CLINICAL

MTAP
CHEMISTRY
BY: GEORGE VINCENT GELLENA, RMT Table 1. – Types of Error
Remedios Trinidad Romualdez Medical Foundation

CONTENTS:
1 Quality Management
Automation Random Error Systematic Error
2 Carbohydrates Description
4 Lipids
7 Proteins Error which varies Error that influences
Enzymes from sample to observations
10 Non-Protein Nitrogens sample consistently in one
12 Liver Function Tests direction
16 Electrolytes
Acid-Base Balance
18 Endocrinology Examples
20 Therapeutic Drug Monitoring
23 Toxicology -Mislabeling of sample
-Pipeting error -Deterioration of rgts.
24 -Temperature & ctrl materials
30 Fluctuations -Contaminated sol'ns
32 -Improper mixing of -Failing Instruments
samples and rgts.
QUALITY MANAGEMENT
Statistics
 Quality Control - Is a system of ensuring accuracy
& precision in the laboratory by including quality control
 Mean – measure of central tendency;
reagents in every series of measurements measure of accuracy; AVERAGE
 Quality Assurance - is a systematic action necessary
 Standard Deviation – measure of dispersion of
to provide adequate confidence that laboratory services
values from the mean; measure of precision; most
will satisfy the given medical needs for patient care
frequently used measure of variation
 Standard – material of kno  Coefficient of Variation - index of precision; percentile
concentrati used in developing a standard curve expression of the mean
and/or instrument calibration  Variance - measure of variability
 Control – sample of known quantity  F-test - determines whether there is a
with
several analytes present
statistically significant difference between the standard
Parameters
 Sensitivity - Is the Ability of an analytical deviations of two groups of data

method to measure the  T-test - determines whether there is a

of the
smallest
analyte of interest statistically significant difference between the means of
 Analytical two groups of data
Specificity - Is the Ability of an analytical
method to measure ONLY the analyte of Interest  Median - MIDPOINT of the distribution;
 Accuracy - Is the Nearness or Closeness of value of the observation that divides the observation into
the Assayed value to the true or target value two equal groups of data
 Precision Mode – most FREQUENT observation
- The ability of an analytical 

method to give repeated results on the same sample  Range - is the difference between the
that
agree with one another highest and lowest score in data
 Practicability - The degree by w/c a method is Quality Control Charts
 Gaussian Curve - data element are centered
easily
 Reliability around the mean with most elements close to the mean
- The ability of an analytical
method to  CUSUM - provides the earliest
maintain accuracy & precision over an
period of time w/c equipment, indication of systematic error (trend); requires computer
extend duri
reagents, & personnel may change implementation
 Diagnostic Youden/ Twin Plot – compare results obtained on
Sensitivity - The Ability of an analytical 

method to detect the proportion of individuals with the a high and low control serum from different laboratories
disease. (Screening tests require high sensitivity)  Shewhart Levey- – most widely used QC
Jennings
 Diagnostic - The Ability of an analytical chart in the clinical laboratory; allows laboratorians to
Specificity
method to detect the proportion of individuals without apply multiple rules without the aid of computer;
the disease. (Confirmatory tests require high specificity) identifies both random and systematic error
 Clerical Error – highest frequency occurs with Table 2. Errors observed in LJ Chart
the use of handwritten labels and request forms TREND SHIFT
Gradual change in Abrupt change in the
the mean mean
COLLEGE OF MEDICAL LABORATORY SCIENCE | 1 | 34
Ctrl value increase or Ctrl values distribute
 Clinical Chemistry

10x 7. Meter/ Read out device – Displays output of the detection

12s (accept) system
SINGLE BEAM SPECTROPHOTOMETER
41s  Simplest Type; Designed to make one measurement at a
time at one specified wavelength
R4s
12s (accept) 22s
13s (reject)

Westgard Errors on LJ Chart

Table 3. Westgard Control Rules

Random Errors Systematic Errors

12s – 1 control value 22s – 2 consecutive
Components of a single-beam spectrophotometer.
exceeds ±2SD; rejection or control values exceed
A, Exciter lamp; B, entrance slit; C, monochromator; D,
warning rule either ±2SD
exit slit; E, cuvet; F, photodetector; G, light-emitting
13s – 1 control value 41s – 4 consecutive
diode (LED) display
exceeds ±3SD control values exceed ±1SD
R4s – Range/ 10x – 10 consecutive DOUBLE BEAM SPECTROPHOTOMETER
difference between the control values fall on 1 side  Splits monochromatic light into two components: one
highest and lowest control or the other side of the beam passes through the sample and the other through
result within an analytical mean a reference solution or blank
run is 4SD 1. Double Beam in Space – 2 photodetectors

AUTOMATION
Automation
 Wavelength – distance between two successive peaks
 400-700 nm – visible spectrum
 <400 nm – ultraviolet region (UV)
 >700 nm – infrared region
 Didymium or holmium oxide filter is used to check
wavelength accuracy
 Neutral density filters and dichromate solution verify Double Beam in Space_. A, Exciter lamp;
absorbance accuracy B, mirror; C, entrance slits; D, monochromators; E,
 Beer-Lambert’s law
exit slits; F, cuvets; G, photodetectors; H, light-
 A = abc = 2 – log%T
emitting
o A: molar absorptivity
diode (LED).
o B: length of light through the solution
2. Double Beam in Time – 1 photodetector
o C: concentration of absorbing molecules
and 1 chopper or rotating sector mirror
o T: transmittance
 One-point calcuation or calibration
𝐶𝑜𝑛. 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝐶𝑠) 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 (𝐶𝑢)
=
𝐴𝑏𝑠. 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝐴𝑠) 𝐴𝑏𝑠. 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 (𝐴𝑢)

SPECTROPHOTOMETRY

 Measurement of light transmitted by a solution to

determine the concentration
PARTS OF A SPECTROPHOTOMETER
1.
Light Source – Provide Polychromatic light Double Beam in Time
2.
Entrance Slit - Minimizes unwanted or stray
light; prevents entrance of scattered light
3.
Monochromator – Isolates specific or individual Flame Emission Photometry
wavelength of light
 Excitation of electrons from lower to higher energy state
4.
Exit Slit – Controls the width of light beam  Measures light emitted by single atom burned in flame;
(bandpass) measures excited ions (Na+ and K+)
5.
Cuvet – Holds the solution whose Atomic Absorption Spectrophotometry
concentration is to be measured
6.
Photodetector – Detects and converts  Element is not excited but merely its
transmitted dissociated
light into photoelectric energy chemical bond and placed in an unionized, unexcited
ground state

COLLEGE OF MEDICAL LABORATORY 2 | 34

 Clinical Chemistry
 Measures light absorbed by atoms dissociated by heat; GAS CHROMATOGRAPHY
measures unexcited trace metals (Ca2+ and Mg2+)
 for naturally volatile compounds or easily converted to
volatile form
Titrimetric/ Volumetric  Mass Spectroscopy – based on fragmentation
and ionization of molecules using a suitable energy
 Unknown sample is made to react with known solution
source
in the presence of an indicator  GC-MS – gold standard for drug testing
 Schales and Schales (Chloride)  Tandem MS/ (MS/MS) – detects 20 inborn
 EDTA titration (Calcium) errors of metabolism from a single blood spot

TURBIDIMETRY LIQUID CHROMATOGRAPHY

 based on distribution of solutes between a liquid mobile
 Determines the amount of light blocked by a
phase and a stationary phase
particulate matter in a turbid solution
 High performance liquid chromatography (HPLC) –
 Used in measuring proteins and bacterial suspensions
used in rapid HbA1c testing
 Liquid chromatography-Mass Spectroscopy (LC-MS)
NEPHELOMETRY – used in detecting non-volatile substances;
complementary to GC-MS
 Determines amount of by a particulate
scattered
matter in a turbid solution FLUOROMETRY/MOLECULAR
 Used in measuring antigen-antibody complexes LUMINESCENCE

 Determines the amount of light emitted by a molecule

after excitation by electromagnetic radiation
 Uses 2 measures amount of light
intensity present over a zero background; affected by
quenching

Optical Arrangements of Nephelometry and

Turbidimetry

ELECTROPHORESIS

 Migration of charged particles in an electric

 Separates proteins on the basis of electrical charge;
Buffer: Veronal/Barbital (pH 8.6)
Components of a Fluorometer

DENSITOMETRY


Chemiluminiscence
Measures absorbance of
 Scans and quantitates electrophoretic pattern;  Chemical reaction yields electronically excited
measures concentration of dye and protein fraction compound that emits light as it returns to its ground
state
ISOELECTRIC FOCUSING  Emission of light is created from a chemical or
electrochemical reaction; usually used in immunoassays
 Migration through a pH gradient
 *pH gradient – created by adding acid to anodic area
OSMOMETRY
and base to the cathode area
 Ideal for separating proteins of identical sizes but with  Based on measuring changes in colligative properties of
different net charges; detects CSF oligoclonal banding solutions
 Freezing-point depression osmometry – most
CHROMATOGRAPHY commonly used method
 Separation of soluble components based on physical
and chemical characteristics

COLLEGE OF MEDICAL LABORATORY 3 | 34

 Clinical Chemistry
ELECTROCHEMISTRY TECHNIQUES
CARBOHYDRATES
POTENTIOMETRY GLUCOSE
 Measurement of electrical potential due to free ion  Glucose is the principal and almost exclusive
activity carbohydrate circulating in the blood
 Use: pH/ pCO2  Glucose is the central, pivotal point of carbohydrate
metabolism
COULOMETRY
 Brain is the most important glucose consumer.
 Measurement of electricity (Coulombs) at fixed
potential CNS consumes about 50% of glucose used by the body
 Use: Chloride  Glucose can be derived from (1) diet, (2) from body
stores like glycogen, and (3) from endogenous synthesis
AMPEROMETRY
from proteins or glycerol of triglycerides.
 Measurement of current flow produced by oxidation
reaction
 REGULATION OF BLOOD GLUCOSE
Use: pO2, glucose, chloride, & p_eroxidase. CONCENTRATION
POLAROGRAPHY
PROCESSES INVOLVED IN CARBOHYDRATE METABOLISM
 Measurement of differences in current at constant 1.
Glycolysis
voltage  Metabolism of glucose molecule to pyruvate or
 Use: Specific for pO2 and glucose lactate to energy
VOLTAMETRY  Decreases blood glucose since glucose is consumed
 Measurement of current after which a potential is to produce lactate/pyruvate
2.
applied to an electromechanical cell Gluconeogenesis
 Formation of glucose-6-phosphate from non-
 Lead and iron testing (anodic stripping voltammetry)
carbohydrate sources
THREE BASIC APPROACHES  Increases blood glucose; new glucoses are formed
from other sources
CONTINUOS FLOW ANALYZER 3.
Glycogenolysis
 Samples flow through a common reaction vessel; uses
 Breakdown of glycogen to glucose for use as energy
a system of continuous tubing; Parallel Testing  Increases glucose due to glycogen degradation
 Mixing of Sample and Reagent: Glass coil inserted into 4.
Glycogenesis
the flow path  Conversion of glucose to glycogen for storage
CENTRIFUGAL ANALYZER  Decreases gluceose since excess glucoses in the
 Uses acceleration and deceleration of rotor to transfer body is stored in the liver and skeletal muscle as
reagents and sample from one chamber to another; glycogen
Batch Analysis 5.
Lipogenesis
 Mixing of Sample and Reagent:  Conversion of carbohydrates to fatty acids
Centrifugal Force (Rotor)
 Decreases glucose since carbohydrates are
DISCRETE ANALYZER converted into fatty acids and stored as fats
 Uses syringe pipettes (positive-liquid displacement) to 6.
Lipolysis
aspirate and dispense samples; most versatile and  Breakdown of fats; fats are used as energy
most popular; Random Access Capability HORMONES INVOLVED
 Mixing of Sample and Reagent: Magnetic driven Teflon 1. Hyperglycemic hormones
stirring bar, etc  Glucagon, Epinephrine, Cortisol, Growth hormone,
 Batch Testing – all samples are Thyroxine
loaded at the same time and a single test is 2. Hypoglycemic hormone
conducted on each sample  Insulin
 Parallel Testing – more than one test
is analyzed concurrently on a given clinical specimen 3. Regulator hormone
 Random Access Testing – any test can be  Somatostatin – inhibits release of growth hormone,
performed on any sample in any sequence insulin, and glucagon
 Sequential Testing – multiple tests are
analyzed one after another on a given specimen
SPECIMEN FOR GLUCOSE DETERMINATION
 Open Rgt System – a system other
 Standard clinical specimen is Fasting Venous Plasma/Serum
than the manufacturer’s reagents can be utilized
for measurement  Fasting Blood Sugar should be obtained after
 Closed Rgt System – a system where the 8-10 hrs of overnight fasting
operator can only use the manufacturer’s
reagent  Venous blood has lower glucose levels compared to
arterial blood

COLLEGE OF MEDICAL LABORATORY 4 | 34

 Clinical Chemistry
 Capillary blood has higher glucose levels compared to Table 4. Comparison Between Type 1 and Type 2
venous blood DM
 Whole blood gives approximately 10 – 15 % LOWER
TYPE 1 DM TYPE 2 DM
glucose levels than serum or plasma -due to blood cells
Pathogenesis
 To convert whole blood glucose into serum or plasma B cell Destruction Insulin Resistance
level, multiply value by 1.15
Incidence rate 5-10% 90-95%
 A serum specimen is appropriate for glucose analysis if
Onset Any; most Any; most
serum is separated from the cells within 30 – 60
common to common with
minutes
childhood/teens advancing age,
 Glucose is metabolized at room temperature at a rate of
race/ethnicity,
7mg/dL/hr
hypertension,
 At 4°C, glucose decreases by approximately
2mg/dL/hr dyslipidemia,
 10% contamination with 5% dextrose will increase polycystic
glucose by 500mg/dL or more ovarian
 CSF glucose concentration is approximately syndrome
60-70% that of plasma concentration Risk Factors Genetic, auto- Genetic, obesity,
 Blood glucose should be obtained 1 – 2 hours before immune sedentary
the spinal tap lifestyle,
 CSF for glucose analysis should be performed polycystic
immediately. If delay in measurement is unavoidable, ovarian
the sample must be centrifuged and stored at 4°C or at - syndrome,
20°C dyslipidemia and
hypertension
CLINICAL SIGNIFICANCE OF GLUCOSE RESULTS C-peptide levels
Undetectable Detectable
HYPOGLYCEMIA
 Glycemic factors such as glucagon are released when Pre-diabetes Auto ab (+) Auto ab (-)
glucose levels reach 65-70 mg/dL
 Observable signs and symptoms of hypoglycemia appear Symptomatology Symptoms Symptoms
when glucose levels reach 50-55 mg/dL develop abruptly develop
 Critical value for glucose is gradually (some
40mg/dL ;
patients are
excessively low glucose values can cause severe CNS
asymptomatic)
dysfunction especially if blood glucose value drops to 20
Ketosis
 – 30 low blood glucose concentration, Common Rare
typical symptoms and symptoms alleviated by glucose
Whipple’s Medication Insulin absolute Oral agents
administration
HYPERGLYCEMIA
GESTATIONAL DIABETES MELLITUS
Laboratory Finidngs in Hyperglycemia  A disorder characterized by impaired ability to
1. Increase glucose in plasma and urine metabolize carbohydrate usually caused by a deficiency
2. Increase in urine specific gravity of insulin, metabolic or hormonal changes
3. Ketones in serum and urine
 It occurs during pregnancy and disappears after delivery
4. Decreased blood and urine pH (acidosis)
 Screening should be performed between 24-28 weeks of
5. Electrolyte imbalance (decrease Na+ and HCO +,
3 gestation
increase K+)
DIABETES MELLITUS (DM)  Screening and diagnostic test: 2-hour OGTT using 75g
glucose load
 Group of metabolic disorders characterized by
 Infants born to diabetic mother are at increased risk for
hyperglycemia resulting from defects in insulin
respiratory distress syndrome, hypocalcemia and
secretion, insulin receptors or both
hyperbilirubinemia
 Fasting plasma glucose concentration
≥126  After giving birth, women with GDM should be
on more than one testing is diagnostic of hypoglycemia evaluated 6-12 weeks postpartum
 Glucosuria occurs when the plasma glucose levels
exceed
9.9mmol/L or with normal renal  GDM converts to DM within 10 years in 30-40% of cases
180mg/dL function
 Severe DM, the ratio of β-hydroxybutyrate to  Diagnostic Criteria for GDM
acetoacetate is 6:1 1. FBS - ≥92 mg/dL

COLLEGE OF MEDICAL LABORATORY 5 | 34

Clinical Chemistry
2. 1-hour OGTT = ≥180 mg/dL
3. 2-hour OGTT = ≥153 mg/dL

COLLEGE OF MEDICAL LABORATORY 6 | 34

 Clinical Chemistry
GLUCOSE METHODOLOGIES  Guidelines for OGTT
CHEMICAL METHODS o Patient is aked to consume a normal to high
carbohydrate intake (150g carbs per day) for 3
OXIDATION REDUCTION METHOD
days prior to the test
1. Alkaline Copper Reduction Test – reduction of
o Patient is asked to fast overnight and to avoid
cupric to cuprous ions forming cuprous oxide in hot
alkaline solution excessive physical activity. Patient should fast
 Folin Wu Method at least 8-10 hrs but not greater than 16 hrs
 Nelson Somogyi Method o OGT testing should be performed on the
 Neocuproine Method morning to prevent hormonal diurnal effect on
 Benedict’s Method (Modified Folin Wu) glucose.
–
uses citrate or tartrate as stabilizing o Patient should be Ambulatory .
agent
2. Alkaline Ferric Reduction (Hagedorn Jensen)
- reduction Patient should refrain from exercise, eating, or
drinking (except water) and smoking
of yellow ferricyanide to colorless ferricyanide
o FBS is measured before giving the glucose load;
(inverse colorimetry)
if the FBS is greater than 140, test should be
CONDENSATION METHOD
terminated; if the FBS is <140 mg/dL, glucose
 AKA Ortho-toluidine/Dubowski method
load should be given to the patient.
o Glucose load for an adult is 75g and the patient
ENZYMATIC METHODS
should finish drinking it within 5 minutes _
GLUCOSE OXIDASE o Patient should not vomit, if they vomit,
 measures B-D glucose ; also discontinue the test
measures glucose in CSF and urine; presence of bleach
o Blood Glucose is taken every 30 minutes for 2
can cause a false increase in glucose
hrs
 Colorimetric (Saifer Gernstenfield) - enzymes used:
 Intravenous Glucose Tolerance Test (IVGTT)
glucose oxidase, peroxidase "Mutarotase"
o Used for patients with GI disorders (eg.
 Polarographic - measures the rate of oxygen
Malabsorption)
consumption which is proportional to glucose
o 0.5 g of glucose/kg body weight (given w/in 3
concentration using an oxygen-sensing electrode;
mins) is administered intravenously
enzymes used: glucose oxidase, catalase
o Second blood collection is 5 minutes after
HEXOKINASE infusion
 Most specific glucose method; reference method; uses 2-HRS POSTPRANDIAL BLOOD SUGAR (2HPPBS)
G6PD which is the
 measures how well the used in the diagnosis of GDM
most specific enzyme/reagent in glucose measurement;
 FBS is measured initially, patient is then given glucose
not affected by ascorbic acid or uric acid
 enzymes used: Hexokinase & G6PD load (75g) and plasma glucose is determeined after 2 hrs
 Normally, blood glucose levels should be back near the
reference limits approximately 2 hrs post load
GLUCOSE DEHYDROGENASE used in the diagnosis of GDM
 glucose is reduced to a chromophore that is measured
spectrophotometrically
 Mutarotase: shorten time necessary to reach
equilibrium
 enzymes used: glucose dehydrogenase and diaphorase

LABORATORY TESTS
Random Blood Sugar
 Blood Glucose taken any time of the day and without
any fasting
 Requested during insulin shock and hyperglycemic
ketonic coma
Fasting Blood Sugar
 Measure of over-all glucose homeostasis
 Requirement: non-per orem (NPO) at least 8 hours
before the test
Oral Glucose Tolerance Test (OGTT)
 determines how well the body metabolizes glucose;
COLLEGE OF MEDICAL LABORATORY 7 | 34
 Clinical Chemistry
GLYCOSYLATED HEMOGLOBIN (HbA1c)
 Reliable method in monitoring long-term glucose control
 Reflects average blood glucose level over the
previous 2- 4 months
 Specimen: Whole Blood
 Methods: Electrophoresis, Immunoassay, HPLC, and
Affinity Chromatography
 For every 1% change in HbA1c, 35 mg/dL is
added to plasma glucose
Table 5. Glucose Test Categories in mg/dL
NORMAL IMPAIRED/ DIAGNOSTIC
HIGH RISK FOR DM
FBS 70-100 100-125 =>126
OGTT <140 140-199 =>200
HbA1c (%) <5.7 5.7-6.4 >6.5
Fructosamine (Glycosilated Protein)
 Short term glucose control (3 – 6 weeks)
 Monitoring DM individuals with Chronic Hemolytic
Anemia and Hb variants

COLLEGE OF MEDICAL LABORATORY 8 | 34

 Clinical Chemistry
VALUES TO REMEMBER o Catalyzes the esterification of cholesterol (HDL)
 Fasting Plasma Glucose: 70-100 mg/dL resulting in the formation of lysolecithin and
 Glycosylated Hgb: 4-6% (Henry’s); 4.5-8% cholesterol ester
(Bishop)
 Conversion Factor: 0.0555  Free Cholesterol (30%)
 Critical Values: <40 mg/dL and >500 mg/dL  Found in plasma, serum and rbcs
 Free cholesterol and phospholipids are found on
LIPIDS the surface of lipoproteins
 Are insoluble in blood and water but soluble in organic
solvents (chloroform and ether) TRIGLYCERIDE (TAG)
FORMS OF LIPIDS
 Contains 3 molecules of fatty acid and one molecule of
FATTY ACID
glycerol by ester bonds; main storage lipid in man
 Linear chains of carbon-hydrogen bonds that terminate  Very hydrophobic and water insoluble – does not
with a carboxyl group contain charged or hydrophilic groups
 Mostly found as constituents of phospholipids or  Evaluates suspected atherosclerosis and measure’s
triglycerides the body’s ability to metabolize fat
 Only a small amount is present in the plasma (free  TAG and cholesterol are the most important lipids in the
unesterified form), most is bound to albumin management of coronary artery disease
 Very important sources of energy  Prior to venipuncture, ideally patients should undergo
 Provide the substance for conversion to glucose fasting for 12-14 hrs
(gluconeogenesis)
LIPOPROTEINS
PHOSPHOLIPID
 Are large macromolecular complexes of lipids with
 Most abundant lipid derived from phosphatidic acid specialized proteins known as apolipoproteins
 Amphipathic: with polar hydrophilic (water-loving) head  Main purpose: transport TAG and cholesterol to sites of
groups and nonpolar hydrophobic (water-fearing) fatty energy storage and utilization
acid side chains  Apolipoprotein: helps keep the lipids in solution during
 Similar in structure in triglycerides, except they contain circulation through the blood stream; contain a
two fatty acids structural motif called an “amphiphatic helix”
PHOSPHOLIPIDS IN THE BODY MAJOR LIPOPROTEINS
1.
 Phosphatidylcholine (70 – 75%) CHYLOMICRONS
 Sphingomyelin (18 – 20%)  Largest and lightest among the lipoproteins;
 Phosphatidylserine, Phosphatidylethanolamine (3 – lipoprotein with LDL
 80-95% TAG by weight
6%)
 Transports Exogenous TAG
 Lysophosphatidylcholine (4 – 9%)
 Causes non-fasting lipemia
 No charge; remains in the origin during
CHOLESTEROL
electrophoresis
 Found on the surface of lipid layers and synthesized in 2.
VLDL
the liver  Pre-beta Lipoprotein
 Precursor of 5 major classes of steroids: progestins,  Transports Endogenous TAG
3.
glucocorticoids, mineralocortocoids, androgens and LDL
estrogens  AKA as the bad cholesterol; beta lipoprotein
 Evaluates the risk for atherosclerosis, myocardial and  Transports cholesterol from
Liver to the cells of the body
coronary artery occlusions
 Transport majority (75%) of the cholesterol
 Direct relationship between elevated serum cholesterol
 Directly proportional to the risk of atherosclerosis
and myocardial infarction
and coronary heart disease (CHD); higher LDL
FORMS OF CHOLESTEROL
means higher risk
 Cholesterol Ester (70%) 4.
HDL or Alpha LPP
 Found in plasma and serum; cholesterol bound in  Smallest and heaviest among the lipoproteins
fatty acid  Fastest towards the anode; alpha lipoprotein; good
 Neutral lipid: located at center of lipid drops cholesterol
 Undergoes esterification by lcat  Inversely proportional to the risk of atherosclerosis
and CHD; lower HDL means higher risk
 Lecithin-Cholesterol Acyl Transferase (LCAT)
 Responsible for reverse cholesterol transport;
transports cholesterol from cells to the liver

COLLEGE OF MEDICAL LABORATORY 9 | 34

 Clinical Chemistry
MINOR LIPOPROTEINS ENZYMATIC METHODS
1.
IDL (Intermediate Density Lipoprotein)  Most common method of quantifying the cholesterol
 Product of VLDL catabolism – VLDL remnant; oxidase reaction is to measure the amount of hydrogen
“subclass of LDL”
peroxide produced
 Major apolipoprotein:
1. Cholesterol Oxidase Method (Spectrophotometric)
 Density: 1.006-1.019 kg/L = floats on the 1.063
 Enzymes used: Cholesterol esterase, Cholesterol
density potassium bromide solution
oxidase, Peroxidase
2.
Lp(a) TRIGYLCERIDE
 Known as sinking pre B LPP
due to electrophoretic mobility same as VLDL but CHEMICAL METHODS
density like LDL 1.
Colorimetric - Van Handel & Zilversmith
 LDL-like particles that have a molecule of Apo (a)  Uses chromotrophic acid
linked to Apo B-100 by a disulfide bond  End product: Blue colored compound
 Independent risk factor for atherosclerosis  modified VHZ
 Density: 1.045-1.080 kg/L (CDC Reference method)
o alkaline hydrolysis (saponification) using
ABNORMAL LIPOPROTEINS alcoholic KOH, solvent extraction with
1.
LpX chloroform and the extract is treated with
 Abnormal lipoprotein found in obstructive jaundice silicic acid (chromatography) to isolate TAG and
and LCAT deficiency a color reaction with chromotropic acid giving
 A specific and sensitive indicator of cholestasis rise to a pink end color
 Lipid content is mostly phospholipid and free
2.
Fluorometric - Hantzsch
cholesterol (90%); contains ApoC and albumin  Uses diacetyl acetone and ammonia
2.
B-VLDL  End product: Diacetyl lutidine compound
 Known floating pre B LPP ENZYMATIC METHODS
as “ –
density of VLDL during ltracentrifugation but  Specific, Rapid, and easy to use
migrates with LDL in the β-region during
 Major Interference: Endogenous Glycerol
electrophoresis
 Glycerol Kinase
 Found in type 3 hyperlipoproteinemia or
 Reaction A - lipase, glycerol kinase, pyruvate kinase
dysbetalipoproteinemia
 Also known as VLDL rich in cholesterol due to and LDH
defective catabolism of VLDL  Reaction B - lipase, glycerol kinase, glycerol PO4
dehydrogenase, diaphorase
METHODOLOGIES LIPOPROTEIN
CHOLESTEROL 1. Ultracentrifugation
 Reference method for quantification of lipoproteins
CHEMICAL METHODS
 Based on protein and triglyceride contents of
 PRINCIPLE: Dehydration and oxidation of cholesterol to lipoproteins
form a colored compound  Expressed on svedverg units
1. Liebermann – end product:
Buchardtt  Reagent: potassium bromide solution (1.063)
Cholestadienyl monosulfonic acid (green end color) 2. Electrophoresis
2. Salkowski - end product:  Electrophoretic pattern (from origin): Chylomicrons,
cholestadienly disulfonic acid (red end color) LDL, VLDL, HDL
 Color Developer Mixture (Liebermann Burchardt  Preferred supporting medium: agarose gel
reagent)  Lipid staining dyes: Oil red O, Fat red 7B, Sudan
 Glacial acetic acid Black B
 Acetic Anhydride 3. Chemical Precipitation
 Concentrated H2SO4 a. HDL
Table 6. General Methods o Uses dextran sulfate (synthetic heparin) with
magnesium (precipitants)
METHOD STEPS OTHER NAME
o CDC reference method: ultracentrifugation,
Pearson, Stern
One-step Colorimetry heparin manganese precipitation and Abell-
and MacGavack
Kendall assay
Two-step Extraction + Colorimetry Bloors
o Homogeneous assays are the most popular
Saponification +
Three-step Abell-Kendall method for measuring HDL-C
Extraction + Colorimetry
b. LDL
Saponification +
Schoenheimer i. β-quantification
Four-step Extraction + Colorimetry
Sperry o EDTA plasma is the preferred sample
+ Precipitation

COLLEGE OF MEDICAL LABORATORY 10 |

 Clinical Chemistry
o Plasma is centrifuged for at least 18 IIb (Familial Combined High LDL & VLDL (High
hours; VLDL and CM accumulate as Hyperlipidemia) Cholesterol & TAG)
floating layer, leaving predominantly LDL III (Familial Presence of β-VLDL, High
and HDL in solution
Dysbetalipoproteinemia) Cholesterol & TAG)
ii. Homogeneous Direct LDL-C method
IV (Familial Hight VLDL (High TAG)
o First reagent: selectively removes non-
Hypertriglyceridemia)
LDL lipoproteins
High VLDL and Presence of
o Second reagent: releases cholesterol
V CM (High Cholesterol and
from LDL so that it can be measured TAG)
enzymatically
LIPID STORAGE DISEASES
4. Chromatographic methods
 Uses either Gel Chromatography or Affinity Table 8. Lipid Storage Diseases
Chromatography Lipid Storage Disease Enzyme Deficient
5. Immunochemical methods Fabry’s disease Alpha galactosidase
 Uses antibodies specific to epitopes on the GM-1 Gangliosidosis Beta galactosidase
apolipoproteins
Gaucher Beta glucosidase
6. Immunoassay or Immunonephelometry
Krabbe Cerebroside beta galactosidase
 Based on the measurement of the turbidity created
Niemann Pick Sphingomyelinase
by apolipoprotein-antibody complexes
 Lp(a) is measured by immunoturbidimetric assay Metachromatic Arylsulfatase A
leukodystrophy
CLINICAL SIGNIFICANCE Sandhoff Total Hexosamindase (A & B)
ARTERIOSCLEROSIS VS ATHEROSCLEROSIS Tay Sach Hexosaminidase
VALUES TO REMEMBER
 Arteriosclerosis – general term for the thickening ATP III CLASSIFICATION FOR LDL, TOTAL
hardening of & HDL
 Atherosclerosis – Hardening of CHOLESTEROL & TAG VALUES
Type of Table 9. LDL CHOLESTEROL
the arteries caused by plaques (made up ofCholesterol,
< 100 mg/dL Optimal
Fatty substances, Cellular Waste products, Calcium and
100 – 129 mg/dL Near Optimal/Above Optimal
Fibrin) that build up inside the arteries
130 – 159 mg/dL Borderline High
CORONARY HEART DISEASE (CHD)
160 – 189 mg/dL High
 Broad spectrum of Heart disease resulting from ≥ 190 mg/dL Very High
Imparied coronary blood flow Table 10. HDL CHOLESTEROL
 Clinical (Non-Laboratory) Risk factors for CHD < 40 mg/dL Low
 Cigarette Smoking ≥ 60 mg/dL High
 Hypertension (BP >140/90 mmHg) Table 11. TOTAL CHOLESTEROL
 Family history of premature CHD < 200 mg/dL Desirable
 Age (Men > 45 years; Women > 55 years) 200 – 239 mg/dL Borderline High
 Obesity ≥ 240 mg/dL High
 Diabetes Mellitus Table 12. TRIGLYCERIDE
 Sedentary lifestyle < 150 mg/dL Normal
ANALPHALIPOPROTEINEMIA
150 – 199 mg/dL Borderline high
 Aka Tangier Disease; HDL deficiency 200 – 499 mg.dL High
ABETALIPOPROTEINEMIA ≥ 500 mg/dL Very High
 Aka Bassen-Kornzweig syndrome; Deficiency of apoB REFERENCE RANGES
(B48 and B100); notable acanthocytes in peripheral  Total Cholesterol – 140 – 200 mg/dL
blood smear
 HDL Cholesterol – (M) 29 – 60 mg/dL; (F) 38 – 75 mg/dL
FREDRICKSON AND LEVY’S
Table 7. Fredrickson Classification of  LDL Cholesterol – 57 – 130 mg/dL
HYPERLIPOPROTEINEMIA
Hyperlipoproteinemia  Triglycerides – 67 – 157 mg/dL
CONVERSION FACTORS

 Cholesterol (mg/dL to mmol/L) – 0.026

TYPE LPP PATTERN
 Triglyceride (mg/dL to mmol/L) – 0.0113
I (Familial LPL deficiency) High CM (High TAG) FORMULA FOR LDL-C
IIa (Familial High LDL (High Cholesterol)
 LDL-C = Total Cholesterol - HDL - VLDL
Hypercholesterolemia)

COLLEGE OF MEDICAL LABORATORY 11 |

 Clinical Chemistry
 Friedewald Method (Indirect)
a1-Fetoprotein (AFP)
 VLDL (mmol/L) Plasma TAG/2.175
 most abundant protein in fetal serum
=
 VLDL (mg/dL) = Plasma TAG/5.0
 detectable in maternal blood up to the 7th or 8th month
 De Long Method (Indirect) of pregnancy
 VLDL (mmol/L) Plasma TAG/2.825
 increased (maternal blood):
= Plasma TAG/6.5 Neural tube defects: Spina bifida & Anencephaly
 VLDL (mg/dL) =
 tumor marker for Hepatic CA & Gonadal CA_
α1 ACID GLYCOPROTEIN/OROSOMUCOID (AAG)
PROTEINS
 Are synthesized in the liver and secreted by the  greatest affinity for progesterone
hepatocyte into the circulation except immunoglobulins  useful diagnostic tool in neonates with bacterial
(plasma cells) infections
 Amphoteric: can bear positive and negative charges α1 ANTI – CHYMOTRYPSIN
because of their acid and basic amino acid compositions
 binds and inactivates prostate specific antigen (PSA)
PLASMA PROTEINS
 major form of PSA found in human sera; associated with
FRACTION SPECIFIC PROTEINS Alzheimer’s disease
Prealbumin Prealbumin GROUP-SPECIFIC COMPONENT (Gc) GLOBULIN
Albumin Albumin  exhibits affinity with vitamin D and actin
α1-Globulin α1antitrypsin, α fetoprotein, α lipoprotein,  method: radial immunodiffusion
α1 acid glycoprotein, α1 antichymotrypsin, ALPHA 2 – GLOBULIN
inter α-trypsin inhibitor, Gc globulin
a2-Macroglobulin (AMG)
α2-Globulin Ceruloplasmin, Haptoglobin, α2
 largest major non-immunoglobulin in plasma
macroglobulin
 increases 10x in Nephrotic Syndrome
β-GlobulinPREALBUMIN (TRANSTHYRETIN)
Transferrin, Hemopexin, β2 microglobulin,
 found principally in the intravascular spaces
 Complement,
Transport protein for T4 &Fibrinogen,
RetinolLDL, VLDL, CRP
Ceruloplasmin
 γ-Globulin Immunoglobulin,
Used to detect malnutrition CRP (In some books)
 copper-binding glycoprotein; imparts blue color to
 Landmark to confirm that the specimen is really CSF protein
ALBUMIN  marker for Wilson’s disease: deposition of copper in
 Protein present in highest concentration in the plasma Rey Julius
skin, liver, brain and corneaBalatay
(Kayser Fleisher rings)
2021-11-15 16:02:34
 decreased: Wilson’s disease, Menkes’ kinky-hair
--------------------------------------------
syndrome 2nd most predominant protein in CSF
Haptoglobin
 General transport protein; maintains osmotic pressure  binds free by its α chain
 Sensitive and highly prognostic marker in cases of cystic  and its constituent iron
prevents the loss of
fibrosis into the urine
 “ Negative Acute Phase Reactant ” BETA - GLOBULIN
 Normal life span in circulation is 15 – 19 days β2 - MICROGLOBULIN

High serum albumin are more often associated
 light chain component of the major human leukocyte
with dehydration or prolonged tourniquet application or
antigen (HLA)
specimen evaporation
 found on surface of most nucleated cells; needed in the
 Low serum albumin levels can be related to:
production of CD8 cells
 Inflammation, Hepatic disease, Urinary loss,
Transferrin
Gastrointestinal loss, Protein-Calorie malnutrition,
 Major Component of the β2 globulin fraction
Burn injury, Edema, and Ascites
 transports iron to its storage sites
 TERMS:
 increased: hemochromatosis (bronze-skin) and IDA
 Analbuminemia – absence of albumin
COMPLEMENT
 Bisalbuminemia – presence of 2 bands in the
 one of the natural defense mechanisms that protects
albumin region
the human body from infection
 Hypoalbuminemia – decreased levels of albumin
ΑLPHA 1 - GLOBULIN  Complement most abundant form in serum

a1antitrypsin (AAT) Hemopexin

 Major inhibitor of protease activity  binds heme released by degradation of hemoglobin;
 Deficiency: emphysematous pulmonary disease and helps in diagnosis of early hemolysis
juvenile hepatic cirrhosis

COLLEGE OF MEDICAL LABORATORY 12 |

 Clinical Chemistry
BETWEEN BETA AND GAMMA
 Can be used to determine if a certain body fluid is a
FIBRINOGEN transudate or an exudate
 most abundant of all the coagulation factors  No to lipemia and hemolysis
 serve as long term marker for prognosis of METHODOLOGIES
cardiovascular disease Kjeldahl
GAMMA GLOBULIN  reference method but not routinely
IMMUNOGLOBULIN  based on the measurement of the nitrogen of
 synthesized in the plasma cells protein (1g of nitrogen=6.54g of protein)

 IgG: most abundant in plasma and lymph  uses serum treated with tungstic acid to form a protein
 IgA: main antibody found in secretions free filtrate
 reagent: H SO
2 4
 IgM: first antibody that appears in response to antigenic
 end product: ammonia
stimulation
 IgD: present mostly on the surface of B cells Biuret
 IgE: antibody associated with allergic and anaphylactic  most widely used method
reactions  requires at least 2 peptide bonds and an alkaline medium
C-Reactive Protein (CRP)  Reagents:
 general scavenger molecule; binds to the C- Rochelle Salt Alkaline CuSO4 NaOH K
polysaccharide of the pneumococcus  Principle: Cupric ions complex the group involved in the
 cardiac marker: early warning for persons at risk for peptide bond forming a which is
violet-colored
coronary artery disease to Plasma Protein
 inflammatory marker: reflect severity of CHD
 rapid test for presumptive diagnosis of bacterial versus
viral infection
MISCELLANEOUS PROTEINS
Myoglobin
 small heme protein found in skeletal and cardiac
muscles that transports and stores oxygen from
hemoglobin to intracellular respiratory enzymes of
contractile cells
 higher affinity for oxygen than does hemoglobin
 marker for chest pain (angina) and early detection of
acute myorcardial infarction (AMI)
 Rises at 1 – 3 hrs; Peaks at 5 – 12 hrs; Returns to normal
in 18 – 30 hrs
TROPONINS

 are regulators of actin and myosin

 most important marker for cardiac injury
Troponin T (Tropomyosin binding sububnit)
 useful for assessment of early and late AMI
 sensitive marker for the diagnosis of unstable angina
 Rises within 3 – 4 hrs; Peak in 10 – 24 hrs; Remains
elevated for up to 10 – 14 days
Troponin I (Inhibitory Subunit)
 only found in myocardium, greater cardiac specificity
than TnT; highly specific for AMI
 Rises within 3 – 6 hrs; Peak in 14 – 20 hrs; Returns to
normal in 5 – 10 days
B-Type Natriuretic Peptide
 A cardiac marker; diagnostic for congestive heart failure
SPECIMEN CONSIDERATIONS AND PATIENT
PREP
 Serum is preferred; 24-hr urine and serous fluids can
also be used
 Protein in CSF is Less than 1% compared

COLLEGE OF MEDICAL LABORATORY 13 |

 Clinical Chemistry
proportional to the number of peptide bonds present
and reflects the total protein level at 545nm.
FOLIN – CIOCALTEU (LOWRY)

 has the highest analytical sensitivity

 Principle: Oxidation of phenolic compounds such as
tyrosine, tryptophan, and histidine to give a deep
blue color
 Main reagent: Phosphotungstic molybdic acid or
phenol reagent
 Color enhancer: Biuret reagent
ULTRAVIOLET ABSORPTION

 Principle: The absorbance of proteins at 210nm due

to the absorbance of peptide bonds at that specific
wavelength
 All proteins have absorbance at 210 except
tryptophan, tyrosine and phenylalanine (280nm)
REFRACTOMETRY

 alternative test to chemical analysis of serum proteins

SALT FRACTIONATION

 Globulins can be separated from albumin by salting-

out procedures using sodium salts
 Reagent: sodium sulfate salts
Table 13. Solubility Property of Proteins

PROTEIN SOLUBLE INSOLUBLE

Albumin Water Saturated Salt Soln
Conc. Salt Soln Highly conc salt soln
Hydrocarbon solvents
Globulin Weak Salt Soln Water
Hydrocarbon solvents Saturated Salt soln
Conc salt soln

COLLEGE OF MEDICAL LABORATORY 14 |

 Clinical Chemistry
SERUM PROTEIN ELECTROPHORESIS
 when the substrate concentration reaches a
 The single most significant clinical application of SPE is maximal value, higher concentration of substrate
for the identification of monoclonal spike of no longer result in increased rate of reaction
immunoglobulins and differentiating them from 3. Cofactors
polyclonal hypergammaglobulinemia  nonprotein entities that bind to enzymes before a
 reaction occurs
“bli in the late α2 or early β zone region: free
hemoglobin  Coenzymes – is an organic compound, which when
 small spikes in the β region: iron deficiency anemia increased in concentration, will result to an increase
(transferrin) in velocity of the reaction
 polyclonal gammopathy: rheumatoid arthritis and  Activato – inorganic ions that alter the
malignancy spatial configuration of the enzyme for proper
ABNORMAL SPE PATTERNS substrate binding
1. Gamma Spike – Multiple Myeloma  Metalloenzym – an organic ion attached to a
2. β-γ bridging – Hepatic Cirrhosis molecule
3. α globulin spike – Nephrotic Syndrome
4. Inhibitors
2
 Competitive Inhibition
4. α1 globulin flat curve – Juvenile Cirrhosis
5. α1, α2 and β globulin spikes - Inflammation o Physically binds to the active site of an
and competes with the substrate for that
o The
si effect of the inhibitor can be counteracted
by adding excess substrate to bind the enzyme

Non Competitive Inhibition
o doesareas
for not compete with
other than thethe substrateallosteric
active but
site)
o increasing substrate concentration does not
Abnormal SPE Patterns
reverse the inhibition
DYES
 Uncompetitive Inhibition
1. Bromcresol Purple – most sensitive, o binds to the enzyme substrate
specific, and precise among the dye-binding assays o increasing substrate concentration results to
2. Bromcresol Green – most commonly increased inhibition
used, measure absorbance
5. Isoenzymes - have the same catalytic reactions but
3. Tetrabromphenol blue – used in urine reagent strip,
slightly different molecular structures
4. Ninhydrin – for amino acids
sensitive to 6. Temperature
5. Methyl orange
 enzymes are active at 25°C, 30°C or 37°C
6. Hydroxybenzeneazobenzoic acid (HABA)
 37°C = optimum temperature
7. Coomassie Brilliant Blue
8. Pyrogallol Red – used for analysis of fluids with lower  45-50°C = enzyme start to denature
protein concentrations such as urine and CSF  60-65°C = inactivation of enzymes
VALUES TO REMEMBER 7. pH - most physiologic reactions occur in the pH range of
 Reference Range: 7 to 8.
 Total Protein: 6.5 – 8.3 g/dL 8. Storage - low temperatures (refrigeration/freezing)
 Albumin: 3.5 – 5.5 g/dL render enzymes visibly inactive
 2° to 8°C = ideal storage temperature for substrates
 Globulin = Total Protein – Albumin
and coenzymes
 Conversion Factor: g/dL to g/L = 10
 -20°C = preservation for longer periods of time
 room temperature = ideal storage for LDH (LD4 and
ENZYMES
LD5)
FACTORS AFFECTING ENZYMATIC
9. Hemolysis - most increases enzyme concentration
REACTIONS 10. Lactescence or Milky serum - decreases enzyme
1. Enzyme Concentration concentration
 The higher the enzyme concentration, the faster is ENZYME NOMENCLATURE
the reaction  1st digit = classification
2. Substrate concentration  2nd and 3rd digits = subclass
 with the amount of enzyme exceeding the amount  4th and 5th digits = serial number
of substrate, the reaction rate steadily increases as
more substrate is added

COLLEGE OF MEDICAL LABORATORY 15 |

 Clinical Chemistry
Table 14. Classification of Enzymes
ENZYMATIC REACTION
CLASS FUNCTION EXAMPLE
Oxidoreductases Catalyze the LDH, MDH, 1. Zero-Order Reaction
– reaction rate
(-dehydrogenase) removal or addition ICD, G6PD depends only on enzyme concentration
of electrons 2. First Order Reaction – reaction rate is
Transferases Catalyze the CK, AST, ALT, directly proportional to substrate concentration
(-transferase, transfer of chemical OCT 1. FixedGENERAL
time/endpoint – reactionTO
METHODS proceeds for a
MEASURE
-kinase) group other than ENZYMATIC
designated time, the reaction is stopped and a
hydrogen from one measurement is made.
substrate to another 2. Continuous Monitoring/ kine ass – multiple
Hydrolases Catalyze hydrolysis Esterases – measurements are made during the reaction; preferred
ACP, ALP, CHS, than fixed-time
or splitting of a
LPS ALKALINE PHOSPHATASE (ALP)
bond by the
Peptidases – CHARACTERISTICS
addition of water Trypsin,
(hydrolytic pepsin, LAP  Catalyzes (or organic
hydrolysis of
reactions) Glycosidase – phosphate esters) into alcohol and phosphate at an
AMS,  Requires activator zinc
galactosidases alkaline pH (9.0 –
ISOENZYMES
Lyase Catalyze removal of Glulatamate
 Normal Isoenzymes: Intestinal, Placental, Bone, and Liver
(-decarboxylase) groups from decarboxylase,
 Liver and Bone ALP are the most predominant fractions
substrates without pyruvate
 Can be differentiated using electrophoresis, heat
hydrolysis decarboxylase,
denaturation, and chemical inhibition
tryptophan
 Carcinoplacental isoenzymes include Regan, Nagao, and
decarboxylase,
Kasahara. They are usually found in patients with
aldolase
malignancy and their characteristics resemble that of
Isomerase Catalyze the Glucose
placental ALP
intramolecular phosphate
ELECTROPHORESIS
arrangement of the isomerase,
substrate ribose  Origin towards anode:
"I Promise to Be Loyal"
compound phosphate  Intestinal - Placental - Bone - Liver
isomerase  Liver and bone fractions are difficult to resolve during
Ligase Catalyze the joining Synthase electrophoresis
of two substrate  To improve separation of bone and liver forms, use
molecules, coupled neuraminidase & wheat germ lectin
with breaking of the HEAT DENATURATION
pyrophosphate
GENERAL PROPERTIES OF ENZYMES  Most Heat Stable to Most Heat
bond in ATP or Labile "Pangako Ikaw Lang Beh"

1. similar compounds
Active site - is a water-free activity, where the substrate 
Placental - Intestinal - Liver - Bone
interacts with particular charged amino acid residues
 Heat stability is determined by heating serum at 56C for
2. Allosteric site - is a cavity other than the active site
10 – 15 minutes
 When bound tightly to the enzyme, the coenzyme is
 Bone – most heat stable of
called a prosthetic groups
all the normal ALP isoenzymes
 Apoenzyme (enzyme portion) and coenzyme forms
 Regan ALP – most heat stable among all the types of ALP
a complete and active system known as
CHEMICAL INHIBITION
holoenzyme (apoenzyme + prosthetic group =
holoenzyme)  Placental and intestinal ALPs are inhibited by
 Digestive enzyme in its inactive form originally phenylalanine reagent and 3M urea inhibits bone ALP
secreted from the organ of production is called a  Levamisole inhibits both liver and bone ALP
proenzyme or zymogen
ENZYME THEORY

1. Emil Fisher’s (Lock and Key Theory) – the shape of

the key (substrate) must fit into the lock (enzyme)
2. Kochland’s (Induced Fit Theory) – based on the
substrate binding to the active site of the enzyme

COLLEGE OF MEDICAL LABORATORY 16 |

 Clinical Chemistry
METHODOLOGIES
Table 16. Summary of ACP Methods
Table 15. Summary of ALP Methods METHODS SUBSTRATE END
PRODUCTS
METHODS SUBSTRATE END
Gutman & Phenyl PO4 Inorganic PO4
PRODUCTS
Gutman
Bodansky
Inorganic Shinowara PNPP p-nitrophenol
Shinowara β-
phosphate + Babson, Read, & α-naphthol α-naphthol
Jones glycerophosphate
glycerol Philips phosphate
Reinhart
Roy & Hillman Thymolphthalein Free
King &
Phenylphosphate Phenol MonoPO4 thymolphthalein
Armstrong
CLINICAL SIGNIFICANCE
Bessy, Lowry, P-nitrophenol or
PNPP  Elevated in patients with prostatic CA, however it is not
& Brock "PNP bows & besso" Yellow
Bowers & Nitrophenoxide specific since it can also be elevated in prostatic
PNPP hyperplasia and prostatic surgery
McComb Iron
Huggins & Phenolphthalein Phenolphthalein  Prostatic ACP (PAP) is used together with prostate
Talalay disphosphate Red specific antigen (PSA) to monitor recurrence of prostatic
α-naphthol cancer
Moss α-naphthol  Useful in forensic clinical chemistry – especially in
phosphate
Buffered medico legal evaluation of rape
Klein, Babson, Free  ACP may also be elevated in bone diseases due to
phenolphthalein
& Read Phenolphthalein osteoclastic activity, as well as in Gaucher disease
phosphate
ASPARTATE AMINOTRANSFERASE (AST)
CLINICAL SIGNIFICANCE CHARACTERISTICS
 Often used in evaluation of Hepatobillia (obstructive  Aka Serum Glutamic Oxaloacetate Transaminase (SGOT)
conditions) and (Osteoblast
Bone
involvement)
Paget’s
 Highest elevation of ALP is seen in:  Involved in the transfer of an amino group between
-bone disorder acids with the formation of
(Osteitis deformans) aspartate and α-
 Increased in: Hyperparathyroidism, rickets &
oxaloacetate and
osteomalacia, fractures, and Malignant tumors  2 isoenzymes: cytoplasmic (predominant form in serum)
 Cirrhosis and Hepatitis produce only slight elevations and mitochondrial
 Biliary obstruction (Cholestasis) – 3 -10 times elevation  Major tissue source: cardiac tissue, liver and skeletal
 Physiologic elevation of ALP can be seen in growing muscle
children due to osteoblastic activity CLINICAL SIGNIFICANCE
ACID PHOSPHATASE (ACP)  used in the evaluation of
CHARACTERISTICS myocardi infarctio
hepatocellul disorde and s kel l muscle
 catalyzes the same reaction made by ALP, except that it involveme
is active at pH 5.0  Often used in conjunction with ALT for hepatocellular
 tissue sources: Prostate (major source), disorders
RBCs, platelets, liver and bone SPECIMEN CONSIDERATION
ISOENZYMES
 Hemolysis should be avoided because it can dramatically
 Electrophoretic Separation increase serum AST concentration
 Erythrocytic ACP remains in Origin METHODOLOGIES
 Prostatic ACP migrates with great mobility Karmen
 – (pH 7.5) uses malate
 Chemical Inhibition
dehydrogenase (MD) and monitors the change in
absorbance at 340nm
 Prostatic ACP is inhibited by L-tartrate ALANINE AMINOTRANSFERASE (ALT)
 Erythrocytic is inhibited by 2% CHARACTERISTIC
ACP
formaldehyde and 1mM cupric sulfate solution Serum Glutamic PyruvicTransaminase (SGPT)
METHODOLOGIES  catalyzes the transfer of an amino group from alanine to
α-ketoglutarate with the formation of glutamate and
 Thymolpthalein Monophosphate = specific
pyruvate
substrate; for endpoint reaction  Major tissue source: Liver
 a-naphthyl phosphate = preferred for for 2 days
 Ifcontinuous
not assayed immediately, serum should be frozen to a
pH lower than 6.5. With acidification, ACP will be stable

COLLEGE OF MEDICAL LABORATORY 17 |

 Clinical Chemistry
CLINICAL SIGNIFICANCE

 Highest elevation is found in

Acute Hepatitis

COLLEGE OF MEDICAL LABORATORY 18 |

 Clinical Chemistry
 ALT is slightly increased in obstructive jaundice but METHODOLOGIES
markedly increased in necrotic jaundice
 uses Olive Oil as the substrate
METHODOLOGIES
 Triolein (purer form of TAG) is also used as a substrate
 Couple Enzymatic reaction: ALT + Lactate for LPS assay
dehydrogenase; pH 7.5, reading at 340nm Table 19. Methods for Lipase
Table 17. Differentiating AST and ALT Determination
AST ALT Methods Principle
Major Organ Cherry Reference method; substrate – 50% olive oil
Affected
Heart Liver Hydrolysis of olive oil after incubation for 24
Crandal
Substrate Alanine & hours at 37°C and titration of fatty acids using
Aspartate &
a-ketoglutarate a-ketoglutarate NaOH
End products Pyruvate & Tietz &
Oxaloacetate &
Glutamic Acid Glutamic Acid Fiereck
Color developer 2,4 DNPH 2,4 DNPH Peroxidase Most commonly used; do not use 50% olive
Color intensifier 0.4 N NaOH 0.4 N NaOH Coupling oil
Methods Reitman & Reitman & Table 20. Acute Pancreatitis Markers
Frankel Frankel Onset of Peak Duration of
Marker
AMYLASE (AMS) Elevation Activity Elevation
CHARACTERISTICS Amylase 2 – 12 hrs 24 hrs 3 – 5 days
Lipase 6 hrs 24 hrs 7 days
 catalyzes the breakdown of starch and glycogen
 Isoenzymes: Salivary (Ptyalin) & Pancreatic (Amylopsin) LACTATE DEHYDROGENASE
CHARACTERIST
 Major tissue source: Acinar Cells of Pancreas & IC
Salivary Glands
CLINICAL SIGNIFICANCE  catalyzes the interconversion of lactic and pyruvic acid
Earliest Pancreatic Marker - Nonspecific  enzyme virtually found in all cells of the body
 uses NAD+ (nicotinamide dinucleotide) as coenzyme

 LD-2 is the major isoenzyme

Three-fold amylase increase with normal 24 hours urine
 LD-1 is relatively abundant in cardiac LD-5 is
amylase – repeat serum AMS after polyethylene glycol
more abundant in skeletal
precipitation
 LD-6 - alcohol dehydrogenase enzyme;
METHODOLOGIES
responsible for the metabolic conversion of methanol
 STARCH – Substrate for all methods and ethylene glycol
Table 18. Methods for AMS Determination  Conc in Serum: LD-2 > LD-1 > LD-3 > LD-4 > LD-5
Method Principle Table 21. LDH Tissue Sources
Saccharogenic Classic Reference Method; Measures the Isoenzyme Chain Composition Tissue Sources
amount of reducing sugars produced by the LD1 & LD 2 Heart, RBCs,
HHHH/ HHHM
hydrolysis of starch & Kidney
Amyloclastic Measures AMS activity following the LD3 Lungs, pancreas,
decrease in substrate concentration HHMM & Spleen
Chromogenic Measures AMS activity by the increase in LD4 & LD5
skeletal muscles,
color intensity of the soluble dye-substrate HMMM/ MMMM liver, & intestines
solution
CLINICAL SIGNIFICANCE
Couple- Measures AMS activity by continuous
 highest serum levels: pernicious anemia and hemolytic
enzyme monitoring technique; Enzymes used: AMS,
disorders
Glucosidase, Hexokinase, G6PD
 10-fold increase in hepatic carcinoma and toxic hepatitis
LIPASE (LPS)
 LD-1 > LD-2 (“flipped pattern”) = seen in myocardial
CHARACTERISTIC
infarction and hemolytic

hydrolyzes ester linkages of fats to produce alcohol and anemia
METHODOLOGIES
fatty acid

major tissue source: Pancreas Method Principle
CLINICAL SIGNIFICANCE Most commonly used; pH 8.8;
Wacker absorbance measured at 340 nm

Most Specific Pancreatic Marker
– secreted Forward Reaction
exclusively in the pancreas
 rises more slowly compared to amylase Preferred method;
Wroblenski La
Due
Reverse Reaction
COLLEGE OF MEDICAL LABORATORY SCIENCE | 15 | 34
 Clinical Chemistry
Table 22. Methods for LDH Determination
 increased: skeletal muscle disease, leukemia, hemolytic
CREATINE KINASE (CK) anemia and hepatic cancer
CHARACTERISTIC  Highest level in progressive muscular dystrophy
 catalyzes the transfer of a phosphate group between OTHER CLINICALLY SIGNIFICANT ENZYMES
5’ NUCLEOTIDASE (5’N)
creatine phosphate and adenosine
 found in high concentrations  marker for hepatobiliary and infiltrative lesions
only in muscle and
 major tissue sources: brain, smooth and skeletal in the liver
and cardiac Gamma Glutamyl Transferase (GGT)
 CK-1 – (BB) most anodal and labile isoenzyme;
 catalyzes the transfer of glutamyl groups between
most dominant isoenzyme found in the brain, intestine peptides or amino acids through linkage at a gamma
and smooth muscle
carboxyl group (Patency of bile duct)
 CK-2 _ (20%) – (MB) present only in the myocardium
 useful in differentiating the increase in ALP
 CK-3 _ – (MM) least anodal; major isoenzyme (94-
 elevated in all hepatobiliary disorders –
100%) -mostly found skeletal muscles
biliary duct obstruction
CLINICAL SIGNIFICANCE
 sensitive indicator of occult alcoholism – most
 a very sensitive indicator of acute myocardial infarction sensitive marker of acute alcoholic hepatitis
(AMI) and Duchenne disorder PSEUDOCHOLINESTERASE (PCHE)
 highest elevation of total CK is seen in Duchenn  marker for insecticide/pesticide poisoning
muscular dystrophy (50x) (organophosphate poisoning) – low serum PchE
Table 23. Acute Myocardial Infarction  monitor the effect of muscle relaxants (succinylcholine)
Markers "MyTropICAL
after surgery
Marker
heart"
Onset of Peak Duration of ANGIOTENSIN-CONVERTING ENZYME (ACE)
Elevation activity elevation  also known as peptidyldipeptidase A or kininase II
Myoglobin 1 – 3 hrs 5 -12 hrs 18 – 30 hrs  possible indicator of neuronal dysfunction (Alzheimer’s
Troponin T 3 -4 hrs 10 – 24 hrs 10 - 14 days disease – CSF)
Troponin I 3 – 6 hrs 12 – 18 hrs 5 – 10 days  for the diagnosis and monitoring of sarcoidosis
CK – MB 4 – 6 hrs 12 – 24 hrs 48 – 72 hrs CERULOPLASMIN
AST 6 – 8 hrs 24 hrs 5 days
 marker for Wilson’s disease (hepatolenticular disease)
LDH 12 – 24 hrs 48 – 72 hrs 10 – 14 days ORNITHINE CARBAMOYL TRANSFERASE (OCT)
METHODOLOGIES
 Adenylate kinase released after red cell lysis interferes
 marker for hepatobiliary disease
with CK assay particularly with hemolysis
 Cleland’s reagent and glutathione – partially restore
NON-PROTEIN NITROGENS
lost activity of CK UREA
Table 24. Methods for CK Determination CHARACTERISTICS
Methods Principle  major end product of protein
pH 9.0; 340 nm  constitutes to 45% of the total
Tanzer- Enzymes used: CK, pyruvate kinase, 
synthesized in the liv via the Kreb’s Henseleit cycle
Gilvarg lactate dehydrogenase
 first metabolite to elevate in kidney
Forward  – pertains to the nitrogen
Blood Urea Nitrogen
Most commonly used; pH 6.8; 340 nm
Oliver- Enzymes used: CK, hexokinase, G-6-PD
content only of This value is often obtained using

Rosalki indirect methods

 Urea Concentration – concentration of
Reverse urea as a
molecule, not just the nitrogen portion. This value is
ALDOLASE
often obtained using the direct methods.
CHARACTERISTIC
METHODOLOGIES
 splits fructose-1,6-diphosphate aldolase into 2 triose
DIRECT METHOD
phosphate molecules in the metabolism of glucose
 ISOENZYMES
1.
Diacetyl Monoxime Condensation Method
 Aka Fearon reaction
 Aldolase A – Skeletal Muscle
 Inexpensive, Lacks
 Aldolase B – WBC, Liver, Kidney
 Aldolase C – Brain tissue  End product: Yellow Diazine
2.
CLINICAL SIGNIFICANCE O-phthaldehyde
 End product: Colored product

COLLEGE OF MEDICAL LABORATORY 16 |

 Clinical Chemistry
INDIRECT METHOD  Normal BUN to Creatinine Ration = 10:1 ; 20:1
 Measures urea by converting it first into ammonium METHODOLOGIES

using ammonium ions formed from urea are CHEMICAL (DIRECT JAFFE)
measur 1.
Folin Wu
 Initial Reaction: Urea (derived from jack bean meal)  Sensitive but non-specific method
converts urea to ammonium ions and carbonate ions 2.
Lloyd or Fuller’s Earth Method
 Secondary Reactions: Quantifies ammonium that  Adsorbent:
form after the initial reaction o Lloyd – sodium aluminum silicate

Nesslerization o Fuller's Earth – aluminum magnesium
o Reagents: KI, HgI silicate
o Products measured: Yellow-orange colloid KINETIC JAFFE
(Ammonium dimercuric iodide)  Principle: Serum is mixed with alkaline picrate solution

Berthelot and the rate of change in absorbance is measured
o Reagents: NaOCl, phenol, sodium nitroprusside between 2 points
o NaOCl chlorinates ammonia into
 Jaffe Reagent: Satd picric acid & 10% NaOH
monochloramine; monochloramine reacts with
 popular, inexpensive, rapid and easy to perform
phenol to form indophenol (blue or green) ENZYMATIC METHOD
o Reaction is maintained at alkaline pH (>10.0)
1. Creatinine aminohydrolase – CK
with sodium nitroprusside acting as catalyst  enzymes used: creatinine aminohydrolase,
o Product measured: indophenol at 630 – 660
creatinine kinase, pyruvate kinase and lactate
nm
dehydrogenase

GLDH-coupled 2. Creatinase – Hydrogen peroxide method
o Ammonia + 2-oxoglutarate + NADH + GLDH ->
 potential to replace Jaffe method (specific than
NAD + Glutamate + Water
Jaffe method)
o Measurement of decrease in absorbance at
 enzymes: Creatininase (creatinine aminohydrolase),
340 nm
creatinase, sarcosine oxidase, peroxidase

Conductimetric CLINICAL SIGNIFICANCE
o Conversion of unionized urea into ammonium
and bicarbonate ion; measure increase in AZOTEMIA
conductivity rate 1. Pre-Renal (slow flow of blood to

Indicator Dye  diminished glomerular filtration with normal renal
o Color change; used in dry reagent strips function
CLINICAL SIGNIFICANCE  cause: dehydration, shock, congestive heart failure
2. Renal
DECREASED BUN LEVELS
 damaged within the
 Decreased protein intake
 acute/chronic renal disease, glomerulonephritis
 Liver disease
3. Post-Renal
 Vomiting and Diarrhea
 usually the result of urinary tract obstruction
 Pregnancy VALUES TO REMEMBER
UREMIA VS AZOTEMIA
 Azotemia – refers to
increa  Reference Value: M = 0.6 – 1.2 mg/dL; F = 0.5 – 1.1 mg/dL
particularly urea in blood Conversion factor: (mg/dL to mmol/L) = 88.4
NP 
 Uremia – increase in NPNs in URIC ACID
blood; defined as increase in NPNs with of CHARACTERISTIC
organ involvement such as renal failure
VALUES TO REMEMBER  major end product of purine catabolism
 final breakdown of nucleic acid catabolism in humans
 Reference Value: 7 – 18 mg/dL
 formed from xanthine by the action of xanthine oxidase
 Conversion Factor: mg/dL to mmol/L = 0.357
in the liver and intestine
 Urea Concentration: BUN x 2.14
 filtered by the glomerulus but 40% of uric acid is
CREATININE
CHARACTERISTIC
 end product of muscle catabolism reabsorbed in the kidneys
METHODOLOGIES
 not affected by protein diet; not easily removed by
dialysis CHEMICAL (CARAWAY/HENRY METHOD)
 Reagent: Phosphotungstic acid (PTA)
 Reaction: Uric Acid + PTA -> Tungsten blue + Allantoin
 Index of Overall Renal Function; used to

COLLEGE OF MEDICAL LABORATORY 17 |

Clinical Chemistry
evaluate fetal lung maturity

COLLEGE OF MEDICAL LABORATORY 18 |

 Clinical Chemistry
 Sodium Cyanide is used to increase color and inhibit
 Right lobe is 6x larger than the left lobe
fading
 1.2 – 1.5 kg in weight
 Sodium carbonate may be used instead
 Smallest functional unit of the liver is known as the
ENZYMATIC
hepatic
 Uricase is the enzyme used  Blood is supplied from two sources: hepatic artery
 Principle: Uricase converts uric acid into portal
FUNCTIONS OF THE LIVER
Allontoin & H2O2 Table 25. Functions of the Liver
 Uric acid has a UV absorbance peak at 293 nm
CLINICAL SIGNIFICANCE Function
Synthesis Plasma proteins, carbohydrates, lipid,
HYPERURICEMIA
lipoproteins, clotting factors, ketone
 Gout – associated with pain and inflammation of the bodies, enzymes
joints; (+) birefringent crystals in the synovial fluid
Conjugation Bilirubin
 Increased nuclear metabolism – seen in leukemia,
Detoxification Toxic substances absorbed in the
lymphoma, multiple myeloma or polycythemia,
and Drug intestine and by-products of metabolism
hemolytic and megaloblastic anemias
metabolism
 Chronic renal disease – due to decreased GFR and
Excretion and Bile acids or salts, bile pigments,
tubular secretion
 Lesch-Nyhan Secretion cholesterol
– deficiency in hypoxanthine
Syndrome Storage Fat and water soluble vitamins, glycogen
guanine phosphoribosyl transferase (HGPT)
HYPOURICEMIA Table 26. Liver Function Tests
 Liver disease, defective reabsorption of uric acid by Function Tests
kidneys (i.e. Fanconi Syndrome) Synthesis
VALUES TO REMEMBER
Total Protein, Albumin, PT
Conjugation
 Reference Value: M = 3.5 – 7.2 mg/dL; F = 2.6 – 6.0 Bilirubin
mg/dL & Excretion
 Conversion Factor: mg/dL to umol/L = 0.0595/ 0.06 Detoxification
Ammonia & Enzyme
AMMONIA
CHARACTERISTIC
 arises from the deamination of amino acids BILIRUBIN
CHARACTERISTIC
 preferred specimen: arterial blood
METHODOLOGIES  end product of hemoglobin metabolism and principal
 Digestion (Kjeldahl) method – nitrogen ion in a protein- pigment in bile
free filtrate of the specimen is converted to ammonia  also formed from destruction of heme-containing
using hot concentrated H2SO4 in the presence of catalyst proteins such as myoglobin, catalase and cytochrome
 Measurement of ammonia oxidase
 – uses Gum Ghatti reagent Table 27. Comparison between Conjugated
Nesslerization
 – uses sodium nitroprusside and Unconjugated Bilirubin
Berthelot
 Glutamate dehydrogenase Bilirubin 1 (B1) Bilirubin 2 (B2)
CLINICAL SIGNIFICANCE
Unconjugated Conjugated
 used in the diagnosis of hepatic failure (hepatic
 elevated levels of ammonia are neurotoxic and are Water-insoluble Water Soluble
and Reye’s
often associated with encephalopathy Nonpolar Polar
VALUES TO REMEMBER Indirect Direct
Hemobilirubin Cholebilirubin
 Reference Range: 19 – 60 ug/dL 1-minute/ Prompt Bilirubin
Slow Reacting
 Conversion Factor: ug/dL to umol/L = 0.587
Post hepatic, hepatic,
Pre hepatic obstructive, regurgufative
LIVER FUNCTION TESTS BILIRUBIN METABOLISM AND EXCRETION
LIVER
ANATOMY OF THE LIVER 1. When RBCs are destroyed in the spleen, hemoglobin is
released
 Largest and the most versatile organ in the body 2. Hemoglobin is broken down into heme and globin
 Has two main lobes, separated from each other by 3. Globin is recycled while heme is divided into iron and
falciform ligament the protoporphyrin ring
4. Iron is transported into the bone marrow to be used for

COLLEGE OF MEDICAL LABORATORY 19 |

Clinical Chemistry
RBC sythesis

COLLEGE OF MEDICAL LABORATORY 20 |

 Clinical Chemistry
5. Protoporphyrin is broken down into biliverdin, and then
 Diazo Reagents
further broken down into bilirubin, specifically the
 Diazo A = 0.1% sulfanilic acid + HCl
unconjugated form
 Diazo B = 0.5% sodium nitrite
6. Unconjugated bilirubin (UB) is transported by albumin
 Diazo blank = 1.5% HCl
towards the site of conjugation, the liver
7. In the liver cells, UB is converted into Conjugated  Principle: Diazo reaction is diazotization of bilirubin to
bilirubin (CB) through the action of the enzyme uridyl produce azobilirubin
diphosphate glucoronyl transferase (UDPGT). The Table 28. Main Bilirubin Methodologies
process occurs in the smooth ER of the liver cells. Evelyn Malloy Jendrassik-Grof
8. Conjugated bilirubin goes out of the liver, down to the Accelerator
50% methanol caffeine Na
biliary tree (bile duct) and emptied into the intestine. benzoate
9. In the intestine, conjugated bilirubin is converted into pH Acidic Alkaline
urobilinogen. Urobilinogen is a colorless substance
Final
formed in the intestine Purple Blue
reaction
10. Urobilinogen is converted into urobilin, an orange
Maximum
brown pigment responsible for the natural brown color 600 nm
absorbance 560 nm
of the stool. It is then excreted through the stool out of
CLINICAL SIGNIFICANCE
the body.
11. Some of the urobilinogen from the intestine is Delta Bilirubin
reabsorbed back into the blood, only to be excreted by  conjugated bilirubin tightly bound to albumin; has
the kidneys into the urine. longer half-life than other bilirubin
 formed due to prolonged elevation of conjugated
bilirubin in biliary obstruction
 helps in monitoring the decline of serum bilirubin
following surgical removal of gallstones
JAUNDICE
 also called icterus or hyperbilirubinemia; characterized
by yellow discoloration of the skin, sclera and mucous
membranes
 clinically evident when bilirubin levels exceeds 2mg/dl
Table 29. Classifications of Jaundice
Schematic summary of the pathway of bilirubin Urine Urine
Jaundice B1 B2
(Bili, in brown circles) transport and metabolism urobilinogen bilirubin
METHODOLOGIES Pre-Hepatic
 Hemolysis will cause increase bilirubin while lipemia can
cause decrease bilirubin
(Hemolytic
Anemia)
↑ N ↑ (-)
Hepatic
 Visible icterisia occurs when >25mg/
 Bilirubin standard solution is usually made up of
(Liver
diseases)
↑ ↑ ↑/+ (-)
unconjugated bilirubin Post
 Conjugated bilirubin produces color in aqueous Hepatic
solution. (Bile duct N ↑ ↓/N (+)
 Unconjugated bilirubin produces color only after obstruction)
the addition of alcohol DERANGEMNTS IN BILIRUBIN METABOLISM
 Total bilirubin is measured 15 minutes after adding
methanol or caffeine solution 1.
Gilbert's – Bilirubin transport
 Caffeine-benzoate is preferred over methanol
deficit
because the latter promotes protein precipitation  characterized by impaired cellular uptake in
and increases turbidity bilirubin; elevated B1
 Jendrassik & Grof – most commonly used
2. Crigler-Najjar – Conjugation deficit
method; more sensitive and not affected by hemoglobin  infants are treated with phototherapy; elevated B1

 Sodium acetate = maintains alkalinity

 Type 1
o deficiency of the enzyme UDPGT resulting to
 Sodium tartrate = provides alkalinity total absence of B2
 Ascorbic acid = terminates initial reaction and o (+) kernicterus
destroys excess diazo reagent  Type 2
o partial deficiency of UDPGT
o small amount of B2 is produced
COLLEGE OF MEDICAL LABORATORY 21 |
 Clinical Chemistry
3.
Dubin Johnson & Rotor
excretion deficit – Bilirubin  Diarrhea, Excess fluid loss
 SIADH, Excess water intake
 blockade of the excretion of bilirubin into the  Adrenal insufficiency
canaliculi caused by hepatocyte membrane defect  Reset osmostat
 elevated B2 and total bilirubin  Acute or Chronic renal failure
4.
Lucy Driscoll – Conjugation inhibition
 Nephrotic syndrome, Hepatic cirrhosis, Congestive heart
 familial form of unconjugated bilirubinemia caused
failure
by circulating inhibitor of bilirubin conjugation
 Pseudohyponatremia (hyperglycemia, hyperlipidemia,
 elevated B1 hyperproteinemia)
VALUES TO REMEMBER VALUES TO REMEMBER
 Total Bilirubin – Direct Bilirubin = Indirect Bilirubin  Reference Value: 135 – 145 mmol/L
 Reference Values:  Threshold Critical Value
 Total Bilirubin: 0.2 – 1.0 mg/dL  Hypernatremia:
 Direct Bilirubin: 0 – 0.2 mg/dL  Hyponatremia:
 Indirect Bilirubin: 0.2 – 0.8 mg/dL  Conversion Factor: mEq/L to mmol/L =
 Conversion Factor: mg/dL to umol/L = 17.1 POTASSIUM
 Critical Value for Bilirubin: > 18 mg/dL CHARACTERISTIC

 otherwise known as “kalium”

ELECTROLYTES
SODIUM  major intracellular cation – only 2% of the body’s total
CHARACTERISTIC potassium circulates in plasma
 Functions: heart contraction, neuromuscular
 also known as “natrium”
excitability, ICF volume regulation and hydrogen ion
 major extracellular anion hence the major contributor of concentration
osmolality
METHODOLOGIES
 principal osmotic pressure outside the cell; depends
greatly on the intake of excretion of water 1. Ion selective electrode (Valinomycin gel)
2. Flame emission photometry (Violet)
HORMONES AFFECTING SODIUM LEVELS
3. Atomic absorption spectrophotometry
1.
4. Colorimetry (Lockhead & Purcell)
 major electro-regulating hormone
CLINICAL SIGNIFICANCE
 promotes sodium retention and potassium
excretion CAUSES OF HYPERKALEMIA
2. 1. Decreased Renal Excretion
 endogenous antihypertensive agent; promotes  Acute or Chronic Renal Failure
natriuresis  Hypoaldosteronism; Addison’s disease
 blocks aldosterone and renin secretion and inhibits  Diuretics
the action of angiotensin II and vasopressin 2. Increased Intake
METHODOLOGIES  Oral or IV potassium replacement therapy
3. Cellular Shift
1. Ion selective electrode (Glass aluminum silicate) – most
 Acidosis, Muscle/Cellular injury
commonly used method
 Chemotherapy
2. Flame emission photometry (Yellow)
 Leukemia (Increased WBC)
3. Atomic absorption spectrophotometry
4. Colorimetry (Albanese Lein)  Hemolysis
CLINICAL SIGNIFICANCE 4. Increased Intake
 Sample hemolysis, Thrombocytosis
CAUSES OF HYPERNATREMIA  Prolonged tourniquet use or excessive fist clenching
 Diabetes Insipidus CAUSES OF HYPOKALEMIA
 Osmotic dieresis 1. GI Loss
 Loss of thirst  Vomiting, Diarrhea, Gastric suction, Intestinal
 Insensible loss of water tumor, Malabsorption
 Gastrointestinal loss of hypotonic fluid  Cancer therapy
 Excess intake of sodium  Large doses of laxatives
CAUSES OF HYPONATREMIA 2. Decreased Intake
 Diuretics, Potassium depletion 3. Renal Loss
 Aldosterone deficiency, Ketonuria  Diuretics – thiazides, mineralocorticoids
 Salt-losing nephropathy, Vomiting  Nephritis, Renal tubular acidosis,
Hyperaldosteronism; Cushing’s syndrome

COLLEGE OF MEDICAL LABORATORY 22 |

 Clinical Chemistry
 Hypomagnesemia CALCIUM
 Acute leukemia CHARACTERISITC
4. Cellular Shift
 present almost exclusively in the plasma
 Alkalosis, Insulin overdose
VALUES TO REMEMBER  involved in blood coagulation, enzyme activity,
excitability of skeletal and cardiac muscles and
 Reference Value: 3.5 – 5.2 mmol/L
maintenance of blood pressure
 Threshold Critical Value
 99% is part of the bones and 1% is in the blood and ECF
 Hyperkalemia:
FORMS OF CALCIUM
 Hypokalemia:
 Ionized (active) calcium - 50%
 Conversion Factor: mEq/L to mmol/L =
 Protein-bound calcium - 40%
EFFECTS OF POTASSIUM TO CARDIAC MUSCLES
 Complexed with anions - 10%
 8mmol/L – lack of muscle excitability
FACTORS AFFECTING SERUM CALCIUM LEVELS
 6-7mmol/L – may alter ECG

 10mmol/L – fatal (cardiac arrest)
 increases intestinal reabsorption of calcium
 3.0-3.4mmol/L – arrhythmia & paralysis
 increases reabsorption in the kidneys
CHLORIDE

CHARACTERISTIC
 activates the process of bone resorption
 major extracellular anion – chief counterion of sodium
 stimulates the conversion of inactive vit. D to active
 promotes maintenance of water balance and osmotic
vit. D3
pressure in conjunction with sodium

 only anion to serve as enzyme activator
 secreted by the parafollicular/C cells of the thyroid
 Functions: maintains osmolality, blood volume and
gland
electric neutrality
 hypocalcemic hormone – inhibits PTH and vit. D3
 Responsible for Chloride shift – an exchange
 inhibits bone resorption
mechanism between chloride and bicarbonate across
METHODOLOGIES
the membrane of RBCs
1. Precipitation and Redox Titration
METHODOLOGIES

 Interferences: bromide, cyanide, and cysteine o end product: oxalic acid (violet color)
 Mercurimetric Titration (Schales & Schales) 
 indicator: Diphenylcarbazone o end product: chloranilic acid (violet)
 endproduct: Mercuric chloride (blue violet) 2. Ortho-Cresolpthalein Complexone dyes
 Spectrophotometric method  Dye: Arzeno III
 Mercuric thiocyanate (Whitehorn titration) 3. EDTA titration method (Bachra, Dawer & Sobel)
 Ferric perchlorate 4. Ion selective electrode (Liquid-membrane)
 Colorimetric amperometric titration – Cotlove 5. Flame emission photometry
Chloridometer CLINICAL SIGNIFICANCE
 – most commonly used HYPERCALCEMIA
method
 Primary Hyperparathyroidism (Most Common PTH –
CLINICAL SIGNIFICANCE
mediated hypercalcemia)
HYPERCHLOREMIA  Familial hypocalciuric hypercalcemia
 Excess loss of bicarbonate  Ectopic secretion of PTH by neoplasms
 Renal tubular acidosis  Malignancy associated (most common non PTH
 Metabolic acidosis mediated hypercalcemia)
HYPOCHLOREMIA  Vitamin D intoxication, Thyrotoxicosis
 Prolonged vomiting, Diabetic ketoacidosis, Aldosterone  Hypoadrenalism, Immobilization with increased bone
deficiency turnover, Milk-alkali syndrome, Sarcoidosis, Multiple
 Salt-losing renal disease (eg Pyelonephritis) Myeloma
 High serum bicarbonate (eg compensated respiratory HYPOCALCEMIA
acidosis or metabolic acidosis)  Primary hypoparathyroidism
VALUES TO REMEMBER  Severe hypomagnesemia
 Reference Values: 98 – 107 mmol/L  Longstanding hypercalcemia
 Conversion Factor: mEq/L to mmol/L =  Pseudohypoparathyroidism
 Vitamin D deficiency, Chronic renal failure
 Renal tubulopathies, Fanconi’s syndrome

COLLEGE OF MEDICAL LABORATORY 23 |

 Clinical Chemistry
 Mutations of Vitamin D receptor MAGNESIUM
 Hypoalbuminemia, Acute pancreatitis CHARACTERISTIC
 Rhabdomyolysis
 intracellular cation second abundance to potassium;
 Tetany
also an enzyme activator
VALUES TO REMEMBER
 majority is stored in bones (53%)
 Reference Values:
 life threatening symptoms occur if the serum levels
 Total Calcium: 2.15 – 2.5 mmol/L reaches 5mmol/L
 Ionized Calcium: 1.16 – 1.32 mmol/L
FORMS OF MAGNESIUM
 Conversion Factor: mg/dL to mmol/L =
 Free Mg2+/Ionized form – 55%
INORGANIC PHOSPHOROUS
 Protein-bound Mg2+ - 30%
CHARACTERISTIC
 Complexed with ions – 15%
 related to calcium; FACTORS AFFECTING MAGNESIUM LEVELS IN BLOOD
maximally absorbed in the
 - increases
 organic phosphorous exists as:
renal and intestinal reabsorption of magnesium
 Organic phosphate – principal anion within  - increases
cells
 Inorganic phosphate – part of the blood buffer renal excretion of magnesium
 Only inorganic phosphate is measured in clinical METHODOLOGIES
laboratory
 Colorimetric methods
FORMS OF PHOSPHOROUS
 Calmagite
1. Free/Unbound form – 55%
 Formazen Dye
2. Complexes with ions – 35%
 Magnesium thymol blue
3. Protein-bound – 10%  – reference
FACTOS AFFECTING PHOSPHATE CONCENTRATIONS
method
1. PTH – decreases phosphate by renal excretion  Dye Lake method – Titan yellow dye (Clayton/Thiazole
2. Calcitonin – inhibits bone resorption
yellow)
3. Growth hormone – increase phosphate renal
CLINICAL SIGNIFICANCE
reabsorption
METHODOLOGIES HYPERMAGNESEMIA

  Acute or Chronic Renal Failure

 most commonly used method to measure inorganic  Hypothyroidism

phosphate  Hypoaldosteronism
 most common reducing agent: pictol  Hypopituitarism
 other reducing agents: elon, ascorbic acid and  Antacids
senidine  Enemas
 endproduct: ammonium-molybdate complex  Cathartics
CLINICAL SIGNIFICANCE  Dehydration
HYPERPHOSPHATEMIA  Bone carcinoma/Bone metastasis
HYPOMAGNESEMIA
 Renal Failure
 Increased breakdown of cells (eg Intravascular  Poor diet; Prolonged magnesium-deficient IV therapy,
hemolysis) Chronic alcoholsim
 Neoplastic disorders (eg Lymphoblastic leukemia)  Pancreatitis, Vomiting, Diarrhea
 Intensive exercise, Severe infections  Laxative abuse, Hyperparathyroidism,
HYPOPHOSPHATEMIA Hyperaldosteronism, Hyperthyroidism
 Hypercalcemia, Diabetic ketoacidosis
 Infusion of dextrose solution
 Diuretics, Excess lactation
 Use of antacids
 Pregnancy
 Alcohol withdrawal
 Tubular disorders, Glomerulonephritis, Pyelonephritis
 Poor diet
 Tetany
 Vomiting
VALUES TO REMEMBER
 Ketoacidosis
VALUES TO REMEMBER  Reference Value: 0.63 – 1.0 mmol/L
 Conversion factor: mEq/L to mmol/L =
 Reference Values: 0.87 – 1.45 mmol/L
 Conversion Factor: mg/dL to mmol/L =

COLLEGE OF MEDICAL LABORATORY 22 |

 Clinical Chemistry
ANION GAP
 the pH decreases by 0.015/each Celsius above 37°C
 is the difference between the unmeasured cations
(sodium and potassium) and unmeasured anions
(chloride and bicarbonate)  Evaluate ventilation by lungs
 form of quality control for the analyzer used to measure  is an index or efficiency of gas exchange and not a
these analytes measure of CO2 concentration in the blood
 Increased: renal failure/uremia, ketoacidosis (starvation
or diabetes), poisoning by methanol, ethanol, ethylene  Evaluate metabolic process
glycol or salicylate, lactic acidosis, hypernatremia and  the kidneys regulate pH by excreting acid and
instrument error reabsorption of HCO3 from the glomerular filtrate
 Decreased: hypoalbuminemia, hypercalcemia,
hyperlipidemia and elevated myeloma proteins
 Evaluate degree of oxygenation
 FORMULA:
 reflects the availability of the gas in blood but not its
 AG =
content
o RV: 8 – 16 mmol/L
Table 30. Levels of Hypoxemia
 AG =
pO2
o RV: 10 – 20 mmol/L
Mild 61 – 80 mmHg
Moderate 41 – 60 mmHg
ACID – BASE BALANCE, & VITAMINS
ORGANS ASSOCIATED WITH ACID-BASE Severe ≤ 40 mmHg
BALANCE MAINTENANCE CLINIAL SIGNIFICANCE
 LUNGS RESPIRATORY ACIDOSIS
 Help maintain acid-base balance through gas  pH: Decreased; HCO3: Decreased
exchange or respiration  Organ Defective: Lungs
 Rapid or short term compensation  Primary Cause: Hypoventilation
 Analyte(s) controlled: O2 & CO2  Organ to Compensate: Kidneys
 KIDNEYS  Primary Compensation: Bicarbonate Reabsorption
 Help maintain acid-base balance through  Example:
reabsorption or excretion of bicarbonate
 Slow but long term compensation RESPIRATORY ALKALOSIS
 Analye controlled: Bicarbonate (HCO -)
3  pH: Increased; HCO3: Increased
BLOOD BUFFERS
1. Bicarbonate and carbonic acid – major extracellular  Organ Defective: Lungs
blood buffer  Primary Cause: Hyperventilation
2. Plasma proteins  Organ to Compensate: Kidneys
3. Hemoglobin  Primary Compensation: Bicarbonate Excretion
4. Inorganic phosphate  Example:
HENDERSON-HASSELBACH EQUATION METABOLIC ACIDOSIS

 Where 𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒  pH: Decreased; HCO3: Decreased

pH = pKa + log
 pKa = 6.1 𝑤𝑒𝑎𝑘 𝑎𝑐𝑖𝑑  Organ Defective: Kidneys
 conjugate base = bicarbonate (HCO ) -
 Primary Cause: Bicarbonate Excretion
3
 weak acid = carbonic acid (H2CO3)  Organ to Compensate: Lungs
𝐻𝐶𝑂3−  Primary Compensation: Hyperventilation
pH = 6.1 + log total CO2 = 𝐻𝐶𝑂323
− + 𝐻 𝐶𝑂
𝐻2𝐶𝑂3  Example:
𝐻𝐶𝑂3− = total CO2 - 𝐻
23𝐶𝑂 𝐻23
𝐶𝑂 = 0.03 x pCO2 METABOLIC ALKALOSIS
PARAMETERS  pH: Increased; HCO3: Increased
PH  Organ Defective: Kidneys
 pH of 7.40 is the optimum level for arterial blood  Primary Cause: Bicarbonate Reabsorption
 the reference range for arterial blood (7.35-7.45) is only  Organ to Compensate: Lungs
0.03 pH unit lower for venous blood owing to the  Primary Compensation: Hypoventilation
buffering effects of hemoglobin known as chloride  Example:
isohydric shift SPECIMEN COLLECTION
 specimen: arterial blood
 anticoagulant: 0.05ml heparin/ml of blood

COLLEGE OF MEDICAL LABORATORY 23 |

 Clinical Chemistry
 syringe and needle for arterial blood must be Table 33. Water Soluble Vitamins
preheparinized
Chemical Name Deficiency
 use of butterfly infusion sets is not recommended
Vitamin B1 Thiamine Infants: Dyspnea,
 liquid form of heparin is not recommended cyanosis, diarrhea,
 Samples should be analyzed immediately (<30mins) vomiting
SPECIMEN CONSIDERATIONS Adults: Beri-beri,
 On standing: Wernicke-Korsakoff
syndrome (apathy, ataxia
 Exposure to air: and visual problems)
Vitamin B2 Riboflavin Angular stomatitis,
 Blood samples should be chilled dermatitis, photophobia
 Glycolysis results to Vitamin B3 Panthothenic acid Depressed immune
METHODOLOGIES system, muscle weakness
GASOMETER Vitamin B6 Pyridoxine, Infants: Irritability,
 Van Slyke Pyridoxal seizures, anemia
 Natelson Adult: facial seborrhea
 mercury – to produce vacuum Vitamin B12 Cyanocobalamin Megaloblastic anemia,
 caprylic alcohol – anti-foam reagent Neurologic abnormalities
 lactic acid Vitamin C Ascorbic Acid Scurvy
 NaOH and NaHSO3 Folic Acid Pterolyglutamic Megaloblastic anemia
ELECTRODES acid
Niacin Nicotinic Pellagra (dermatitis,
 pH (potentiomentry)
acid/Nicotinamide disorientation, weight

loss)
– internal reference electrode
 –
ENDOCRINOLOGY
external reference electrode
 Endocrine System: network of ductless glands that
 pO2 – (polarography-amperometry)
secrete hormones directly into the blood; regulatory
 pCO2 – (potentiometry)
system of blood
VALUES TO REMEMBER
Table 31. Normal Values  Hormones: are chemical signals produced by specialized
cells secreted into the blood stream and carried to
Normal Value
target tissue; Major function: to maintain the constancy
pH
of chemical composition of extracellular and intracellular
pO2
fluids and control metabolism, growth, fertility and
pCO2
- responses to stress.
HCO3
FEEDBACK MECHANISMS
Total CO2 (arterial)
 Positive feedback mechanism –an increase in the
Total CO2 (venous)
product results to elevation of the activity of the system
and the production rate.
VITAMINS
Table 32. Fat Soluble Vitamins  Negative feedback mechanism –an increase in the
product results to decreased activity of the system and
the production rate.
Chemical Name Deficiency CLASSIFICATION OF HORMONES
Vitamin A Retinol Night blindness, growth Table 34. Classification of Hormones
retardation, abnormal Proteins/ Glycoprotein Steroids Amino acid
taste response, Peptides derivative
dermatitis GHRH, CRH, TSH, LH, FSH, Cortisol, Melatonin,
Vitamin E Tocopherols Mild hemolytic anemia TRH, GnRH, hCG, EPO Aldosterone, Serotonin,
(newborn), RBC fragility, Somatostatin, Estrogen, T3 , T4,
ataxia PRF, ADH, Progesterone, Epinephrine,
Vitamin D Calciferol Rickets and Osteomalacia oxytocin, GH, Testosterone, Nor-
Vitamin K Phylloquinones, Bleeding disorder, ACTH, PRL, Vitamin D epinephrine
Menaquinones Hemorrhage Calcitonin,
hPL, PTH,
Insulin,
Glucagon

COLLEGE OF MEDICAL LABORATORY 24 |

 Clinical Chemistry
HYPOTHALAMUS
GONADOTROPINS (FSH – FOLLICLE STIMULATING
 above the pituitary gland and is connected to the
HORMONE/LH – LUTEINIZING HORMONE)
posterior pituitary by the infundibulum (pituitary stalk)
 important markers in diagnosing fertility and menstrual
 supraoptic and paraventricular nuclei produce
cycle disorders
vasopressin and oxytocin
 aids in spermatogenesis in males
HYPOTHALAMIC HORMONES
 helps Leydig cells to
1. Thyrotropin-releasing hormone (TRH) produce testosterone (male) and for female, it is
2. Gonadotropin-releasing hormone (GnRH) necessary for ovulation and the final follicular growth
3. Somatostatin/Growth hormone-inhibiting hormone  also acts on thecal cells
(GH- IH)
to cause synthesis of androgens
4. Growth hormone-releasing hormone (GH-RH)
THYROID STIMULATING HORMONE (TSH)
5. Prolactin-inhibiting factor (PIF)
6. Vasopressin (supraoptic)  also known as thyrotropin
7. Oxytocin (paraventricular nuclei)  main stimulus for the uptake of iodide
PINEAL GLAND  stimulates thyroid hormone synthesis
 secretes ADRENOCORTICOTROPIC HORMONE (ACTH)
that decreases pigmentation of the skin  produced in response to low serum cortisol
PITUITARY GLAND  highest levels is between 6:00am to 8:00am; lowest
 also known as the “master gland” level is between 6:00pm to 11:00pm
 located in a small cavity called the sella turcica or  increased: Addison’s disease, ectopic tumors, after
Turkish saddle protein-rich meals
 All pituitary hormones have circadian rhythms  best time for collecting specimen: 8:00am to 10:00am
ADENOHYPOPHYSEAL CELLS PROLACTIN (PRL)
1. – secrete growth hormone  functions in the initiation and maintenance of lactation
2. – secrete prolactin  major inhibitor:
3. – secretes TSH  consequence of prolactin excess: hypogonadism
4. – secretes LH and FSH
 specimen requirement: blood should be collected 3-4
5. – secretes proopiomelanocortin
hours after the individual has awakened; fasting sample
(cleaved to produce ACTH, β-endorphin and β-
lipotropin) POSTERIOR PITUITARY GLAND
ANTERIOR PITUITARY GLAND  capable of releasing hormones’ oxytocin and vasopressin
(ADENOHYPOPHYSIS) but not capable of producing it
GROWTH HORMONE (GH/SOMATOTROPIN) OXYTOCIN
 most abundant of all pituitary hormones  stimulates contraction of the gravid uterus at term –
 controlled by GH-RH (the amount released) and “Fergusson reflex”
somatostatin (governs the frequency and duration of ANTI-DIURETIC HORMONE (ADH)/ARGININE VASOPRESSIN
secretory pulse) (AVP)
 major stimulus:  acts on the distal convoluted tubule and collecting duct
 Disorders:  major function: maintain osmotic homeostasis by
 Idiopathic GH deficiency – most common cause of regulating water balance
pituitary dwarfism in children  promotes reabsorption of water by the renal tubules –
 Pituitary adenoma – most common etiology in maintains water homeostasis
adult-onset GH deficiency  DIABETES INSIPIDUS (DI) is deficiency of ADH; results in
 Acromegaly – due to overproduction of GH severe polyuria
Table 35. Diagnostic Test for Growth  Clinical Picture for Diabetes Insipidus
Hormone o Normoglycemia, Polyuria with low SG,
Screening Tests Confirmatory Tests Polydipsia, Polyphagia
GH deficiency Physical activity Insulin tolerance 
(Exercise) test test (gold standard)
Arginine stimulation o deficiency of ADH with normal ADH receptor
test (2nd 
confirmatory test) o normal ADH but abnormal ADH receptor –
Acromegaly Somatomedin-C or Glucose suppression renal resistance to ADH action
Insulin-like growth test – OGTT (75g o failure of the kidneys to respond to normal or
factor glucose) elevated ADH levels

COLLEGE OF MEDICAL LABORATORY 25 |

 Clinical Chemistry
 Diagnostic test for DI – REIDEL’S THYROIDITIS

THYROID GLAND  thyroid turns into a woody or stony-hard mass

 also known as the “butterfly-shaped’ gland SUBCLINICAL HYPERTHYROIDISM
 consist of two lobes connected by the isthmus located  shows no clinical symptom but TSH is low and FT3 and
at the lower part of the neck FT4 are normal
 follicle is the fundamental unit of the thyroid gland
 2 types of cells: follicular cells (T3 and T4) and
parafollicular cells/C cells (Calcitonin)  associated with neck pain, low grade fever and swings in
 is a glycoprotein thyroid function test
stored in the follicular colloid of the thyroid gland HYPOTHYROIDISM
 is the most
 Signs and symptoms: bradycardia, weight gain,
important element in the biosynthesis of thyroid
coarsened skin, cold intolerance and mental dullness
hormones
PRIMARY HYPOTHYROIDISM
MAJOR THYROID HORMONES
 primarily due to deficiency in elemental iodine
TRIIODOTHYRONINE (T3)  also caused by destruction or ablation of the thyroid
 gland
 principal application of this hormone is in the diagnosis 
of T3 thyrotoxicosis  most common cause of primary hypothyroidism
  characterized by thyroid replaced by a nest of
 an increase in the plasma level of T3 is the first lymphoid tissue
abnormality seen in cases of hyperthyroidism  lab result: high TSH and positive TPO antibody
TETROIODOTHYRONINE (T4)  Myxedema
  myxedema coma is the severe form of primary
 major fraction of organic iodine hypothyroidism
 the amount of serum T4 is a good indicator of thyroid  peculiar non-pitting swelling of the skin
secretory rate  clinical features: “puffy” face, weight gain, slow
THYROID-HORMONE BINDING PROTEINS speech, eyebrow thinned, dry and yellow skin and
1. anemia
 transports majority of T3 (affinity for T3 is lower than Table 36. Autoimmune Disorders of the
T4 ) Thyroid Hormone
 transport 70-75% of total T4 Hashimoto’s Thyroiditis Grave’s Disease
2. Thyroxine-binding prealbumin (transthyretin)
 transports 15-20% of total T4
 T3 has no affinity for prealbumin
3. Thyroxine-binding albumin
 transports T3 and 10% of T4
HYPERTHYROIDISM

 Signs and symptoms: tachycardia, tremors, weight loss, SECONDARY HYPOTHYROIDISM

heat intolerance, emotional lability and menstrual  due to pituitary destruction or pituitary adenoma
changes
TERTIARY HYPOTHYROIDISM
THYROTOXICOSIS
 due to hypothalamic disease
 high levels of free thyroid hormones in the circulation
CONGENITAL HYPOTHYROIDISM
 T3 thyrotoxicosis/Plummer’s disease: FT3 increased
 defect in the development or function of the gland
but FT4 normal with low TSH
 symptoms: retarded physical and mental development
 T4 thyrotoxicosis: T3 normal or low but T4 is increased
of the child
with low TSH
Table 37. Clinical Presentation of Patients
with Hypothyroidism & Hyperthyroidism
 most common cause of thyrotoxicosis
 antibodies are produced which are active against the Hypothyroidism Hyperthyroidism
TSH receptor Basal metabolic
 features: exophthalmos (bulging eyes) and pretibial rate
myxedema Sympathetic
 diagnostic test: TSH receptor antibody test response
Weight

COLLEGE OF MEDICAL LABORATORY 26 |

 Clinical Chemistry

Temperature  inversely related to TBG – decreased T3 uptake results

tolerance to elevated TBG result
THYROXINE-BINDING GLOBULIN (TBG)

GI function  used to confirm results of FT 3 and FT4 or abnormalities in

the relationship of the total thyroxine (TT4) and THBR
test
Cardiovascular  useful in distinguishing between hyperthyroidism
function (elevated T4 + N TBG) and euthyroidism (elevated T4 +
elevated TBG)
Table 38. Thyroid Gland Disturbances
Respiratory T3 & T4 TSH
function Primary Hypothyroidism
General Secondary Hypothyroidism
appearance Primary Hyperthyroidism
Secondary Hyperthyroidism
Table 39. Secondary vs Tertiary
General behavior Mental Hypothyroidism
Restlessness
retardation; Hypothyroidism Secondary Tertiary
Irritability
Mental & T3 & T4
Anxiety
Physical TSH
Hyperkinesis
sluggishness TRH
Wakefulness
Somnolence TRH Stimulation/ TSH before TSH before
Others Increased Administration administration: administration:
Cholesterol & Increased ALP Test
TAG TSH after TSH after
THYROID FUNCTION TESTS administration: administration:

TRH STIMULATION TEST PARATHYROID GLAND

 used to differentiate euthyroid and hyperthyroid  located on or near the thyroid capsule; most people
patients who both had undetectable TSH levels have 4 parathyroid hormones
 used to confirm borderline cases and euthyroid  smallest endocrine gland; secretes parathyroid hormone
Grave’s disease – hypercalcemic hormone
PARATHYROID HORMONE

 most important thyroid function test – best method for  to prevent hypocalcemia (regulates blood calcium)
detecting clinically significant thyroid dysfunction  preserve calcium and phosphate within normal range
 promotes bone resorption – release calcium in
 most clinically sensitive assay in the detection of primary the bloodstream
thyroid disorders  increases renal calcium reabsorption
REVERSE T3 (rT3)  stimulates conversion of inactive vitamin D to activated
 formed from the removal of one iodine from the inner vitamin D3
ring of T4 HYPERPARATHYROIDISM
 rd
3 major circulating thyroid hormone  Primary Hyperparathyroidism
FREE THYROXINE INDEX (FTI OR T7)  physiologic defect lies within the parathyroid gland
 indirectly assesses the level of T4 in the blood  most common cause of hypercalcemia
 FTI = TT4 x THBR  due to the presence of a functioning parathyroid
 FTI = TT4 𝑇3𝑈 (%)
adenoma
x 100
TOTAL T3 (TT3), FREE T3 (FT3) AND FREE T4 (FT4)  Secondary hyperparathyroidism
 used to differentiate drug-induced TSH elevation and  develops in response to decreased calcium
hypothyroidism  diffuse hyperplasia of all 4 glands
 the value of TT3 or FT3 is in confirming hyperthyroidism  Tertiary hyperparathyroidism
T3 UPTAKE  phosphate levels are normal to high; calcium
 measures the number of available binding sites of the phosphates precipitate in soft tissues
thyroxine-binding proteins (TBG)
 does not measure the level of T3 in serum but it reflects
the serum level of TBG

COLLEGE OF MEDICAL LABORATORY 27 |

 Clinical Chemistry
Table 40. Hyperparathyroidism Levels
 signs and symptoms: obesity but with thin extremities
Primary Secondary (“buffalo hump”), hirsutism, hyperglycemia, thinning
of the skin, poor wound healing, hypertension,
Serum Calcium
hypercholesterolemia, low WBC count (lymphocytes)
Serum
Phosphate  – current

PTH reference method for measuring urinary free cortisol

 – most sensitive
Urine Calcium
and screening test
Urine Phosphate
Table 43. Diagnostic Tests for
VALUES TO REMEMBER
Cushing’s Syndrome
 Calcium level = laryngeal
Screening Tests Confirmatory Tests
stridor and tonic clonic seizures
 Calcium level = tetany and 24-hour urinary free Low dose dexamethasone
altered neuromuscular activity cortisol test suppression test
Overnight dexamethasone Midnight plasma cortisol
ADRENAL GLAND suppression test Corticotropin-releasing
 has pyramid-like shape located above the kidneys Midnight salivary cortisol hormone (CRH) stimulation
 composed of distinct but conjoined glands, the outer test test
adrenal cortex (yellow) and inner adrenal medulla (dark
HYPOCORTISOLISM
mahogany)
 screening test:
ADRENAL CORTEX

 major site of steroid hormone production  confirmatory test: Insulin tolerance test
Table 41. Adrenal Cortex Layers  – gold
Layer Function standard for secondary and tertiary hypocortisolism
 Overnight metyrapone test – alternative diagnostic or
Zona glomerulosa (Outer
confirmatory test for seconda
10%)
 ry or tertiary adrenal insufficiency
Zona fasciculata (Middle
 Primary hypocorticolism (primary adrenal insufficiency)
75%)
 due to decreased cortisol production – 90%
Zona reticularis (Inner 10%)
destruction of adrenal cortex
 disorder: Addison’s disease – hypotension,
hyponatremia, hyperkalemia, weight loss,
CORTISO
hyperpigmentation
 is the principal glucocorticoid
 Secondary hypocorticolism (secondary adrenal
 mostly bound to glycoprotein – transcortin insufficiency)
 stimulates gluconeogenesis in the liver resulting in  due to hypothalamic-pituitary insufficiency with
hyperglycemia loss of ACTH
 is the only adrenal hormone that inhibits the secretion  absence of hyperpigmentation
of ACTH
 highest level and
 results in the deficiency of enzymes necessary for the
lowest at night
synthesis of cholesterol which will result to decreased
 specimen: serum (red top), urine; blood sample should
plasma cortisol and increased ACTH and androgen levels
be drawn at 8:00 AM
 definitive tests:
 urine free cortisol levels are sensitive indicators of
 17-OHP measurement in amniotic fluid
adrenal hyperfunction (endogenous hypecorticolism) –
 Genotyping cells from chorionic villous sampling –
24 hour urine collection
preferred
Table 42. Cortisol Urinary Metabolites
ALDOSTERONE
Method Reagent
17- Phenylhydrazine  most potent mineralocorticoid (electro-regulating
hydroxycorticosteroid in H2SO4 + hormone)
alcohol  helps regulate water and electrolytes (sodium, chloride
17-ketogenic steroids Meta- and potassium) and blood pressure
dinitrobenzene  acts on renal tubular epithelium to increase retention of
Na+ and Cl- and excretion of K+ and H+
Hypercortisolism (Cushing’s syndrome)  method: RIA and chromatography
 caused primarily by excessive production of cortisol and
ACTH but decreased aldosterone and renin

COLLEGE OF MEDICAL LABORATORY 28 |

 Clinical Chemistry
PRIMARY HYPERALDOSTERONISM (CONN’S DISEASE) EPINEPHRINE (ADRENALINE/SECONDARY AMINE)
 caused by aldosterone-secreting adrenal adenoma
 most abundant medullary hormone
 associated with elevated plasma aldosterone and low
 produced from norepinephrine and only from the
plasma renin
adrenal gland
Table 44. Tests for Conn’s Disease
 called the “flight or fight” hormone: release in
Screening Test Confirmatory Test
response to physiologic (injuries) or psychologic
Plasma aldosterone Saline suppression test
(stress, anxiety) threats
concentration/plasma renin Oral sodium loading test
Fludrocortisone  major metabolite: :
activity ratio
suppression test major (60%) catecholamine metabolite in the urine
 >30 ratio: Captopril challenge test  minor metabolites: metanephrine, normetanephrine
and homovanillic acid
DOPAMINE (PRIMARY AMINE)
 >60 ratio:
 catecholamine produced via the decarboxylation of 3,4-
dihydroxyphenylalanine
SECONDARY HYPERALDOSTERONISM  major intact catecholamine in the urine
 major metabolite:
 occurs as a result of excessive renin production
PHEOCHROMOCYTOMA
 accompanied by elevated plasma levels of aldosterone
and renin  tumor of the adrenal medulla or sympathetic ganglia
 associated disorders: Liddle’s syndrome  due to the overproduction of catecholamine
(pseudohypoaldosteronism), Barterr’s syndrome  screening test: plasma metanephrines and
(bumetanide-sensitive chloride channel mutation), normetanephrines by HPLC (four-fold increase)
Gitelman’s syndrome (thiazide-sensitive transporter  diagnostic test: 24-hour urinary excretion of
mutation) metanephrines and normetanephrine
HYPOALDOSTERONISM  pharmacologic test: clonidine test and glucagon
 due to the destruction of the adrenal glands and stimulation test
deficiency of glucocorticoid Patient preparation in the diagnosis of Pheochromocytoma
 also associated with 21-hydroxylase deficiency  Avoid caffeine, nicotine, alcohol and acetaminophen,
 Tests for hypoaldosteronism monoamine oxidase inhibitors and tricyclic
 Furosemide stimulation test or upright posture antidepressants for at least 5 days before testing
 Saline suppression test NEUROBLASTOMA
WEAK ANDROGENS
 fatal malignant condition in children resulting to
 serve as precursors for the production of more potent excessive production of norepinephrine
androgen and estrogen in tissues  (+) high urinary excretion of HVA or VMA or both and
 example: dehydroepiandrosterone (DHEA) and dopamine
androstenedione METHODOLOGIES
 : principal adrenal
androgen; converted into estrone  Specimen:
 Patient preparation:
 excessive production of androgens results in virilization
(pseudohermaphroditism)  Patient should undergo overnight fasting
 Avoid smoking or drinking coffee at least 4 hours
ADRENAL MEDULLA
prior to blood collection
 composed primarily of chromaffin cells that secrete  Blood is collected on pre-chilled EDTA tubes
catecholamines  Specimen considerations:
 L-tyrosine is the precursor of catecholamines  Catheterization is the preferred method of blood
 ratio of norepinephrine to epinephrine is 9:1 collection
NOREPINEPHRINE (PRIMARY AMINE)
 Urine is preserved with 10ml of 6N HCl
 produced by the sympathetic ganglia 1. Chromatography – HPLC or GC-MS (VMA and
 highest concentration is in the brain (CNS) metanephrines)
 major metabolite in CNS is MHPG 2. Radioimmunoassay – sensitive screening test for total
MAJOR METABOLITES OF NOREPINEPHRINE plasma catecholamines
 3-methoxy-4-hydroxyphenylglycol (MHPG) – CSF and  >2000pg/ml of plasma catecholamines: diagnostic
urine for pheochromocytoma
 vanillylmandelic acid

COLLEGE OF MEDICAL LABORATORY 29 |

 Clinical Chemistry
REPRODUCTIVE HORMONES
 prime secretory product of the ovary
 testes and ovaries produce sex steroids such as
 single best hormone to determine whether ovulation
androgens and estrogens from cholesterol
has occurred
 major transport proteins: sex-hormone binding globulin  deficiency: failure of implantation of embryo
(SHBG), corticosteroid-binding globulin (CBG) and  metabolites: pregnanediols (most easily measured
albumin metabolite), pregnanediones, pregnanalones
TESTOSTERONE HUMAN CHORIONIC GONADOTROPIN (HCG)

 principal androgen hormone in the blood – most potent  produced by the trophoblast cells of the placenta during
male androgen pregnancy
 synthesized by the Leydig cells of the testis of the male;  same α-subunit as LH, FSH and TSH; similar β-subunit
controlled primarily by the FSH and LH to LH – “LH-like” hormone
 function: growth and development of the reproductive  intact HCG (α and β) is the predominant form
system, prostate and external ganglia throughout pregnancy
 tests for male fertility: semen analysis, testosterone,  method: immunometric (sandwich) method – serum
FSH and LH and urine samples
 transport proteins: SHBG (60%) and albumin (40%) INHIBIN A
Table 45. Types of Testicular
 Reproductive hormone which inhibits FSH activity
Infertility (Hypogonadism) METHODOLOGIES
Testos- FSH & Description/ other  Porter-Silber – for 17-OHCS
terone LH information  Zimmerman – steroids with 17-keto structure
Pre-  Pisano – quantitating metanephrines and
Due to hypothalamic
testicular normetanephrines
or pituitary lesions
(Secondary)  Kober – estrogen
May be congenital TESTS FOR MENSTRUAL CYCLE DYSFUNCTION
(e.g. Klinefelter’s 
Testicular
syndrome) or TESTS FOR FEMALE INFERTILITY
(Primary)
acquired (e.g.

varicocele)
MARKERS FOR DOWN SYNDROME
Disorders of sperm
Post-  Increased:
transport and
testicular  Decreased:
function GASTRIN
DEHYDROEPIANDROSTERONE (DHEA)
 peptide secreted by the G cells of the antrum of the
 principal androgen formed by the adrenal cortex; weak stomach
androgen  causes secretion of the HCl by parietal cells in the
ESTROGEN stomach
 functions: promotion of breast development,  diagnostic marker for Zollinger-Ellison syndrome (islet-
maturation of the external genitalia, deposition of body cell tumor)
fat and termination of linear growth (secondary sexual SEROTONIN (5-HYDROXYTRYPTAMINE)
characteristics in the female)  derived from the hydroxylation and decarboxylation of
 precursor: acetate, cholesterol, progesterone and tryptophan
testosterone  synthesize by argentaffin cells, primarily in the GI tract
FORMS OF ESTROGEN  diagnostic marker for carcinoid tumor
1. – most abundant estrogen in post-  tests: Ehrlich’s aldehyde test – (+) purple color
menopausal women
2. – most potent estrogen; major THERAPEUTIC DRUG MONITORING
estrogen  a quantitative procedure performed for drugs with
 most abundant estrogen in pre-menopausal women narrow therapeutic index
 transport proteins: albumin (60%), SHBG (38%)  the half-life of the drug determines the time to reach
3. – estrogen found in maternal urine the steady-state or average concentration
 major estrogen secreted by the placenta during  Mixed Function Oxidase (MFO) system: biochemical
pregnancy pathway responsible for the greatest portion of drug
PROGESTERONE metabolism

 produced mainly by the granulose (lutein) cells of the

corpus luteum in the female

COLLEGE OF MEDICAL LABORATORY 30 |

 Clinical Chemistry
PHARMACOLOGICAL PARAMETERS THAT ANTIBIOTICS
DETERMINE DRUG CONCENTRATION Aminoglycosides
1. Liberation – release of the drug
 Gentamicin, tobramycin, amikacin, kanamycin,
2. Absorption – transport of the drug from the site of
neomycin, streptomycin
administration to the blood
 Used for treatment of Gram-negative bacterial infection
3. Distribution – delivery of the drug to the tissues
Lucy Driscoll  Toxic effects: nephrotoxocity and ototoxicity
4. Metabolism – process of chemical modification of the
Vancomycin
drug by cells
 Used against Gram-positive bacilli and cocci
5. Excretion – process by which the drug and its
 Toxic effects: “ red man syndrome ”,
metabolites are excreted from the body
nephrotoxicity, ototoxicity
TERMINOLOGIES
1. Bioavailable fraction (f) – fraction of the dose that CHLORAMPHENICOL
reaches the blood  Toxic effects: blood dyscrasia, cytoplasmic vacuolation
2. First pass hepatic metabolism – drugs that are (erythroid and myeloid cells)
transported to the liver lost a fraction of its ANTI-EPILEPTIC DRUGS
bioavailability before the drug reaches the general Phenobarbital
circulation
3. Half-life (t1/2) – time required to reduce a drug level to
 Long acting barbiturate that controls grand-mal tonic
half its original value
4. peak concentration – highest clonic seizures and focal epilepsies
concentration of a drug obtained in the dosing interval Phenytoin (Dilantin)
5. trough concentration – lowest  Controls tonic-clonic, simple partial seizures; a short
concentration of a drug obtained in the dosing term prophylactic agent in brain injury
interval
6. pharmacodynamics – relationship Valproic Acid
between the drug concentration at the target site and VERAPAMIL
response of the tissues  Treatment of angina, hypertension and supraventricular
7.
pharmacokinetics _ – mathematical expression arrhythmia
of the relationship between drug dose and drug blood
level
8. Pharmacogenomics – study of genes that affect the
performance of a drug in an individual
THERAPEUTIC DRUGS
CARDIOACTIVE DRUGS

 Class I – rapid Na+ blockers

 Class II – beta receptor blockers
 Class III – K+ channel blockers
 Class IV – Ca2+ channel blockers
DIGOXIN
 Treatment of atrial arrhythmia and congestive heart
failure (CHF)
LIDOCAINE (XYLOCAINE)
 Correct ventricular arrhythmia and treatment of MI
QUINIDINE
 Treatment of arrhythmia
procainamide
 Treatment of ventricular arrhythmia
 Toxic effects: reversible lupus-like- syndrome
DISOPYRAMIDE
 Has anti-cholinergic effects – dry mouth and
constipation
PROPANOLOL
 Used in treatment of thyrotoxicosis
AMIODARONE
 Iodine-containing drug which can cause hypo or
hyperthyroidism

COLLEGE OF MEDICAL LABORATORY 31 |

 Clinical Chemistry
 Treatment of petit mal (absence seizures), atomic
seizure and grand mal
Carbamazepine
 Effective for grand mal seizures and treating
seizures accompanied by pain
ETHOSUXIMIDE
 Drug of choice in controlling petit mal (absence) seizure
Gabaentin
 Chemically similar to neurotransmitter
gamma aminobutyric acid
 Adverse effects: dizziness, ataxia, fatigue and nystagmus
PSYCHOACTIVE DRUGS

LITHIUM
 Used for treatment of manic-depressive bipolar
disorders
TRICYCLIC ANTI-DEPRESSANTS
 Used for treatment of depression, insomnia and
extreme apathy
 Major metabolite: desipramine
FLUOXETINE (PROZAC)
 Treatment of obsessive-compulsive disorders
BRONCHODILATOR
thoeophylline
 Treatment of asthma and chronic obstructive
pulmonary disease
IMMUNOSUPPRESIVE DRUGS

CYCLOSPORINE
 For suppression of acute graft-versus-host disease
(GVHD)
 Specimen of choice: whole blood

COLLEGE OF MEDICAL LABORATORY 32 |

 Clinical Chemistry
TACROLIMUS
 Peak concentrations are drawn one hour after an orally
 100x more powerful than cyclosporine administered dose and immediately after infusion for an
 Toxic effects: thrombus formation, nephrotoxicity, IV administered drug; best specimen
neurotoxicity COLORIMETRY
RAPAMYCIN
 Acetaminophen in urine is detected by boiling to form p-
 Similar to tacrolimus with major side effects of
amphenol which then reacts with o-cresol to form
thrombocytopenia and lipid abnormalities
indophenol blue
MYCOPHENOLATE MOFETIL
 Trinder assay for salicylate using ferric nitrate forming a
 Decreases renal allograft rejection
colored complex
LEFLUNOMIDE (LFM)
IMMUNOASSAY METHODS
 Inhibits lymphocyte proliferation; treatment of RA
ANTINEOPLASTIC DRUGS  can detect drug levels in nanomolar range; sensitive and
specific methods
METHOTREXATE
 Enzyme-mediated (multiplied) immunologic technique
 Inhibits DNA synthesis in all cells
(EMIT)
 Leucovorin rescue: reverse action of methotrexate
 Fluorescence polarization immunoassay (FPIA)
BUSULFAN CHROMATOGRAPHIC METHODS
 Treatment of leukemias and lymphomas prior to
 best specimen: urine
bone marrow transplantation  Thin Layer Chromatography
ANTI-INFLAMMATORY/ANALGESICS
 quantitatively identifies drugs by means of their Rf
Acetyl salicylic Acid/ salicylates(aspirin) values
 Commonly used analgesic, anti-pyretic and anti-  extraction of drugs is pH dependent – acidic drugs
inflammatory drug at pH 4.5 and alkaline drugs at pH 9.0
 Has anticoagulant property by cylooxygenase  HPLC
inhibiting
 Acute aspirin intoxication: common cause of fatal drug  ideal for separation of tricyclic antidepressants and
poisoning in children its metabolites
 Method: Trinder assay, HPLC, EMIT, Enzymatic assay  GC-MS
–
Acetaminophen (tylenol)(paracetamol) gold standard
 Commonly used analgesic and anti-pyretic drug
 Over dosage leads to hepatotoxicity TOXICOLOGY
antidote: N- acetylcysteine ALCOHOLS
 Method: HPLC
ETHANOL (GRAIN ALCOHOL)
Ibuprofen
 Lower risk of toxicities than salicylates and  most common abused drug
IN USA: ETHANOL
IN PHIL: CAFFEINE
acetaminophen  antidote for chronic intoxication: diazepam (for
alcoholic mania)
NEUROLEPTICS (ANTI-PSYCHOTIC
MAJOR  specimen: serum (capillary and arterial blood are
 Block the action of dopamine and serotonin in the limbic preferred)
system  methods for testing: enzymatic, gas-liquid
 Used in the treatment of acute schizophrenia chromatography and electrochemical oxidation
 2 classes: phenothiazines (chlorpromazine) and  preferred method: enzymatic using alcohol
butyrophenones (haloperidol) dehydrogenase reagent
> 0.05 % W/V (50
 Examples: risoperdal, olanzapine, quetiapine, mg/dL) = symptoms of

aripiprazole alcohol intoxication begin to manifest
 > 100 mg/dL
METHODOLOGIES FOR TDM = legally intoxicated
 > or = to 0.10%
SPECIMEN CONSIDERATIONS w/v
= presumptive
 Trough concentrations are drawn immediately or 30
 Specimen of choice: serum or plasma minutes before the next dose; lowest level of drug in blood
 Whole blood EDTA sample is required for cyclosporine
and
 tacrolimus test
 Timing of the specimen collection is the single most
important factor in TDM

COLLEGE OF MEDICAL LABORATORY 33 |

 Clinical Chemistry
evidence of driving under influence of alcohol
Table 46. Stages of Ethanol Impairment
Alcohol (%, w/v) SxS
0.01 – 0.05 No obvious impairment, some changes
observable on performance testing
0.03 – 0.12 Mild euphoria, decreased inhibitions,
some impairment of motor skills

COLLEGE OF MEDICAL LABORATORY 34 |

 Clinical Chemistry

0.09 – 0.25 Decreased inhibitions, loss of critical MERCURY

 inhibits catecholamine-0-methytransferase
judgment, memory impairment,
diminished reaction time  has the ability to “amalgamate” – mix or merge with
other substances
0.18 – 0.30 Mental confusion, dizziness, strongly
impaired motor skills (staggering,  forms of mercury: elemental/metallic mercury,
mercurous, mercuric and alkyl mercury
slurred speech)
 major toxic effect of elemental mercury: pink dse (acrodymia)
0.27 – 0.40 Unable to stand or walk, vomiting,
and erethism
impaired consciousness
 major toxic effect of alkyl mercury: congenital
0.35 – 0.50 Coma and possible death
Minimata disease
METHANOL (WOOD ALCOHOL)
 samples: whole blood and 24-hour urine
 commonly used solvent and a contaminant of
homemade liquors  method: Reinsch test

 Symptoms of intoxication: frank blindness (ocular LEAD

 blocks delta aminolevulinic acid (ALA) synthetase
toxicity) and metabolic acidosis , pyrimidine 5' nucleotidase and Na K dependent ATPase
 screening test: computation of osmolal gap
 preferred method: GC-MS
 source: paints and gasoline
ISOPROPANOL (RUBBING ALCOHOL)
 indications of toxicity: urinary aminolevulinic acid, free
 metabolized by hepatic alcohol dehydrogenase into RBC proporphyrin and presence of
acetone Basophilic Stippling in RBC
 preferred method: gas chromatography  lead chelators: EDTA and dimercaptosuccinic acid (DMA)
 antidote: activated charcoal  characteristic “wrist drop” or “foot drop”
ETHYLENE GLYCOL (1,2 – ETHANEDIOL)
manifestation (peripheral neuropathy)
 common constituent of hydraulic fluid and anti-freeze  samples: whole blood (quantitative testing), urine
 converted to oxalic acid and glycolic acid by hepatic (recent lead exposure), hair
alcohol dehydrogenase LABORATORY TESTS FOR LEAD
 indication of toxicity: deposition of calcium oxalate
1. Screening test - Zinc protoporphyrin test
crystals in renal tubules (monohydrate/ dumbell) (Fluorometric) δ-ALA dehydratase test (sensitive)
 major metabolite: glycolic acid (cause of acute toxicity 2. In-vivo x-ray fluorescence of bones
and death) 3. Atomic absorption spectrophotometry
 preferred method: HPLC 4. Inductively couples plasma emission spectrophotometry
SPECIMEN PRECAUTION
5. Anodic stripping voltammetry
 Specimen must be capped at all times to avoid alcohol DRUGS OF ABUSE
evaporation  Designer drugs – are modified forms of established
 prior to collection, alcohol-free skin cleanser must be drugs of abuse
used instead of isopropanol Amphetamine
CARBON MONOXIDE  therapeutically used for the treatment of narcolepsy
 is a colorless, odorless, tasteless gas; very toxic and attentional deficit disorder
substance
 cause the release of dopamine from brain leading to
 binds with heme proteins: cytochromes, hemoglobin
“pleasant feeling” or “high” among users
and myoglobin
 3,4-methylenedioxymethamphetamine (MDMA or
 higher affinity for hemoglobin that does oxygen (200x
‘ecstasy’) – a derivative of methamphetamine, is a
faster than oxygen)
popular recreational abused drug
 indication of acute toxicity: “ cherry red_” color of the
 cause of false positive reaction: presence of
face
 sample for testing: EDTA whole blood
 definitive method: COOximetry antihistamine
(carboxyhemoglobin measurement) ANNABOLIC STEROIDS
CYANIDE
 super toxic substance (fast acting toxin) and death may  chemically related to testosterone; improves athletic
occur in less than an hour performance by increasing muscle mass
 inhibits cellular respiration, electron transport and ATP CANNABINOIDS
formation  naturally-occurring cannabinoids: marijuana and
 indication of toxicity: “ odor of bitter almonds ” hashish

breath and altered mental status  Tetrahydrocannabinol (THC) : most potent

COLLEGE OF MEDICAL LABORATORY 35 |

 Clinical Chemistry
 antidote: sodium thiosulfate, amyl and sodium component or the psychoactive substance of marijuana;
lipophilic

COLLEGE OF MEDICAL LABORATORY 36 |

 Clinical Chemistry
 THC-COOH can be detected in urine for 3-5 days; up to 4
weeks for chronic users
1. METHODS
Enzymatic – FOR IDENTIFYING
alcohol is measured AND
from blood using
 urinary metabolite: 11-nor-deltatetrahydrocannabinol MEASURING ABUSED
(THC-COOH) alcohol dehydrogenase as reagent
2. Capillary electrophoresis – different analyte selectivity
COCAINE (CRACK)
is based on different physicochemical principles of
 used as local anesthetic for nasopharyngeal surgery separation without changes in instrumental hardware.
 derived from coca plant (Erythroxylon) and used as 3. Homogenous immunoassay – assay is done in one
additive to some foods solution without separation
 inhibitor: Prozac
 treatment for cocaine addiction: Benzodiazepine
 urine metabolite: Benzoylecgonine  Enzyme mediated immunologic technique (EMIT) –
uses enzyme-labeled drug that competes with the
 not considered as an addictive drug – does not reflect
drug in sample
true dependence
4. Chromatographic methods
 easily passes the placenta and mammary glands
 Thin layer chromatography – uses serum, urine or
resulting to mental retardation, slow mental
gastric fluid for analysis
development and drug dependence in newborns
 Liquid chromatography-Mass spectroscopy (LC-MS)
 can cause sudden death due to direct toxicity in
– used to confirm positive test results from a
myocardium (cardiac toxicity)
screening assay
OPIATES
 High performance liquid chromatography (HPLC) –
 capable of analgesia, sedation and anesthesia used as an alternative to GC-MS in definitive
 derived chemically from opium poppy identification of drugs
 naturally occurring opiates: opium, morphine and  Gas chromatography
codeine o Gas liquid chromatography (GLC) – legally
 chemically modified opiates: heroine, hydormorphone accepted method for ethanol testing
and oxycodone o GC with infrared spectroscopy – detection of
 commonly tested opiates: morphine and codeine amphetamines
 major metabolites of heroine: N-acetylmorphine o Gas chromatography-Mass spectroscopy – gold
(heroin) and morphine standard for confirmation of screening tests;
 antagonist for opiate overdose: naloxone (narcan) allows for detection of low levels of drugs
 Codeine is anti-tussive drug .
 Morphine is a metabolite of heroin used in the REFERENCES
treatment of congestive heart failure.  Rodriguez, Maria Teresa. Clinical Chemistry Handbook
 Heroin is highly addictive (true physical dependence) for medical technologists. Cattleya Star Copy Center and
PHENCYCLIDINE Book Binding, 2014
 physiologic effects: analgesia and anesthesia  Coderes, Errol. March 2018 Medical Technology Board
 major metabolite: phencyclidine HCl Exam Final Coaching Notes. Pioneer Educational Review
 mode of treatment: isolation (kept in quiet, dark room) Center, Manila, 2018
SEDATIVE HYPNOTICS  Policarpio, Judea Marie. March 2018 Medical
 examples: barbiturates and benzodiazepines Technology Board Exam Final Coaching Notes. ACTS
 Commonly abused barbiturates: secobarbital, Review Center, Manila, 2018
pentobarbital, phenobarbital and thiopental  Plaza, Xenia. March 2019 Medical Technology Board
 commonly abused benzodiazepines: diazepam (valium), Exam Clinical Chemstry Review Notes. Remedios
chlordiazepoxide (Librium) and lorezepam (Ativan) Trinidad Romualdez Medical Foundation, 2019
 major metabolite: secobarbital (barbiturates)
 barbiturate chemoadsorbent: activated charcoal
LYSERGIC ACID DIETHYLAMIDE (LSD,
LYSERGIDE)

 one of the most potent pharmacologic materials known

 most common adverse reaction: panic reaction - “bad
trip”
 toxic effects: blurred/ “undulating” vision and
synesthesia

COLLEGE OF MEDICAL LABORATORY 37 |