Secretion stop
by increase ionized calcium.
3) Calcitonin
 Is thyroid hormone, secreted by
the parafollicular C cells of the
thyroid gland.
 It inhibits PTH and vitamin D3—
hypocalcemic hormone
 It inhibits bone resorption
 It promotes urinary excretion of
calcium
HOMEOSTASIS:
 Ionized Ca concentration of the
extracellular fluid (ECF) is kept constant
through the action of parathyroid
hormone (PTH) & 1.2.5
dihydroxyvitamin D3
 PTH, which secreted by the parathyroid
gland, acts on the bones and Ca into the
ECF when plasma ionized calcium levels
decreases. It also acts on the kidney and
stimulates increased phosphate
secretion and calcium reabsorption
returning the ionized calcium
concentration to normal.
 Vitamin D almost exclusively activated
by the kidney stimulates calcium
absorption
 Calcitonin possible plays a role in
regulating process. It has a lowering
effect on calcium.
CLINICAL SIGNIFICANCE:
 INCREASED CALCIUM LEVELS seen in:
periods of rapid growth in children;
pregnancy; lactation
 DECREASED CALCIUM LEVELS seen in:
old age
FACTORS THAT INFLUENCE CALCIUM LEVEL:
A. INCREASED CALCIUM ABSORPTION
 Vitamin D (major stimulus)
 Growth hormone
 Increased dietary protein
 HYPOCALCEMIA:
B. DECREASED CALCIUM ABSORPTION
 Severe hypocalcemia leads to tetany
 Formation of insoluble salts
with phosphorus
 CAUSES: Hypoparathyroidism;
 Phytic acid
Pseudohypoparathyroidism; deficiency
 Dietary oxalate
in Vitamin D; chronic renal failure;
 Fatty acids
hypomagnesemia; acute pancreatitis
 cortisol
 CHARD (Calcitonin,
Hypoparathyroidism, Alkalosis, Renal
C. INCREASED URINARY CALCIUM
Failure, vitamin D deficit)
EXCRETION
 Hypercalcemia
 Phosphate deprivation ANALYTICAL METHOD:
 Acidosis
 Glucocorticoids A. TOTAL CALCIUM
a. Spectrophotometric analysis
D. DIMINISHED URINARY CALCIUM with the methaliochromic
EXCRETION: indicators
(Orthocresolphthalein
 PTH
complexone & arsenazo III)
 Certain diuretics
b. Titration of fluorescent calcium
 Vitamin D
complex with ethylene diamine
ABNORMALITIES: tetra acetic acid (EDTA_ or
ethylene glycol tetra acetic acid
HYPERCALCEMIA (EGTA)
c. AAS
 Associated with anorexia, nausea,
d. Clark and Collip method (redox
vomiting constipation, hypotonia,
titration method)
depression and coma

B. IONIZED CALCIUM: ISE (ION SELECTIVE

 CAUSES: primary hyperthyroidism;
multiple endocrine neoplasia; familial
hypocalciuric hypercalcemia; Vitamin D
intoxication
 Thyrotoxicosis: hypoadrenalism;
multiple myeloma
 CHIMPS (Cancer, Hyperthyroidism,
Iatrogenic Cause, Multiple Myeloma,
HyperParathyroidism, and Sarcoidosis)
ELECTRODE) FOR CALCIUM
REFERENCE LEVELS:
 TOTAL CALCIUM - SERUM, PLASMA
o Child: 12 years 2.20-2.70
mmol/L (8.8-10.8 mg/dL)
o Adult: 2.15-2.50 mmol/L (8.6-
10.0 mg/dL)
  IONIZED CALCIUM – SERUM
o Child: 1.20-1.38 mmol/L (4.8-
5.5 mg/dL
o Adult: 1.16-1.32 mmol/L (4.6-
5.3 mg/dL
 IONIZED CALIUM – PLASMA
o Adult: 1.03-1.23 mmol/L (4.1-
4.69mg/dL)
 IONIZED CALCIUM – WHOLE BLOOD
o Adult: 1.15-1.27mmol/L (4.6-5.1
mg/dL)
 TOTAL CALCIUM – URINE (24h)
o 2.50-7.50 mmol/day (100-300
mg/day); varies with the diet
MAGNESIUM (Mg2+)
 It is essential for the function of cellular
enzyme and energy metabolism.
 Has an important role in membrane
stabilization, nerve conduction, ion
transport and calcium channel activity.
 Role in maintenance of intracellular K
concentration.
DISTRIBUTION:
 4th most abundant cation in the body
and the 2nd most prevalent intracellular
cation.
 Magnesium is found in bones and in
soft tissue.
 In serum, 50% Magnesium is in the form
of a free ion (ionized or free
magnesium)
 30% associated with protein, primarily
albumin.
 15% is complexed with phosphate,
citrate and other anions
FACTORS AFFECTING PLASMA MAGNESIUM
LEVEL:
1. Parathyroid Hormone
 It increases renal reabsorption
of magnesium.
 It increases intestinal
absorption of magnesium
2. Aldosterone and Thyroxine
 It increases renal excretion of
magnesium
ABNORMALITIES:
HYPERMAGNESEMIA:
 Increased in blood that is rare and
usually iatrogenic
 Elderly and patients with bowel
disorder and renal insufficiency at risk.
 Clinical manifestation includes,
hypotension, bradycardia, respiratory
depression, depressed mental status
and electrocardiographic (ECG)
abnormalities.
 Common cause renal failure
 Severe result combine effects decrease
renal function and increase intake of
commonly prescribe magnesium
containing medication such as antacids,
enemas or catartics
HYPOMAGNESEMIA:
 Seen in magnesium in the GI tract as in
chronic diarrhea and malabsorption
steatorrhea
 Diabetes mellitus secondary to
glycosuria and osmotic diuresis.
 Alcohol
  Stress
 Frequently observe in hospitalized
patients
 Very rare for non-hospitalized
patients/out-patients
ANALYTICAL METHOD:
A. TOTAL MAGNESIUM
a. AAS (Reference method) –
routinely done in the clinical
laboratory
b. Photometric method on
automated analyzers
c. These methods employ
metallochromic indicators or
dye like calgamite, formazan
dye, magon, and titan yellow
dye.
B. IONIZED (FREE) MAGNESIUM: ISE FOR
MAGNESIUM
C. INTRACELLULAR MAGNESIUM
a. Flourescence measurement
using Furapate (Magnesium
binder)
b. Nuclear magnetic resonance
Spectroscopy
c. Electro probe microanalysis
d. ~Intracellular magnesium
measurement are not currently
employed in clinical lab;
research purposes
DYE BINDING METHODS
Calgamite ( reddish violet complex at 532 nm)
Formazan ( Blue complex at 660nm)
Methylthymol Blue ( 600nm)
O-cresolpthalein complexone ( 570 nm)
REFERENCE INTERVAL:
 TOTAL MAGNESIUM: 0.75-0.95nmol/L
(1.7-2.2 mg/dL or 1.5-1.9
mmol/L)
 IONIZED MAGNESIUM: 0.44-0.60
mmol/L
PHOSPHOROUS* other book uses phosphate*
 An important constituent in nucleic
acid, phospholipid and
phosphoproteins.
 It forms high energy compounds such as
ATP and cofactor (NADP) and is involved
in intermediary metabolism and
various enzyme system
 Essential for muscle contractility,
neurologic function, electrolyte
transport, and oxygen carrying by
hemoglobin.
 In blood, organic phosphate is located
in the red cells, while plasma contains
the inorganic ones.
 In serum, 65% is free, 10% is bound to
protein such as albumin and 35% is
complexed with sodium, calcium, and
magnesium
 Only organic phosphorus is measured
routinely
DISTRIBUTION:
 85% of the total body phosphorus is
present in the skeleton and remaining
15% is in the extracellular fluid (ECF)
and soft tissue.
 The skeleton contains primarily
inorganic phosphates.
 Soft tissues contain primarily organic
phosphates
  In blood, phosphate is located in red
cells; plasma contains organic
phosphate
ABNORMALITIES:
HYPERPHOSPHATEMIA:
CAUSES:
 Decreased level excretion in acute and
chronic renal failure
 Increased in intake with excessive oral,
rectal, intravenous administration
 Increase extracellular load due to
transcellular shift in acidosis
 Secondary to over medication with
Vitamin D and production of Vitamin D
by granulomatous tissue
HYPOPHOSPHATEMIA:
SEEN IN:
 Alcohol abuse
 Intestinal loss due to vomiting, diarrhea,
and use of phosphate binding antacids.
 Induced by a shift of phosphorus from
extracellular fluid in cells.
 Increased urinary excretion, secondary
to hyperparathyroidism, renal tubular
defects and diuretic therapy,
Avitaminosis D)
 D deficiency and steatorrhea
PHOSPHOROUS HAS INVERSE RELATION SHIP
WITH CALCIUM
ANALYTICAL METHOD:
 Reaction of phosphate with Ammonium
Molybdate (Fiske-Subbarow)
Phosphate + ammonium molybdate →
phosphomolybdate complex (measured at
direct UV absorption @ 340 nm)
 Reduction of phosphomolybdate to
molybdenum blue measured at 600-700
nm spectrophotometrically
 Enzymatic method
FISKE-SUBBAROW METHOD
 Most commonly used method to
measure serum organic phosphate
 Most common reducing agent: pictol
(Amino Napthol Sulfonic Acid)
 Other reducing agents: elon (methyl
amino phenol), ascorbic acid, senidine
(N-phenyl-p-phenylene diamine
hydrochloride)
 End product: ammonium-molybdate
complex (unstable)
 The unreduced complex at 340nm is the
most accurate measurement of
inorganic phosphorus in serum
 The pH must be maintained in the acid
range because higher range (alkaline)
can result in reduction of the complex.
 The reduced form of the end product
yields a blue color and determined
between 600nm to 700nm; pag di
reduced 340nm accurate
REFERENCE LEVEL:
 Adult: 2.3-4.7 mg/dL (0.74-1.52
mmol/L)
 Children: 4.0-7.0 mg/dL (1.29-2.26
mmo/L)
OTHERS: BICARBONATE
 Quantitatively second most important
anion fraction in serum.
  Production in the body results from the
dissociation of H2CO3 produced from
the formation of CO2 during
metabolism
 Reconverted to H2CO3 and hence to
H2O and CO2 ad the blood perfuses in
the lungs.
MEASUREMENT:
 Direct titration with an acid.
 Indirectly using PCO2 and pH (H) in an
equation or nomogram
 As total CO2 since 89-90%of all CO2 in
the serum is in the form of HCO3
 Total CO2 determinations are useful
along with pH and PCO3 to evaluate
acid-base disorder
REFERENCE VALUE: 22-32 mmol/L
ANION GAP
 It is difference between sodium and the
sum of chloride and bicarbonate which
is around 26 mEq/L
 Used to monitor recovery from diabetic
ketoacidosis
 An indirect measure of the molar
concentration of acid added to the
system.
 An increase anion gap is due to
replacement of bicarbonate with anion
organic acid.
 The change in anion gap should equal
the change in bicarbonate.
 The presence of a widened anion gap
signifies the presence of metabolic
acidosis due to a non-chloride
containing acid.
 Low anion gap signifies the presence of
high levels of basic proteins, often a
myeloma protein
 Abnormal anion gaps in sera from
healthy person indicate an instrument
problem
 NO TRUE ANION GAPS JUST A
CALCULATED MEASURE.
 NO/NEVER ACTUAL GAP BETWEEN
CATIONIC AND ANIONIC CHRGES.
THERE ARE TWO COMMOLNY USED MTHODS
FOR CALCULATING ANION GAP (AG):
1. AG= Na^+ - (Cl^- + HCO3-)
REFERENCE VALUE: 7-16 mmol/L
2. AG= (Na^+ + K^+) – (Cl^- + HCO3-)
REFERENCE VALUE: 10-20 mmol/L
SWEAT CHLORIDE
SWEAT TEST is performed for the diagnosis of
cystic fibrosis
 Pilocarpine is introduces into the skin
by iontophoresis to stimulate locally
increased sweat secretion.(sweat
inducer) (Gibson and Cooke)
 The resulting sweat is absorbed by filter
paper, weighed, diluted with water and
analyzed for sodium and chloride
 Concentration of over 60 mmol/L of
sweat in least 2 occasions is diagnostic
of cystic fibrosis in children
 Levels of 50-60 mmol/L are suggestive
of cystic fibrosis.
CYSTIC FIBROSIS (mucoviscidosis) of the
pancreas is an autosomal recessive disease
characterized by abnormal secretion of the
various exocrine glands including the sweat
glands. In cystic fibrosis, there is an increased
demonstration of sodium and chloride in the
sweat.

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BITCHES!!!
MODULE 3- LABORATORY
THYROID HORMONES
THYROID GLAND
POSTERIOR VIEW OF THYROID GLAND
 Located near the thyroid capsule
(region of the thyroid gland);
sometimes within the thyroid gland
 May also be found outside their normal
anatomic site- between the hyoid bone
in the neck and mediastinum
 Butterfly shaped gland
 Located in the lower part of neck and
just below the voice box or larynx
 Connected by a narrow band called
isthmus
 11 weeks of gestation, hormone
produce---the measurable hormone or
the thyroid hormone
 Follicle - fundamental unit of thyroid
gland
 2 types:
o FOLLICULAR CELLS or
SECRETORY (T3 & T4)
o PARAFOLLICULAR or C CELLS
(CALCITONIN)--polypeptide
 THYROGLOBULIN – acts as preformed
matrix containing tyrosyl group;
glycoprotein- stored in the follicular
colloid of the thyroid gland
 THYROGLOBULIN – building blocks for
the active thyroid hormones Thyroxine
(T4) and Triiodothyronine (T3)
 T4 has half-life of one week in
circulation; T3 has half-life of one day
 (T4) N.S. 5.5-12.5 ug/dL ; T3 100-200
ng/dL
 T3 exerts majority of thyroidal hormone
effects
 Both T3 and T4 are bound in serum
proteins mostly with thyroxine binding
globulin (TBG)
 3 binding proteins: thyroxine-binding
globulin (TBG), thyroxine-binding
prealbumin (transthyretin), thyroxine-
binding albumin (TBA).
 TBG- transports the majority of T3;
transports 70-75% of total T4.
 TBP/Transthyretin- transports 15-20%
total T4; and it has no affinity for the T3
 TBA- transports T3 and 10% of the T4
BIOSYNTHESIS OF THYROID HORMONES
(formation)
 Thyroid gland synthesizes its
hormone from iodine and essential
amino acid tyrosine
 Iodine enters through the
alimentary tract as iodide (I), lungs
and skin.—also enters thyroid
gland.
 1/3 (of iodine) enters thyroid gland,
2/3(of iodine) leaves body in urine.
 Enzymes oxidize iodide to organic
iodine into monoiodotyrosine (MIT)
and diodotyrosine (DIT).
 Iodination of tyrosine residues in
thyroglobulin results in formation of
MIT and DIT
TYROSINE + IODINE = MIT & DIT
 T3 = MIT + DIT
T4 = DIT + DIT
 99.97% of T4 circulates in bound form
with TBG
 99.7% of T3 circulates in bound form
with TBG
 Main stimulus of biosynthesis of thyroid
hormone is when TSH stimulates the
synthesis of thyroid hormone.
 Note: TSH stimulates synthesis of
thyroid hormone
 T4 secreted 100% in the thyroid gland
 Conversion of T4 to T3 takes place in
many tissues particularly liver and
kidney.
 Protein bound hormone are
metabolically inactive and free
hormones (FT4 and FT3) are
physiollogically active portions of the
thyorid hormones.
 Protein bound hormones do not enter
cells, thus considered to be biologically
inert and functions as storage sites of
circulating thyroid hormones.
 Reverse T3 (rT3) produces by removal
of one iodine from the inner ring of T4,
metabolically inactive product of T4
metabolism
 Iodine is the most essential in the
thyroid hormone synthesis
 recommended daily intake of iodine is
150 ug/day;
 Iodine intake has a below 50 ug/day it is
an indicative of defiency on hormone
secretion.
 Thyroid hormone is very important
because it affects synthesis,
degradation, and intermmediate
metabolism of adipose tissue and
circulating lipids.
PITUITARY – HYPOTHALAMIC RELATIONSHIP
 Thyroid gland responds with the
production of thyroxine on stimulation
of anterior pituitery hormone TSH
(thyrotropin)
 TSH follows stimulation of the gland by
the Hypothalamic peptide TRH –
responds to active levels of T3 and T4
 Low hormone levels TRH provokes TSH
secretion thus, increased thyroidal
iodine metabolism and hormone
production
 The Hypothalamic-pituitarry-thyroid
axis (HPTA) is neuroendocrine system
that regulates the production and
secretion of thyroid hormones
FUNCTION OF THYROID HORMONE
1. For tissue growth
2. For development of the central nervous
system (CNS)
3. Elevated heat production
4. Control of oxygen consumption
5. Influences carbohydrate and protein
metabolism
6. For energy conversation
EFFECTS OF THYROID HORMONE
 Control of oxygen consumption
 Measured by basal metabolic rate
 Influence CHO and protein metabolism,
mobilization of electrolytes, conversion
of carotene to vitamin A
 Essential for development of CNS-
deficient in infant causes irreversible
 mental damage CRETINISM; in adults 14 years old -105-245 ng/dL or 1.8-3.8
slows deep tendon reflexes, diffuse nmol/L
psychomotor retardation
 Thyroid hormone can be a treatment on
Hormone Replcement Therapy (HRT)

TRIIODOTHYRONINE (T3)/ 3,5,3’

TRIIODOTHYRONINE
HINDI KO ALAM KUNG SERYOSO SI MA’AM
 Also known as 3,5,3’ PAREHO ANG DEFINITION NG T3 AT T4 SA PPT
TRIIODOTHYRONINE NIYA , NALOKO NA. yung nasa lower part nang
 Principal secretory product, originates T3 pang 6th bullet ayun mga sinabi niya, di ko
in the thyroid gland inalis ang mga informations from ppt niya at
 Major fraction of organic iodine in the baka malintikan pero information/definition
circulation yun for T4 TALAGA– YNOR QT  ♥
 A prohormone for T3 production
 Serum T4 - indicator of thyroid TETRAIODOTHYRONINE (T4)/ 3,5,3,5’
secretory rate TETRAIODOTHYRONINE
 Elevated Thyroxine causes inhibition of
 Also known as 3,5,3,5’
TSH secretion and vice versa.
TETRAIODOTHYRONINE/THYROXINE
 Major/most active thyroidal hormonal
 Most abundant thyroid hormone
activity
 Principal secretory product, originates
 Almost 75-80% is produced from the
in the thyroid gland
tissue deiodination of T4 – conversion
 Major fraction of organic iodine in
of T4 to T3 takes place in many tissues,
circulation
particularly the liver and the kidneys
 A prohormone for T3 production
 The principal application of this
 Serum T4 - indicator of thyroid
hormone is in diagnosing T3
secretory rate
thyrotoxicosis
 Elevated Thyroxine causes inhibition of
 T3 is a beta indicator of recovering from
TSH secretion and vice versa.
Hyperthyroidism and reccurence of
 All circulating T4 hormone originates in
hyperthyroidism
the thyroid gland – it is secreted 100%
 T3 is helpful in confirming the diagnosis
in the thyroid gland.
of Hyperthyroidism, especially with the
 Amount of Serum T4 is a good insicator
patients with no or minimally elevated
of the thyroid secretory rate.
levels of T4
 REFERENCE VALUE: ADULT: 5.5-12.5
 Increase in the plasma level of T3 is the
ug/dL or 71-161 nmol/L; NEONATE:
first abnormality seen in cases of
11.8-22.6 ug/dL or 152-292 nmol/L
hyperthyroidism.
 REFERENCE VALUE: ADULT- 60-160 THYROID AUTOANTIBODIES RESPONSIBLE FOR
ug/dL or 0.9-2.46 nmol/L; CHILDREN- 1- AUTOIMMUNE THYROID DISORDERS:
 1. Thyroperoxidase (TPO)
-is involved in the tissue destructive
process (Hashimoto’s disease-
autoimmiune thyroid disease)
2. Thyroglobulin (Tg)
3. TSH receptor (TR)
-it is involved in Grave’s disease
FACTORS THAT DECREASE THYROID ACTIVITY
 Enzyme deficiencies may interfere with
iodine metabolism causing congenital
goiter (enlargement of thyroid gland)
 Exogenous thyroid hormone suppresses
hormone production by depressing TSH
levels.
 Thiocyanate and Perchlorate interfere
with iodide concentration
 Thiourea and Thiouracil prevent
incorporation of thyroidal iodine into
organic compounds.
 Anti-thyroid effects of iodine includes
inhibition of iodine binding and
hormonal release
 Radioactive iodine (RAI) has the
additional effect of selectively
irradiating hormonally active tissue,
owing to its uptake by the actively
functioning gland.; also use for the
treatment of thyroid cancer.
NON-SPECIFIC INDICATORS OF THYROID
ACTIVITY
 All thyroidal effects is to stimulate
overall metabolism
 Earliest test for thyroid function was
Basal Metabolic Rate (BMR )- measures
oxygen consumption.
BLOOD CHEMISTRY VALUES
 Thyroid hormones affects synthesis,
degradation and intermediate
metabolism of adipose tissue and
circulating lipids.
 Hyperthyrodism- degradation and
excretion
 Increase than synthesis, resulting to low
levels of cholesterol, phospholipids and
triglycerides
 Hypothyroidism slow catabolism more
then affecting synthesis,
hypercholesterolemia and
hypertriglyceridemia—high levels of
cholesterol and triglyceride
 Increased serum cholesterol levels may
indicates impending hypothyroidism
 Cholesterol levels drops to normal
within 2-3 weeks after successful
therapy for hypothyroidism
 Severe hypothyroidism serum levels of
muscle-associated enzymes tend to rise.
CK and LDH. isoezyme partition reveals
skeletal muscle as the source
EFFECTS OF THYROID DYSFUNCTION ON
NONTHYROID TEST
HYPOTHYROIDISM
 Increased serum cholesterol,
triglycerides
 Increased serum carotene (yellow skin
discoloration)
 Increased serum levels of muscle
enzyme: CPK, AST, LDH
 Increased serum prolactin
 Increased capillary fragility
 Increased spinal fluid protein
 Normochromic anemia, hemoglobin
around 10 g/dL
 Decreased urinary excretion of 17KS,
17—OHCS
HYPERTHYROIDISM
 Increased skin temperature, pulse rate,
pulse pressure
 Decreased serum cholesterol,
triglycerides
 Increased serum level of
aminotransferases and alkaline
phophatase
 Increased retention of BSP
 Altered glucose insulin relationship
 Increased proportion of lymphocytes in
differential white count
 Increased urinary calcium excretion
MEASURING THYROID ACTIVITY
IODINE LEVELS
 Iodine plays one physiologic role in
serum- as a vital constituent of thyroid
hormones, thus, iodine measurements
were used as indicator of thyroid
function
1. PROTEIN BOUND IODINE (PBI)
 Earliest measurement of iodine, but
severely affected by organic and
inorganic iodine contaminants
 Useful to document nonhormonal
iodine, physical damage to the thyroid
gland liberating iodine to the blood
stream.
 Diagnosis of iodine overload or toxicity
like burn victims.
2. BUTANOL-EXCTRACTABLE IODINE
(BEI)
 Technically more difficult
 Does not offer great accuracy
 High levels of iodinated radiographic
contrast dyes causes interference
 Iodine is also a component of
radioactive machines; CT-Scan,
3. THYROXINE (T4)
 Radioimmunoassays (RIA)/ Enzyme-
Linked immunoassay (ELISA)
 Widely available as antibody specifities
have no significant cross activity
between T4 and T3 in assays for each
hormone.
 Uses TBG as the specific binder in place
of an anti-T4 antibody
 Measurement of T4 is the first round
test in screening thyroid disease.
TRIIODOTHYRONINE (T3)
 Most active thyroid hormonal activity
 75-80% produced from tissue
deiodination of T4
 Useful in the confirmation of a
suspected thyroid abnormality (T3
thyrotoxicosis)
 Indicator of recovery from
hyperthyroidism, recognition of
recurrence
 Triiodothyronine levels are necessary to
monitor thyroid replacement therapy
consist of T3
THYROXINE- BINDING GLOBULIN & FREE
HORMONE
 Evaluated when T3 and T4 values are
abnormal
 Use RIA or T3 uptake test.
o For HYPERTHYOIDISM, both T3
and T4 have HIGH values.
o For HYPOTHYROIDISM, both T3
and T4 have LOW values.
 Increase in the plasma level seen in
cases of hyperthyroidism
  REFERENCE VALUES: 80-200 ng/dL or
1.2-3.1 nmol/L (ADULT); 105—245
ng/dL or 1.8-3.8 nmol/L (CHILDREN 1-14
yrs. Old)
TETRAIODOTHYRONINE/THYROXINE (T4)
 Principal secretory product, originates
in the thyroid gland
 Major fraction of organic iodine in the
circulation
 A prohormone for T3 production
 Serum T4 – indicatorr of thyroid
secretory rate
 Elevated Thyroxine causes inhibition of
TSH secretion and vice versa
 REFERENCE VALUES: 5.5-12.5 ug/dL or
71-161 nmol/L (ADULT); 11.8-22.6
ug/dL or 152-292 nmol/L (NEONATES)
FACTORS AFFECTING THYROXINE- BINDING
GLOBULIN (TBG)
 Estrogens- increase the amount of TBG
 Androgens and glucorticoids-
decrease/depress TBG
 Pregnant women and patients taking
exogenous estrogens, or endocrine
secreting tumors,- T3 uptakes are low
due to increase TBG concentration
 Low TBG concentration, high T3 uptake,
protein levels are low.
Example: Liver disease,
nephrotic syndrome
 Drugs like salicylates and phenytoin,
anticonvulsants binds TBG, competes
with thyroxine. Thus gives hish residual
T3 uptake occupied by the
nonhormonal agents.
 Administration of heparin causes
elevation of free fatty acids from
breakdown of triglycerides by post-
heparin lipoprottein lipase activation.
 False High T3 uptake
 Lithium also afects thyroid hormones
STIMULATION OFTHYROID-STIMULATING
HORMONE (TSH)
 Thyroid activity is regulated by the
body’s perceived need for hormone.
 If in-adequate thyroid hormone
circulates in free fraction,
hypothalamus produces, TRH that
provokes rising TSH levels to stimulate
thyroid output.
 Immunoassays
 VALUES: 0-10 ulU/ml
 SIGNIFICANCE:
o TSH is increased in primary
hypothyroidism.
o TSH levels distinguish true
hypothyroidism from euthyroid
condition with low thyroid
hormone levels
CLINICAL DISORDERS
 SCREENING: recommended at the age
of 35 and every 5 years thereafter
THYROID DISORDERS
HYPOTHYROIDISM
 Hypometabolism called myexedema
Example: Lethargy ,
constipation, dry skin, and hair,
premenopausal women
(excessive bleeding)
  SYMPROTMS: Slowed tendon reflexes,
coarse skin texture, facial puffiness,
cold intolerance, decreased sweating,
impaired memory, slowing of speech
and motor activity
 Systolic and distolic is high but slow
heart rate
 CONGENITAL HYPOTHYROIDISM –
irreversible, severe mental retardation,
characteristic body changes, Cretinism.
All newborn are screened by heel-stick
blood sample for low T4, confirmed by
TSH assay.
 Treatment- lifelong administration of
thyroid hormone soon after birth
HYPERTHYROIDISM
 Thyroid produces excessive hormone
from nodular areas of:
o Hyperfunctioning – ex:
adenomas and toxic nodular
goiter.
o Overall hyperactivity – diffuse
toxic goiter. Ex: Grave’s disease
 Manifest nervousness, fatigue, weight
loss, heat intolerance, increased
sweating
 TOXIC NODULAR GOITER- excessive
prominence of eyes (exopthalmos),
widening of palpebral fissures.
 T3T4 are high T3 uptake and high free
T4 index.
 Subclass, T3 thyrotoxicosis. Normal T4
and Ft4 but increased T3.
 STIMULATION OF CHRONIC
GONADOTROPIN (same chemical
structure with TSH): Px with high hCG
levels from hydatid form mole,
choriocarcinoma and embryonal
carcinoma of the testis causes
hyperthyroidism
 Thyroid cancer is compatible to life; also
it is a friendly cancer.
THYROIDITIS
 Lymphocytic infiltration and fibrosis
occur
 Histologic appearance: that of
mediated chronic inflammation
 CONDITION: Lymphocytic
thyroiditis or Hashimo’s disease
(thyroglobulin is the auto antibody).
Often with glandular enlargement.
High titer of thyroglobulin and
microsomal antigen
SUBACUTE THYROIDITIS
 May indicate viral agent
 Disorder begins as sore throat with
fever, progresses involving one or both
lobe, large and tender on palpation or
on swallowing
 Thyroid Biopsy- inflammatory cell
infiltrates
 Atrophy - leads to irreversible
hypothyroidism
 Can recover with treatment of aspirin:
Steroids
EUTHYROID SICK SYNDROME
 Severe illness that indicates
hypothyroidism. Patients with
neoplastic disease, diabetes mellitus,
burn, trauma, cardiovascular
conditions, liver disease, renal failure or
prolonged infection
 2 changes underlie abnormal hormonal
measurement. Serum albumin drops on
severe illness, fall of prealbumin
decreases hormone binding capacity.
TSH normal, reverse T3 elevated.
CLINICAL DISORDERS
1. HYPERTHYROIDISM
 Excess circulating TH
 Signs and symptoms/ Features:
Tachycardia, tremors weight loss, heat
intolerance, emotional changes,
menstrual changes.
 Primary Hyperthyroidism (PM Hy):
Elevated T3 and T4 decreased TSH;
major hormone
 Secondary Hyperthyroidism(Sec. Hy).:
Increased TSH and FT4 (due to lesion in
pituitary gland or other gland)
A. THYROTOXICOSIS
 High levels of free TH in circulation
 Is applied to a group of syndrome
caused by high levels of free thyroid
hormones in the circulation
 T3 thyrotoxicosis or Plummer’s
disease: FT4 increased but FT4 normal
with low TSH
 T4 thyrotoxicosis: T3 normal or low but
T4 increased with low TSH
B. GRAVES’ DISEASE (DIFFUSE TOXIC
GOITER)
 Common cause of thyrotoxicosis
 Autoimmune disease in which
antibodies are produced that inactive
the TSH receptor
 More in women; occurs 6x more
commonly in women than in men
 SS/ Features: exophthalmos (bulging
eyes), pritibial myxedema
 Diagnostic Test: TSH receptor antibody
test.
C. REIDEL’S THYROIDITIS
 Thyroid turns to woody or stony hard
mass.
D. SUBCLINCAL HYPERTHYROIDISM
 Shows no clinical symptums BUT Low
TSH, FT3 & FT4 normal
E. SUBACUTE
GRANULOMATOUS/SUBCACUTE
NONSUPPURATIVE THYROIDITIS/DE
QUERVAIN’ THYROIDITIS (PAINFUL
THYROIDITIS)
 SS/Features: neck pain, low grade
fever, swings in thyroid function test.
 Thyroidal peroxidase (TPO) antibodies
absent; ESR and thyroglobulin levels are
elevated
2. HYPOTHYROIDISM
 Develops whenever insufficient
amounts on thyroid hormone are
available to tissues.
 It is treated with thyroid hormone
replacement therapy (levothyroxine)
 Signs and symptoms Features:
bradycardia, weight gain, coarse skin,
cold intolerance, and mental dullness
1. PRIMARY HYPOTHYROIDISM
 Deficiency in elemental iodine (↓T3
and T4, ↑ TSH)
 Cause by destruction or ablation of
thyroid gland.
 Signs and symptoms/Features: surgical
removal of gland, used of radioactive
iodine for hyperthyroidism treatment;
radiation exposure; drugs like lithium
 a. HASIMOTO’S DISEASE
(CHRONIC AUTOMIMMUNE
THYROIDISTIS
 Most common cause of primary
hypothyroidism
 Characterized by a thyroid replaced
by a nest of lymphoid tissue –
sensitized T
lymphocytes/autoantibodies bind
to cell membrane causing cell lysis
and inflammatory reaction.
 It is associated with enlargement if
the thyroid gland (goiter)
 METHOD OF TESTING:
o TPO antibody= +result
o TSH= increased
b. MYXEDEMA
 Describes the peculiar nonpitting
swelling of the skin
 Skin becomes infiltrated by
mucopolysaccharides
 Myxedema coma is the severe
form of primary hypothyroidism
 Signs and Symptoms/ Features:
“puffy” face, weight gain, slow
speech, eyebrows thinned, dry and
yellow skin, andanemia
2. SECONDARY HYPOTHYROIDISM
 Due to pituitary destruction or pituitary
adenoma
 Lab results: T3 and T4 low levels, TSH
decreased
3. TERTIARY HYPOTHYROIDISM
 Due to hypothalamic disease
 Lab results: T3 and T4 low levels, TSH
decreased
4. CONGENITAL HYPOTHYROIDISM/
CRETINISM
 Defect in development or function of
the gland
 Symptoms: physical and mental
development of the child are retarded
 Lab results: T4 decreased, TSH
increased
5. SUBCLINICAL HYPOTHYROIDISM
 Lab results: Tt3 and T4 normal but TSH
is slightly increased
THYROID FUNCTION TEST
1. TRH Stimulation Test
2. TSH test
3. Radioactive Iodine Update (RAIU
4. Thyroglobulin (Tg) Assay
5. Reverse T3 (rT3)
6. Free Thyroxine Index (FTI or T7)
7. Total T3 (TT3) Free T3, Free T4
8. T3 uptake test
9. Thyroxine binding globulin (TBG) test
10. Fine needle Aspiration
11. Recombinant Human TSH
12. Tanned Erythrocyte Hemmaglutination
Method
13. Serum Calcitonin Test tumor marker for
detecting residual thyroid metastasis in
Medullary thyroid carcinoma (MTC)
14. Pentagastrin (Pg) Stimulation test
TRH STIMULATION TEST (THYROTROPIN
RELEASING HORMONE)
 Measures the relationship between the
TRH and TSH secretions
 Most specific and sensitive test for the
diagnosis of thyroid diseases
 Used to differentiate euthyroid and
hyperthyroid patients who both have
undetectable TSH levels.
 May also be helpful in the detection of
thyroid hormone resistance syndromes
  Confirm borderline cases of euthyroid
Graves’s disease.
DOSE NEEDED: 500ug TRH by IV
INCREASED LEVEL: primary
hypothyroidism
DECREASED LEVEL: hyperthyroidism
TSH TEST
 Most important thyroid function test –
best screening test; the best method for
detecting clinically significant thyroid
dysfunction
 Most clinically sensitive assay for the
detection of primary thyroid disorders
 Helps in the early detection of
hypothyroidism
 Used to differentiate primary
hypothyroidism from secondary
hypothyroidism
 Used to monitor and adjust thyroid
hormone replacement therapy
 Sensitivity of the third-generation TSH
assays has led to the ability to detect
what is termed subclinical disease – or
mild degree of thyroid dysfunction (due
to the large reciprocal change in TSH
levels seen for even small changes in
free T4)
 REFEENCE VALUES: 0.5-5 uU/mL
INCREASED TSH DECREASED TSH
1. Primary Hypothyroidism 1. Primary hyperthyroidism
2. Hashimoto’s disease 2.Secondary/Tertiary
Hypothyroidism
3. thyrotoxicosis due to 3.Treated Grave’s disease
pituitary tumor
4. TSH antibodies 4.Euthyroid sick disease
5. Thyroid hormone 5.Over replacement of thyroid
hormone in hypothyroidism
THYROXINE BINDING GLOBULIN (TBG)
 Useful to distinguish between
hyperthyroidism causing high thyroxine
levels and euthyroidism with increased
binding by TBG and increased thyroxine
(↑ T4 + N TBG)
 Total serum T3 and T4 are dependent
on the amount of TBG
 Used to confirm results of FT3 and FT4
or abnormalities in the relationship of
the total thyroxine (TT4) and THBR test
 Helps in the diagnosis of patients having
elevated T3 and T4 levels but no
correlation with the other thyroid
function tests, or not compatible with
clinical findings
 TBG excess leads to increased serum
levels T3 and T4 but the unbound or
free form of these hormones in the
blood remain unchanged
HORMONAL EFFECT: estrogen increases
TBG while androgens depresses TBG
INCREASED LEVELS: hypothyroidism,
pregnancy, estrogen surge
DECREASED LEVELS: anabolic steroids
nephrosis
REFERENCE VALUE: B13-39 ug/dL (150-
360 nmol/L)
FINE-NEEDLE ASPIRATION
 Is the most accurate tool in the
evaluation of thyroid nodules
RECOMBINANT HUMAN TSH
 Used to test patients with thyroid
cancers for the presence of residual or
recurrent disease
TANNED ERYTHROCYTE HEMMAGLUTINATION
 Measure of antithyroglobulin antibodies
 SERUM CALCITONIN
 Tumor marker for det4ecting residual
thyroid metastasis in medullary thyroid
carcinoma (MTC)
 Should be measured before and 6
months after surgery
 High uptake indicates metabolically
active gland (active hormone
production)
 High uptake + TSH deficiency =
autonomous thyroid activity
THYROGLOBULIN (Tg) ASSAY

PENTAGASTRIN (Pg) STIMULATION TEST  Normally used as a postoperative

marker of thyroid cancer
 Used for the diagnosis of MTC
 Used in monitoring the course of
 PROCEDURE: an intravenous Pg (0.5 metastatic or recurrence of thyroid
ug/kg body weight) is given within 5 cancer (a well-differentiated tumors
seconds; blood samples are collected at typically display a 10-fold increase in Tg
baseline and 1, 2, 5, an 10 minutes after in response to a high TSH)
the start of the infusion
 When checking Tg as a tumor marker
DISORDERS T3 T4 TSH FT rT3 Tg TBG for thyroid cancer, Always check a
4 simultaneous sample for thyroglobulin
GRAVE’S DISEASE ↑ ↑ ↓ ↑ ↑ ↑ N antibodies
 Differentiates subacute thyroiditis
PRIMARY N/ ↓ ↑ ↓ ↓ N N
HYPOTHYROIDISM ↓ / (↑Tg) from thyrotoxicosis factitia (↓
↓ Tg)
HASHIMOTO N/ N/ ↑ N/ ↓ N N  INCREASED: untreated and metastatic
THYROIDITIS ↓ ↓ ↓ / differentiated thyroid cancer, nodular
↓ goiter and hyperthyroidism
NONTHYROIDAL ↓ N/ V V N/ N N
 DECREASED: infants with goitorous
ILLNESS ↓ ↑
hypothyroidism and thyrotoxicosis
THYROID HORMONE ↑ ↑ N/ ↑ ↑ ↑ N factitia
RESISTANCE ↑
 REFERENCE VALUE: (ADULT)= 3-42
NEONATAL ↓ ↓ ↑ ↓ ↓ N N
HYPOTHYROIDISM / ng/mL or ug/mL; (INFANT) = 38-48
↓ ng/mL or ug/mL
N-NORMAL V- VARIABLE  METHODS FOR TESTING: double-
antibody RIA, ELISA, IRMA,
MGA HINDI BINANGGIT SA PPT PERO KASAMA immunochemiluminescent assay (ICMA)
SA THYROID FUNCTION TEST:
REVERESE T3 (rT3)
RADIOACTIVE IODINE UPTAKE (RAIU)
 Formed by the removal of one iodine
 Used to measure the ability of the from the inner ring of the T4
thyroid gland to trap iodine  An end product of T4 metabolism; the
 Helpful in establishing the cause of 3rd major circulating thyroid hormone
hyperthyroidism  Identifies patients with euthyroid sick
syndrome (elevated rT3)
  Used to assessed borderline or
conflicting laboratory results
 REFERENCE VALUES: 38-44 ng/dL
FREE THYROXINE INDEX (FTI OR T7)
TERMINOLOGIES
 Indirectly assesses the level of free T4 in
blood
 Based on the equilibrium relationship of
bound T4 and FT4
 Important in correcting euthyroid
individuals
 It is elevated in hyper thyroidism and
decreased in hypothyroidism
 REFERENCE VALUES: 5.4-9.7
TOTAL T3 (TT3) FREE T3, FREE T4
 FT4 is used to differentiating drug
induced TSH evaluation and
hypothyroidism
 The value of TT3 or FT3 is in confirming
hyperthyroidism
 DIRECT/REFERENCE METHOD:
Equilibrium dialysis
T3 UPTAKE TEST
 Measures the number of available
binding sites of the thyroxine-binding
proteins, most notably TBG; a test for
TBG
 Does not measure the level of T3 in
serum but it reflects the serum level of
TBG
 Inversely related to TBG- decr4eased T3
uptake results to elevated TBG result
and vice versa
INCREASED: hyperthyroidism,
euthyroid patients, chronic liver disease
DECREASED: hypothyroidism, oral
contraceptives, pregnancy; acute
hepatitis
REFERENCE VALUE: 25-35 %
1) AMENORRHEA
 Absence of menstruation; Primary-
never occurred
 Secondary absence of menses for 6
months
2) CARCINOID SYNDROME
 Constellation of symptoms like
diarrhea, flushing, tachycardia and
hypotension which are produced by
the release of amines (histamine,
kallokrein, and prostaglandine) into
the circulation
3) CUSHING DISEASE
 Metabolic disorder characterized by
abnormal increased secretion of
ACTH results to cortisol production
4) CUSHING’S SYNDROME
 Chronic excessive production of
cortisol by adrenal cortex or
administration of large doses of
glucocorticoids.
COMMON CAUSE: pituitary
hormone
5) GYNECOMASTIA
 Development of breast tissue in
males. Related balance of estrogen
to androgen
6) HIRSUTISM
  Excessive growth of hair with a
male distribution pattern in a
female. Endocrine disorder to
women

7) TURNER’S SYNDROME
 Short stature. “webbed neck” low
hairline on the neck and broad
shield-like chest
 Amenorrhea with increased levels
of FSH and LH

8) VIRLIZATION
 Secondary male sexual
characteristic are acquired by a
female. Result of adrenal
dysfunction or hormonal
medication

9) ZOLLIGER-ELLISON SUNDROME
 Severe peptic ulcer of the stomach
MODULE 4- LABORATORY
PANCREATIC HORMONES
PANCREAS
 Digestive gland in the
gastrointestinal system.
 Both an endocrine and exocrine
gland
o ENDOCRINE GLAND –
insulin, glucagon, and
somatostatin
o EXOCRINE GLAND -
amylase and Lipase;
Digestive enzymes
 The specialized tissue called islets
of Langerhans
 Islets of Langerhans represent
approximately 1-2% of the pancreas
 TYPES OF CELLS ARE RECOGNIZED
IN THESE ISLETS:
o A CELLS (alpha) – Producing
glucagon (25% of all islets
FUNCTIONS OF PANCREAS
cells)
o B CELLS (beta)– producing
insulin (60% of all islets
cells)
o D CELLS (delta)– producing
somatostatin (10% of all
islet cells)
o F CELLS (known now as
PANCREATIC POLYPEPTIDE
or PP CELLS) – producing
pancreatic polypeptide (5%
of all islet cells)
 Islets of Langerhans play a crucial role in
carbohydrate metabolism and so in a
plasma glucose concentration. It
involves:
o GLYCOLYSIS – the anaerobic
conversion of glucose to
lactate. Occurs in the red blood
cells, renal medulla and skeletal
muscles.
o GLYCOGENESIS – the synthesis
of glycogen from glucose.
Glucose is stored (in liver,
muscle) in the form of glycogen
and this serves to maintain a
constant plasma glucose
concentration
o GLYCOGENOLYSIS- the
breakdown of glycogen to
glucose
o GLUCONEOGENESIS- the
production of glucose from
non-sugar molecules (amino
acids, lactate, glycerol)
o LIPOLYSIS- the breakdown of
triacylglycerols into glycerol and
fatty acids.
o LIPOGENESIS- the synthesis of
triaglycerols.
 Pancreatic hormones are responsible
for storage of fat and glucose, as
glycogen, after meal
 Enables the mobilisation of energy
reserves as a result of food deprivation,
stress, physical activity
 Maintain the constant plasma glucose
concentration.
  Promote growth

PANCREATIC HORMONE

 Insulin – primary hormone responsible

for decreasing the glucose levels;
synthesize by beta cell of islets of
Langerhans; release when there is a
high glucose levels; only hormone that
HYPOGLYCEMIC; stored in the liver, fat
and muscles; reciprocal relationship
with glucagon
 Glucagon – primary hormone
responsible for increasing glucose
levels; HYPERGLYCEMIC AGENT;
synthesize by the alpha cell; release
during stress and fasting state
 Somatostatin- produce by the delta
cells of islets of Langerhans; primarily
inhibits the action of insulin growth
hormone and glucagon

PANCREATIC ENZYMES

 Amylase
 Lipase

Note: Please see your notes in ENZYMES

LECTURE DISCUSSION the AMYLASE and LIPASE
to be exact; MODULE 1
MODULE 5- LABORATORY
GONADS
REPRODUCTIVE HORMONES/ SEX HORMONES
 Testes and Ovaries: Produces sex
hormones- Androgens and Estrogens
from cholesterol
 OVARIES- converts testosterone to
estradiol and androstenedione to
estrone
 Peripheral tissues converts or reduce
testosterone to dihydrosterone (DHT)
hydroxylate estradiol to estriol, convert
adrenal androgens to testosterone and
androgens to esterone and estradiol
 Major transport proteins: Sex
hormone-binding globulin (SHBG) 60%,
Corticosteroid-binding globulin (CBG)
and albumin 40%
 Concentration of binding protein
determines the level of total
testosterone but not free testosterone
level during laboratory estimation
SEX HORMONES
 SHBG: transports androgens and
estrogens;
 CHG: delivers progesterone and
glucocorticoids
 1-2% sex steroids are unbound or free.
Remaining is bound to proteins
(ALBUMIN, SHBG, AND CHG)
 Free fractions are active, and only the
ones can diffuse into the vascular
system and interact with the target cells
TESTOSTERONE
 Principal androgen hormone in the
blood -- Most potent male androgen,
 synthesized by the Leydig cells of the
testis from progesterone,
 controlled primarily by FSH and LH
 FUNCTIONS: growth and development
of the reproductive system, prostate
and external genitalia
 Demonstrate circadian pattern: peak at
time of awakening (8am), lowest at
8pm
 Gradual reduction of testosterone at
the age of 30, average decline of about
110 ng/dL every decade
 Obesity may cause decrease plasma
testosterone concentration
 After age 50, men experience a
decrease in secretion rate and
concentration of testosterone, and
women have an increase in pituitary
gonadotropins, especially follicle-
stimulating hormone.
 Infertility testing: semen analysis,
testosterone, FSH,LH
 Reference value: 3.9-7.9 ng/mL (serum)
 TYPES OF TESTICULAR INFERTILITY
(HYPOGONADISM)
o PRETESTICULAR INFERTILITY
(SECONDARY
HYPOGONADISM):
HYPOTHALAMIC/ PITUITARY
LESIONS- Normal to decrease
levels in testosterone, FSH and
LH levels
o TESTICULAR INFERTILITY
(PRIMARY HYPOGONADISM) :
may be congenital
(cryptorchidism, Kinefelter’s
syndrome and 5-a-reductase
deficiency) or acquired
(varicocele, tumor, orchitis).;
Decreased testosterone levels
 and increased FSH and LH
levels.
o POST-TESTICULAR
INFEERTILITY: due to disorders
of sperm transport and function
Normal blood levels:
testosterone, FSH and LH
OTHER DISORDERS:
 TESTICULAR FEMINIZATION
SYNDROME
o most severe form of androgens
resistance syndrome resulting
in lack of testosterone action in
target tissues.
o Physical development pursues
the female phenotype, with
fully developed breast and
female distribution of fat and
hair
o No utility or response to
administration of exogenous
testosterone
o Lab tests: normal levels of
testosterone with elevated FSH
and LH levels
 SERTOLI-CELL-ONLY SYNDROME
o lack of germ cells,
o men with small testis, high FSH
levels, azoospermia and normal
testosterone levels
o testicular biopsy is the only
procedure to confirm this
diagnosis
 KALLMANN’S SYNDROME
o Result of inherited, x-linked
recessive trait that manifest
hypogonadism during puberty
o ASSOCIATED DEFECTS: anosmia
(inability to smell) and midline
defects (cleft palate and lip)
DHEA: DEHYDROEPIANDROSTERONE/
ESTROGEN
 DHEA
o formed by adrenal cortex;
weak androgen;
o Androgen is derived from
adrenal gland.
o Valuable in the assessment of
adrenal cortical function
 ESTROGEN
o Carbon-18 steroid hormone
that have a phenol A ring.
o structural alteration of
testosterone molecule;
o produce by ovaries after
menopause
o FUNCTION: promotion of breast
development, maturation of the
external genitalia, deposition of
body fat and termination of
linear growth. (secondary
sexual characteristic in the
female)
o PROGESTERONE: function in
uterine growth and regulation
of menstrual cycle and
maintenance of pregnancy
o DEFICIENCY: irregular and
incomplete development of the
endometrium
o PRECURSOR: acetate,
cholesterol, progesterone, and
testosterone
ESTROGEN
 Estrone and Estriol are metabolites of
intraovarian and extra glandular
conversion
3 FORMS:
 ESTRONE (E1):
o most abundant estrogen in
post-menopausal women
 ESTRADIOL (E2):
o most potent estrogen secreted
in ovary, major estrogen;
o most abundant in
premenopausal women; low
levels in menopausal stage;
o synthesized from testosterone
then diffuses out of the thecal
cells of the ovaries in female;
o precursors of E1 and E3
o used to assess ovarian
functions; serves as negative
feedback for FSH
o TRANSPORT PROTEIN: albumin
(60%); SHBG (38%)
o Free form of E3 is
approximately 2%
 ESTRIOL (E3)
o metabolite of estradiol;
o found in maternal urine;
o major estrogen secreted on
fetal and placental during
pregnancy-formation in a
pregnant women is dependent
on fetal and placental function
o Used to assess fetoplacental
unit postdate gestations and
intrauterine retardation;
o promotes uteroplacental blood
flow as potent as estrogen;
o used as marker for Down
syndrome (together with AFP
and HCG)
o PREFFERED SPECIMEN: plasma
PROGESTERONE
 Carbon-21 compound in the steroid
family
 Produced by the Lutein cells of the
corpus luteum in the female
 Prime secretory product of ovary.
 Dominant hormone responsible for the
luteal phase cycle among females.
 Single best hormone in the
determination of ovulation if occurred.
 Prepares the uterus for pregnancy and
lobules of breast for lactation
 Intermediate in the synthesis of adrenal
steroids and androstenedione.
 DEFICIENCY: failure of implantation of
embryo
 METABOLITES: pregnanediols (easiest
measured metabolite), pregnanediones,
pregnanalones
SPECIFIC TESTS:
 Test of menstrual cycle dysfunction
and anovulation: Estrogen,
progesterone, FSH and LH
 Test for female infertility: HCG (human
Chorionic Gonadotropin), PRL, FT$, TSH,
FSH, LH, ESTRADIOL, and
PROGESTERONE
 Test of infertility male comes first.
MODULES IN THE CANVAS
THYROID GLAND
Thyroid Function Test
Introduction
The thyroid gland is a butterfly-shaped
endocrine gland that is normally located in the
lower front of the neck. The major thyroid
hormone secreted by the thyroid gland is
thyroxine, also called T4 because it contains
four iodine atoms. To exert its effects, T4 is
converted to triiodothyronine (T3) by the
removal of an iodine atom. This occurs mainly in
the liver and in certain tissues where T3 acts,
such as in the brain. The amount of T4
produced by the thyroid gland is controlled by
another hormone, which is made in the
pituitary gland located at the base of the brain,
called thyroid stimulating hormone
(abbreviated TSH). The amount of TSH that the
pituitary sends into the bloodstream depends
on the amount of T4 that the pituitary sees. If
the pituitary sees very little T4, then it produces
more TSH to tell the thyroid gland to produce
more T4. Once the T4 in the bloodstream goes
above a certain level, the pituitary’s production
of TSH is shut off.
Principle of the test:
The principle of the following enzyme
immunoassay test follows a typical one-step
capture or ‘sandwich’ type assay. The assay
makes use of two highly specific monoclonal
antibodies: A monoclonal antibody specific for
TSH is immobilized onto the microwell plate and
another monoclonal antibody specific for a
different region of TSH is conjugated to horse
radish peroxidase (HRP). TSH from the sample
and standards are allowed to bind
simultaneously to the plate and to the HRP
conjugate. The washing and decanting steps
remove any unbound HRP conjugate. After the
washing step, the enzyme substrate is added.
The enzymatic reaction is terminated by
addition of the stopping solution. The
absorbance is measured on a microtiter plate
reader. The intensity of the colour formed by
the enzymatic reaction is directly proportional
to the concentration of TSH in the sample. A set
of standards is used to plot a standard curve
from which the amount of TSH in patient
samples and controls can be directly read.
Materials:
Manufacturer: Labor Diagnostika Nord GmbH
TSH TEST ELISA
1. Precision pipettes to dispense 50, 100, 150
and 300 µl
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microwell plate reader with a filter set at
450nm and an upper OD limit of 3.0 or
greater*
Procedure:
1. Prepare working solutions of the anti-TSH-
HRP conjugate and wash buffer.
2. Remove the required number of microwell
strips. Reseal the bag and return any
unused strips to the refrigerator.
3. Pipette 50 µl of each calibrator, control and
specimen sample into correspondingly
labelled wells in duplicate.
4. Pipette 100 µL of the conjugate working
solution into each well. (We recommend
using a multichannel pipette).
5. Incubate on a plate shaker (approximately
200 rpm) for 90 minutes at room
temperature.
6. Wash the wells 3 times with prepared wash
buffer (300 µL/well for each wash) and tap
the plate firmly against absorbent paper to
ensure that it is dry (The use of a washer is
recommended).
7. Pipette 150 µL of TMB substrate into each
well at timed intervals.
8. Incubate the plate on a plate shaker at
room temperature for 15-20 minutes. (or
until Calibrator A attains dark blue colour
for desired OD).
9. Pipette 50 µl of stopping solution into each
well at the same timed intervals as in step
7. 10. Read the plate on a microwell plate
reader at 450 nm within 20 minutes after
addition of the stopping solution.
a. TSH test
 A high TSH level most often means you
have hypothyroidism, or an underactive
thyroid.
 This means that your thyroid isn’t making
enough hormone. As a result, the pituitary
keeps making and releasing TSH into your
blood. A low TSH level usually means you
have hyperthyroidism, or an overactive
thyroid.
 This means that your thyroid is making too
much hormone, so the pituitary stops
making and releasing TSH into your blood.
b. T4 tests
 A high blood level of T4may mean you have
hyperthyroidism.
 A low level of T4may mean you have
hypothyroidism.
 changes in binding protein levels don’t
affect free T4levels, many healthcare
professionals prefer to measure free T4.
c. T3 test
 If your health care professional thinks you
may have hyperthyroidism even though
your T4 level is normal, you may have a
T3 test to confirm the diagnosis.
 Sometimes T4 is normal yet T3 is high, so
measuring both T4 and T3 levels can be
useful in diagnosing hyperthyroidism.
d. Thyroid antibody tests
 Measuring levels of thyroid antibody (Links
to an external site.)may help diagnose an
autoimmune thyroid disorder such as
Grave's disease—the most common cause
of hyperthyroidism—and Hashimoto's
disease the most common cause of
hypothyroidism.
 Thyroid antibodies are made when your
immune system attacks the thyroid gland
by mistake.
Pancreatic Hormones
Introduction
Pancreas is both exocrine and endocrine gland.
The exocrinal part secretes pancreatic fluid into
the duodenum after a meal. The endocrinal part
secretes various types of hormones. These are
produced by a specialized tissue in the pancreas
and then released to the capillary system and
reached the liver by the portal venous
circulation. The specialized tissue is called islets
of langerhans. Islets of Langerhans represent
approximately 1-2 % of the pancreas.
Principle of the test:
The human insulin assay used is a sandwich
ELISA. The solid phase microplate is coated with
a blend of monoclonal antibodies directed
against distinct epitopes of the human insulin
peptide. Samples, standards and controls are
pipetted into these wells, along with a
conjugate monoclonal antibody against human
insulin, which has horseradish peroxidase (HRP)
covalently linked to it. During the incubation
the insulin antigen binds to the immobilized
capture antibodies. At the same time the
conjugate antibody is binding to the insulin
present in the well forming the sandwich. After
washing to remove unbound materials, a
chromogenic-substrate for HRP, consisting of
tetramethylbenzidine (TMB) and hydrogen
peroxide (H2O2) is added. The substrate is
converted to a colored compound by the action
of HRP. The reaction is halted by the addition of
H2SO4 and the microplate is read
spectrophotometrically. The absorbance of the
solution is proportional to the amount of insulin
present in the original sample.
Materials
1. BioTek (Machine and commercially
prepared reagent)
2. Serum sample
3. Pipettes
Procedure:
1. Samples and standards were prepared off
line and 50 µL of each was pipetted
manually into the assay plate.
2. After loading samples and standards, 50 µL
of assay conjugate was added to all
wells. The plates were incubated at room
temperature for 30 minutes on a
microplate rotary shaker.
3. After incubation the plates were
transferred to ELx405 Automated
Microplate Washer and washed three times
with 400 µL of washer buffer with a 15
second soak period between wash
cycles. After washing, 100 µL of substrate
solution was added.
4. Plates were returned to the microplate
shaker and the color development allowed
for 7.5 minutes.
5. After color development, 100 µL of Sulfuric
acid stop solution was added and the
absorbance of each well at 450 (650 nm
reference wavelength) was determined
using Synergy™ Mx Multi-Mode Microplate
Reader (BioTek Instruments).
Results of the test:
Normal values for Insulin Determination
Optimal: 3-8 uIU/mL (18-48 pmol/L)
Low: <3 uIU/mL (<18 pmol/L)
High: >8 uIU/mL (>48 pmol/L)
Low Insulin
Many labs cannot detect insulin lower than 2
uIU/mL. In such cases the result is reported as
“below detectable limits.” If there is no
detectable fasting insulin ( <2 uIU/mL, or <12
pmol/L) and blood sugar is significantly elevated
(ex HgbA1C >6.4%, or >46 mmol/L), the patient
could have type 1 diabetes or Latent
Autoimmune Diabetes of Adulthood (LADA) and
should be immediately assessed.
High Insulin
High insulin on a fasting blood test indicates a
chronically anabolic state – effectively storing
sugars while building fat and muscle. Practically,
high insulin results in central obesity and high
blood pressure. High fasting insulin is rare in a
thin or frail person. Future risk of type 2
diabetes begins to go up in those with fasting
insulin above 8 uIU/mL (48 pmol/L).
Sex Hormones
Introduction
Sex hormone, a chemical substance produced
by a sex gland or other organs that has an effect
on the sexual features of an organism.
Progesterone, testosterone, androgens,
estrone, and estradiol are steroid hormones
synthesized either in the testes, ovaries, or
adrenal glands. Each of these steroid hormones
has cholesterol as its precursor.
Principle of the test
A. Total testosterone
Generally draw blood for testosterone lab tests
between 7 a.m. and 10 a.m. Total testosterone
measures free, albumin bound and SHBG bound
testosterone in the blood and is the most
commonly used measure of testosterone levels.
It is the test usually used to assess testosterone
levels in men with suspected hypogonadism or
testosterone deficiency. There are several
methods available to measure total
testosterone.
B. Free Testosterone
A test for free testosterone measures only the
2% of testosterone which remains unbound to
proteins in blood. Free testosterone levels may
be measured in men whose total testosterone
test results showed testosterone levels which
are on the borderline of levels considered to
indicate testosterone deficiency.
Materials
1. Morning blood sample for men
2. reagents for immunoassays
Procedure
A. Total testosterone
Automated immunoassay systems
Automated platform immunoassay systems are
testosterone tests in which most steps (e.g.
adding the blood sample, adding reagents) are
performed automatically by a machine, rather
by people in the laboratory. These tests are the
fastest method of assessing testosterone levels.
They are considered accurate enough for
assessing testosterone levels in men but not in
women.

B. Free Testosterone

Direct immunoassay test kits

Direct immunoassay test kits are the easiest and

fastest available methods of assessing free
testosterone. They are test kits in which
reagents containing testosterone antibodies
and tracer particles (either radioactive or non-
radioactive) which bind to free testosterone in
the sample. The tracer particles are easily
identifiable and can be counted to measure the
concentration of free testosterone in the
sample. The results are less accurate than other
methods though and lower results are typically
obtained compared to equilibrium dialysis.

Results of the test

Testosterone level range:

MEN: 300 to 1,000 nanograms per deciliter

(ng/dL).

WOMEN: 15 and 70 ng/dL.

Testosterone levels can decrease naturally due

to your age or other health conditions. After the
age of 40, men’s testosterone levels decrease
by an average of at least 1 percent every year.
Some symptoms of low testosterone,
particularly erectile dysfunction, are commonly
seen in men over 40. Low testosterone levels
have often been observed in people with
obesity, no matter their age. The most common
testosterone-related problem in men is
hypogonadism, also called low testosterone.
CC2-MODULE 6- LABORATORY
TOXICOLOGY AND THERAPEUTIC DRUG
MONITORING
TOXICOLOGY
 Absorption of toxins in the GIT is by
passive diffusion – this process requires
that the substance cross cellular
barriers
 Toxins that are not absorbed from the
GIT not produce systemic effects but
may produce local effects- diarrhea,
bleeding and malabsorption of
nutrients
 In cases of drug overdose, CBC, serum
electrolytes, BUN, glucose, urinalysis
and blood gas must be determined
 The study of the adverse effects of
xenobiotics in humans
o XENOBIOTICS are chemicals
and drugs are not normally
found in or produced by the
body
THREE MAJOR DISCIPLINES:
 MECHANISTIC TOXICOLOGY
o Elucidates the cellular,
molecular, and biochemical
effects of xenobiotics
o Effect of a toxin
TERMINOLOGIES
 DESCRIPTIVE TOXICOLOGY
o Uses the results from animal
experiments to predict what
level of exposure will cause
harm in humans (risk
assessment)
 REGULATORY TOXICOLOGY
o Combined data from
mechanistic and descriptive
studies are used to establish
standards that define the level
of exposure that will not pose a
risk to public health or safety
SPECIALTIES WITHIN TOXICOLOGY
 FORENSIC TOXICOLOGY
o Is primarily concerned with the
medical and legal consequences
of exposure to chemicals or
drugs; used in medico legal
o Used in establishing and
validating the analytic
performance of test method
 CLINICAL TOXICOLOGY
o Focuses on the relationships
between xenobiotics and
disease states; not only for
diagnostic but also for
therapeutic intervention
 ENVIRONMENTAL TOXICOLOGY
o Includes the evaluation of
environmental chemical
pollutants and their impact on
human health
 POISONS- are any substances that
affect biological function in other
organism.
 TOXINS- are biologically produce
chemical substances that affect
biological functions of programs
  TOXICANT- synthesized chemical
substances that affects other organism
 ACUTE TOXICITY- single, short term
exposure to a substance
 CHRONIC TOXICITY- repeated exposure
for extended period of time
 TD50- is the dose that would be
predicted to produce a toxic response in
50% of the population
 LD50- is the dose that would predict
death in 50% of the population
 ED50- is a dose that would be predicted
to be effective or have a therapeutic
benefit of 50% of the population
 POISONOUS ORGANISM- secretes
chemical substances that affect
biological function of other organism
 VENOMOUS CREATURES- injects
chemical substances that gives impact
to biological function in other organism
XENOBIOTICS, POISONS, AND TOXINS
XENOBIOTICS
 Define as exogenous agents that can
have an adverse effect on a living
organism
 More often used to describe
environmental exposure to chemical or
drugs
 EXAMPLES: antibiotics and
antidepressants; chemical (per
fluorinated and brominated
compounds)
POISONS
 Exogenous agents that have an adverse
on a biological system
 More often used when describing
substances from an animal, plant,
mineral, or gas.
 EXAMPLES: venoms from poisonous
snakes or spiders, poison hemlock,
arsenic, lead, and carbon monoxide
TOXINS
 Endogenous substances biologically
synthesized either in living cells or in
microorganisms
 EXAMPLE: botulinum toxin produced
from clostridium botulinum,
hemotoxins produces from venomous
snakes, wasps, and bees, and
mycotoxins produces from fungus;
Plants- Psilosybin- mushrooms, cacti
POISONIG CASES (WHO, 2012)
ROUTES OF EXPPOSURE
 Toxins can enter the body via several
routes
o Ingestion
o Inhalation
o Transdermal absorption
 ***For most toxins to exert systemic effect, o Can be used to evaluate the
they must be absorbed into circulation doses of therapeutic drugs
 The ED50 is the dose that would be
***Toxins that are not absorbed from the
predicted to be effective or have a
gastrointestinal tract do not produce systemic
thera peutic benefit in 50% of the
effects, but may orduce local effects (diarrhea,
population; effective dose
bleeding, and malabsorption) whichc may cause
 The therapeutic index is the ratio of
systemic effects secondary to toxin exposures
the TD50 (or LD50) to the ED50
DOSE-RESPONSE RELATIONSHIP o Drugs with a large therapeutic
index demonstrate fewer toxic
 The concept that all substances have adverse effects when the dose
the potential to cause harm, even of the drug is in the therapeutic
water is a central theme in toxicology range.
 Paracelus (1493 to 1591) pionereed
the use of chemicals in medicine and
coined the term “the dose makes the
poison”
 Fundamental and essential to modern
toxicology
 To enable assessment of substances’
potential to cause pathololgic effects, it
is necessary to establish an index of the
relative toxicities of the substancs

TOXICITY RATING SYSTEM

DOSE-RESPONSE RELATIONSHIPS MAY APPLY
TOXICITY RATING LETHAL ORAL DOSE IN
TO AN INDIVIDUAL OR A POPULATION
AVERAGE ADULT
Super toxic <mg/kg  INDIVIDUAL DOSE-RESPONSE
Extremely toxic 5-50 mg/kg RELATIONSHIP relates the individual’s
Very toxic 50-500 mg/kg health status as well as changes in
xenobiotic exposure levels
Moderately toxic 0.5-5 g/kg
 QUANTAL DOSE-RESPONSE
Slightly toxic 5-15 g/kg RELATIONSHIP describes the change in
P:ractically nontoxic >15 g/kg health effects a defined population
based on changes in the exposure to
 The TD50 is the predicted dose that
the xenobiotic
would produce a toxic response in 50%
of the population; toxic dose ACUTE AND CHRONIC TOXICITY
 The LD50 is the predicted dose that
would result in death in 50% of the Are terms used to relate the duration and
population; lethal dose frequency of exposure to observed toxic effects.
  ACUTE TOXICITY is usually associated
with a single, short-term exposure to a
substance in which the dose is sufficient
to cause immediate toxic effects
 CHRONIC TOXICITY is generally
associated with repeated and frequent
exposure for exrtended periods of time
(monthe to years) at doses that are
insufficient to cause an immediate
acute response
***Dose-response relationship may differ for
acute and chronic exposures for the same
xenobiotic
SITES OF TOXIC ACTION
 LOCAL (NON-SPECIFIC) : wherever the
poison contacts the biological system it
starts its harmful effects, does not
require specific site or receptos to elicit
its effect as toxicity by acids or alkaline
 REMOTE ( SYSTEMIC) : the poison
affects a system far from portal of entry
 LOCALL AND REMOTE : the poison has
the capacity of acting locally and
systematiclally; EXAMPLe: Oxalic Acid
TYPES OF TOXIC MECHANISM
A. DIRECT: poison itself can cause toxic
effects as in corrosives
B. INDIRECT: Toxicity results from the
interaction of the poison with the
biological activity of the poison within
the system
 Binding to cell membrane to
change in their function or
structure thus affecting their
normality
 Interference with enzymatic
actions
 Formation of metabolites which
are more toxic than the parent
poison
 Effects on DNA.
CLASSIFICATION OF TOXIC AGENTS
 According to the target organ they are
acting upon. E.g hepatoxic, nephrotoxic
 According to their use: E.g. food
additives, drug, pesticides
 According to source: animal or plant
 According totheir effefct: Carcinogen or
mutagen
 According to physical state: gas or liquid
 According to their chemistry: amines or
hydrocarbons.
 According to their poisoning
potentiality: extremely toxic, slightly
toxic
 According to their biochemical
mechanisms of action: alkalating agent
FACTORS AFFECTING ACCTION OF POISON
FACTORS RELATED TO THE POISON:
 Dose- basic principle of toxicology;
interference/increase dose, will
increase severity of toxicity
 Physical status- gaseous state is more
toxic than liquid state than solid state
 Purity- this depends on the impurity of
the poison. If the impurity is more toxic
than the poison, the toxic will be more
and vice versa
 FACTORS RELATED TO THE
INDIVIDUAL:
 Age
 Health
 Sensitivity
 Gender
FACTORS RELATED TO MODE OF
EXPOSURE:
 Inhalation > IntraMuscular>
ingestion> skin contact
FACTORS RELATED TO ENVIRONMENT:
 Temperature pressure,
humidity, radiation can cuse
alterations on poisons’ status
METHODS OF ANALYSIS
SCREENING TESTS
 Is a rapid, simple, qualitative
procedure intended to detect
the presence of specific
substance or classes of
toxicants.
 Have good analytic sensitivity
by lack specificity
 A negative result can rule out a
drug or toxicant and a positive
result should be considered a
presumptive positive until
confirmed by a second, more
specific method
CONFIRMATORY TEST
 Is generally quantitative and
report the concentration of the
substance in the specimen in
contrast to qualitative
screening tests that provide a
result of positive (drug is
present) or negative (drug is
absent).
 More specific method
QUALITATIVE METHODS
COLOR TESTS-rapid, easilyperformed but
not specific
 Ferric chloride test for salicylates (pink
purple)
 Zwikker test for barbiturates (purple
color)
 Formaldehyde-Sulfuric Acid test for
benzodiazipine (orange)
 Mandalin Test for opioid 9brown)
CHEMICAL TEST
 Reinsch Test- an initial indicator to
detect presence of one or more of the
following heavy metalas
 Antimony, Arsenic, Bismuth, Selinium,
Thalium, Mercury
QUANTITATIVE METHODS;
CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY TEST
 Mobile phase (a mixture of organic
solvents such as chloroform and
methanol is run across a stationary
phase) silica gel spread on a glass plate.
 Stahl provided methods for 264 stains
or dyes that can be applied for the
required component such as ninhydrin
will react with amphetamine to give a
pink color.
 Flourescent dye can be incorporated in
the solid phase, so that ultraviolet light
 can reveal the sample components as
dark spots against the bright
background.
GAS CHROMATOGRAPHY-MASS
SPECTROMETRY
 Stationary phase is a liquid and the
mobile phase (carrier gas) is an inert gas
like helium or nitrogen
 Use 2 types of columns in GC:
o Packed column: liquid is
coateed onto particles packed
into a stainless steel or glass
column
o Capillary column: the liquid is
coated onto the walls of the
column itself, which is narrow
and made of glass.
 Samples are injected into a heated port,
where they are vaporized and carried
into the column along with thae carrier
gas. A detector then produces a signal
components exit the column.
 This is aplot of electronic signal versus
time (Rt- retention time) which shows
series of peaks that corresponds to the
components of the sample.
 Mass spectrometry: as sample
components exit the GCcolumn, they
are routed into a vacuum chamber
where they hit with a beam of
electrons, This knocks electrons off the
sample molecules, creating positive
electrons and breaking them into
fragments. These fragments are then
passed through an electromagnetic
field, which separates them by their
mass/charge ratio. The resulting
spectrum plotting the abundance and
mass/charge ratio of each fragment is
specific for a given substance
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 Stationary phase is acolumn packed
with solid particles and the mobile
phase is a liquid solvents.
 As the mobile phase is injected. A
detector then identifies the
components a s they exit the column.
 Components are identified by their Rt,
the length of time they take to pass
through the column and the results are
compared with standards.
IMMUNOASSAYS;
 Enzyme Multiplied Immunoassay
Technique/EMIT- drugs in URINE
 Polarization Immunoassay
TOXICOLOGY OF SPECIFIC AGENTS:
ALCOHOL
 Common Central Nervous System
depressants
 May cause disorientation, confusion,
euphoria, progresses to
 unconciousness, paralysis. High level
exposure may end with death.
 Symptoms of alcohol intoxication begin
when the concentration in > 0.05% w/v
(>50mg/dL blood alcohol)
1. ETHANOL (GRAIN ALCOHOL)
 Inhibits ADH → Cause diuresis
 Most common abused drug; a CNS
depressant
 Causes diuresis by inhibiting ADH
 Readily absorbed in GIT and diffuses
easily in tissue.
 Ethanol abuse causes acidosis through
accumulation of ketones and lactate
and also through direct generation of
hydrogen ions as alcohol is ozidized
 Ethanol abuse: Lipid accumulation in
hepatocytes; alcoholic hepatitis; toxic
hepatitis; cirrhosis
 Intoxication SS: blurred vision;
incoordination; slurred speech and
coma; hangover symptoms-due to the
effect of acetaldehyde
 Antidote: Diazepam (for alcoholic
mania)
 SPECIMEN Precaution: spx must be
capped at all times to avoid evaporation
of alcohol; prior to blood collection,
alcohol-free skin cleanser must be used
instead of isopropanol
 SPECIMEN: serum
 MAJOR METABOLIC PATHWAY:
conversion of ethanol to acetaldehyde
and acetyl coenzyme A by hepatic
alcohol dehydrogenase
 METHOD: enzymatic using alcohol
dehydrogenase reagent
INDICATORS OF ETHANOL BLUE
TEST COMMENTS
GGT > Increase can be seen before the onset of
pathologic consequences
>Increases in serum activity can occur in many
non-ethanol related conditions
AST >Increases in serum activity can occur in many
non-ethanol related conditions
AST/ALT > A ratio of >2.0 is highly specific for ethanol-
ratio related liver disease
HDL > High serum HDL is specific for ethanol
consumption
MCV > Increase erythrocyte MCV is commonly seen
with excessive ethanol consumption
> Increases are not related to folate or Vitamin
B12 deficiency
2. METHANOL (WOOD ALCOHOL)
 Commonly used solvent and
contaminant of homemade liquors
 Formaldehyde to formic acid (alcohol
dehydrogenase) severe acidosis
 Formic acid causing optic neuropathy to
blind ness
 Symptoms of intoxication: frank
blindness (ocular toxicity) and
metabolic acidosis
 Screening test: osmolal gap
 METHOD: GC-MS
 FATAL DOSE: 60-250 mL
 TOXIC BLOOD LEVEL: >50 mg/dL
3. ISOPROPANOL (RUBBING ALCOHOL)
 Rapidly absorbed by GIT
 Metabolized by hepatic alcohol
dehydrogenase to acetone (long life)
severe acute phase
 Depressant effect. Activated charcoal
 SYMPTOMS INTOXICATION: CNS
depression and hypertension
 INDICATION OF TOXICITY: elevated
levels of acetone in the blood and urine
 METHOD: gas chromatography
  ANTIDOTE: activated charcoal  PHASES OF PROGRESSION OF
 FATAL DOSE: 250 mL ALCOHOLISM:
o Pre-alcoholic phase
4. ETHYLENE GLYCOL (1,2-ethenediol) o Prodomal phase
 Common component of hydraulic fluid o Crucial phase
and anti-freeze o Chronic phase
 Metabolism resulting to oxalic acid and  POSSIBLE OUTCOMES OF
glycolic acid causing severe metabolic ALCOHOLISM: (BAD)
acidosis o B- rain damage
 Complications: rapid deposition of o A- lcoholic hallucination
calcium oxalate crystals in renal o D- eath
tubules-renal tubular damage.  COMMON BEHAVIORAL PATTERNS OF
 SYMPTOMS OF INTOXICATION: ALCOHOLIC PATIENTS (5D)
metabolic acidosis, depressed reflexes, o Denial
anuria and necrosis o Dependency
 INDICATION OF TOXICITY: deposition o Demanding
of calcium oxalate crystals in renal o Destructive
tubules o Domineering
 MODE OF TREATMENT: inhibit the  COMMON WITHDRAWAL SIGNS AND
action of alcohol dehydrogenase SYMPTOMS (HITS)
 MAJOR METABOLITE: glycolic acid o Hallucination (visual or tactile)
(cause of acute toxicity and death) o Increased vital signs
 METHOD: HPLC o Tremors
 FATAL DOSE: 100g o Sweating and seizures
 COMMON DEFENSE MECHANISM BY
ALCOHOLISM ALCOHOLIC PATIENTS (DRIP)
o Denial
 A chronic disease or a disorder
o Rationalization
characterized by excessive alcohol
o Isolation
intake
o Projection
 Causes interferences in:
o The individual’s health TREATMENT:
o Interpersonal relationships
o Economic functioning  DRUG OF CHOICE: DISULFERAM
 LEVEL OF INTOXICATION (ANTABUSE)
o 0.1-0.2%- Low coordination  ACTION: delays metabolism of alcohol
o 0.2-0.3%- Presence of ataxia,  SIDE EFFECT: nausea, abdominal
tremors, irritability cramps, sweating, and more severe
o 0.3 and above- includes neuritis, and polyneuritis,
Unconsciousness drowsiness, skin eruptions
 IMPORTANT NOTE: instruct the patient to
avoid alcohol based substances for at least SYMPTOMS OF CARBOXYHEMOGLOBINEMIA
12 hours before giving Antabuse COHb (%) SYMPTOMS AND COMMENTS
0.5 Typical in non-smokers
 ETHANOL ENZYMATIC METHOD 5-15 Range of values seen in smokers
PRINCIPLE: 10 Shortness of breath with vigorous exercise
20 Shortness of breath with moderate
exercise
30 Severe headaches, fatigue, impairment of
40-50 Confusion, fainting on exertion
CARBON MONOXIDE 60-70 Unconsciousness, respiratory failure,
death with continuous exposure
 A colorless, odorless, tasteless gas; very 80 Immediately fatal
toxic substance CARBON MONOXIDE SPOT TEST
 Produced by incomplete combustion of  A qualitative test to identify the
carbon-containing substances like presence of Carboxyhemoglobin
gasoline.
 Odorless, colorless, incomplete
 Improperly ventilated furnaces, wood combustion
or plastic fire, and rubber. This includes
 Sources:
cigarette smoking o Tobacco or cigarette smoking
 CO binds with heme proteins o Grilling
(cytochromes, hemoglobin and o Vehicles
myoglobin) CO + Cytochrome A3 results  SIGNS AND SYMPTOMS OF CO
to inhibition of cellular respiration and POISONING: headache, dizziness,
electron transport
contusion, muscle pain, hallucination,
 CO + Hgb (myoglobin) reduces oxygen depression, impaired mental state. Fatal
supply to cardiac and skeletal muscles episode is manifested with vomitous
or direct muscle damage with whitish fluid
 CO + Hgb → COHb (200-225 x >  REACTION:
oxygen); Air 20% oxygen by volume o NaOH + Oxyhemoglobin →
 Causes decrease concentration of BROWN COLOR
oxyhemoglobin o NaOH + Carboxyhemoglobin
 High affinity for hemoglobin than does →CHERRY RED COLOR
oxygen (200x faster than oxygen)  PROCEDURE:
 SIGN OF TOXICITY: Cherry red colored 1. To 20 ml distilled water
face; affects brain and heart 2. Add 0.5 ml whole blood
 SAMPLE FOR TESTING: EDTA whole 3. Add 0.5 ml 1M NaOH
blood 4. Mix and observe
 DEFINITIVE METHOD FOR TESTING:  RESULT:
cooximetry (carboxyhemoglobin o BROWN COLOR: NORMAL
measurement) oxyhemoglobin is present
 o CERRY RED COLOR:
CARBOXYHEMOGLOBIN IS
PRESENT
 TREATMENT: Potassium Ferricyanide
“CLEAN AIR = INCREASE OXYGEN: CLEAR
MIND”
NOTE: FOR THE DEGREE OF EXPOSURE AND
EFFECT
CYANIDE
 Exists as solid, gas or in solution
 Super toxic; fast acting death may occur
in less than an hour
 Components of insecticides and
rodenticides; common suicidal agent
 Also a pyrolysis product- burning
plastics
 Binds with iron (ferric or ferrous form) –
tissue or cellular hypoxia
 Inhibits cellular respiration, electron
transport and ATP formation by
preventing reoxidation of cytochrome
A3- inhibition of cellular respiration
leads to metabolic acidosis due to
increased lactic concentration in the
blood
 Toxic effect is inhibition of electron
transport chain and cell death
 INDICATION OF TOXICITY: Odor of
bitter almonds breath and altered
mental status
 ANTIDOTE: sodium thiosulfate, amyl
and sodium nitrite
 Toxic symptoms: tachypnea,
convulsions and coma
 > 2 ug/MI Enough to be Toxic
HEAVY METAL TOXICITY
 All metals can be toxic if ingested in
large quantities and absorbed in their
ionized forms.
 Heavy metal poisoning can be acute or
chronic and may be caused by the
following:
o Lead, Mercury, Iron, Arsenic
o Cadmium, Thallium, Bismuth
 Metals may enter the body by
ingestion, inhalation, absorption
through the skin or mucous membranes
 Most common heavy metal poisoning:
Lead
 Mercury can be found in the elemental
state (dental amalgam, thermometers),
inorganic (industrial processes), and
organic compounds (pesticides, wood
preservatives, some medicines and
contaminated fish)
ARSENIC
 Arsenic poisoning: depending on form
 3 MAJOR GROUPS OF ARSENIC
o Arsine gas
o Inorganic (trivalent or
pentavalent)
o Organic form (arsenobetaine &
arsenocholine)
 Found in sea foods as easily absorbed in
GIT by passive diffusion.
 Component Ant poison, rodenticides,
paints and metal alloys
 Common suicide and homicide agent;
common agent of heavy metal
poisoning
 Expresses its toxicity by high affinity
binding to the thiol groups in proteins.
  Inhibits sulfhydryl enzymes throughout
the body; crosses the placenta, high
affinity to thiol groups in proteins
 King of poisons an poison of Kings-
bioaccumulation
LEAD
 SYMPTOMS: Headache, confusion,
severe diarrhea and drowsiness
 As the poisoning develops, convulsions
and changes in fingernail pigmentation
called leukonychia may occur; use hair
and nails as specimen for long term
exposure “mees line”
 CHRONIC EXPOSURE: Vitamin A
deficiency, heart disease, night
blindness.
 INDICATION OF TOXICITY: “odor of
garlic” breath and metallic taste
 TOXIC EFFECTS: intravascular
hemolysis, hemoglobinemia,
nephroxicity and multi-organ
involvement
 ANTIDOTE: British anti-Lewisite (BAL)
 METHOD: AAS, Reinsch test
CADMIUM
 Utilized In electroplating and
galvanizing
 A significant environmental pollutant
 Poisoning can result from ingestion of
acidic foods stored or prepared in metal
containers made up of cadmium
 Significant environmental pollutant –
pigment on piant and plastics
 Poisoning can result from ingestion of
acidic foods stored or prepared in metal
containers made up of cadium
 Toxicity may result to destruction of
type 1 epithelial cells in the lungs and
decreased resistance to bacterial
infections
 It may also accumulate in renal tubules
causing tubular damage
 TOXIC INDICATOR: (+) GGT in urine
sample
 A potent enzyme inhibitor- it blocks
delta aminolevulinic (ALA) synthetase,
pyrimidine-5’-nucleotidase and Na-K-
dependent ATPase
 Lead poisoning- Age- dependent
differences in the absorption: adult
absorb 10% of a n ingested dose;
Children 40%
 Initially distributed to the soft tissues
and slowly redistributed to bone, teeth
and hair
 Detected by x-ray
 HALF-LIFE: 1-2 months in blood; 20-30
year in the bone;
 CNS: headache, confusion, clumsiness,
insomnia, fatigue, impaired
concentration, disease progresses,
clonic convulsions and coma occur
 Treated with chelation therapy
 BLOOD: 5-20 ug/dl in children result to
lower IQ levels:
 TOXIC DOSE: >0.5mg/day
 FATAL DOSE: 0.5g
 CDC CUT-OFF LEVEL IN CHILDREN: <10
ug/dL
 TOXIC BLOOD LEVELS: <10 ug/dL
 REQUIRES CHELATION THERAPY
(CHILDREN): <25 ug/dL
 LEAD CHELATORS: EDTA and
dimercaptosuccinic (DMA)
 GIT: discomfort, constipation. Higher
exposures can produce painful
intestinal spasms
  BLOOD: hypochromic, microcytic
anemia. Inhibit the heme synthesis
enzymes.
 SOURCE: paints and gasoline
 MODE OF ACQUISITION: ingestion or
inhalation
 INDICATION OF TOXICITY: urinary
aminolevulinic acid, free RBC
proporphyrin and presence of
basophilic stippling in RBC
MERCURY
 Binds with sulhydryl proteins.
 A potent enzyme inhibitor- inhibits
catecholamine-0-methyltransferase, an
enzyme essential in the metabolism of
“catecholamines”
 FORMS OF MERCURY: elemental or
metallic mercury, mercurous, mercuric
and alkyl mercury
 MODES OF ACQUISITION: inhalation,
skin absorption, and ingestion
 SYMPTOMS OF TOXICITY:
hypertension, tachycardia, and
sweating-“cardinal signs” of
pheochromocytoma or mimics that
adrenal gland disorder
 GENERAL TOXIC EFFECT: organ
dysfunction- lungs, kidney, and CNS
 MAJOR TOXIC EFFECT OF ELEMENTAL
MERCURY: pink disease (acrodynia) and
erethism
 MAJOR TOXIC EFFECT OF ALKYL
MERCURY: congenital Minimata disease
 Ability to “Tremors, depression,
memory loss, decreased verbal skills
and inflammation of the kidneys. Non-
selective pulmonary toxicity
 An exposure to inorganic salts of
mercury (mercuric chloride) leads to
renal damage.
 Consumption of food contaminated
with methyl-mercury (fish) cause
organic mercury toxicity
 Visual disturbances, Parenthesias,
ataxia, hearing loss, mental
deterioration, muscle tremors,
movement disorders, paralysis and
death
 SAMPLES: whole blood and 24-hour
urine
IRON
 Iron is a vital mineral in the human
body
 Toxicity causes hemosiderosis and
hemochromatosis
 Iron toxic serum level:
o Mild toxicity: 450-500 ug/dL
o Severe toxicity: 800-1000 ug/dL
 Toxic effect due to hemorrhagic
necrosis in GIT
TREATMENT
 Chelation therapy: Chelation agent
plus metal = chelate
 A chelating agent is a chemical
compound or drug molecule capable of
forming a heterocyclic ring with a metal
ion as its closing member.
METALS ANTIDOTES
LEAD Dimercapto succinic acid, Calcium
disodium edetate
MERCURY, Dimercaprol
ARSENIC, GOLD
IROON Deferoxamine
COPPER/MERCURY Penicillamine
CYANIDE Sodium nirite, Sodium thiosulfate,
Amyl Nitrite pearls
REINSCH TEST FOR HEAVY METALS
A qualitative screening test for the presence of
the following metals: Sb, As, Bi, Hg
 Specimen:
o Gastric washing or lavage for
acute poisoning
o Urine for chronic poisoning
o Tissue biopsy (if observed that
it has reach the tissues Reinsch
test is not used)
 MATERIALS:
o 10cm COIL of Copper wire
heavy gauge
o 2M Hydrochloric acid
o Water bath
 PROCEDURE: (USING EARLY MORNING
URINE/RANDOM)
1. Using the 10cm coil of a heavy
gauge of copper wire
2. Ad 10 ml of urine and 10 ml of
2M HCI “acidification” to
enhance the reaction
3. Boil in the water bath for 10
minutes and examine the
copper coil for any stain
4. Remove from the water bath
and let it stand for 1 hour. If no
stain in the copper coil is found,
This could be due to delayed
reaction
5. Detection: up to 5 microgram of
heavy metals in urine
6. After the incubation period and
there is no stain observed then
can be reported as NEGATIVE
 RESULT:
 ANTIMONY: Blue/ Black Purple
 ARSENIC: Flat Black
 BISMUTH: Shiny Black is seen
with light
 MERCURY: Silver Gray
THERAPEUTIC DRUG MONITORING
 Must be monitored to determine what
doses are inadequate or excessive in
the treatment of the patient. Often, the
ingested drug (called the “parent” drug)
is metabolized to form an active
metabolite that produces an effect
similar to the parent drug
 Involves the analysis, assessment and
evaluation of circulating concentrations
of drugs in serum, plasma or whole
blood.
 Also it is a quantitative procedure
performed for drugs with a narrow
therapeutic index.
 It allows for the safe use of drugs that
would otherwise be potentially toxic.
 Ensures that a given drug produces
maximal therapeutic benefit and
minimal side effects; to achieve a
constant serum level of the drug that
will be therapeutic
 Most drugs have a half-life independent
of their concentrations.
 Half-life of the drug determines the
time to reach the steady-state or
average concentrations
INDICATIONS FOR TDM
1. The consequences of overdosing and
under-dosing are serious.
2. There is a small difference between a
therapeutic and toxic dose
3. There is a poor relationship between
the dose of drug and circulating
concentrations but a good correlation
 between circulations and therapeutic
and toxic effect
4. There is a change in the patient’s
physiologic state that may be
unpredictably affect circulating drug
concentrations
5. A drug interaction is or may be
occurring
6. It helps in monitoring patient
compliance
ROUTES OF ADMINISTRATION:
 Circulatory system is a convenient
route that can effectively deliver most
drugs to its site of action
 Intravenous
 Oral -0.7
 Intramuscular
 Subcutaneous
 Inhalation
 Suppository
 Transcutaneous
PHARMACOLOGICAL PARAMETERS THAT
DETERMINES SERUM DRUG CONCENTRATION:
A. LIBERATION – is the release of the
ingredient, followed by the process of
the drug passing into solution
B. ABSORPTION – is the process by which
the drug molecule is taken up into
systemic circulation. Following
absorption through the intestinal
mucosa, a drug traverses the hepatic
system, where some drugs undergo
substantial metabolism and elimination.
This called first-pass elimination or
metabolism
C. DISTRIBUTION- Drug molecules can be
confined to the blood, leave the blood
stream, and enter the extravascular
space, or they can migrate into various
tissues. This is referred to as
distribution, a process that typically
occurs between a period of 30 minutes
and 2 hours.
**the bioavailability of a drug is the amount of
drug that is absorbed into the system and is
available for distribution.
D. METABOLISM is the process of
transformation of the parent drug
molecule to its metabolite(s).
Metabolites are usually water soluble
and can be easily excreted; most of the
metabolism occurs in the liver, where
enzymes catalyze oxidation, reduction,
or hydrolysis of the drug.
E. ELIMINATION is the process of
excretion of the drug from the body.
Drugs are typically excreted in the urine
but also can be eliminated in the feces,
sweat, expired air, and saliva
 ABSORPTION
o Most drugs are absorbed by
passive diffusion
Intestine → hepatic portal system (liver)
→general circulation
o Factors affecting absorption:
changes in intestinal
movement, pH, inflammation,
and presence of food and other
drugs
 DISTRIBUTION
o The location where drugs are
effective are in the body tissues
not generally in blood
 o Drugs diffuse widely throughout
the body, frequently reaching
higher concentrations in tissues
than in blood
 EXCRETION
o All drugs are excreted-either
unchanged in the urine or
excreted as metabolite of the
parent drug.
o Rate at which a particular drug
is cleared from the circulation is
dependent not only on the type
of drug itself, but also on a
patient’s capacity to metabolize
and excrete it
Basic principles. TDM measures drug
concentrations during therapy with
pharmaceutical agents.
a) A steady-state drug level (complete
with peaks and troughs) exists for each
drug. When a single dose of a drug is
administered orally, the blood level
changes markedly over time and, at
some time, the concentration in the
plasma reaches its peak (highest point)
and then declines. Immediately before
the next dose of medication, a trough
level occurs
1) For single-dose administration, the
rate of decline in concentration is
expressed in terms of half-life,
which is the time required for the
concentration of the drug to
decrease by 50%.
2) The half-life is different for each
drug. At steady-state levels, the
rate administration of the drug is
allowing the drug level to remain
constant.
PHARMACOKINETICS is the mathematical
interpretation of drug overtime to determine
proper dosing amounts of a therapeutic drug.
Pharmacokinetic responses are typically graphic
plots of blood concentration of the drug versus
time, such as a dose-response curve.
Three kinetics processes are used to describe
the fate of drugs in the body over a period of
time and can be illustrated in a dose-response
curve
1) FIRST-ORDER KINETICS describe
absorption, distribution, and
elimination of drugs. This means that
the rate of change of concentration of a
drug is dependent on the drug
concentration. It is represented by the
first phase of the dose-response curve.
2) ZERO-ORDER KINETICS describes the
rate of change of concentration of a
drug that is independent of the
concentration of the drug. That is a
constant amount of drug is eliminated
per unit of time. This typically depends
on the ability of the liver to metabolize
the drug. This is illustrated by the
second phase of the curve.
3) MICHAELIS-MENTEN KINETICS state if a
drug concentration in a system exceeds
the capacity of the system, the rate of
change of concentration proceeds
according to the Michaelis-Menten
equation.
CAUSES OF DRUG TOXICITY
1) Elevated concentration of free drug
2) Abnormal response to drug after
administration
3) The presence of active metabolites
TERMINOLOGIES:
 BIOAVAILABLE FRACTION (F)
o It is a fraction of dose that
reaches the blood
 Vd OF A DRUG
o Represents the dilution of the
drug after it has been
distributed in the body
o It is used to estimate the peak
drug blood level expected after
loading dose is given
o It is the principal determinant
of the dose
 FIRST-PASS HEPATIC METABOLISM
o Drugs that are transported to
the liver lost a fraction of its
bioavailability before the drug
reaches the general circulation
 FIRST-ORDER ELIMINATION
o Represents a linear relationship
between the amount of drug
eliminated per hour and the
blood level of a drug.
 HALF-LIFE (t1/2)
o Time required to reduce a drug
level to half of its initial value
 PEAK CONCENTRATION
o The highest concentration of a
drug obtained in the dosing
interval
 PHARMACODYNAMICS
o The relationship between the
drug concentration at the target
site and response of the tissues
 PHARMACOGENOMICS
o Refers to the study of genes
that affect the performance of a
drug in an individual
 PHARMACOKINETICS
o The mathematical expression of
the relationship between drug
dose and drug blood level
 THERAPEUTIC INDEX
o Ration between minimum toxic
and maximum therapeutic
serum concentration
 THERAPEUTIC RANGE
o the difference between highest
and lowest effective dosages
 TROUGH CONCENTRATION
o The lowest concentration of a
drug obtained in the dosing
interval
CARDIOACTIVE DRUGS
 These drugs are divided into two
categories: the cardiac glycosides and
the antiarrhythmic drugs. These agents
serve to maintain normal heart
function.
 Drugs are used for treatment of
arrhythmias and congestive heart
failure.
CLASSIFICATION OF CARRDIOACTIVE DRUGS:
 CLASS I – rapid sodium channel blockers
o Quindine, Procainamide,
Lidocaine
 CLASS II - beta receptor blockers
o Propanolol
 CLASS III- K+ channel blockers
o Amiodarone
 CLASS IV- calcium channel blockers
o Verapamil
DIGOXIN
 The major cardiac glycoside and alters
the force of contraction through its
 effect on the ATPase pump in heart
muscle
 Blood specimens must be collected 8
hours after a dose of digoxin is
administered, because its peak
concentration in tissue occurs 6 to 10
hours after administration
 Digoxin toxicity produces symptoms of
nausea, rapid heart rate, and visual
impairment. Digoxin is excreted as
digoxigenin in the urine.
 Inhibits membrane Na-K-ATPase, thus it
decreases K+ and Mg+, and increases
Ca+ (cardiac contractility-inotropic
effect, 0.8-2 ng/mL)
 25% is protein bound; free (nonbound)
form is sequestered into muscle cells
 Hyperthyroid individuals are resistant to
the action of digoxin
 Timing of blood collection after last
dose can be critical to interpretation of
the drug levels.
 HALF-LIFE: 38 hours (average adult)
 THERAPEUTIC LEVEL: 0.5-2 ng/mL
 TOXIC LEVEL: > 2 ng/mL
 The antiarrhythmic drugs are
prescribed to treat irregular heart beat
that produces inappropriate ventricular
contraction or tachycardia (increased
heart rate)
 ELIMINATION: renal filtration of the
plasma free form
 PEAK SERUM LEVEL: 8 hours after an
oral dose
 TOXIC EFFECTS: nausea, vomiting, visual
disturbances, premature ventricular
contractions and atrioventricular node
blockage.
The antiarrhythmic drugs are prescribed to
treat irregular heart beat that produces
inappropriate ventricular contraction or
tachycardia (increased heart rate)
LIDOCAINE (XYLOCAINE)
 Used for the treatment of faulty
ventricular contractions and
arrhythmias. It binds to a1-acid
glycoprotein and is metabolized in the
liver, producing two active metabolites
monoethylglycinexylidide and
glycinexylide
 Used to correct ventricular arrhythmia
for treatment of acute myocardial
infarction
 Administered by continuous IV infusion
after a loading dose
 Can be used as a local anaesthetic
 Bound to albumin and AAG
 Cannot be administered orally due to
almost complete hepatic removal of the
absorbed drug
 THERAPEUTIC LEVEL: 1.4-4.0 ug/mL
 TOXICITY LEVEL: > 4.0 ug/mL
 CNS DEPRESSION: > 4-8 ug/mL
 SEIZURE AND DECREASED BP AND
CARDIAC OUTPUT: >8 ug/mL
 TOXIC EFFECT: CHF and heart block
 ELIMINATION: by hepatic metabolism;
changes in renal function have little
effect
 PRIMARY PRODUCT OF HEPATIC
METABOLISM: monoethylglycinexy
lidide (MEGX); glycinexylide
QUINIDINE
 A myocardial depressant that decreases
that heart’s ability to conduct current. It
is metabolize in the liver to produce
 several active metabolite, including 3-
hydroxyquinidine.
 If quinidine is added to digoxin therapy
regimen, an interaction occurs that
induces an increase in digoxin
concentration
 A naturally occurring drug for the
treatment of arrhythmia
 85% protein-bound; GIT absorption is
complete and rapid for the sulfate.
 PEAK SERUM LEVEL:2 hours after oral
dose (sulfate); 4-5 hours (gluconate)
 THERAPEUTIC LEVEL: 2.3-5 ug/mL
 TOXIC LEVEL: >5 ug/mL
 TOXIC EFFECTS: cinchonism, blood
dyscrasia and hepatitis
 ELIMINATION: hepatic metabolism
 ROUTE OF DELIVERY: oral
administration
 COMMON FORMULATIONS: quinidine
sulfate and quinidine gluconate
PROCAINAMIDE (PRONESTYL)
 Used to treat inappropriate ventricular
contractions and tachycardia. This drug
is metabolized in the liver to form an
active metabolite, N-
acetylprocainamide, which produces
the same effect as its parent drug.
Therefore, serum levels of both drugs
must be analyzed
 Administered orally
 GIT absorption is rapid and complete
 20% protein-bound; eliminated by renal
filtration and hepatic metabolism
 PEAK SERUM LEVEL: 1 hour after dose
 THERAPEUTIC LEVEL: 4-10 ug/mL
 TOXIC LEVEL: >12 ug/mL
 TOXIC EFFECTS: reversible lupus-like
syndrome (ANA), nephrotic syndrome,
urticarial
 COMMON ROUTE: oral
 HEPATIC METABOLITE: N-acetyl
procainamide (NAPA)
DYSOPYRAMIDE
 It stabilizes the heartbeat. It is both
excreted by the renal system as the
unchanged drug and is metabolized in
the liver to form an inactive metabolite
 Used to treat cardiac arrhythmias; used
as substitute for quinidine
 Administered orally; GIT absorption is
complete and rapid
 Has anticholinergic – dry mouth and
constipation (> 4.5 ug/mL)
 THERAPEUTIC LEVEL: 3-5 ug/mL
 TOXIC LEVEL: 10 ug/mL
 TOXIC EFFECTS: bradycardia and
atrioventricular node blockage
 ELIMINATION: renal filtration
PROPANOLOL
 Prescribed for atrial and ventricular
arrhythmias and hypertension. It is
considered to be a beta-blocker
 A beta-receptor blocking drug; used in
the treatment of angina pectoris,
hypertension and coronary artery
disease
 Suppresses the conversion of T4 and T3-
used in the treatment of throtoxicosis
 THERAPEUTIC LEVEL: 50-100 ng/mL
 TOXIC EFFECTS: bradycardia, arterial
insufficiency (Raynaud’s type), platelet
disorder and pharyngitis
AMIODARONE (CODARONE)
 Blocks potassium channels in the
cardiac muscle and used for treatment
of ventricular arrhythmias
  An iodine-containing drug which can
cause hyperthyroidism or
hypothyroidism
 THERAPEUTIC LEVEL: 1.0-2.5 ug/mL
 TOXIC LEVEL: >2.5 ug/mL
 TOXIC EFFECTS: bradycardia, hepatitis,
photodermatitis and thyroid
dysfunction
VERAPIMIL
 For treatment of angina, hypertension
and supra ventricular arrhythmias
 THERAPEUTIC LEVEL: 80-400 ng/mL
 TOXIC EFFECTS: hypotension, peripheral
edema and ventricular fibrillation
ANTIBIOTICS
AMINOGLYCOSIDES (GENTAMICIN,
TOBRAMYCIN, AMIKACIN, KANAMYCIN,
NEOMYCON, STREPTOMYCIN)
 Used for treatment of gram-negative
bacterial infections; not given to
outpatient
 Administered IM or IV; not well
absorbed from the GIT
 May cause damage to the 8th cranial
nerve at toxic level (hearing loss)
 TOXIC LEVEL: > 30 ug/mL (amikacin and
kanamycin)- peak levels; 12-15 ug/mL
(gentamicin and tobramycin)- peak
levels
 TOXIC EFFECTS: nephrotoxicity and
ototoxicity
VANCOMYCIN
 Glycpeptide effective against gram-
positive cocci and bacilli
 Poor oral absorption; administered by
IV infusion
 Toxic side effects occur in the
therapeutic range (5-10 ug/mL)
 Only the trough levels are monitored to
ensure the serum drug concentration is
within the therapeutic range.
 TOXIC LEVEL: >1ug/mL- nephrotoxicity;
>40 ug/mL - ototoxicity
 TOXIC EFFECTS: red-man syndrome,
nephrotoxicity and ototoxicity
 ELIMINATION: renal filtration and
excretion
CHLORAMPHENICOL
 It distributes to all tissues and it
concentrations in CSF
 50% protein bound; rapidly absorbed in
the GIT
 TOXIC LEVEL: >25 ug/mL
 TOXIC EFFECTS: blood dyscrasia,
cytoplasmic vacuolation (erythroid and
myeloid cells)
ANTICONVULSANTS/ANTIEPILEPTIC DRUGS
 Function to alter transmission of nerve
impulses within the brain to minimize
the seizures of epilepsy.
 general seizure- both sides of brain;
petit mal/absence seizure- rapid
blinking or few seconds of space; grand
mal seizure- loss of consciousness
PHENOBARBITAL
 Long-acting barbiturate that controls
grand mal tonic-clonic seizure and focal
epileptic; not used for petit mal seizure;
 Used for treating withdrawal symptoms
in infants – mothers are addicted to
opiate or barbiturate
  Used to treat cases of congenital
hyperbilirubinemia – this drug enhances
bilirubin metabolism
 Absorption is slow but complete; 50%
orotein-bound; majority is stored in the
brain
 Used to treat all types of seizures
except absence seizures. It is effective
in children and neonates. It is
metabolized in the liver, and serum
concentrations increase during the
administration of valporic or salicylic
acid.
 INACTIVE FORM: primidone (mysoline)
 HALF-LIFE: 70-100 hours
 PEAK SERUM LEVEL: 10 hours after an
oral dose
 THERAPEUTIC LEVEL: 20-440 ug/mL
(phenobarbital); 5-12 ug/mL
(primidone)
 TOXIC EFFECTS: nystagmus, stupor,
ataxia and respiratory depression
**PRIMIDONE is metabolized in the liver to
form phenobarbital. Therefore, dual analysis
must be performed to determine the proper
dosage of this drug. It is used to treat both
grand mal and complex-partial seizures.
PHENYTOIN (DILANTIN)
 Corrects grand mal seizures. It is
metabolized by the liver and can
interact with several drugs that induce
increased serum concentration or
increased metabolism or phenytoin.
 Controls seizures (tonic-clonic, simple
partial seizures); a short-term
prophylactic agent in brain injury
 Not used for petit mal and atomic
seizures
 Decreases sodium and calcium influx
into hyperexcitable neurons
 IV administered; GIT absorption is
incomplete
 87-97% protein-bound; free ( unbound)
form is the biologically active portion
 INJECTABLE PERFORM: fosphenytoin
 THERAPEUTIC LEVEL: 10-20 ug/mL; 1-2
ug/mL (free form)
 TOXIC LEVEL: >20 ug/mL
 MAJOR TOXICITY: initiation of seizures;
tetratogenic action (cleft lip and palate)
andynystagmus
VALPROIC ACID (DEPAKENE)
 Used for treatment of petit mal
(absence seizure(, atomic seizure and
grand mal
 Orally administered; Git absorption is
rapid and complete
 93% highly protein bound
 Hepatic dysfunction is observable which
requires monitoring after 6 months of
therapy
 Prescribed for absence (petit mal)
seizures. Valproic acid affects many
others anticonvulsants by inhibiting
their metabolism in the liver, thus
increasing serum concentration.
 THERAPEUTIC LEVEL: 50-100 ug/mL
 TOXIC LEVEL: >100 ug/mL (nausea,
lethargy, weight gain); >200 ug/mL
(pancreatitis, hallucinations and
hyperammonemia)
CARBAMAZEPINE (TEGRETOL)
 Typically used for treatment of various
seizures and facial pain.
 Tricyclic compound related to
imipramine (TCA)
  Effective for grand mal seizures and
treating seizures accompanied by pain
 Has antineuralgic action; 70-80%
protein-bound
 Has serious effects and not frequently
used; orally administered
 IDIOSYNCRATIC EFFECT: rashes,
leukopenia, nausea, vertigo, febrile
reaction
 THERAPEUTIC LEVEL: 4-16 ug/mL
 TOXIC LEVEL: >12 ug/mL
 TOXIC EFFECT: hematologic dyscrasias,
aplastic anemia, irregular pulse and
ataxia
ETHOSUXIMIDE (ZARONTIN)
 Prescribed for the treatment of petit
mal seizures;orally administered
 Free serum and not protein bound
 THERAPEUTIC LEVEL: 40-100 ug/mL
 TOXIC LEVEL: >100 ug/mL
 TOXIC EFFECT: GI disturbances, ataxia,
SLE, aplastic anemia and pancytopenia
GABAPENTIN (NEUROTIN)
 Chemically similar to neurotransmitter
gamma aminobutyric acid (GABA)
 Used for partial seizures; for adjunctive
therapy
 Administered orally; it is unbound to
plasma proteins
 Excreted unchanged in the urine; not
metabolized in humans
 ADVERE EFFECTS: dizziness, ataxia,
fatigue and nystagmus
 THERAPEUTIC LEVEL: 2-15 ug/mL
 TOXIC EFFECT: somonolence
(drowsiness) and ataxia
TOPIRAMATE
 Used as an adjunct drug for partial
seizures
LAMOTRIGINE (LAMICTAL)
 Used as an adjunct drug for partial
seizures
FELBAMATE
 Monotherapy or adjunct in refractory
partial seizures with or without
secondary generalization
PSYCHOACTIVE DRUGS
 Are used to treat psychotic patients
 They can be categorized in two classes:
lithium and the antidepressants
LITHIUM
 Treats manic-depressive illness (bipolar
disorders). The mechanism of action of
lithium as a mood stabilizer remains
unknown, although effects on synaptic
neurotransmission are thought to be
cause
 Lithium is filtered by the renal
glomerulus and eliminated as the
unchanged drug.
 Drug of choice of the prevention of
chronic cluster headache
 Inhibits thyroid hormone synthesis and
release- inhibits iodine uptake
 Cationic metal that does not bind to
proteins
 Orally administered; absorption is rapid
and complete
 Subject to reabsorption
 Distribution of this drug is uniform
throughout the body water.
  THERAPEUTIC DRUG: 0.8-1.2 mmol/L
 TOXIC LEVEL: 1.2-2 mmol/L- apathy,
lethargy, speech difficulties; >2 mmol/L-
seizures, muscle rigidity and coma
ANTIDEPRESSANTS, OR TRICYCLIC
ANTIDEPRESSANTS
 Used to treat depression that has no
apparent organic or social cause.
Antidepressants include imipramine,
nortriptyline, amitriptyline, and
desipramine, all of which are
metabolized by the liver to form active
metabolites.
 The active metabolites include
desipramine (parent is imipramine;
major metabolite), nortriptyline (parent
is amitriptyline), and 2-hydroxy-
desipramine (parent is desipramine)
 Used for the treatment of depression,
insomnia, extreme apathy and loss of
libico
 Orally administered with variability in
absorption; highly protein-bound
 PEAK SERUM CONCENTRATION: 2-12
hours
 THERAPEUTIC LEVEL: 100-300 ng/mL
 TOXIC EFFECTS: drowsiness, blurred
vision, memory loss, seizure, cardia
arrhythmia, parkinsonian syndrome and
unconsciousness
FLUOXETINE (PROZAC)
 Not chemically related to the tricyclic
antidepressants, but has a similar effect
by blocking serotonin uptake by nerve
terminals in the CNS and by platelets
 Blocks the re-uptake of serotonin in
central serotonergic pathways
 Used for treatment of obsessive-
compulsive disorders
 THERAPEUTIC LEVEL: 90-300 ng/mL
 TOXIC EFFECTS: attempted suicide,
decreased libido and sexual function
BRONCHODILATORS
 Act to relax bronchial smooth muscle
for relief or prevention of asthma.
Theophylline is the most common in
this category of therapeutic drugs and is
metabolized in the liver to produce
several metabolites, including caffeine
THEOPHYLLINE
 belongs to methylated xanthine class
 action is to relax bronchial smooth
muscle
 used for treatment of asthma and
chronic obstructive pulmonary disease
 drug of primary apnea prematurity-
absence of respiratory effort in
newborn infants
 action is inhibitory to the release of
histamine and other proinflammatory
agents
 It crosses the placenta and may be
tetratogenic in pregnant females
 Best predictor of toxicity is the blood
level of the drug and not early signs or
symptoms toxicity
 THERAPEUTIC LEVEL: 10-20 ug/mL
 TOXIC LEVEL: >20 ug/mL
 TOXIC EFFECTS: GI bleeding, seizures,
tachycardia and syncope
IMMUNOSUPPRESSIVE DRUGS
CYCLOSPORINE
 Inhibits the cellular immune response
by blocking production of interleukin-2
  Used to prevent rejection of allogenic
organ transplants
 Used for suppression of acute graft-
versus-host disease (GVHD)
 Organs such as heart, liver and pancreas
require high dosage (300 ng/mL)
 Marked affinity with RBC; RBC-
cyclosporine is temperature dependent
 TOXIC LEVEL: > 500 ng/mL
 TOXIC EFFECTS: renal tubular and
glomerular dysfunctions, GI
disturbances, hirsuitism, and
hematologic dyscrasias
TACROLIMUS (PROGRAF/ FK-506)
 It is a molecule lactine antibiotic and
100x more powerful than cyclosporine
 Macrolide lactone antibiotic; GIT uptake
is variable
 Elevated levels are observed in
cholestasis
 TOXIC EFFECTS: thrombus formation,
nephrotoxicity and neurotoxicity
RAPAMYCIN (SIROLIMUS)
 Similar to tacrolimus
 Major side effects are lipid
abnormalities and thrombocytopenis
MYCOPHENOLATE MOFETIL
 Decreases renal allograft rejection
LEFLUNOMIDE (LFM)
 Inhibits lymphocyte proliferation
 Treatment for rheumatoid arthritis
ANTINEOPLASTIC DRUGS
 Used in the management of certain
tumors, including those found in breast,
testicular, pharyngeal, and sometimes
lung cancer
 These agents work by inhibiting DNA
synthesis
METHOTREXATE
 Inhibits DNA synthesis in all cells by
blocking dihydrofolate reductase
 Effective etherapy for a variety of
neoplastic conditions; also an
immunosuppressive agent
 Leucovorin is used to reverse the
action-leucoverin rescue
 TOXIC LEVEL: 0.01 umol/L
 TOXIC EFFECTS: leukopenia, GI
ulceration, thrombocytopenia, cirrhosis
BUSULFAN
 Alkalyting agent used to treat leukemias
and lymphomas prior to bone marrow
transplantation
 OVERDOSAGE: hepatic occlusive
disease
ANTI-INFLAMMATORY/ANALGESICS
SALICYLATES/ASPIRIN (ACETYLSALICYLIC ACID)
 Commonly used analgesics, antipyretic
and anti-inflammatory drug
 Direct stimulator of the respiratory
system and an inhibitor of the Kreb’s
cycle
 Acute aspirin intoxication- a common
cause of fatal drug poisoning in children
 An anticoagulant property (antiplatelet
activity( by inhibiting the action of
cyclooxygenase
  FUNCTION: Decreases thromboxane
and prostaglandin formation through
inhibition of cyclooxygenase
 SIDE EFFECTS: gastrointestinal
disturbance and interference with
platelet aggregation
 THERAPEUTIC LEVEL: 5mg/dL
(treatment of headache)
 TOXIC LEVEL: >30 mg/dL
 TOXIC EFFECTS: mixed acid-base
disturbances (metabolic acidosis and
respiratory alkalosis), Reye’s syndrome
and hypoglycemia
 METHOD: Trinder assay: enzymatic
assay (salicylate hydroxylase), HPLC and
EMIT)
ACETAMINOPHEN (TYLENOL)
 Inhibitor of prostaglandin metabolism
 Commonly used as analgesic and
antipyretic drug
 OVERDOSAGE: hepatoxocity
 THERAPEUTIC LEVEL: 25 ug/mL
 TOXIC LEVEL: >50 ug/ml; 100-300
ug/mL (hepatic necrosis)
 TOXIC EFFECTS: cyanosis due to
methemoglobinemia, CNS depression
and seizure
 METHOD: HPLC
IBUPROFEN
 Analgesic and anti-inflammatory actions
 Has a lower risk of toxicities than
salicylates and acetaminophen
 THERAPEUTIC LEVEL: 10-50 ug/mL
 TOXIC LEVEL: >100 ug/mL
 TOXIC EFFECTS: nausea, vomiting,
blurred vision, abdominal pain and
edema
NEUROLEPTICS (ANTIPSYCHOTIC MAJOR
TRANQUILIZERS)
 Blocks the action of dopamine and
serotonin in limbic system
 Used for treatment of acute
schizophrenia
 Monitoring of these drugs in serum is
difficult due to abundant metabolites
for each drug resulting to extensive
metabolism in the liver
 2 Classes: phenothiazines
(chlorpromazine) and butyrophenones
(haloperidol)
 TOXIC EFFECTS: cholestasis, orthostatic
hypotension, aplastic anemia, muscle
rigidity
 EXAMPLES: risperidal, olanzapine
(Zyprexa), quetiapine (Seroquel),
ariprazole (abilify)
METHODS
 SPECIMEN OFCHOICE: serum or plasma
 CYCLOSPORINE AND TACROLIMUS
TEST: whole blood EDTA sample
 Sample for TDM should not be
collected in SST or gel separators (some
gels absorbs certain drugs (phenytoin,
phenobarbital, lidocaine, quindine and
carbamazepine) → false decreased
results
 SPECIMEN CONSIDERATIONS: through
concentrations and peak concentrations
 No changes occurred in theophylline
and salicylate levels when blood is
collected in serum separator tube (SST)
 Measurement of serum concentration
should be done only after steady state
has been achieved
 Trough concentrations
 o Drawn immediately(or 30
minutes) before the next dose
 Peak concentrations
o Drawn one hour after orally
administered dose (except
digoxin)
o Determine after IV infusion is
completed
COLORIMETRY
 Acetaminophen in urine is detected by
boiling to form p-amphenol which
reacts with o-cresol to from indophenol
blue
 Trinder assay for salicylate using ferric
nitrate forming a (+) colored complex
IMMUNOASSAY METHODS
 Rapid analysis of blood and urine
samples
 Detect drug levels in the nanomolar
range; sensitive and specific methods
 Uses an antibody specifically reactive
with a particular drug- the drug must
bind to antibody before it is identified
a. Enzyme-Mediated (Multipled
Immunologic Technique /EMIT
 Enzyme activity is directly
proportional to the amount of
drugs present in the sample
b. Fluorescence Polarization
Immunoassay (FPIA)
 The binding of the marker drug
to antibody can be quantitated
by the angle at which the
emissions occurs
 The drug is attached to a
fluorescent label or fluorophore
CHROMATOGRAPHIC METHODS
 BEST SPECIMEN: urine
a. Thin Layer Chromatography
 It qualitatively identifies drugs
by means of their Rf values
 Extraction of drugs is pH
dependent: acidic drugs (pH
4.5); alkaline drugs (pH 9.0)
b. High Performance Liquid
Chromatography (HPLC)
 It is a highly quantitative
procedure
 It is ideal for separation of
tricyclic antidepressants and its
metabolites
 Measurement depends on the
type of column used, the
solvent and detector systems
c. Gas Chromatography-Mass
Spectrometry (GC-MS)
 Used for quantitation of many
drugs
 Drugs must be volatile in form
or can be chemically derivatized
into valuable form.
 MS can added onto effluent end
of GC, enhancing its capability
to quantify drugs
CC2-MODULE 7- LABORATORY
DRUGS OF ABUSE
DRUGS OF ABUSE
 Almost all drugs of abuse are basic
drugs (amine derivatives) which
contain benzene rings; barbiturates are
acidic drugs
 Many of the abuse drugs directly act on
dopaminergic neurotransmitter system
(limbic system; smell brain)
 A positive drug screening test cannot
differentiate casual user from chronic or
habitual user, likewise detect the time
frame of using the drug dose of the
drug taken
 Designer drug- modified forms of
established drugs of abuse
PHARMACOLOGICAL CLASSSIFICATION OF
DANGEROUS DRUGS
1. Hypnotics
2. Sedatives and tranquilizers
3. Hallucinogens and Psychotomimetic
drugs
4. Stimulants
5. Inhalants and Intoxicants
6. Cannabis/Cannabinoids
7. Anabolic steroids
CANNABIS/CANNABINOIDS
 Naturally occurring cannabinoids:
marijuana and hashish
INHALANTS
MARIJUANA
 Also known as: Pot grass, Weed, Mary
Jane or MJ, Acapulco gold, :Damo”
 Hemp, plant (Cannavis sative), Hashish
(Hash), Hashish oil (Hash oil)
 Tetrahydrocannabinol (THC)- most
potent components or psychoactive
substance of Marijuana
 THC is a lipophilic substance, distributes
in the adipose tissues; it is easily enters
the brain; it induces a sense of well-
being and euphoria; it is a hallucinogen;
also associated with the impairment of
memory and intellectual functions
 After a single use, THC-COOH can be
detected in urine for 3-5 days; up to 4
weeks for chronic user
 Principal psychoactive component of
cannabis is delta 9-tetra-
hydrocannabinol (also referred to as
delta—9THC)
 Urinary metabolite is 11-
nordeltatetrahydrocannabinol
 Symptoms- wide array
 Effects of low-moderate doses
includes: loquacious euphoria, changes
of perception of time and space,
impaired coordination, judgement and
memory, increased visual and auditory
sensitivity, conjunctivitis and bronchitis
 Psychologic effect: reddening of
conjunctiva and increased pulse rate
 Higher dosage: illusions, delusions,
depression, confusion, alienation and
hallucinations, impaired psychomotor
cognitive and endocrine functions.
 Toxic effects: paranoia, disorientation,
altered physical senses and
bronchopulmonary disorders
 Produces psychoactive vapors
1. Nitrous oxide - laughing gas,
whippets
2. Amyl nitrite - poppers,
snappers
 3. Butyl nitrite – bolt, locker
room, bullet
4. Chlorohydrocarbon - aerosol
spray
5. Hydrocarbon - solvents
 It includes: aerosols, gasoline, some
glues, solvents, Butyl nitrites (room
odorizers)
 Short time sniffing may cause: disturb
vision, impair judgement, reduce
muscle and reflex control
 Death without warning due to
suffocation, respiratory collapse or
heart failure
 Abused inhalants and solvents
includes: rugby, glue, gasoline,
acetone, paint thinner
 SS: odor of substance in breath and
clothing; excess nasal secretions and
lacrimations, poor muscle control,
slurring of speech
 Deep inhaling of vapors causes
disorientation and violent behaviors.
 Long period of sniffing concentrated
vapors: permanently damage the
central nervous system
STIMULANTS
1. Cocaine- coke, flakes, white blow
2. Crack or cocaine crack or rocks
3. Amphetamines- speed, uppers, black
pep pills, bumble bees, co-pilot
4. Methamphetamine- crack, crystal
meth, methedrine, speed or “shabu”
COCAINE (CRACKS)
 Derived from coca bush (Erythroxylon
coca) evergreen shrub grown in South
America
 Alkaloid salt (ecognine) that can be
taken directly (insugfflation of IV) or by
inhalation/snorting
 Potent CNS stimulant that elicits a sense
of excitement and euphoria; increase
physical activity
 Cocaine can be sniffed, smoked or
injected
 Smoking coca leaves
 A stimulating drug can induce euphoric
excitement and hallucinatory
experience, sense of being “super
powerful” combined with paranoid
delusions and auditory visual and tactile
hallucination. When induced user can
commit serious anti-social acts.
 Used as local anaesthetic for
nasopharyngeal surgery
 Can cause sudden death due to direct
toxicity on myocardium- it
vasoconstriction, platelet aggregation
and synthesis of plasminogen activator
inhibitor
 Cause malformations in uterus
 Not considered as an addictive drug- it
does not reflect the true dependence
commonly seen in abusers of
barbiturates and opiates
 Easily passes the placenta and
mammary glands (readily passed from
mothers to infants) resulting mental
retardation, slow mental development
and drug dependence in newborns
 Overdosage: violent behavior; high
abuse potential
 Single use: can be detected in urine for
up to 3 days
 Chronic user: up to 20 days
 Inhibitor: Prozac
 Treatment and addiction:
Benzodiazepine
  Toxic effects: hypertension,
arrhythmias, seizures and myocardial
infarction
 Urine metabolite: benzoylecgonine
AMPHETAMINE AND METHAMPHETAMINES
 Therapeutically used for treatment of
narcolepsy and attention deficit
disorder
 Increases mental alertness and physical
capacity, and has no anorectic property
 It is structurally related to dopamine
and catecholamine
 Have a capacity to elevate mood and
dispel fatigue
 Cause the release (together with
cocaine) of dopamine from the brain
leading to a “pleasant feeling” (so called
“high”_ among users
 Most cases taken for euphoric effect
 Used to be treatment for narcolepsy
(sleep episode) and to calm down
hyperkinetic children or overactive
children with attention deficit disorder
 3,4 methylenedioxymethamphetamine
(MDMA or ‘ecstacy’) - drug derivative
of methamphetamine; designer drug
 Amphetamine like compounds:
ephedrine, pseudoephedrine and
phenylpropanolamine
 Signs of acute intoxication:
hyperpyrexia
 Acute psychotic syndrome: auditory
and visual hallucinations, suicidal
tendency and paranoia
 Toxic effects: palpitation, hypertension,
cardiac arrhythmias, convulsion,
pancytopenia, mental impairment and
teeth grinding
DEPRESSANTS
 DEPRESSANTS: acts on central nervous
system. Close pharmaceutical relative
to sedatives that higher doses induce
sleeps.
o Examples: Demerol
(meperidine hydrochloride)
cough syrups (codeine)
1. BARBITURATES: Downers, barbs, red
devils, yellow jackets, Nembutal,
seconal, amytal
2. METHAQUALENE: Qualudes, Ludes,
Soppers, Mandrakes
3. TRANQUILIZERS: Valium, Librium,
Equanil, Miltown, Serax, Trnxene
 Most abused: Barbiturates and
Tranquilizers
 SS: signs of alcoholic intoxication but no
alcoholic breath, Drowsiness and
appears disoriented, difficulty of
concentration, Pupils dilated; all vitals
are down/decreased
 Large doses: produce respiratory
depression, slurring of speech and
altered perception
 Taken with alcohol multiplies the risk.
Babies born to depressant drug
dependent mother may produce mental
and physical defects
SEDATIVE HYPNOTICS
 Have therapeutic roles and they are
CNS depressants
 Used also to potentiate the effects of
heroine
o EXAMPLES: Barbiturates and
benzodiazepine
 Commonly abused barbiturates:
secobarbital, phentobarbital,
phenobarbital and theopental
  Commonly abused benzodiazepine:
diazepam (Valium), chlordiazepoxide
(Librium), and Lorazepam (Ativan)
 Toxicity: initiated by ethanol
 Major metabolite: secobarbital
(barbiturates)
 Barbiturate chemoadsorbent: activated
charcoal
 Toxic effects: Cheyne-Stokes
respiration, depression, cyanosis,
areflexia, stupor, coma
 Barbiturates are condensation products
of urea and malonic acid
 Benx=ziodiazepines are used for the
treatment of cocaine addictions
 Diazepam has a clinical use for rapid
control of acute seizure activity; a minor
tranquilizer
 Phenobarbital structurally resembles
phenytoin (Dilantin)
METHAQUALONE (QUALUDE)
 A 2,3 – distubstituted quinazoline with
anesthetic, antihistamine and
antitussive properties
 It has also sedative-hypnotic properties;
a hallucinogen
 Has a similar symptoms of toxicity to
barbiturates, as well as pyramidal signs
(hypertonicity, hyperreflexia and
myoclonus)
 CHEMOADSORBENT: activated charcoal
HALLUCINOGEN
 Are chemically diverse group which
produce mental changes like euphoria,
anxiety, sensory distortion, vivid visual
and auditory hallucination
o Example: DOM (dimethoxy-4-
methylamphetamine) PCP
(Phecyclidine), most powerful is
LSD (Lysergic acid diethylamide)
1. PHENCYCLIDINE- PCP, angel dust, hog,
killer weed, Love boat
2. LYSERGIC ACID DIETHYLAMIDE- LSD,
Acid, Green or Red dragon, White
lightning, Blue heavens
3. MESCALINE AND PEYOTE- Mesc,
Buttons, Cactus
4. PSILOCYBIN- Magic mushrooms,
mushrooms (classified also as
tryptamines are derivatives of serotonin
and components of some components
in plants)
 MESCALINE- the most active
component of cactus peyote
(Loporophora Williamsii) grows wild in
Mexico, Southern and Western US.
Favorite ritualistic hallucinogen.
 DOM- effects heightened awareness of
sensory input. The phenomenon as a
result to use of other hallucinogen:
Psilon or Psilocybin from Psilocybin
Mexicana
 PIPTADENIE PERIGRINA (found in
Orinoco region of Amazonia) SOURCE
OF TRYPTEMINES: a. DMT
(Dimethyltryptamine) b. DET
(Diethyltryptamine)
 EFFECTS: finds difficulty in
distinguishing between fantasy and
reality, one object to another, even
oneself to surroundings
 PCP- KILLER WEED or Angel Dust: is
usually smoked with tobacco can
provoke serious toxic psychosis,
schizophrenic in character, violent
aggressive or self-destructive behavior
 LSD- users sits quietly in a dream-like
state. Altered senses of sight, hearing
and touch as well as images. Drug
 blocks the pain receptor of the brain
and may cause self-inflicted injuries.
Large doses may produce convulsions,
coma and death.
TRYPTAMINES
 Derivatives of serotonin; some
compounds are present in palnts
 Examples: N,N-dimethyltrypthamine
(DMT), psilocin
 DMT is a short-term hallucinogen and
taken by smoking; businessman’s lunch
 Psilocyn is a component of “magic
mushrooms” (psilocybe); a hallucinogen
 Monoamine oxidase inhibitor (b-
carboline) enhances the hallucinogenic
effect of tryptamines
 Ayahuasca- a tea which contains
tryptamines
 Antagonist: benzodiazepine
 Toxic effects: tachycardia,
hypertension, dystonia, seizures,
rhabdomyolysis and paralysis
PHENCYCLIDINE (ANGEL DUST/ANGEL HAIR)
 A depressant, stimulant, has
hallucinogen and anaesthetic properties
 Can be ingested or inhaled by smoking
 About 10-15% is unchanged when
excreted in urine – acidification of the
urine helps in immediate excretion
 Physiologic effects: analgesia and
anaesthesia
 Major metabolite: Phencyclidine HCI
 Mode of treatment: Isolation (kept in
quiet and dark room)
 Toxic effects: tachycardia, seizure and
coma (eventually death)
LYSERGIC ACID DIETHYLAMIDE
(LSD/LYSERGIDE)
 Semi-synthetic indolalkalylamine
 A hallucinogen
 One of the most potent pharmacologic
materials known
 Produces effects at low doses: 20 ug (IV
or ingestion)
 Most common adverse reaction: panic
reaction- “bad trip”
 Toxic effects: blurred or undulating
vision and synthesis
NARCOTICS
 HEROIN- Smoke. Horse, brown sugar,
black Tar
 METHADINE- Dolly, Dolophine,
Methadose, Amidone
 MORPHINE- Pectoral syrup, sweet
amorphous
 CODEINE- Empirine compound with
codeine, codeine in cough medicines
 MEPERIDINE- Pethidine, Demerol
Mepergan
 OPIUM- Paregoric, Dakers powder,
parapectolin
 OTHER NARCOTICS- Fentanyl, Darvon,
Valium, Lomotil
 Narcotics also classified as Opiates
 HEROIN- POPPY PLANT (Papaver
somniferum L.) – coagulated juice from
the unripe capsule. Originated in the
Mediterrenean and spreads to Persia,
Egypt, China and Europe
 PHILIPPUS PARACELSUS, the 16th
century alchemist and physician who
started use and preparation of Opium
for treatment of various illness.
 MORPHINE- main active principle of
opium. Contains 10% morphine.
 Extracted wither from opium or directly
from poppy straw. Still the classical
effective analgesic for the relief of
severe pain;
 CODEINE (METHYLMORPHINE) – used
extensively as an effective cough
suppressant (anti-tussive). And a mild
analgesic with a low addictive potential.
 SS OF NARCOTICS ABUSER: (Heroin,
Morphine, Opium) feeling of euphoria,
followed by drowsiness, nausea and
vomiting, constricted pupils water and
itchy eyes.
 Overdose: may produce slow and
shallow breathing grayish color of skin,
convulsion], coma, and death, multiple
scar marks in arm and due to
contaminated needles may acquire
AIDS and other infectious diseases
 Addiction to pregnant women may
result to premature delivery, still birth
and addicted infants who experience
severe withdrawal symptoms
OPIATES
 Capable of analgesia, sedation and
anesthesia
 Derived chemically from opium poppy
 Naturally occurring opiates: opium,
morphine and codeine
 Common modified opiates: heroin,
hydromerphone and oxycodone
(Percodan)
 COMMON SYNTHETIC OPIATES:
MERPERIDINE (Demerol), methadone
(Dolophine), Propoxyphene (Darvon),
pentazocine (Talwin) and fentanyl
(Sublimaze)
 Codeine is an antitussive drug
 Methadone is a nonbicyclic drug that
binds with morphine in the brain
 Fentanyl “lollipops” or “patches” are
more potent analgesics than morphine
 Commonly tested opiates: morphine
and codeine
 Major cause of drug related death:
darvon overdose combine with alcohol
 Major metabolites of heroin: N-acetyl
morphine (heroin) and morphine
 Withdrawal symptoms of heroin: cold
seweats, nightmares and hypothermia
 Antagonist for opiate overdose:
naloxone (narcan)
 Toxic effects: respiratory acidosis,
myoglobinuria, cardiopulmonary failure
and pupillary constriction (“pin-point
pupils”)
MORPHINE AND HEROIN
 Morphine is a metabolite of heroin; a
powerful analgesic; used in treatment
of acute congestive heart failure
 Morphine binds to mu-receptors in the
limbic system( CNS) producing analgesic
effect.
 Morphine and meperidine increase liver
and pancreatic enzymes.
 Heroin is highly addictive (true physical
dependence)
 Heroin crosses the blood-brain barrier-
elevated levels in the CNS
 Heroin is taken by IV administration
ANABOLIC STEROIDS
 Chemically associated to male
hormone, testosterone
(dihydotestosterone and testosterone.)
 Improves athletic performance by
increasing muscle mass.
  Toxic effects: chronic hepatitis,
atherosclerosis, abnormal platelet
aggregation and cardiomegaly
SPECIMEN CONSIIDERATIONS:
 Alcohol in blood samples may be
analysed even after a moment of delay
provided the samples in tubes remain
sealed
 Urine temperature is a vital factor to
assure it is freshly voided (abused
drugs)
 Aspiration of gastric contents or
vomitus may reveal tablets or capsules
from which the ingested drug can be
determined
SAMPLRE REQUIREMENTS:
 Urine, serum, hair, nails, whole blood or
plasma (alcohol), sweat, saliva and
exhales breath (alcohol intoxication)
 Urine specimen collected →
concentration and extraction
procedures; least expensive; within the
week
 Drugs deposited in hair are generally
present in relatively low levels
 Sweating testing- parent drug is
increased than metabolites
 Saliva testing- drug concentration in it
reflects the free or active fraction
 2 basic techniques for identification of
drugs: immunochemical and
chromatography
 Hair specimen twice more sensitive,
requires 1.5x1.5
 Urine 60 ml single container
CHECK AUBF BOOK FOR MORE INFO
STRASINGER 6TH ED.
WAYS OF TAMPERING SPECIMEN
 Adulteration- specimen containing a
substance a not normal constituent for
that type of spx/ containing
endogenous substance that is not
normal physiological concentration;
EXAMPLES: bleach, salt, vinegar
 Dilution (internal or external)- with <
normal physiological contents; internal-
drinking plenty of water EXAMPLE:
diuretics; external- addition of water to
the specimen.
 Substitution
METHODOLOGY
 SCREENING METHOD:
o FDA-DOH registered kits using
immunoassay technique
o Instrumented screening
method: ELISA, FPIA
(immunoassay), TLC, HPLC
(chromatographic)
 CONFIRMATORY METHOD
o GC-MS- gold standard
 ENZYMATIC TEST
o Alcohol is measured from blood
using alcohol dehydrogenase as
reagent
o Quantitates the sum of all
alcohols present in a sample
o Does not distinguish alcohols
from its metabolites during
quantitation
 CAPILLARY ELECTROPHORESIS
o Different analyte selectivity is
based on different
physochemical principle of
separation without changes in
instrument hardware, a distinct
advantage of this technique.
 oRecent variant of TLC that
includes the advantages of
HPLC
 HOMOGENOUS IMMUNOASSAY
o Done in one solution without
separation
o EMIT- method uses enzyme
labeled drug that competes
with the drug in the sample; the
active site of the enzyme is
blocked with the anti-drug
antibosy, resulting to decreased
enzymatic activity; free drug
(analyte in the serum or urine)
competes with the antibody-
drug-enzyme complex,
 CHROMATOGRAPHIC METHODS
 TLC
o Uses serum, urine, or gastric
fluid for analysis
o Extraction of drugs is pH
dependent – acidic drugs at pH
4.5 (barbiturates) and alkaline
drugs (opiates, amphetamines)
at pH 9.0
 LC-MS
o Used to confirm positive test
results from a screening assay
(non-volatile compounds)
o Used for detection of poisons in
acute or chronic intoxication,
therapeutic drug identification
and quantitation,
pharmacokinetics and drug
metabolism studies
 HPLC
o Allows quantitative
measurement of drugs as well
as separation of these same
drugs, especially tricyclic
antidepressants including its
active and inactive metabolites
oMay be used as an alternative
to GC-MS in definitive
identification of drugs
 Gas Chromotography (GLC, GC-MS)
o Gas Liquid Chromotography
(GLC) - Legally accepted in
ethanol testing
o GC With Infrared Spectroscopy
– detection of amphetamines
o Gas Chromatography-Mass
Spectroscopy (GC-MS)- gold
standard for confirmation of
screening methods such as TLC
and EMIT; allows for detection
of low levels of drugs like
cocaine and drug metabolites
FACTORS REESULTING TO INNOCENT DRUG
TESTING RESULTS
 Presence to detergents will be result to
alkaline pH
 Use of sodium chloride (table salt)
 Low specific gravity (diluted urine)
 High pH (alkaline urine)
 Blood in urine (hematuria)
URINE IS THE PREFERRED SAMPLE ESPECIALLY
IN DRUG TESTING BECAUSE OF THE
FOLLOWING REASONS:
1. Drugs that their metabolites are present
in higher concentrations in urine than in
blood
2. Larger sample volumes are easily
collected
3. There is no pain or discomfort when
collecting the sample
4. The process of obtaining the sample is
noninvasive
DRUG TEST KIT (DKT)