Name:_____________________________________

Date: ______________
Clinical Chemistry Lecture Part 1
Prepared By: Prof. Jenny S. Gayondato, RMT, MSMT, MLS (ASCPi)CM

Quality Assurance
I. Basic Concepts
■ Descriptive Statistics: Measures Center, Spread and Shape
■ Random and Systematic Error
■ _______________ is used to verify the acceptability of new methods prior to reporting patient results.
■ laboratory ensures it remains valid over time; this is achieved by a process known as __________________.
I.I Descriptive Statistics: Measures Center, Spread and Shape
--foundation for monitoring performance
_________: most common.
_________: more powerful

I.I Descriptive Statistics: Measures Center

• Median: used with skewed data
• Mode: data that have 2 centers (bimodal)
• Mean: X bar
I.I Descriptive Statistics: Measures Spread
✓ _________
✓ _______________ (“s,” SD, or σ): The SD and,
more specifically, the variance represent the “average”
distance from the center of the data (the mean) and
every value in the dataset Summation of x sub 1 – mean
squared divided by n-1
✓ ____________: represents the difference
between each value and the average of the data.
• ____________________ compare SDs with
different units
• SD/mean X 100

• Gaussian distribution (also called normal distribution

_______: 1SD
_______: 2SD
_______: 3SD

1.2 Random and Systematic Error

■ Error: difference between test and reference method results
■ _________ Error: present in all measurements. Can be positive or negative.
■ _________ Error: influences observations consistently. In one direction (higher or lower).
– Consistent: continual difference regardless of the concentration
– Proportional

II. Reference Interval Studies

■ Reference Interval: A pair of medical decision points that span the limits of results expected for a defined healthy
population.
 – 2 types:
■ Establishing a reference interval
■ Verifying a reference interval
– Selection of Reference Interval Study Population (Inclusion and Exclusion Criteria)
– Pre-Analytic and Analytic Considerations
– Data Analysis to Establish a Reference Interval (120 individuals)
– Data Analysis to Establish a Reference Interval (20 Individuals)
■ ___________________: represents the boundary between different therapeutic approaches.
■ ___________________: Range of results between two medical decision points
■ ___________________: Reference interval applied to a therapeutic drug.
■ Nonparametric method: makes no specific assumption
■ Parametric method: assumes the observed values, or follow a (normal) Gaussian distribution
■ ___________________: Range of values that include a specified probability, usually 90% or 95%.
■ ___________________: Difference between the observed mean and the reference mean. Constant systematic
error

III. Measures of Diagnostic Efficiency

■ To determine how good a given test is at detecting and predicting the presence of disease
■ ___________________ refers to the lower limit of detection

■ ___________________refers to the proportion of individuals with that disease who test positively with the test
■ ___________________ is defined as the proportion of individuals without a condition who have a negative test
for that condition

■ ____: the probability of an individual having the disease if the result is abnormal
■ ____: the probability that a patient does not have a disease if a result is within the reference range
IV. Methods of Evaluation
IV.I. Imprecision vs Inaccuracy

Imprecison: _______ error

Inaccuracy: _______ error

IV.II. Measurement of Imprecision

■ Performed over a 10- to 20-day
period
■ two controls are run twice a day for
10 days

IV. III. Measurement of Inaccuracy

■ _____________: to determine how
much of the analyte can be detected (recovered) in the presence of all the other compounds in the matrix.
■ _____________________: to determine if specific compounds affect the accuracy of laboratory tests
■ COM: compare a method being evaluated (test method) with a reference method.
■ 40 to 100 specimens be run by each method on the same day over 8 to 20 days (preferably within 4 hours)

Quality Control
■ Control Limits: The expected values are represented
by intervals of acceptable values with upper and lower
limits.
■ Control: Specimens analyzed for QC purposes
Quality Control Charts
■ _____________________: Graphical representation of
observed values of a control material over time in the
context of the upper and lower control limits.
Operation of Quality Control System
1. Establishing allowable statistical limits of variation for each
analytic method
2. Using these limits as criteria for evaluating the QC data
generated for each test
3. Taking action to remedy errors when indicated
a. Finding the cause(s) of error
b. Taking corrective action
c. Reanalyzing control and patient data
Westgard Multirule
■ Shewhart in the 1920s
 Carbohydrates
Introduction
• Primary energy source stored primarily as glycogen
• Disease status involved hyperglycemia and hypoglycaemia
• Contain C, H, and O (Cx (H2O)y with C=O and –OH functional groups
Classification Definition

Monosaccharides or simple ▪ Cannot be hydrolyzed to a simpler form

sugars ▪ Fructose, glucose, galactose
▪ Formed by interaction of two monosaccharides
Disaccharides
▪ Maltose, Lactose, Sucrose
▪ Linkage of many monosaccharide units
Polysaccharides
▪ Starch, cellulose and glycogen

Glucose Metabolism
(1) ______________ (EMP Pathway) ▪ Glucose –Insulin→ Glycogen
▪ Metabolism of glucose to lactate or pyruvate for
production of energy (4) ______________
▪ Glucose –Insulin→ ATP +lactate/pyruvate ▪ Breakdown of glycogen to G-6-P
▪ Glycogen –Glucagon→ Glucose
(2) ______________
▪ Formation of G-6-P from non-carbohydrate
source (5) ______________
▪ Fats (ketone bodies), Protein (urea nitrogen) • Conversion of carbohydrates to fatty acids
(6) ______________
(3) ______________ • Decomposition of fat
▪ Conversion of glucose to glycogen
 HORMONAL ACTIVITY AFFECTING SERUM GLUCOSE
Hormone Source Action
Insulin _________________________ Decrease serum glucose
_________________________ Stimulates glucose uptake by cells
Glucagon ___________________________ Increase serum glucose
______________________ Glycogenolysis (breakdown of
glycogen to glucose)
ACTH ___________________ Increase serum glucose
Insulin antagonist
GH _____________ Increase serum glucose
Insulin antagonist
Acromegaly=hyperglycemia
Cortisol __________________ Increase serum glucose
Gluconeogenesis (formation of
glucose from non-carbohydrate
source)
Human Placental Lactogen _______________ Increase serum glucose
Insulin antagonist
Epinephrine __________________ Increase serum glucose
Glycogenlysis
Phaeochromocytoma:tumor of
adrenal medulla→hyperglycemia
T3 and T4 __________________ Increase serum glucose
glycogenolysis

Diabetes Mellitus
▪ Metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or
both

1. ________ (IDDM) 2. ________ (NIDDM)

▪ β-Cell destruction ▪ 90% of all cases of diabetes
▪ Absolute insulin deficiency ▪ Adult onset
▪ Autoantibodies

Laboratory Findings
Diagnostic Criteria for Diabetes Mellitus
1. ↑ glucose in plasma and urine
1. Random plasma glucose _________, + symptoms
2. ↑ urine specific gravity
of diabetes
3. ↑ serum and urine osmolality
2. Fasting plasma glucose _________
4. Ketones in serum and urine (ketonemia and
ketonuria) 3. 2-h plasma glucose _________ during an OGTT
5. ↓ blood and urine pH (acidosis)
6. Electrolyte imbalance (↓ __,↑ __)

Categories of Fasting Plasma Glucose (FPG)

Provisional diabetes diagnosis FPG ___________

Impaired fasting glucose FBG ___________

Normal fasting glucose FBG ___________

 Hypoglycemia
• No coexisting disease • Compensated coexistent
o Insulinoma, Islet hyperplasia o Drugs/disease
o Factitial hypoglycemia • Patients Appears ill
(insulin/sulfonylurea) o Drugs, Predisposing illness, Hospitalized
o Severe exercise, Ketotic hypoglycemia patient

Diagnosis of Glucose Metabolic Alterations

Considerations:

1. WB glucose concentration is 11% _______ than plasma

2. Serum / plasma must be refrigerated and separated from the cells within 1 hr
3. ____________ (gray-top) can be used to inhibit glycolytic enzymes
4. FBG should be obtained in the morning after _______________ fasting (not longer than 16 hours)

Methods: Comments

1. Fasting Blood Glucose

2. POC
3. 2-Hr Post Prandial Sugar
4. OGTT
5. HbA1C
6. Microalbuminuria

Lipids and Lipoproteins

Lipid Chemistry
• Composed of mostly carbons-hydrogen (C-H) bonds
Classification: 3. Cholesterol
1. Fatty acids 4. Phospholipids
2. Triglycerides Transported by lipoproteins (VLDL, LDL, HDL)
Lipoprotein Structure
Apolipoprotein Major LPP Location Function

Apo A-I HDL LCAT activator, ABCA1 lipid acceptor

Apo A-II HDL inactivates LCAT
Apo A-IV Chylos, VLDL, HDL
____________ LDL, VLDL LDL receptor ligand
____________ Chylos Remnant receptor ligand

Apo C-I Chylos, VLDL, HDL

Apo C-II Chylos, VLDL, HDL LDL cofactor
Apo C-III Chylos, VLDL, HDL LDL inhibitor
Apo E VLDL, HDL LDL receptor Ligand
____________ Lp(a) plasminogen inhibitor
 LIPOPROTEIN CLASSES
Characteristics Chylo. VLDL LDL HDL

Density (g/mL) <0.93 0.93-1.006 1.019-1.063 1.063-1.21

Diameter (nm) 80-1,200 30-80 18-30 5-12
Total lipid (%) 98 89-96 77 50
Triglyceride (%) 84 44-60 11 3
Cholesterol (%) 7 16-22 62 19
Major Protein Apo B-48 Apo B-100 Apo B-100 Apo A-1

LIPOPROTEIN CLASSES
• 4 classes based on particle size, chemical composition, flotation characteristics and electrophoretic mobility; more
protein, higher density; more lipid, lower density.

Lipoprotein Physiology and Metabolism

SPECIMEN COLLECTION AND HANDLING
1. _____________ hours fasting required
2. Non fasting may cause:
Turbid serum with layer of ____________ on top following refrigeration on abnormal finding in fasting
specimens)
Increased ______________

LIPID METHODS
1. Enzymatic methods (measure ____________) have replaced colorimetric (Liebermann-Burchardt) method
2. Triglycerides
a. Enzymatic methods: best
Involves liberation of glycerol with __________
Glycerol contamination from stoppers of evacuated tubes or ingestion of glycerol-coated medications can cause
false increased results.
3. HDL-cholesterol
A. Chylomicrons, VLDL and LDL are removed by precipitation
B. HDL-cholesterol is analyzed directly in the ___________ by cholesterol method.
4. Friedewald method for the calculation of LDL and VLDL
a. LDL= ________________
b. VLDL=TAG/5 in mg/dL

Non-Protein Nitrogens
Compound Approximate Plasma Concentration Approximate Urine Concentration (%
(% of Total NPN) of Excreted N)

Urea 45-50 86.0

Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8

(1) Urea
✓ Physiology
• Major end product of protein (Dietary) and amino acid catabolism - 45% of the total NPN
• First metabolite to elevate in kidney diseases
• Good indicator of nitrogen intake and the state of hydration
• To obtain the concentration of urea from BUN: _______ X BUN = urea (mg%)
✓ Clinical Application
• Clinically, BUN rises in response to renal dysfunction
• Low BUN are not generally considered abnormal renal function
• Serum urea levels drop in severe hepatic disease because of a decline in the capacity of the liver to generate urea
from ammonia
✓ Method
• Chemical Method (Direct Method) non-specific (__________ method)
• Diacetyl Monoxime Method
Urea + DAM → yellow diazine derivative
• Enzymatic Method (Indirect Method)
• Hydrolysis of urea by urease
Urea + urease → NH₃ + CO₂
• After urease reaction, the ammonia produced can be treated with _________ reagents
• Ammonia and carbon dioxide produced are measured by various methods to calculate the concentration
of urea in the original sample. Measurement of ammonia is most often used.
• Isotope Dilution Mass Spectrophotometry (IDMS)– reference method
(2) Uric Acid
✓ Physiology
• Major end product of _________ catabolism primarily in the liver
• Present in plasma as monosodium urates
• Concentration >6.8 mg/dL urate crystals may precipitate in tissues
✓ Clinical Application
• Assess inherited disorders of purine metabolism
• Confirm diagnosis and monitor treatment of _______
• Diagnosis of renal calculi
• Prevent uric acid nephropathy during chemotherapy
• Detect kidney dysfunction
✓ Method
Chemical Method Principle

Phosphotungstic (____________ Method) Uric Acid + H₃PW₁₂O₄₀ + O₂-Na₂CO₃/OH⁻→ Allantoin + tungsten blue
+ CO₂

(3) Creatinine
✓ Physiology
• Chief product of muscle metabolism
• Not affected by protein diet
✓ Clinical Application
• Determine sufficiency of kidney function
• Determine severity of kidney damage
• Monitor the progression of kidney disease
• Measure completeness of 24-hour urine
Renal Clearance and Glomerular Filtration Rate

Glomerular Filtration Rate (Creatinine Clearance) Volume of plasma filtered (V) by the glomeruli per unit of
time (mL/minute)

✓ Method of Analysis
Chemical Method Principle

Direct Jaffe Reaction Creatinine + picrate → ____________complex

Jaffe Kinetic (rate of change of absorbance) Rate of change of absorbance (color formation) is
detected to avoid interference of noncreatinine
chromogens
Jaffe with adsorbent Creatinine in protein-free filtrate is adsorbed onto
Fuller’s earth (___________________________);
Lloyd’s regent (___________________________),
then eluted and reacted with alkaline picrate
Jaffe without adsorbent Creatine in protein-free filtrate reacts with alkaline
picrate to form colored complex
• BUN/Creatinine Ratio
• Comparison of the BUN and creatinine levels is a better indicator of the source of elevation of either
substance
• The normal BUN:Crea ratio is ___________
• Increased concentration
• Renal failure (glomerular function)
• ↑ plasma concentration= ↓ GFR

(4) Ammonia
✓ Physiology
• By product of amino acid deamination
• Remove from the circulation and converted to ____ in the liver
 ✓ Clinical Application
• Diagnosis of hepatic failure and hepatic coma
• ________________= acute metabolic disorder of the liver
• Inherited deficiencies of urea cycle
✓ Method
Chemical Method Principle

Ion-selective electrode Diffusion of NH₃ through selective membrane into NH₄Cl

causing pH change, which is measured
potentionmetrically
Spectrophotometric NH₃ + bromphenol blue → blue dye

Enzymatic Method Principle

__________________ NH₄⁺ + 2-oxoglutarate + NADPH + H⁺ --GLDH →

Decrease in absorbance is measured at 340nm Glutamate + NADP⁺ + H₂O

AMINOACIDOPATHIES
Disease Enzyme deficient

___________ Phenylalanine hydroxylase

___________ Fumarylacetoacetate hydrolase

___________ Tyrosine aminotransferase

___________ 4-Hydroxyphenylpyruvate oxidase
___________ Homogentisate oxidase
___________ Branched-chain α-ketoacid decarboxylase
___________ Isovaleryl-CoA dehydrogenase
___________ Cystathionine-β synthetase
Citrullinemia Arginosuccinic acid synthetase
Arginosuccinic aciduria Argininosuccinic acid lyase
Cystinuria Defective AA transport system

PROTEINS
Plasma Proteins
• Most frequently analyzed of all proteins
• Divided into 2 groups:
• Albumin
• Globulin • _______________________
• with 4 major types • A general transport protein
• _______________________ • Major contributor to oncotic pressure
• Sensitive marker of nutritional status • Highest concentration in plasma
• Binds thyroid hormones (T3, T4) • Negative APR
• Binds retinol-binding protein
• α1 -Globulins • Oxidase activity;
• _______________ contains copper
• Main globulin of a1 fraction (90 • Marker for wilson’s
%) disease (O.1 g/L of
• Main plasma inhibitor of serine ceruloplasmin)
proteases (trypsin, elastase) • _______________
• Deficiency causes • Largest non-
emphysematous pulmonary immunoglobulin in
disease plasma
• _______________ • Inhibits proteases
• Principal fetal protein • Inc 10x in nephrotic
• Gestational marker syndrome
• Screening test for NTDs and DS • β-Globulins
• Tumor marker and Post surgical • _______________
marker • Transports Lipids (VLDL,
• _______________ Triglycerides)
• Also known as orosomucoid • _______________
• Acute phase reactant • Transports iron
• Diagnostic tool for neonates • _______________
with bacterial infection • Binds heme
• _______________ • Diagnosis of early hemolysis
• Transports lipids (HDLs) (Intravascular)
• _______________ • _______________
• Inhibits serine proteases • Transports lipids (LDL)
• _______________ • _______________
• Acute phase reactant • Component of Leukocyte
• Inhibits serine proteases antigen molecules
• Major form of PSA found in • _______________
human sera • Immune response
• INC: Infection, malignancy, burn • _______________
and Alzheimer’s dse • Precursor of fibrin clot
• _______________ • _______________
• Transports vitamin D and binds • One of the first acute phase
actin reactants to rise in response to
• α2 –Globulins inflammation
• _______________ • g-Globulins
• Acute Phase reactant • Immunoglobulin G
• Binds hemoglobin • Immunoglobulin A
• _______________ • Immunoglobulin M
• Acute phase reactant • Immunoglobulin D
• Immunoglobulin E

Laboratory Methods of Determination

• _________
• Reference method but not routinely
used
• Principle: Based on the measurement
of the Nitrogen content of
protein
• Uses serum treated with an organic
acid or Tungstic acid.
• Assumes average nitrogen content of
16 %
• 15.1% to 16.8% -> actual nitrogen
content
• Reagent: H2SO4 (digesting agent)
• _________
• Rapid and simple
• Only requires a small volume of serum.
• Principle: Measurement of Refractive
index due to solutes in serum.
• _________
• Most widely used
• Recommended by the International
Federation of Clinical Chemistry
• Principle: Cupric ions complex the
groups involved in the peptide
bond forming a violet-colored
chelate which is proportional to
the number of peptide bonds
present.
• Reagents:
• Alkaline Copper Sulfate
• Rochelle Salt
• NaOh & Potassium
Iodide
Identification of Specific Protein
• Salt Fractionation
o Fractionation of Proteins is done using Precipitation.
o Globulins can be separated from albumin by salting-out procedures using sodium salts.
o Reagent: Sodium salt
o The albumin that remains in the supernatant can be measured by any of the routine total protein
methods
▪ Globulin is insoluble in water but not in dilute salt solution
• Albumin
✓ Dye binding procedures are the most widely used for determining albumin.
METHOD COMMENT
____________________ Non-specific for albumin
____________________ Many interferences (salicylates, bilirubin)
____________________ Most commonly used
Sensitive; overestimates low albumin dyes
____________________ Specific, Precise and sensitive
Name:_____________________________________
Date: ______________
Clinical Chemistry Lecture Part 2
Prepared By: Prof. Jenny S. Gayondato, RMT, MSMT, MLS (ASCPi)CM

LIVER
• Largest internal organ
• Weighs 1.2 – 1.5 kg
• Vascular System
Hepatic Artery (_____) o2 rich blood from the heart to the liver
(from the circulation)

Portal vein (______) nutrient rich blood from the digestive tract
to the liver

Microstructure of the Liver

• _____________________
✓ Functional units
✓ Six sided with central vein and portal triads
The Functions of the Liver
(1) Excretion of waste products
• Hemoglobin = Heme and globin + iron
• ____________________
• The major heme waste product
• The principal pigment in bile
• Highly insoluble in water

Differences between Conjugated and Unconjugated Bilirubin

Unconjugated Bilirubin Conjugated Bilirubin

Solubility in water

Affinity to serum albumin

Renal excretion

Van den Berg Reaction

(2) Synthesis of CHO, CHONS and Lipids

• CHO metabolism • CHON metabolism
• Glycolysis • Synthesis of albumin, alpha and beta
• Glycogenesis globulins
• Glycogenolysis • Coagulation proteins (except)
• Gluconeogenesis • Fat metabolism
• Synthesis of bile salts and lipids from
acetyl-CoA
• Metabolism of lipids by LPL
(3) Detoxification
• Allow the substances to reach the circulation: ______________________
• Excretion of steroid hormones, drugs and foreign compounds.
• Usually occurs in the microsomes of the liver via _____________________ isoenzymes
Liver Function Alterations During Disease
(1) Jaundice
✓ Jaundice (_______________)
• Yellow discoloration of skin, eyes and mucous membranes (>3 mg/dL)
• NV= 0.5 – 1.0 mg/dL
• Classification:
• Prehepatic, Hepatic and Post hepatic

✓ 1.1 Pre-hepatic (Unconjugated hyperbilirubinemia)

• Excessive destruction of RBC
• ↑____________ (↑ ____, Normal ____)
• Acute and chronic hemolytic anemia
• HDN
• HTR
• Malaria

✓ 1.2 Hepatic (intrinsic liver defect)

➢ A. _____________________
o Unconjugated hyperbilirubinemia due to defective conjugation system
o Reduced expression of UGT1A1
o ↑ Tot. Bili (↑ B1, Normal B2)

➢ B. _____________________
o Molecular defect of gene
o Type 1 – absence of UDP-GT
o Type 2 – deficiency of UDP-GT
o ↑ TB (↑ B1, ↓ B2)

➢ C. _____________________________
o Deficiency of MDR2/cMOAT
o ↑ TB(Normal B1, ↑ B2)
o Bilirubinuria
o Dark stained granules
o Presence of Delta Bilirubin)

➢ D. _____________________
o Deficiency of Ligandin
o ↑ TB (Normal B1, ↑ B2)
o No Dark stained granules

➢ E. _____________________
o Deficiency in the glucoronyl transferase
o ↑ TB (↑ B1, Normal B2)
o Leads to Kernicterus
o Treated with phototherapy

✓ 1.3 Post-Hepatic Jaundice

• Biliary obstructive disease
• Physical obstructions which prevent flow of B2 into bile canal.
• Gall stones
• Tumors
• ↑ ___ (Normal ____, ↑ _____) Bilirubinuria
Changes in Concentration of Bilirubin with Jaundice
Total Bilirubin B1 B2

HDN, HTR
Gilberts Syndrome
Crigler-Najjar Syndrome
Dubin Johnson
Rotor
Gall Stones, Tumors

Other Liver Disorders

(1) Cirrhosis Alcohol Related Disorder
• Scar tissue replaces liver tissue resulting to (1) Alcoholic Fatty Liver
blockage of blood flow through the liver • Inc. AST, ALT and GGT
• Chronic alcoholism and chronic hepatitis C • Fatty infiltrates in vacuoles of liver
(2) Tumors (2) Alcoholic Hepatitis
• Primary or Metastatic • Increased (2X) AST, ALT, GGT and ALP
• Benign • Bilirubin 30mg/dL, dec. albumnin, inc. PT
Hepatocellular adenoma (3) Alcoholic Cirrhosis
hemangioma • Increased (3x) AST, ALT, GGT and ALP
• Malignant • Inc. Bilirubin, dec. albumin, inc. PT
Hepatocellular carcinoma
Hepatocarcinoma and hepatoma Drug Related Disorder
Predisposes from cirrhosis and HBV/HCV • Acetaminophen
(3) Reye Syndrome • Hepatic necrosis
• Disorders preceded by infectious (viral) or drug • Tranquilizers
(aspirin) related disease in children. • Antineoplastic Agents
• Noninflammatory encephalopathy and fatty liver • Lipid-Lowering Medication
degeneration • Anti-Inflammatory Drugs

Excretory Function Test

Direct Van den Bergh Indirect Van den Bergh

B2 + Diazo reagent → Azobilirubin B1 + Accelerator + Diazo reagent → Azobilirubin

Specimen Collection and Transport

• Serum specimen
• Hemolysis - False ____
• Lipemia - False ____
• Photosensitive - ____ by 30-50% / hour
Methods of Bilirubin Analysis
Malloy-Evelyn Jendrassik-Grof

Accelerator

Stopper

pH

End Color

Urobilinogen
• _____________
• Clinical significance:
1. Hemolytic Disease
2. Hepatic Disease
3. Biliary obstruction

Liver Markers : Enzymes

AST, ALT, LDH, ALP, 5’NT, GGT

Test for the Synthetic Function of Liver

Disease TP Alb Glob
Hepatic Damage ______ _______
• Cirrhosis β-γ bridging _______
• Hepatitis ↑ γ-globulins
• Immunoglobulin
primary biliary cirrhosis ______

chronic active hepatitis ______

alcoholic cirrhosis ______

• Clotting Factors
• Prothrombin Time (PT)
• Increased in liver disease
• Disruption of bile flow resulting in inadequate absorption of Vitamin K in intestine

Test For Detoxification • Hepatic failure and hepatic coma

• Test the ability of the liver to convert ammonia • Reye syndrome
to urea • Inherited deficiencies of urea cycle
• By product of amino acid deamination • Test: GLDH: decrease in absorbance at 340 nm

Enzymes
Functions
• Catalyzation is essential to physiologic function such as:
• Hydration of CO2 (respiration)
• Nutrient degradation (digestion)
• Nerve conduction
• Muscle contraction
• Energy use
Introduction
• Biologic proteins that catalyze biochemical reactions
• Not consumed or changed in composition
• Protein in nature
• Found in all body tissue and is ↑ in serum after cell injury (intracellular)

Components of Enzyme
___________________ ___________________

A cavity of an enzyme where substrates bind and A cavity other than the active site that binds regulatory
undergo a chemical reaction. (effector) molecules.

Enzyme theories
• Lock and Key theory: __________
• Induced fit theory: _____________

Definition of Terms
(1) ___________________ Classification of Enzymes
• Substances acted upon enzymes (1) ___________________
• Specific for each of their particular • Catalyze redox reaction between two substrates
enzyme (2) ___________________
• Cofactors • Catalyze the transfer of C, N or P-containing
• Non protein substances groups
added in the enzyme (3) ___________________
needed for catalytic • Catalyze the cleavage of bonds by addition of
activity water
(2) ___________________ (4) ___________________
• Similar enzymatic activity but differ in • Catalyze the removal of groups from substrates
physical, biochemical and immunologic without hydrolysis; the product remain double
characteristics bonds
(3) ___________________ • They catalyze the cleavage of C-C, C-S and
• The protein portion of the enzyme certain C-N bonds
• Subject to denaturation, in which (5) ___________________
enzyme losses its activity • Catalyze the interconversion of geometric,
(4) ____________________ optical or positional isomers
• An active substance formed by (6) ___________________
combination of a co-enzyme and an • Catalyze the joining of two substrate molecules,
apoenzyme. coupled with breaking of pyrophosphate bond in
(5) ____________________ ATP
• An inactive enzyme precursor • Bond formation coupled with ATP hydrolysis
• E.g. Coagulation factors and digestive
enzymes
Nomenclature
• Enzyme commission standard
• Example:
• ACP = E.C. 3.1.3.2
• LDH = E.C. 1.1.1.27
• AST = E.C. 2.6.1.1

Enzyme Kinetics
• Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach
Relationship between Enzyme, Substrate and
Product Catalytic Mechanism of Enzymes
• Enzyme catalysis is a two step process: (1) Absolute specificity
(1) ___________________ • E Combines with only one S and catalyzes only one
• The S combines with the E to reaction (E.g. _______)
form a noncovalent ES complex (2) Group specificity
(2) ___________________ • E Combines with all S containing a particular
• E S complex decomposes into P chemical group (E.g. _______)
and a free Enzyme (3) Bond specificity
• E Specific to chemical bonds (E.g. _______)
(4) Sterioisometric specificity
• E Combine with one optical isomer (E.g. _______)

Factors that Influence Enzymatic Reactions

(1) Substrate Concentration
• First order Kinetics
• Zero order kinetics
✓ __________________________
o Mathematically represents the relationship between the reaction velocity and substrate concentration
o Vo= Vmax (S)
Km + S
(2) Enzyme concentration
• The higher the enzyme concentration, The faster the reaction will proceed because more enzyme is present to
bind with the substrate.
(3) pH
• Common range ___________________
• pH affects ionization / charge of amino acids in the active site.
(4) Temperature
• Enzyme is active at 25°C, 30°C, 37°C.
• Increase in T’ increases the rate of chemical reaction by increasing the movement of the molecule
(5) Cofactors
A. Coenzyme
B. Activator

(6) Inhibitors
_______________________ Compete with S for the A-site

_______________________ Binds with E at a place other than the A-site

_______________________ Binds with E-S complex

Enzyme Measurement General Methods of Measuring Enzymatic

• Enzymes are measured in terms of their activity
rather than in terms of their concentration.
• Basis of measurement could be:
✓ Decrease in substrate concentration
✓ Increase/Decrease in Coenzyme concentration
(NADH)
✓ Increase in Product concentration
✓ Increase in Altered Concentration
Reactions
(1) Fixed time (Two point) Assay
• Simplest and most widely used
(2) Continuous-monitoring (Kinetic) assays
• Measurements at specific time intervals
(continuous measurement of change in
concentration)
• Rate of change in substrate, cofactor,
product.
Source of Errors for Enzyme Assays
• Hemolysis
• Causes false increase
• Anticoagulants
• Causes enzyme inhibition; serum is
preferred than plasma
• Milky serum
• Causes variable absorbance readings in
spectro.
Clinically significant enzymes
Cardiac Enzymes
(1) CK (Creatine Kinase) EC. 2.7.3.2
• catalyzes the transfer of PO4 grp
between creatine PO4 and ADP.
• Involved in the storage of high-energy
creatine PO4 in the muscles
• small amount in the entire body but is
found in high conc. only in muscles and
brain.
✓ Other Isoenzymes
o Macro-CK
▪ Migrate midway CK-MM and CK-
MB
▪ CK-BB complexed with IgG/IgA
▪ CK-MM with Lipoprotein
o Mitochondrial CK (CK-Mi)
▪ Migrates cathodal to CK-MM
▪ Bound to mitochondrial
membranes
▪ has been detected in cases of
malignant tumor and cardiac
abnormalities
✓ Significance
• sensitive indicator of AMI and Duchenne
disorder
• In AMI, CKMB begin to rise within 4-8
hrs, peak at 12-24 hrs, normalize within
48-72hrs.
• Markedly elevated Total CK = crush
injury, convulsion, tetany, surgical
incision, intramuscular injection
✓ Lab methods
• Tanzer-Gilbarg Assay
• Oliver-Rosalki
✓ Source of errors
• Hemolysis
• Adenylate kinase released after
RBC lysis that hydrolyzes ADP.
Add'n of AMP inhibits AK
activity.
Calculation of Enzyme Activity
• IU (EC): ______________
• Kat (SI) : ______________
• 1.0 IU = ______________
Measurement of Enzyme Mass
• Quantification of Enzyme concentration by:
1. Immunoassays
2. Electrophoresis
(2) AST (Aspartate aminotransferase)
• transfer amino group between aspartate
and alpha keto acids with the form'n of
oxaloacetate and glutamate.
• Major sources: cardiac, liver, and
skeletal muscle.
• Other source: kidney, pancreas, RBC
✓ Isoenzymes
• Cytoplasmic AST = predominant in
serum
• Mitochondrial AST
✓ Significance
• MI, hepatocellular disorders, skeletal
muscle involvement
• In AMI, AST begin to rise 6-8hrs, peak at
24hrs, normalize within 5 days.
✓ Lab method
• ______________ (Kinetic method)
• __________________ (Colorimetric)
✓ Source of error
• Hemolysis (10x)
(3) LDH (Lactate dehydrogenase) EC 1.1.1.27
• catalyzes the interconversion of lactic
acid and pyruvic acid
• hydrolase that uses coenzyme NAD+
✓ Isoenzymes
• LDH2 = major isoenzyme in healthy
person.
• Flipped pattern = LDH1 > LDH2 (AMI and
hemolytic anemia)
• In AMI, LDH begins to rise w/in 12-24
hrs, peaks at 48-72 hrs, remains
elevated for 10-14 days.
✓ Isoenzymes
• 10 fold increase in hepatocarcinoma and
toxic hepatitis
• LDH4 and 5 = heat labile
 •
LDH 6
• alcohol dehydrogenase enzyme • _________________
• elevated in hepatotoxicity and • 2x faster than forward reaction
obstructive jaundice (LDH5)
• migrates cathodic to LDH 5. • ↓ in absorbance is monitored at
✓ Lab methods 340 nm
• _________________ • Optimal pH is 7.1 to 7.4
• ↑ in absorbance is monitored at • (reverse/indirect)
340 nm • Sources of errors
• Optimal pH is 8.3 – 8.9 • Hemolysis
• most commonly used (LDH1) • Decreased values are observed when
samples are frozen.

MARKER ELEVATION PEAK DECREASED

(NORMAL)
_____________ 1-4 hours 6-9 hours 18-24 hours
_____________ 4-6 hours 12-18 hours 6 days
_____________ 4-8 hours 12-24 hours 3 days
_____________ 6-8 hours 24-48 hours 4-5 days
_____________ 12-24 hours 48-72 hours 5-10 days

❖ B Natriuretic Peptide
➢ Synthesized in and secreted from myocardial ventricles in response to ventricular volume expansion and pressure
overload
➢ Increase in _________________
➢ NV: <100pg/mL

Liver Enzymes
(1) ALT (Alanine aminotransferase)
o transfer amino group from alanine to alpha ketoglutarate with form'n of glutamate and pyruvate.
o Major source: _____________________
o Other source: kidney, pancreas, RBC, heart, skeletal mucles, lungs
 Significance
o Evaluation of hepatic disorders, effect of drug therapy
o _______________
▪ AST and ALT ratio
▪ >2.0 = alcoholic hepatitis, hepatocellular carcinoma
▪ < 1.0 = viral hepatitis
▪ >1.0 but <2.0 = cirrhosis
 Lab methods
o Walker method (Kinetic), Reitman and Frankel (Colorimetric)
AST/SGOT ALT/SGPT
Organ Affected Heart Liver
Aspartate Alanine
Substrate
α-ketoglutarate α-ketoglutarate
Glutamic acid Glutamic acid
End Products
Oxaloacetic acid Pyruvic acid

Karmen Walker
Test
Reitman-Frankel Reitman-Frankel
(2) ALP (Alkaline phosphatase) EC 3.1.3.1
• non specific enzyme capable of reacting with many different substrate.
• liberate inorganic PO4 from an organic PO4 ester with the concomitant production of an alcohol.
Liver Bone Placental Intestinal
Electrophoretic migration (anodal)
Heat stability (56 deg celsius for 10-
15mins.)
Chemical inhibition (phenyalanine)
 Significance
• Obstructive jaundice
• Paget's disease (Osteitis deformans)
• Rickets
• Sprue
LIVER PAGETS
ALP _______ _______
GGT _______ _______
 Lab methods
• Bowers and Mc Comb (Continuous-monitoring tech.)
Methods Substrate End Product
1. Bodansky
2. Shinowara
β-glycero-phosphate ________________
3. Jones
4. Reinhart
5. Bessy, Lowry & Brock
p-nitrophenyl phosphate ________________
6. Bowers & McComb
7. King and Armstrong Phenyl phosphate ________________

 Sources of errors
• fatty meal = Blood type __________
• hemolysis
• Stored in low temperature = increase
(3) GGT (Gamma glutamyl transamine peptidase) EC 2.3.2.1
 It catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at a gamma carboxyl
group
 It is located in the canaliculi of the hepatic cells and particularly in the epithelial cells lining the biliary ducts
 Sensitive indicator of alcoholism (___________________)- most sensitive marker of acute alcoholic hepatitis
 Biliary tract obstruction
 Lab methods
• __________________
(4) 5’NT
• phosphatase catalyzing the hydrolysis of nucleoside-5′-phosphate esters.
• Major source: Liver
• elevated in hepatobiliary disease.
• more sensitive to metastatic liver disease than is ______.
• increase may be noted after abdominal surgery.
Pancreatic Enzymes
(1) Amylase EC 3.2.1.1
o catalyzes the breakdown of starch and glycogen
o Normally filtred by kidneys (smallest enzyme)
✓ Isoenzymes
o salivary origin - S1, S2, and S3
o pancreatic origin - P1, P2, and P3
✓ Significance
o Early marker for acute pancreatitis
o In AP, levels rise 2-12 hrs after onset, peak at 24 hrs, and normalize within 3-5 days.
o Parotitis, ectopic pregnancy, peptic ulcer
✓ Lab methods
o Substrate: _____________
METHODS
_____________________ -measures the amt. of reducing CHO by hydrolysis of starch
_____________________ -measure decrease in substrate concentration.
_____________________ - measure increase in color intensity of the solution produced
_____________________ - continuous-monitoring technique.
✓ Sources of errors
o Serum AMS may be inhibited by TAG
o Macroamylasemia = AMS + Ig
(2) Lipase
o hydrolyzes the ester linkages of fats (TAG) to produce alcohols and fatty acids.
o Major source: Pancreas
✓ Significance
o Specific marker for acute pancreatitis
o In AP, level rise 6hrs after onset of attack, peak at 24hrs., remains elevated for 7 days, and normalize in 8-
14days.
✓ Lab methods
o Peroxidase coupline (Most commonly used)
Cherry Crandal (REF. MTD.) Tietz and Fiereck
Substrate ________________
Titrating Agent ________________
Indicator phenolphthalein Tymolpthalein+ veronal
End product ________________
End color ________________ ________________
• Sources of Error
o Hemolysis should be avoided.
Other Enzymes
(1) Acid phosphatase
✓ reaction same as ALP
✓ pH: approximately ________
✓ Major sources: Prostate, RBC, plts, bone, spleen, kidneys
• Significance:
• Investigation of rape cases, prostatic carcinoma
• Breast, lung, thyroid carcinoma
• _______________, _______________
• Lab methods
METHODS SUBSTRATE END PRODUCTS
Gutman and Gutman Phenyl PO4 Inorganic PO4
Shinowara PNPP p-nitrophenol
Babson, Read, and Philips alpha-naphthyl PO4 alpha-naphthol
(continuous)
Roy and Hillman (end-point) ______________________ free thymolpthalein

• Sources of errors
o Decreases w/in 1-2hrs if left at RT.
o Hemolysis
o Inhibitors
▪ _____________ = prostatic ACP
▪ _____________ and _____________= red cell ACP

(2) G6PD (4) ALDOLASE EC 4.1.2.13

• Catalyzes the oxidation of G6P to 6- • Catalyzes the splitting of fructose 1,6
phosphogluconate diphosphate to glyceraldehyde-3-phosphate and
• It is found in adrenal cortex, spleen, RBC and dihydroxyacetone phosphate
lymph nodes • skeletal muscles, heart muscles, liver and red
• Newborn screening marker cells
• _____________________ • Increased: _____________________

(3) CHOLINESTERASE (5) ORNITHINE CARBAMOYL TRANSFERASE EC 2.1.3.3

• Hydrolyzes a number of esters with
acetylcholine to choline and acetic acid
• A. True cholinesterase (acylcholine
acylhydrolase) EC 3.1.1.7
• B. Pseudocholinesterase (acetylcholine acetyl-
hydrolase) EC 3.1.1.8
• Decreased value in __________________
• Catalyzes the reversible conversion of ornithine
to citrulline (synthesis of urea)
• Liver
• _____________________ and
_____________________
(6) LEUCINE AMINOPEPTIDASE EC 3.4.4.1
• Catalyzes the hydrolysis of N-terminal residues
from certain peptides and amides containing
free amino groups
• Urine, serum and bile
• Increased: Hepatobiliary diseases like hepatitis,
cirrhosis, obstructive jaundice, metastatic
carcinoma of liver and pancreatitis
 Osmolality and Water Balance
Electrolytes
• Ions capable of carrying an electric charge
Anions Cations

______________________ ______________________

Cl-, HCO3-, PO4- ,SO4 Na+, K+, Mg2+, Ca2+

-

Functions of Electrolytes
• Maintenance of osmotic pressure and hydration
(__________) and volume regulation
Water
• Buffering functions (__________) Acid-base
• 40 – 75% total body wt. (ave. water content)
balance
• The fractional water content:
• Activators in enzyme reactions ((__________)
• ↑ infants and children
• Normal neuromuscular excitability
• ↓ progressively with aging.
(__________), myocardial rhythm and
• Constitutes the medium by which solutes are
contractility (__________)
dissolved and by which metabolic reactions take
• Redox reaction / Electron transport (_____)
place
• ATPase pump (_____)
• Transports nutrients to cells
• Blood Coagulation (__________)
• Removes waste products (through urine)
• Production and use of ATP from glucose (Mg,
PO4)
Active transport vs. Passive transport
• monitoring and maintaining electrolyte concentrations.
___________________ ___________________

✓ Requires energy ✓ Diffusion process

✓ Against their concentration gradient ✓ Following their concentration gradient
✓ ATPase ion dependent pump ✓ Hormonal or physiologic
Osmolality vs. Osmolarity
___________________ ___________________

Dissolved solutes per kilogram of body weight Dissolved solutes per liter of solution

Mmole of solute mOsm

Kg of solvent Liter of solution
Regulation of Osmolality
• Thirst sensation
• ADH /AVH (secreted by pituitary gland)
• ANP: increases Na and water excretion in the
kidneys (B-type natriuretic peptide and ANP
act together in regulating blood pressure and
fluid balance)
• GFR increases with volume expansion and
decreases with volume depletion
• RAAS- Renin-angiotensin-aldosterone system
(regulates blood flow)
Determination of Osmolality
• Any substance dissolved in a solvent will
• ↓ ____________ by 1.858°C
• ↑ ____________ by 0.52°C
• ↓ ____________ (dew point) by 0.3 mmHg
• ↑ ____________ by 17,000 mmHg
✓ Main contributors for Osmolality are Na, Cl, Urea and Glucose
• Osmometers: supercooled to -7 degree celsius
• Osmolal gap: difference between the measured and calculated osmolality
Reference Ranges

Serum 275-290 mOsm/kg

Urine (24h) 300-900 mOsm/kg
Random Urine 50-1200 mOsm/kg
Urine/Serum ratio 1.0-3.0
Osmolal gap 5-10 mOsm/kg

Electrolytes
Concentration of Cations and Anions in Extracellular and Intracellular Water

Extracellular Intracellular Extracellular Intracellular

Cation Anion
(mmol/L (mmol/L) (mmol/L (mmol/L)

Na+ 136-145 15 HCO3- 23-29 10

K+ 3.5-5.1 150 Cl- 98-107 1

Ca2+ 2.15-2.5 1 HPO42- 0.78-1.42 50

Mg2+ 0.63-1 13.5 SO42- 0.5 10

(1) Sodium
• Major Extracellular cation in the ECF (90%) • Sodium levels are regulated by:
• Most Abundant cation (in the ECF) 1. Diet
• Functions: 2. Kidney
1. Water pull • 60 – 75% Na is reabsorbed in
2. Blood volume regulation PCT
3. Neuromuscular excitability • Others are reabsorbed in DCT
• K is secreted in exchange of Na under the and DLOH
influence of aldosterone 3. RAAS
• Na+, K+ -ATPase ion pump moves 3 Na+ ions out • Aldosterone promotes retention
of the cell in exchange for 2 K+ ions of Na in exchange of secretion
of K and Hydrogen ions

✓ Hyponatremia
• Depletion of body sodium (less than 135 mmol/l)
• This may be caused by:
1. Increased Na+ loss
2. Increased H2O retention
3. Water imbalance
• Depletion of body sodium may lead to symptoms of hypovolemia with hypotension
 ❖ Causes of Hyponatremia
Increased Sodium loss Increased water retention Water imbalance

Hypoadrenalism Renal failure Excess water intake

Potassium deficiency Nephrotic syndrome SIADH
Diuretic use Hepatic cirrhosis Pseudohyponatremia
Ketonuria CHF
Salt-losing nephropathy
Prolonged vomiting or diarrhea
Severe burns

A. Increased Na Loss
• Diuretics
• Thiazides
• K deficiency C. Water imbalance
• Due to K exchange with Na • Occur as a result of polydipsia
• Ketonuria • Increased thirst
• Na lost with ketones • SIADH
B. Increased water retention • Due to increased ADH production
• Urine Na is >20mmol/day • Pseudohyponatremia
• ARF or CRF • Patients with hyperproteinemia or
• Urine Na is <20mmol/day hyperlipidemia
• Nephrotic syndrome, Hepatic cirrhosis
or CHF

❖ Symptoms and Treatment of Hyponatremia

Sodium level Symptoms

_______________ Gastrointestinal symptoms

_______________ Neuropsychiatric symptoms

_______________ Emergency case

• Treatment
• May be treated acutely with IV administration of saline solution

✓ Hypernatremia B. Decreased water intake

• Increased in serum Na+ due to: • Adults with altered mental status
A. Excess loss of water • Infants
B. Decreased water intake • Patients with renal insufficiency
C. Increased Na+ intake or retention • Older persons
• Hypernatremia is seen in excessive sweating, • Those patients who cannot obtain water / ask
diarrhea and renal loss and d.i. for water (i.e. infants)
A. Excess loss of water C. Increased Na+ intake or retention
• Diabetes insipidus • Hypertonic Salt Solution
A. Nephrogenic D.I. • Dialysis (Sodium bicarbonate or hypertonic
• Kidney cannot respond to ADH dialysis)
B. Central D.I. • Hyperaldosteronism
• ADH secretion is impaired ❖ Symptoms and Treatment of Hypernatremia
• Profuse sweating • Altered mental status, muscle problems and
• Severe burns increased thirst
• Renal tubular disease • Treatment with rehydration with hypotonic IV
• Prolonged Diarrhea fluids
 ❖ Determination of Sodium
• Methods:
1. Emission Flame Photometry (EFP)
2. Atomic Absorption Spectrophotometry (AAS)
3. Ion selective electrodes (ISE) = ______________
4. Colorimetric method = ______________
• Sodium combined with Zinc uranyl acetate to produce sodium uranyl acetate precipitate.
• Addition of water produces Yellow solution
• ___________- the most commonly used method; uses glass ion exchange membrane for Na measurement

(2) Potassium
• The major intracellular cation ❖ Functions of K
• ____________ (1’ regulator) • Neuromuscular excitability regulator
• Reabsorption at PCT • Contraction of the heart
• Secretion at DCT • ICF volume
• GUT (minor regulator) • H+ concentration
• Secreted in gastric juice ✓ ↑ K+, ↑ cell excitability (________________)
• Reabsorbed by the small intestine ✓ ↓ K+, ↓ cell excitability (________________)

❖ Reference Range for Potassium

Serum 3.5 – 5.1 mmol/L

Plasma Males: 3.5 – 4.5 mmol/L

Females: 3.4 – 4.4 mmol/L

24-Hour Urine Collection 25 – 125 mmol/day

❖ Causes of Hypokalemia
GI Loss Renal Loss Cellular shift Decrease Intake

Vomiting Diuretic (most common Alkalosis

Diarrhea
Intestinal tumor
Laxative abuse
Gastric Suction
❖ Symptoms of Hypokalemia
3.0 – 3.4 mmol/L
therapy) Insulin overdose
Kidney disorder (RTA)
Hyperaldosteronism
Cushing Syndrome
Asymptomatic

Below 3.0 mmol/L Muscle paralysis

Increased risk of arrhythmia
❖ Treatment of Hypokalemia
• Oral KCl replacement of K+ or IV replacement of K+
❖ Hyperkalemia
Increased K intake Decreased K+ excretion Cellular shift

High potassium diet Renal failure Acidosis

Oral potassium supplements Hypoaldosteronism Muscle / Cellular injury
IV infusion with potassium Diuretics that block DCT Chemotheraphy
High doses of K penicillin Addison’s disease Leukemia
Transfusion of aged blood
 ❖ Determination of Potassium
• Methods: _____________ Alters electrocardiogram
1. Emission Flame Photometry (EFP)
2. Atomic Absorption Spectrophotometry
(AAS) _____________ Muscle weakness, tingling, numbness
3. Ion selective electrodes (ISE) = use or mental confusion by altering
_____________ neuromuscular conduction, fatigue
4. Colorimetric method = _____________
✓ Uses cobaltinitride to produce sodium potassium _____________ Cardiac arrest
cobaltinitrite
✓ Addition of phenol reagent produces blue color

(3) Chloride
• The major extracellular anion ❖ Chloride shift
• It is present in the diet as salts of Na, K, Ca or Metabolic Decreased
Mg. acidosis bicarbonate is
• Readily absorbed in the intestine and excreted accompanied
via urine and sweat by a high
• Only known anion to serve as an activator chloride
❖ Functions:
o Maintenance of electrolyte balance / Metabolic Increased
electrical neutrality (Chloride shift) alkalosis bicarbonate,
o Hydration low chloride
• _________
o CO2 generated by cellular metabolism Hyperchloremia Dehydration,
within the tissue diffuses out into both Renal tubular
plasma and red cell. acidosis,
o HCO3 diffuses out into the plasma CHF,
o Cl2 diffuses into the red cell to maintain Metabolic
electric balance of the cell acidosis,
• Buffering system of the blood (for acid-base Cushing’s
balance) syndrome,
• HCO3- ion is pulled out of the erythrocyte Anemia
• Cl- ion moves into the erythrocyte Hypochloremia Respiratory
• Resulting in decrease serum Cl- acidosis,
❖ Chloride concentration Metabolic
Plasma, 98 – 107 alkalosis,
serum mmol/L diarrhea,
Profuse
24 hr. 110 – 250 sweating,
Urine mmol/day, Increased
varies with gastric juice
diet secretion,
Salt-losing
nephritis /
pyelonephritis,
Addison’s
disease,
Severe
vomiting,
severe burns
 ❖ Specimen Consideration • Cl ions combine with mercury
Specimens WB, Serum, plasma, ion of Mercuric nitrate to form
24 hour urine and mercuric chloride (Soluble) with
Sweat drop of Mercuric nitrate, the
indicator will change from
Anticoagulant _________________ colorless or faint pink to violet
of Choice ✓ D. _________________
• Colorimetric procedure
Hemolysis False _________ • Reagent contains Mercuric
❖ Determination of Chloride thiocyanate and ferric nitrate
✓ A. _________________ • Chloride displace thiocyanate
• The most common method ions and reacts with ferric ions
• Uses silver sulfide and silver chloride membrane to produce reddish brown ferric
✓ B. _________________ thiocyanate
o Accurate ❖ Sweat Chloride
o Gives direct read-out chloride concentration • 50 mg sweat sample is needed
o Adaptations: Cotlove (digital) titrator or • Sweat is collected using iontophoresis and the
chloridometer pilocarpine is introduced in the skin and uses
• Titration with Mercuric Nitrate electric current.
✓ C. _________________ • >60 mmol/L Cl+ = _________________
• Mercuric nitrate titration

(4) Bicarbonate
• 2nd Most Abundant anion in the ECF
• Accounts for more than 80% of total CO2
• Major component of the buffering system of the blood
Metabolic Acidosis Metabolic Alkalosis

HCO3- deficit HCO3- Excess

Decrease pH, CO2 and pCO2 Increased pH, CO2 and pCO2

Hyperventilation = Dec. pCO2 Hypoventilation = Inc. retention of CO2

Diabetic ketoacidosis = Inc. acid production Vomiting = HCl loss, there is GI loss of HCO3
❖ Specimen Consideration and CO2 Determination
• Serum, Plasma (_________)
• False ↓ if left uncapped
• ↓ ____________ per hour
• Methods
• Enzyme method
(5) Magnesium
• 4th most abundant cation; 2nd major intracellular cation
• 53% (Bone), 46% (muscle, soft tissues), <1% (Blood
• In serum: 60 – 70% is ionized; 30% is bound to protein; 5% complexed with phosphate and citrate
• Neuromuscular conduction, Enzyme cofactor and ATPase ion pump
• Majority of this mineral is stored in the bones in complex with Ca and PO4
• Cofactor of Phosphatases, Phosphorylases, Kinases and Enolases
❖ Regulation of Mg
_________ Increases renal reabsorption of Mg
Enhances intestinal reabsorption of Mg (in the intestine)

_________ and _________ Increases renal excretion of Mg

 • To regulate metabolism by the rate of the ✓ Hypermagnasemia
oxidation in cells • Increase intake (Medication, Magnesium sulfate
✓ Hypomagnesemia therapy)
 Reduced Intake • Hypoparathyroidism
• Poor diet/starvation • Hypoalderosteronism
• Prolonged magnesium-deficient IV • Renal Failure (Decrease excretion)
 Others • Renal Ca / Bone metastases
• Excess lactation • due to ↑ in bone loss
• Pregnancy ❖ Specimen Consideration
 Decreased Absorption • Serum , Plasma (lithium heparin), 24 hour urine
• Malabsorption syndrome • Hemolysis cause False ↑
• Diarrhea • Because Mg is 20x greater inside
• Vomiting RBC
• Laxative • Urine must be acidified with HCl (to avoid
 Renal disorders precipitation)
• Tubular disorders ❖ Determination of Magnesium
• Pyelonephritis • A. ___________
• Glomerulonephritis o Mg2+ + Calmagite → Reddish-violet (532 nm)
 Drug induced • B. ___________
• Diuretics and Cardiac glycosides o Mg2+ + Dye → Colored complex (660 nm)
(DIGITALIS) • C. ___________
 Endocrine disorders o Mg2+ + Chromogen → Colored complex
• Hyperparathyroidism • D. ___________ / Dye Lake Method/Clayton
• Hyperaldosteronism Yellow/Thiazole Yellow
• Hyperthyroidism o Mg + Titan Yellow → Red lake colloidal
• Hypercalcemia precipitate
• Diabetic ketoacidosis • E. ___________ Method
o Mg + 8-hydroxy-5-quinoline sulfonic acid →
Fluorescence (380-410nm)
• F. AAS
• G. ISE

(6) Calcium
• The most abundant cation in the body
• 99% in bone and teeth and 1% in blood and ECF
• For muscle contraction Total Calcium
• For blood coagulation (2.15 – 2.50 mmol/L)

❖ Regulation
• A. Bone Resorption
o Cause increase Ca in blood
Ionized Ca+2 Protein bound Ca+2 Complex Ca+2
o PTH mobilizes Ca from the bone Unbound or free, Bound to protein (E.g. Bound to anions (E.g.
• B. Bone deposition physiologically active albumin) HCO3-, PO4- and lactate)
45% of Total Calcium 40% 15%
o Cause decrease Ca in blood
o Calcitonin inhbits PTH and Vit.
D3
• C. Intestinal absorption
o Vit. D3 increase Ca in the intestine
 ❖ Conditions that affect Serum Calcium
Hypercalcemia Hypocalcemia

Hyperparathyroidism Hypoparathyroidism

Increased Vit. D Hyper/Hypomagnesemia

Diuretics Hypoalbuminemia

Malignancy Vit. D deficiency

Prolonged skeletal immobilization Acute pancreatitis

Muscle weakness / disorientation Tetany

Rhabdomyolysis
❖ Specimen Consideration
• Serum, Plasma (Dry lithium heparin), 24 hour urine
• Hemolysis cause False _____
❖ Determination of Calcium
✓ AAS, ISE, Flame photometry
✓ _______________ (Redox Titration method)
• Precipitation of Ca+2 as Calcium Oxalate (CaC2O4)
• CaC2O4 + H2SO4 → oxalic acid (H2C2O4)
• H2C2O titrated with KMNO4 → pink color
✓ _______________ (Precipitation with Chloranilic acid)
• Ca+2 + Sodium chloranilate → Ca Chloranilate
• Ca Chloranilate + EDTA → Chloranilic acid
✓ Ortho-cresolphthalein complexone (CPC)
• Formation of colored complexes between calcium and the dye with absorbance of 578nm.
• Uses _______________ to inhibit Mg
✓ Other Methods:
• Alizarin Dye, Arsenzo III dye, Methyl phenol blue

(7) Phosphate
• Major intracellular anion
• ____ decrease renal excretion of phosphate
• Exist as inorganic phosphate or organic phosphate esters
• 80% - bone, 20% - soft tissues, 1% - serum/plasma
❖ Functions of Phosphate
• Component of phospholipids, DNA, creatine phosphate and ATP
• Phosphate homeostasis is closely linked with calcium regulation
• Insulin-mediated entry of glucose into cell (by process involving phosphorylation of glucose and co-entry of K)
❖ Regulation
✓ PTH
o Lowers Phosphate concentration by increasing renal excretion
✓ Vit. D
o Increases Phosphate concentration by reabsorption in the kidney
✓ GH
o Increases Phosphate concentration by regulation of skeletal growth
 ❖ Clinical Application
Hypophosphatemia Hyperphosphatemia

Hyperthyroidism Renal failure

Vitamin D deficiency Increased phosphate intake

Increased cellular phosphate release

Increased breakdown of cells

❖ Specimen Consideration
• Patient must be in fasting state
• Serum, Plasma (Dry lithium heparin), 24 hour urine
• Hemolysis cause False ____
• Commercial heparin preparations may also contain phosphatases
❖ Methods
• Formation of ammonium phosphomolybdate complex (_______________)
• Read-out at 340nm
• _______________ method
• Conversion of inorganic phosphate to heteromolybdenum blue by reacting with ammonuim molybdate
• Read-out at 700 nm

(8) Lactate
• A by-product of anaerobic metabolism
• Liver converts lactate back to glucose “Gluconeogenesis”
• Indicator of severity of oxygen deprivation (hypoxia)
• Conversion of glucose from other non-carbohydrate products
❖ Specimen Consideration
• Tourniquet should not be used (Ideally)
• Patient should not exercise the hand before or during collection
• Transport with ice or separate plasma immediately (heparinized)
• Iodoacetate or fluoride inhibits glycolysis
❖ Determination of Lactate
• Enzymatic Method
• Uses lactate oxidase to produce pyruvate and H2O2
• Peroxidase may be used to produce a colored chromogen from H2O2
Anion Gap
+ - -
(Na ) – (Cl + HCO3 )
• The difference between the sum of Na+ and K+ and the sum of Cl- and HCO3-
• Useful in indicating an increase in one or more of the unmeasured anions
• Used as a QC (to determine an instrument problem)
• Inc Anion Gap = ____________________
• Normal Value: 10 – 20 mmol/L
❖ ↑ Anion gap
• Uremia or Renal Failure ❖ ↓ Anion gap
• Ketoacidosis (Starvation / diabetes) • Hypoalbuminemia
• Lactic acidosis (Hypoxia) • Hypocalcemia
• Hypernatremia • Erroneous Reporting
• Instrument Error
 ❖ Cystic Fibrosis
• Inherited disorder of exocrine glands causing:
–abnormally thick secretions of mucus
–Elevated sweat electrolytes
• Diagnostic test: Sweat test Coulometry
❖ Sweat inducer: ____________
❖ (+) increased Na and Cl in sweat
• Ref. Mtd.: Gibson and Cooke Pilocarpine Iontophoresis
• (+) >65 mmol/L of sweat electrolytes
• RV: 5-40 mmol

TRACE ELEMENTS
• Elements present in the body in very low amounts: 1 microgram per gram of tissue
• Most are metals, except iodine, bromine and fluorine
Trace elements Ultratrace elements

Body fluids
Tissue
❖ Specimen Type or Source
Element Specimen Determination
As LC-ICP-MS, HG-GFAAS and AAS, AES

Cu SEC – ICP- MS
(Size exclusion chromatography)

Pb AAS, ICP-MS, AES, GC-GFAAS

Fe IRMA, ELISA, Chemiluminescene

Zn AEC-ICP-MS

Hg ICP-MS
❖ Instrumentation
Most sensitive and the widely used method __________
(Flame or Flameless (i.e. graphite furnace)

Now the method of choice __________

(Inductively coupled plasma)
❖ Collection
• Plain glass containers
• Lithium heparin
• Anticoagulant suitable for most analyses
• Run dilute acid blanks
• To ensure free from contaminants
• Polyethylene bottles with glacial acetic acid
• 24 hour urine

(1) Aluminum (Al)

• Crystalline-white ductile metal
• Most abundant metal
• Found in: antacids, astringents, buffered aspirins, food additives, cosmetics and antiperspirants
 • Toxicity: encephalopathy (Alzheimer’s disease), osteomalacia (hypercalcemia), renal insufficiency (dialysis)
• Specimen: ____________________
• Lab methods: ____________________

(2) ARSENIC
• Arsenic has both metallic and nonmetallic properties
• The predominant natural sources are volcanoes and weathering of minerals
• insecticides, pesticides and herbicides
• Shellfish
• Used in homicidal intent
✓ Toxicity
• Arsenic inhibits sulfhydryl enzyme
• Ingestion causes GIT irritation with vomiting and diarrhea
• Seizure, coma and death (Neurologic impairment)
• Socks and Glove neuropathy
• Arsenic has high affinity of binding for keratin, leading to high concentrations in hair and nails
• Mees lines
• White striate across the fingernails
✓ Determination of Arsenic
• Arsenic toxicity is best detected by urine due to the short half-life of arsenic in blood
• Specimens:
• ___________________________
❖ Method:
__________________________ Urine, Serum Acute or recent poisoning

Hair or nail clippings Chronic exposure

(3) Cadmium (Cd)

• Soft, bluish-white metal that is easily cut with a knife
• Pigments and batteries, smoke and tobacco

(4) Chromium (Cr)

• Makes rubies red and emeralds green
• 21st most abundant element
• ________________: better absorbed and more toxic
• ____________________: maintaining normal metabolism of glucose, fat and cholesterol

(5) COPPER
❖ Dietary Sources
❖ Most foods contain appreciable quantities of • Legumes
copper • Organ meats
• Grains • Cocoa
• Shellfish • Water

• component of several metalloenzymes including ceruloplasmin and cytochrome C oxidase and clotting factor V
and keratin
❖ Function
• Seen in RBC
• Bound to transport proteins (Albumin and ceruloplasmin)
• Ceruloplasmin
• Necessary for the absorption of iron by oxidizing ferrous iron to ferric state
• It has peroxidase activity
• Erythropoiesis and catalytic activity of several enzymes (cytochrome oxidase and uricase)
 • copper transport protein
• a cofactor for oxidase
• Enzymes
❖ Hypocupremia
• Anemia in infants
• ________________ syndrome
• A genetic defect of Copper absorption
• Leads to progressive brain disease in infants
• Kwashiorkor, nephrosis, sprue, celiac disease
• It is related to malnutrition and malabsorption
❖ ________________ (Copper accumulation disease)
• Hepatolenticular degeneration
• A genetic abnormality in which excessive copper accumulates in liver, eye, brain and
other organs
• Deficiency in Ceruloplasmin (copper-transport protein)
• ___________________
• A pigmented ring around the iris
• Green- brown discoloration in the cornea
• __________________ is pathognomonic for the Wilson’s disease condition

(6) IRON
• It is in hemoglobin, mostly in RBCs and RBC precursor
• 25% (stored in ferritin and hemosiderin)
❖ Transport and Storage
• It is bound to ______________
• A plasma beta-globulin
• _____________
• A spherical molecule consisting of apoferritin
• ______________
• An amorphous iron stained by Prussian Blue
• _______________- a specific iron transport protein
❖ Forms of Iron
_________________ Oxyhemoglobin and reduced hemoglobin

_________________ Ferritin, hemosiderin, transferrin and methemoglobin

❖ Iron Transport
_________________ Binds hemoglobin and serves to facilitate disposal of the iron from this molecule

_________________ Binds heme to aid its removal from the circulation

❖ Measurement of Serum Iron ❖ Reference Range

• _____________ method Men 65 – 165 ug/DL
• Ferrozine (Chromogen) (11.6 – 29.5 umol/L)
• Thioglycolic acid (Reduces ferric to
ferrous ion) Women 45 – 160 ug/dL
• Product:Highly colored ferrous complex (8.1 – 28.6 umol/L)
• Wavelength: 56 nm

• Serum Iron: measures iron bound to transferrin

• Values are higher in infant and then falls below adult levels
 • Higher values obtained in the morning due to diurnal
variations
❖ Peripheral Blood Smear
• _________________
• The most simple cost effective method
• Measures the capacity of Transferrin to bind to iron
❖ Measurement of Total Iron Binding Capacity (TIBC)
• Measures the capacity of Transferrin to bind to iron
• Transferrin Saturation = Serum Fe x 100 (%
Saturation) TIBC
• Ratio of serum to TIBC
• Normal range = 20 – 45 % Saturation
Condition Serum Iron Transferrin/TIBC Ferritin Percent Saturation

Iron Deficiency

Iron Overload

Hemochromatosis

Malignancy

Chronic Infection
(7) LEAD
❖ Sources
• Paints, gasoline, storage batteries (released on
burning), eating utensils, plates and ceramics,
lead pipes (drinking water), ammunition
• Lead exposure is usually occupational
• Pica
• A tendency to consume inedible
material
• Toys produced in china may contain high lead
contents
• Depresses enzyme activity of heme synthesis
• Defective Delta aminolevulinic dehydratase
activity causes delta aminolevulinic acid to
accumulate
• Depression of heme synthetase causes RBC to
accumulate excessive protoporphyrin
• at the beginning, middle and end of heme
synthesis
• Soft tissues: liver, kidney and brain
❖ Lead is contained largely in erythrocytes
• Lead Toxicities:
• Kidney damage
• Brain (Encephalopathy)
• Interference with the biosynthesis of
heme (heme is required in hgb
synthesis)
• Causes __________________________
❖ Lead determination
• Specimen
• ________________
• Method
• Flameless AAS (graphite furnace)
• ________________
• Urine is used for detecting recent exposure to
lead
❖ Laboratory Findings in Lead Poisoning
• PBS _____________________
• Mild anemia with reticulocytosis
• Moderate anisocytosis and poikilocytosis
• BM with Ringed sideroblast
• increase free erythrocyte protoporphyrin- used
to screen occupational exposures;
the best lab test to quantify lead toxicity
• Increased erythrocyte protoporphyrin, excretion
of gamma aminolevulinic acid and excretion of
coproporphyrin
• Normal porphobilinogen
(8) MERCURY Organic Inorganic Mercury
✓ Quicksilver Mercury
✓ Heavy Silvery Metal
• Fungicide, dental amalgams, mascara, ointment, Seafood industry
antiseptics (diet)
• Mode of Entry
• Inhalation: pulmonary damage Affects Affects more peripheral (GIT and kidney)
the CNS
• Ingestion: GIT damage
• Liquid elemental mercury is essentially nontoxic but Found Urine is used to demonstrate inorganic
elemental mercury vapor is toxic in WB / mercury exposure
• Most of the dietary intake comes from RBC
consumption of meat and fish products
(9) Manganese (Mn) (12) ZINC
• 12th most abundant • The second most abundant of the trace metals
• Manganese depletion: blood clotting defects, in humans
hypercholesterolemia, dermatitis, • Zinc is used in treating Wilson’s disease
hypercalcemia, Increased ALP • Zinc is relatively nontoxic
• Toxicity: locura manganica • Poor zinc nutrition is associated with old age
(___________________) • Poor zinc nutrition is associated pregnancy and
lactation
(10) Molybdenum (Mo) • Co-factor or component of more than 300
• Can cross the placenta metalloenzymes
• Toxicity in NB: seizures, anterior lens dislocation, • Involved in insulin and porphyrin metabolism
decreased brain weight, and usually death prior • Significant for growth and sexual maturation
to 1 year of age • Involved in wound healing and sensory
perception
(11) Selenium (Se) • Zinc Measurement
• Antioxidant and anticarcinogenic • _________
• Deficiency: ____________ (endemic • Zinc chloride causes tissue necrosis
cardiomyopathy in China), • Zinc oxide fumes causes chemical pneumonitis
_____________(endemic osteoarthritis in China) ❖ Dietary sources
• The most available dietary sources of Zinc are
red meat and fish
• Other sources are seafoods, meat, milk and eggs

Acid – Base Balance and Blood Gases

Introduction
• Acid: a substance that can yield a H+ when dissolved in water
• Base: a substance that can yield OH−
❖ pKa
• negative log of the ionization constant
• pH in which the protonated and unprotonated forms are present in equal concentrations

❖ Buffer
• combination of a weak acid or weak base and its salt
• a system that resists changes in pH
• pH= ________
(1) ____________________
• The major buffer localized inside the RBC
• Plasma CHONS exert buffering effect through the charges on their surfaces
• Hgb binds with CO2, binds and transports O2 and participates in the chloride shift
(2) ____________________
 • Present in RBC
• 2nd most abundant protein
• Accelerates uptake of H+ by hgb
• An enzyme that catalyzes the reaction between CO2 and water to form carbonic acid
• Others: histidine
(3) ____________________
• Involve in exchange of sodium ion in the urine H+ filtrate

❖ Henderson-Hasselbalch Equation
• pH = pK + log [A-] [HCO3]
[HA] [H2CO3]
• The concentration of H2CO3 is proportional to the partial pressure exerted by the dissolved CO2
• 0.0307
• The factor to convert mmHg -> mmol/L

❖ Acid-Base Balance
• (1) H2CO3 dissociates into CO2 and H2O, allowing CO2 to be eliminated by the lungs and H+ as water
• (2) changes in CO2 modify the ventilation (respiratory) rate
• (3) HCO3- concentration can be altered by the kidneys
✓ Regulation of CO2 by the Lungs
• 1. Chloride shift occurs to maintain electroneutrality (equal number of negative and positive ions on the red cell
membrane)
• 2. The plasma BUFFERS combine with H+ to maintain a stable pH
• Hyperventilation
• ↑ CO2 release
• Hypoventilation
• ↓ CO2 release

✓ Regulation of HCO3 by the kidneys

• The kidney controls:
1. The bicarbonate concentration / conservation
2. The excretion of the acid (by excreting the H+ in exchange to Na)

❖ Definition of Terms
• Compensation: the body tries to restore acid–base homeostasis whenever an imbalance occurs
• Fully compensated: implies that the pH has returned to the normal range (the 20:1 ratio has been restored)
• Partially compensated: implies that the pH is approaching normal

Acidemia
(1) Metabolic Acidosis
❖ Causes:
A. Direct administration of acid-producing substance (e.g. ammonium chloride and calcium chloride)
B. Excessive formation of organic acids (e.g. diabetic ketoacidosis and starvation)
C. Reduced excretion of acids (e.g. RTA)
D. Excessive loss of HCO3 (e.g. diarrhea or drainage from biliary or pancreatic fistula)
❖ Compensation: _________________________
(2) Respiratory Acidosis
❖ Causes:
A. Decreased alveolar ventilation (hypoventilation)
B. Bronchopneumonia
C. Drugs (e.g. barbiturates, morphine and alcohol)
D. Decreased cardiac output (e.g. CHF)
❖ Compensation: _________________________
Alkalemia
(1) Metabolic Alkalosis
❖ Causes:
A. Excess administration of bicarbonate-producing salts (e.g. sodium lactate, citrate and acetate)
B. Excessive loss of acid (e.g. vomiting, nasogastric suctioning or prolonged use of diuretics)
❖ Compensation: _________________________
(2) Respiratory Alkalosis
❖ Causes:
A. Hypoxemia
B. Early salicylates
C. Increase in environmental temperature
D. Fever
E. Hysteria (hyperventilation)
F. Pulmonary emboli/fibrosis
❖ Compensation: _________________________

❖ Reference Range At 37°C

• pH: _____________
• pCO2 (mm Hg): _____________
• HCO3(mmol/L): _____________
• Total CO2 content (mmol/L): 23–27
• pO2 (mmol/L): 80–110
• SO2 (%): >95
• O2Hb (%): >95

❖ Definition of Terms
• Partial pressure: each gas in the atmosphere is equal to the BP at a particular altitude times the appropriate
percentage for each gas.
• O2Hb describes O2 reversibly bound to hemoglobin
• Oxygen saturation (So2) represents the ratio of O2 that is bound to the carrier protein, hemoglobin, compared
with the total amount of hemoglobin capable of binding O2
• Partial pressure of oxygen dissolved in plasma (po2) accounts for little of the body’s O2 stores

Case Study 17-1

• A 50-year-old man came to the emergency department after returning from foreign travel. His symptoms
included persistent diarrhea (over the past 3 days) and rapid respiration (tachypnea). Blood gases were drawn
with the following results:
• pH 7.21
• pco2 40 mm Hg
• po2 96 mm Hg
• HCO3 − 7 mmol/L
• So2 96% (calculated) (reference range, >95%)
Question
1. What is the patient’s acid–base status?
2. Why is the HCO3 − level so low?
3. Why does the patient have rapid respiration?
❖ Blood Gas Analyzers
• pO2: ____________________
microammeter: between anode and cathode and measures current
• pH: glass membrane placed around an internal Ag–AgCl electrode to form a measuring electrode
• pCO2: modified pH electrode: ____________________