CARBOHYDRATES

What are Carbohydrates?

A. Characteristics of Carbohydrates
composed of three elements carbon, hydrogen and oxygen
hydrates of carbon; general formula: C«(H20)
water soluble; most are reducing sugar, except for SUCR0SE
B. Functions of Carbohydrates
Major component of human diet, an important source of body energy
Found as part of the cell membrane
Found as antigens in RBCs to determine blood type (ABO blood antigens)
C. Classification of Carbohydrates

Ribose and deoxyribose are part of RNA and DNA respectively.

Fructose is also known as levulose or the fruit sugar
Lactose is known as the milk sugar. It is found in dairy products (milk and cheese).
Sucrose is known as the common table sugar. It is
obtained from beets and sugar cane.
Maltose is found in cereals, wheat and malt products.
Glycogen is the storage form of glucose in
the body. It is stored in the liver and skeletal
muscles
MONOSACCHARID E DISACCHARIDE POLYSACCHARIDDE

Pentose (5 carbon) Sucrose Glucosans

ribose, deoxyribose = fructose +glucose (starch, glycogen)
Hexose (6 carbon) Maltose Fructosans (inulin)
glucose, galactose, = glucose + glucose Cellulose
fructose Lactose Chitin
galactose + glucose

D. Glucose (Dextrose)
ucose is the principal and almost exclusive carbohydrate circulating in the blood
Glucose is the central, pivotal point of carbohydrate metabolism
Brain is the most important glucose consumer. CNS consumes about 50% of glucose
used by the body
Glucose can be derived from (1) diet. (2) body stores like glycogen and (3)
endogenoussynthesis from proteins or glycerol of triglycerides
E. Carbohydrate Metabolism
1. Carbohydrate Digestion
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Polysaccharides and disaccharides from diet enter the body through mouth
Carbohydrate digestion starts in the mouth through the enzyme salivary
amylase (aka ptyalin)
Only partial digestion of carbohydrates occurs in the mouth.
Partially digested carbohydrates go from the mouth to the esophagus and
into the stomach
No carbohydrate digestion occurs in the stomach due to the acidic pH.
From the stomach, carbohydrates go toward the small intestines.
Alkaline pancreatic secretions increase the pH of the intestines, enabling
carbohydrate digestion through pancreatic amylase (aka amylopsin).
Enzymes such as maltase, sucrase and lactase hydrolyzes the disaccharides
to form monosaccharides (glucose, galactose and fructose)
Monosaccharides are absorbed from the duodenum and ileum into blood
Carbohydrate Metabolism in the Blood
Metabolism of hexoses in the blood can result to:
a. Energy production by conversion to carbon dioxide and water
b. Storage as glycogen in the liver
C.Storage as triglycerides in the adipose tissues
d. Conversion to ketoacids, amino acids or proteins
F. Regulation of Glucose Concentration in the Blood

1. Processes involved in carbohydrate metabolism

Glycolysis
metabolism of glucose molecule to pyruvate or lactate to energy
decreases blood glucose since glucose is consumed to produce
lactate/pyTuvate
Gluconeogenesis
formation of glucose-6-phosphate from non-carbohydrate sources
increases blood glucose; new glucoses are formed from other sources
Glycogenolysis
breakdown of glycogen to glucose for use as energy
increases glucose since glycogen is degraded into glucose molecules
Glycogenesis
conversion of glucose to glycogen for storage
decreases glucose since excess glucoses in the body is store in the liver
and skeletal muscle as glycogen
Lipogenesis
conversion of carbohydrates to fatty acids

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decreases glucose since carbohydrates are converted into fatty acids and
stored as fats
Lipolysis
Breakdown of fats
increases glucose because fats are converted into consumable glucose
2. Hormones involved in carbohydrate metabolism
a. Hormones that increase blood glucose
Glucagon, epinephrine, cortisol, growth hormone, thyroxine
b. Hormones that decrease blood glucose
Insulin
C. Regulator hormone: Somatostatin inhibits release of growth hormone,
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insulin and glucagon

INSULIN GLUCAGON

Produced by beta cells of pancreas Produced by the alpha cells of pancreas

proins ulin Stimulus: de crease in plasma glucose
Proinsulin is converted into ins ulin triggers an increase in glucagon
through the removal of Cpepti de Action:
fragment a. Promotes glycogenolysis
Stimulus: increase in plasma gl ucose b. Promo tes gluconeogenesis
causes an increase in insulin
Action:
a. Increases gluco se entry into cells
b. Promotes glycogenesis
c. Promotes lipogenesis
d. Promotes glycolysis
E. Promotes synthesis of amino acids
from pyruvate

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Il. Specimen for Glucose Determination

Whole blood, serum, plasma, CSF, serous fluids and urine can be used as sample
Standard clinical specimen is venous plasma glucose.
Fasting Blood Sugar should be obtained after 8 to 10 hours of overnight fasting
Venous blood has lower glucose levels compared to arterial blood
Capillary blood has higher glucose levels compared to venous blood
Whole blood gives approximately 10 to 15% LOWER glucose levels than serum or
plasma.
To convert Whole blood glucose into serum or plasma level, multiply value by 1.15
According to old studies: plasma glucose is 5% lower compared to serum.
Recent studies reported that plasma glucose is essentially the same as serum
glucose
A serum specimen is appropriate for glucose analysis if serum is separated from
the cells within 30 to 60 minutes
Glucose is metabolized at room temperature at a rate of 7 mg/dL/hr (0.4
mmol/L/hn
At 4°C glucose decreases by approximately 2 mg/dL/hr.
Gray top tubes are preferred for plasma glucose collection. Gray top tubes usually
contain sodium fluoride which serves as anti-glycolyticagent, and potassium
Oxalate which serve as anticoagulant
Sodium fluoride is binds calcium but is a weak anticoagulant, and thus often
combined with potassium oxalate. 2mg of sodium oxalate and 2mg of sodium
fluoride is often used.
2 mg of sodium fluoride per mL of whole blood prevents glycolysis for 48-72
hours
Fluoride binds magmesium, which causes inhibition of the enzyme enolase.
CSF gucose concentration is approximately 60 to 70% that of plasma

concentrations.
Blood glucose should be obtained 1-2 hours before the spinal tap
CSF for glucose analysis should be performed immediately. If delay in

measurement is unavoidable, the sample must be centrifuged and stored at 4°C or

at-20°C.

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IlI. Methodologiesfor Glucose Analysis

A. Non-enzymatic or Chemical Methods
1. Copper Reduction
Most carbohydrates are reducing sugars, meaning, they are capable of
decreasing the oxidation state of copper, from the cupric form (Cu3-) into
cuprous (Cu2)
Reducing
Substances
Cm34
L(cupric)
(glucose) Culs
(cuprous)
Amount of cuprous ions that forms is directly proportional to the amount of the
reducing sugar present. The more cupric ions are reduced to cuprous, the more
reducing sugars present
Cuprous can then be quantified using any of the following methods:
a. Folin Wu Reagent: phosphomolybdate
Positive Result: phosphomolybdenum blue
b. Nelson-Somogyi Reagent: arsenomolybdate
Positive Result: Arsenomolybdenum blue
C.Neocuproine method Reagent: Neocuproine
Positive Result: orange-red
(or yellow to orange) color
Copper reduction IS NOT SPECIFIC FOR GLUCOSE since other carbohydrates
such as fructose and galactose are also reducing sugars
Sucrose is NOT DETECTED by copper reduction methods since it is a non
reducing sugar
2. Ferricyanide method (Ferric Reduction)
"Also known as Hagedorn Jensen method; negative/inverse.colorimetry
Used in AutoAnalyzers (Technicon)
Reagent: hot alkaline solution of potassium ferricyanide
"Reducing sugars can reduce ferricyanide into ferrocyanide
Reduction is accompanied by disappearance of color: from yellow into
colorless, measured at 400nm
Reduction in the color is related to the glucose concentration
Non-specific for glucose since other reducing sugars can also react
3. Condensation Method (o-toluidine method/ Dubowski method)
Reagents: o-toluidine + glacial acetic acid + 100°C heat
Positive Result: bluish green (or green) color measured at 620-630nm

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B. Enzymatic Methods
1. Glucose Oxidase

Step 1: Glucose oxidase

glucose oxidase
GLUCOSE +OXYGEN GLUCONIC ACID + H202

Step 2:
a. Polarographic Method
Measures oxygen depletion through electrodes
For each molecule of glucose that reacts, one molecule of oxygen is consumed
The rate of decrease in oxygen is proportional to the concentration of glucose
Source of error: H202, if left alone, can be broken down to form oxygen,
leading to erroneous results. To prevent this, hydrogen peroxide should be
destroyed by:
Adding ethanol to hydrogen peroxide to form acetaldehyde
AAdding iodide to form molecular iodine
b. Colorimetric Method
Reagents: Glucose oxidase + Peroxidase + Chromogens/Indicators
peroxidase
CH202 + chromogen- H20+oxidized chromogen
Chromogens/Indicators
o-dianisidine (positiveblue)
aminoantipyine (positive rose-pink)
o-tolidine
diethyl-p-phenylenediamine
0-anisidine
3-methyl-2-benzothiazolinone hydrazone & N,N-dimethylaniline
A 4-aminophenazone

Glucose oxidase is specific to the beta-D-glucose

A Two forms of glucose exists in the body:

alpha-D-glucose = 35-36% beta-D-glucose 64-65%

Glucose oxidase can only detect for the presence of beta-D-glucose
Addition of mutarotase can convert alpha-D-glucose to beta-D-glucose
Sources of Error:
Oxidizing agents (bleach/detergent) causes false increase
AReducing agents (ascorbic acid, uric acid, bilirubin, hemoglobin) causes
falsedecrease
Addition of potassium ferrocyanide decreases interference with bilirubin

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2. Hexokinase with Glucose-6-phosphate dehydrogenase

Reference method for glucose analysis
Step 1:
GLUCOSE+ ATPNES GLUCOSE-6-PHOSPHATE+ADP

Step 2:
GLUCOSE-6-PHOSPHATE 6-PHOSPHoGLUCONATE
G6PD
+

NAD or NADP NADH or NADPH

Absorbance of NADH or NADPH is measured at 340nm

Increasein absorbance is proportional to the amount of glucose present
NAD is required if the G6PD used is obtained from bacteria (Leuconostoc
mesenteroides)
NADP is required if the G6PD used is obtained from yeast
Product of the G6PD reaction is 6-phosphogluconate (or 6
phosphogluconolactone in some references)
The specificity of this enzymatic system does not lie on the use of hexokinase
since hexokinase reacts with any hexoses (glucose, galactose, fructose).
It is the G6PD thatmakes the reactionspecificfor glucose determination
Some modification adds an additional colorimetric step.
NADH or NADPH from the 2nd step can be reacted with a chromogen such as
phenazine methosulfate with nitrophenyl tetrazolium derivative.
Absorbance is then measured at 520nm.
Sources of Error: hemolyzed and icteric sample can cause a false decrease

3. Glucose dehydrogenase
Variations
a. Spectrophotometric
Step 1:
alpha-D-glucose terolEsebeta-D-glucose
Step 2:
glucose dehydrogenase
beta-D-glucose +NAD D-gluconolactone+ NADH
Step 3: (MTT is a color reagent)
MTT+NADH diaphorase MTTH (blue) + NAD

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b. Through Electric current:

Step 1:
Glucose + Pyrroloquinoline quinone (PQQ)
glucose dehydrogenase Gluconolactone
+ PQQH
Step 2:
PQQH + ferricyanidePQQ+ ferrocyanide +2H
Step 3:
ferrocyanide ferricyanide +2e
Glucose dehydrogenase is derived from bacteria (Bacillus cereus)
Highly specific for glucose, shows no interference from common anticoagulants
and from substances normally found in serum
Sources of error: maltose, icodextrin and galactose can cause false increase

IV. Methodologies for Other Carbohydrates

A. Seliwanoff/ Selivanoff's Test for FRUCToSE
Reagent: hot hydrochloric acid and resorcinol
Positive result: red within 30 seconds
B Bial's Test for PENTOSES
Reagent: Hydrochloric acid, orcinol and ferric chloride
Positive result: green

V. Laboratory Tests
A. Random blood glucose
blood glucose taken any time of the day and without any fasting; often used for
emergency cases
B. Fasting blood glucose
taken after at least 8-10 hours of overnight fasting; usually done in the morning.
to prevent the effects of diurnal variation caused by hormones
CATEGORIES OF FASTING PLASMA GLUCOSE
Normal fasting glucose = <100mg/dL
Impaired fasting glucose/Prediabetes = 100-125 mg/dL

Provisional diabetes diagnosis = 2126mg/diL

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D. 2-hours Post Prandial Blood Glucose

fasting blood sugar is initially, patient is then given glucose load (usually 75g) and
plasma glucose is determined after 2 hours
normally, blood glucose levels should be back near the reference limits
approximately 2 hours post load
CATEGORIES OF 2-HOURS PLASMA GLUcoSE
Normal glucose tolerance = <140mg/dL
Impaired glucose tolerance/Prediabetes = 140-199mg/dL
Provisional diabetes diagnosis = 2200mg/dL

E. Oral Glucose Tolerance Test

a series of glucose testing; a fasting blood glucose is determined after an 8-10

hours of fast
glucose load is given, and a series of blood samples for glucose assays are then
collected 30 minutes, 1 hour and 2 hours after glucose load intake
Guidelines for Oral Glucose Tolerance Test
Patient is asked to consume a normal to high carbohydrate intake (150g
carbohydrates per day) for 3 days prior to the test
2. Patient should discontinue, if possible, medications known to affect glucose
tolerance
3. Patient is asked to fast overnight and to avoid excessive physical activity.

Patient should fast at least 8-10 hours but not greater than 16 hours.
4. OGT testing should be performed on the morning to prevent hormonal
diurnal effect on glucose. Blood glucose is taken every 30 minutes for 2
hours.
5. Patient should be ambulatory. Patient should refrain from exercise, eating
or drinking (except water) and smoking
6. Fasting blood glucose is measured before giving the glucose load; a fasting
glucose of greater than 140, test should be terminated; fasting glucose of
less than 140mg/dL, glucose load should be given to the patient.
7. Glucose load for an aduit is 75g. Children receive 1.75g per kg of body
weight, max of 75g.
8. Patient should finish drinking the glucose load within 5 minutes.
9. Patient should not vomit. If patient vomits, discontinue the test

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G. Glycated (glycosylated) hemoglobin

used for long term monitoring of glucose control over the previous 2-3 months
(18-20 weeks period in some references), reflecting lifespan of RBCs (120 days)
it is formed by attachment of glucose at the end or both N-terminal valines of
the beta chains of normal adult hemoglobin
for every 1% increase in HbA1c, there is a 35mg/dL (2mmol/L) change in
plasma glucose.
patients with shorter RBC lifespan such as in cases of hemolytic anemia, will
have decreased glycated hemoglobin
specimen is EDTA-whole blood
values can be determined by
(1) assays based on structural differences, such as affinity chromatography
(2) based on differences on electrical charges, such as in ion-exchange
chromatography; electrophoresis and high-pressure liquid chromatography
HPLC can measure all glycosylated hemoglobins (HbA1a, HbA1b, HbA1c)
labile Pre-HbA1c (also known as aldimine) interferes with isoelectric focusing
Affinity chromatography is NOT affected by other hemoglobins and
temperature
ideal value for HbA1c is less than 7%
H. Fructosamine (Glycated Albumin)
used for monitoring glucose control over the previous 2 to 3 weeks
albumin has a life span of 20 days in the circulation;
affected by changes in albumin levels; patients with hypoalbuminemia has
decreased glycated albumin results
I. AGE (Advanced Glycation End-products)
Non enzymatic attachment of glucose to long-lived proteins, lipids or nucleic
acid can produce Amadori early-glycated products
These undergo a series of rearrangements, dehydration and fragmentation
reactions resulting in the stable AGEs
AGEs are irreversible and they accumulate continuously
Patients with diabetes mellitus have more AGE than healthy subjects

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VI. Clinical Significance of Glucose Results

A. Hypoglycemia
Decreased glucose levels
Glycemic factors such as glucagon are released when glucose levels reach 65
70mg/dL
Observable signs and symptoms of hypoglycemia appear when glucose levels reach
50-55mg/dL
Symptoms of hypoglycemia can be classified as:
(1) Neurogenic triggered by autonomic nervous system; include trembling,
-

sweating, nausea, rapid pulse, lightheadedness, hunger and pigastric

discomfort
(2) Neuroglycopenic- caused by diminished glucose supply to the central
nervous system; include headache, confusion, blurred vision and dizziness
to seizures, loss of consciousness and even death
Critical value for glucose is 40mg/dL; excessively low glucose values can cause
severe CNS dysfunction especially if blood glucose value drops to 20-30mg/d
Whipple's Triad is used to assess patients with episodes of low plasma glucose
levels. This refers to symptomsconsistentwith hypoglvcemia associated with
documented low plasmaglucose level and relief of symptoms with correction
of hypoglycemia
Hypoglycemia can be:
(1) Reactive (postprandial) caused by a stimulus; not usually related to
-

underlying disease
(2) Fasting (post-absorptive) often related to an underlying disease
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Causes of Hypoglycemia include:

(1) Drug-induced (eg insulin, alcohol, etc)
(2) Hormonal deficiencies (e.g. glucagon, epinephrine, cortisol, growth
hormone)
(3) Endogenous Hyperinsulinism (eg. insulinoma)
(4) Autoimmune hypoglycemia (e-g. insulin antibodies or insulin-receptor
antibodies)
(5) Hypoglycemia of Infancy and Childhood (eg. GSDs)
(6) Alimentary Hypoglycemia (e-g post gastric bypass surgery)
(7)1diopathic or Functional Postprandial Hypoglycemia

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B. Hyperglycemia
oratory Findings in Hyperglycemia
Increased plasma glucose levels
Positive urine glucose
Urine glucose becomes positive when plasma gucose levels exceed the renal
threshold imit or value for glucose
Renal threshold limit for glucose is 140-160mg/dL (or 160-180mg/dL in
some references)
Increased urine specific gravity due to the presence of glucose in urine
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Increased serum and urine osmolality due to the presence of increased glucose
-

in serum and urine

Ketones in serum and urine (ketonemia and ketonuria)
Decreased blood and urine pH (acidosis)
Electrolyte imbalance
Hyperglycemia leads to hyponatremia
Diabetes mellitus is a group of diseases in which blood glucose levels is elevated.
This is often associated with polyphagia (excessive hunger), polycdipsia (excessive
thirst) and polyuria (excessive secretion of urine)
Diabetes mellitus vs Diabetes insipidus
DIABETES MELLITUS DIABETES INSIPIDUS
Involves insulin Involves anti-diuretic hormone
Increased water excretion with Increased water excretion, no
hyperglycemia hyperglycemia
Polyuria with high urine specific Polyuria with low urine specific
gravity gravity

Criteria for Diagnosis of Diabetes Mellitus

(1) Symptoms of diabetes plus random (casual) plasma glucose concentration
2200 mg/dL (11.1 mmol/L). Random is defined as any time of day without
regard to time since last meal. The classic symptoms of diabetes include
polyuria, polydipsia, polyphagia, and unexplained weight loss.
(2) Fasting plasma glucose (FPC) >126 mg/dL (7.0 mmol/L). Fasting is defined
as no caloric intake for at least8 hours.
(3) 2-Hour postload glucose 2200 mg/dL (11.1 mmol/L) during an oral glucose
tolerance test, using a glucose load containing the equivalent of 75 g
anhydrous glucose dissolved in water.
"These criteria should be confirmed by repeat testing on a different day.

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Classification of Diabetes Mellitus

1. Type I Diabetes Mellitus (e-g. immune-mediated or idiopathic insulin
deficiency)
2. Type I Diabetes Mellitus (insulin resistance)

Frequency
I
Type Diabetes Type II Diabetes
5-10% 90-95%
Age of Onset Any (most common in More common in advancing
children&young adults) age
Risk factor Genetic, autoimmune, Genetic, obesity, sedentary
environmental lifestyle, race/ethniity,
hypertension, dyslipidemia,
polycystic ovarian syndrome
Pathogenesis Destruction of pancreatic No autoimmunity, insulin
beta cells, usually resistance and progressive
autoimmune insulindeficiency
C-peptide levels Very low or undetectable Detectable
Pre-diabetes Autoantibodies (GAD65, IA- Autoantibodies absent
2, 1AA) may be present
Medication Insulin absolutely necessary, | Oral agents, insulin
therapy multiple daily injections or commonly needed
insulin pump
Therapy to None known Lifestyle (weight loss and
prevent or increased physical activity);
delay onset of Oral medications can be
diabetes helpful
Previous names Insulin-dependent DM Non-insulin dependent DM
LJuvenile-onset DM Adult-onset DM

3. Gestational Diabetes Mellitus (GDM)

Glucose intolerance that develops in pregnancy, often caused by hormonal
and metabolic changes
Can lead to increased perinatal complications such as respiratory distress,
hypocalcemia, hyperbilirubinemia and fetal macrosomia
A large percentage of women with GDM develop diabetes within 5 to 10
years
To detect GDM:
One-step approach for high-risk individuals
Step 1-0GTT
OCTT two-step approach for average-risk individuals
Step 1- Initial Screening (1-hr plasma glucose after 50g glucose load)
Step 2-0GTT

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4. Other Specific Types

Endocrinopathies, genetic defects, pancreatic diseases, drug- or chemical-
induced, infections
C. Glycogen Storage Diseases (GSDs)
results from deficiency of specific enzyme that causes an alteration in the glycogen
metabolism
patients exhibit hepatomegaly (due to increased liver glycogen stores) and
hypoglycemia (due to inability to convert glycogen back to glucose)
Von Gierke disease is the most common glycogen storage disorder
GSD ENZYME DEFICIENT
la Von Gierke
-
glucose-6-phosphatase
II- Pompe acid alpha glucosidase/ acid maltase
IlI -
Cori-Forbes amylo-1, 6 glucosidase/ debrancher enzymne
IV- Andersen amylopectinase/ glycogen branching enzyme
V-McArdle muscle phosphorylase
VI -
Hers liver phosphorylase or phosphorylase kinase
VII Tarui -
muscle phosphofructokinase
XI -
Fanconi-Bickel glycogen transporter 2
giycogen synthetase

D. Inborn Errors or Carbohydrate Metabolism

1. Disorders of Galactose metabolism galactosemia -

Galactose-1-phosphate uridyl transferase deficiency

Uridine diphosphate glalactose-4-epimerase deficiency
Galactokinase deficiency
2. Disorders of Fructose metabolism
Fructokinase deficiency (Essential fructosuria)
Fructose-1-phosphate aldolase (Hereditary Fructose Intolerance)
Hereditary fructose-1, 6-diphosphatase deficiency
3. Disorders of Pentose metabolism
Alimentary pentosuria
L-xylulose reductase deficiency (Essential Pentosuria)
4. Other Urinary Sugars
Lactosuria - found in women during lactation and occasionally towards the
end of pregnancy, and in patients with lactase deficiency

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VII. Values to Remember

Fasting Plasma Glucose: 70 to 100mg/dL
Glycosylated hemoglobin: 4-6% (Henry 21st ed); 4.5-89% (Bishop)
Conversion Factor: 0.0555
Critical Values: <40mg/dL and >500mg/dL.

co

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 LIRIDS cO

What are Lipids?

A. Characteristics of Lipids
Body's petroleum industry; contains carbon, hydrogen and oxygen
Water insoluble, non-polar; transported in the body by lipoproteins
B. Lipid Metabolism from Diet
During digestion in the intestines, pancreatic lipase cleaves off fatty acids to convert lipids into
more polar compounds or amphipathic lipids
Triglycerides are converted into monoglycerides & diglycerides; cholesterol esters are
transformed into free cholesterol & phospholipids are converted into lysophospholipids first
Amphipathic lipids aggregate with bile acids and forms micelles
Micelles are absorbed from intestines into the blood
These micelles are re-esterified into triglycerides and cholesteryl esters which are then
transported by lipoproteins
C. Forms of Lipids
1. Triglycerides (Triacylglycerol)
possesses 3 molecules of fatty acids and a molecule of glycerol, which serves as the
backbone
hydrophobic and water insoluble; serves as the main storage form of lipid in man (as found
in adipose tissues)
Most triglycerides from plant sources are rich in unsaturated fatty acids and exist as oils,
whereas triglycerides from animal sources contain mostly saturated fatty acids and are
usually solid at room temperature
Triglycerides do not have any charge they are therefore known as neutral fats/lipids
serves as:
energy source as integral part of the cell membrane
insulation or shock absorber
2. Cholesterol
unique in that, unlike other lipids, it is not readily catabolized by most cells and, therefore,
does not serve as a source of fuel.
serves asS
part of cell membrane
converted in the liver to primary bile acids, such as cholic acid and chenodeoxycholic
acid, which promote fat absorption in the intestine by acting as detergents (fat
emulsification)
can be converted by some tissue (adrenal gland, testis, and ovary) to steroid hormones
(such as glucocorticoids, mineralocorticoids, and estrogens)
can be transformed to vitamin D3 in the skin by irradiation from sunlight
 CHECKPOINT! Clinical Chemistry by Jude

exists in the body in two different forms:

Cholesterol (cholesteryl) Esters or Esterified Cholesterol
accounts to approximately 60-709% of the total cholesterol in the body
composed of cholesterol ring and a fatty acid
Free cholesterol
accounts to approximately 30-40% of the cholesterol in the body
unesterified cholesterol; cholesterol ring only, no fatty acids attached
3. Phospholipids
structurally similar to triglyceride except that is 2 fatty acids and a phospholipid head
group is attached to the glycerol backbone instead of 3 fatty acids
Phospholipids in the body:
phosphatidylcholine (70%-75%)
sphingomyelin (18%-206)
phosphatidylserine, phosphatidylethanolamine (3%-6%)
lysophosphatidyl choline (4%-9%)
phospholipid head groups are hydrophilic and include: such as choline, inositol, serines
and ethanolamine
two fatty acids in phospholipid: one fatty acid is commonly saturated and the other is
unsaturated
important constituent of cell membrane (phospholipid bilayer), contains a polar and
nonpolar regions
can be found in the lungs as surfactant during respiration
4. Fatty acids
building blocks of lipids, hydrocarbon chains with terminal COOH group
usually found in triglycerides, phospholipids and cholesterol
in plasma, they may exist in the free or unesterified form, most of which is bound to
albumin
Forms of Fatty acids:
Saturated fatty acids with no double bonds
Unsaturated - fatty acids with double bond(s), usually in the cis form, making them
more fluid
Trans fatty acid- fatty acids with double bond(s) in the trans form
II. Lipoproteins
Transports lipids
Chemical Composition of the Different Lipoprotein Classes:
In percent Protein Cholesterol Chole Esters Triglyceride Phospholipid
Chylomicrons 1-2 1- 3 2-4 80-95 3-6
LDL
6-10 8 16-22 45- 65 15-20
IDL Intermediatebetween VLDL and LDL
LDL 18-22 6-8 45-50 28-24
HDL 45- 55 3-5 15-20 2-7 26-32

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Apolipoproteins- protein portion of the lipoprotein

FOUND IN: FUNCTIONS
ApoA-I HDL activates LCAT that esterifies cholesterol in plasma
Synthesized in the liver and intestine
ApoA-II HDL May inhibit lipoprotein and hepatic lipases and
increases plasma triglyceride
ApoA-IV HDL, CM and ~ May be a cofactor for LCAT;
increases during fat
freein plasma absorption; HDL biosynthesis
Apo B-100 VLDL and LDL Very large structural protein; synthesized in liver
with lipids of endogenous origin
ApoB-488 CM Synthesized in intestine
Apo C-I CM and VLDL ~ May inhibit hepatic uptake of VLDL and cholesterol

ester transfer protein

Apo C-II CM and VLDL Activates lipoprotein lipase
Deficiency causes reduced clearance of triglyceride-
rich lipoproteins
Apo C-III VLDL, HDL Inhibits lipolysis of triglyceride-rich lipoproteins;
decreases clearance rate of remnant particles
Deficiency causes reduced clearance of triglyceride-
richlipoproteins
Apo D HDL Activates LCAT
Apo E CM, VLDL, IDL, Recognition factor that targets CM and VLDL
remnants and remnants to hepatic receptor; also binds to cell
HDL surface LDL receptors and proteoglycans
E-2, E-3, and E-4 isoforms; E-4 is associated with
high LDL-C, higher risk of CHD and Alzheimer's
disease; E-2 associated with type 3
hyperlipoproteinemia
Apo F HDL, LDIL, VLDL Regulates CETP function
Apo H VLDL Related to activation of LPL; triglyceride
metabolism
Apo Cell-aggregating factor in Sertoli cells; inhibitor of
the C5 7 complement complex; beta-amyloid
clearance in glial cells; cholesterol traffickingin
brain
Involved in apoptosis; linked to neurologic diseases
like Pick's and Alzheimer's; also known as
clusterin
Apo L HDL May be linked to reverse cholesterol transport
Apo M HDL, CM, LDL, May be inked to HDL remodeling
VLDL
Apo (a) Lpla) Homologous to plasminogen, may be
prothrombotic;
bound to apoB-100 by disulfide linkage

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D. McCullough Jr.
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CHECKPOINT! Clinical Chemistry by Jude

Normal Lipoproteins
Chylomicron
Largest and lightest among the lipoproteins; lipoprotein with lowest density
80-95% triglyceride by weight
Transports exogenous triglycerides (triglycerides derived from the diet/
food)
Causes non-fasting lipemia (turbidity of serum in non-fasting patients)
No charge; remains in the origin during electrophoresis
Very Low Density Lipoproteins
Pre-beta lipoprotein
Transports endogenous triglycerides (triglycerides produced by the body)
Low Density Lipoprotein
Also known as the bad cholesterol; beta lipoprotein
Transports cholesterol from the liver to the cells of the body
Transports majority (75%) of the cholesterol
Directly proportional to the risk of atherosclerosis and coronary heart
disease (CHD); higher LDL means higher risk
High Density Lipoprotein
Smallest and heaviest among the lipoproteins
Fastest towards the anode; alpha lipoprotein; good cholesterol
Inversely proportional to the risk of atherosclerosis and coronary heart
disease (CHD); lower HDL means higher risk
Responsible for reverse cholesterol transport; transports cholesterol from
the cells to the liver
Abnormal Lipoproteins
Lp(a)
also knowm as the sinking prebeta lipoprotein
found in the LDL density range in ultracentrifugation, but moves in the pre-
beta region during electrophoresis
associated with increased risk for coronary heart disease, cerebrovascular
disease, stroke and atherosclerosis; may be prothrombotic and can interfere
with normal thrombolysis due to its similarity to plasminogen
Beta-VLDL
also known as the floating beta lipoprotein; a VLDL that is richer in cholesterol
than in triglycerides
found in the VLDL density range but migrates electrophoretically with or near
LDL
typically seen in patients with type Ill hyperlipoproteinemia

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 CHECKPOINT! Clinical Chemistry by Jude

LpX
abnormal lipoprotein that is rich in lipids, primarily unesterified cholesterol
and phospholipids
found in patients with obstructive biliary disease; regurgitation of biliary
contents in the bloodstream results in the buildup of LpX
migrates towards the cathode during electrophoresis
Identification of Lipoproteins
Chromatographic methods (i.e. gel chromatograph affinity)
Immunochemical methods use of antibody directed towards specific
-

apolipoproteins (i.e. anti-apoA1, anti-apo-C, etc.)

Electrophoresis
separates lipoproteins based on their electric charge
lipoproteins carry an electric charge at pH 8.6
lipoproteins can be stained using fat stains: Fast red 7b, 0il red 0, Sudan Black B
Ultracentrifugation - separates lipoproteins based on their densities; reference
method for lipoprotein analysis
uuumum
ELECTROPHORETIC MOBILITY DENSITIES OF LIPOPROTEINS
OF LIPOPROTEINS (ULTRACENTRIFUGATION)
Origin Density Lipoproteins
-Chylomicrons 0.96 1. Chylomicrons

B-Lipoproteins r2. Very-low

<1.006 density
lipoproteins
(VLDL)
Pre-B-lipoproteins
r3. Low-density
1.006 lipoproteins
|1.063 (LDL)

1.006- r4. High-density

-Lipoproteins ipoproteins
1.21 (HDL)

from Henry's 22nd ed from Henry's 22nd ed

aan* ****

III. Methodologies
A. Assays for Triglycerides
1. Chemical Method
Methods Van Handel and Zilversmit method Hantzsch
= Modified Van Handel Zilversmit method

Generalized Steps in the Chemical Method of Triglyceride Analysis

STEP 1: Extraction
PURPOSE: Pretreatment of the sample to remove the lipids from proteins
REAGENT: organic solvent (e-g. chloroform, isopropanol, diethyl ether)

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CHECKPOINT! Clinical Chemistry by Jude

Additional step: Adsorption

PURPOSE: to remove non-triglyceride glycerols such as phospholipids,
monoglycerides and diglycerides as well as interfering substances such
as glucose (carbohydrates) and bilirubin
REAGENT: alumina adsorbent mixture, zeolite, florisil or silicic acid
STEP 2: Hydrolysis or Saponification
PURPOSE: To cleave triglyceride molecules into fatty acids and glycerol
REAGENT: potassium hydroxide in ethanol (alcoholic potassium hydroxide) or
sodium methoxide
KOH
triglycerides+3H,o glycerol+fatty acids
STEP 3:0xidation
PURPOSE: To convert glycerol to a measurable compound
REAGENT: oxidizing agent such as sodium periodate
glycerol + sodium perio date (Nal04) > formaldehyde + fomic acid
STEP 4: Colorimetry
PURPOSE: Aids in the measurement of the compound that forms after oxidation;
measured by its absorbance after addition of color reagent
REAGENT: color reagent
chromotropic acid &H;SO, = pink chromophore measured at 500-600nm
diphenylhydrazone = product measured at 500-600nm
acetylacetone and ammonia (Hantzsch) = reactant of choice; formation of
diacetyl dihydrolutidine, a yellow product that can be measured either:
spectrophotometrically 412nm
fuorometrically (Excitation light: 400nm; Emitted light: 485nm)
CDC Reference Method for Triglycerides
New reference method is Gas Chromatography Isotope Dilution Mass Spectrometry
Original Reference Method:
1. Extraction with chloroform 4. Oxidation with periodate
2. Adsorption with silicic acid 5. Color development using chromotropic acid
3. Hydrolysis with KOH 6. Absorbance is measured at 570nm

2. Enzymatic Method
a. INITIAL ENZYMATIC REACTION (Lipase and Glycerokinase)

Triglycerides Glycerol + Fatty acids

ulycerokinase
Glycerol + ATP Glycerophosphate++ ADP

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CHECKPOINT! Clinical Chemistry by Jude

b. THE INITIAL ENZYMATIC REACTION CAN BE FOLLOwED BY ANY OF THE

FOLLOWING COUPLED ENZYMATIC REACTIONS:
Lipase-Glycerokinase coupled with glycerophosphate dehydrogenase and
diaphorase
Glycerophosphate + NAD Glycerophosphate
dehydrogenase
Dihydroxyacetone phosphate+ NADH+ H+

NADH+Tetrazolium dye Diaphorase Formazan + NAD'

Measurement of absorbance of
Absorbance of NADH can be measured at 340nm, after the
slycerophosphate dehydrogenase reaction
Absorbance of formazan dye can be measured between 500-600nm
Lipase-Glycerokinase coupled with glycerophosphate-oxidase and peroxidase
Glycerophosphate O, epospna
oxidase
Dihydiroxyacetone+ H,O

H,0+ Phenol + 4-aminoantipyrine Peroxidase

Quinoneimine dye + 2H,0|
Absorbance of the red quinoneimine dye can be measured
spectrophotometrically at 500-505nm
Hemoglobin and ascorbic acid can also interfere with the peroxidase step
Bilirubin interference can be minimized by adding potassium ferrocyanide to
the reaction mixture with peroxidase. Potassium ferrocyanide reduces
bilirubin into an inactive compound.
Lipase-Glycerokinase coupled with pyruvate kinase and lactate
dehydrogenase

ADP+ Phosphoenol pyruvate Pyru v ATP+ Pyruvate

kinase

Lactate
Pyruvate+ NADH+ H. Lactate+NAD
dehydrogenase
NAD can be measured at 340nm spectrophotometrically or
NAD Can be measured fluorometrically: Excitation light at 355nm
Emitted light at 460nm

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 CHECKPOINT! Clinical Chemistry by Jude

B. Assays for Total Cholestero

1. Chemical Method
Methods
METHODS PROCEDURE COMMENTS
Zlatkis, Zak & Boyle One-step Rapid method; subject to protein and
Colorimetry chromogen interference; color differences
between free cholesterol and cholesteryl
ester is a problemin the method
Carr& Drekter Two-stepP Chromogen interference and color
Extraction differences between free cholesterol and
Colorimetry cholesteryl ester are problems in the
method; protein interference removed
Abell Three-step Protein interference removed; color
Extraction differences between free cholesterol and
Saponification cholesteryl ester is removed
Colorimetry Chromogeninterference partially removed
Schoenheimer/ Four-stepp Protein and chromogen interferences were
Sperry Extraction removed; color differences between free
Saponification cholesterol and cholesteryl ester is
Purification removed
Colorimetry

General Steps in Chemical Method of Total Cholesterol Analysis

STEP 1: Extraction
PURPOSE: cholesterol is separated from protein
REAGENT: Bloor's reagent (composed of ethanol-ether at a 3:1 ratio)/
chloroform/ hexane
Additional Step: Adsorption
PURPOSE: extracts all forms of cholesterol, leaving behind most of the sterols
REAGENT: zeolite
STEP 2: Saponification/Hydrolysis
PURPOSE: cholesteryl esters after extraction are hydrolyzed into free cholesterol
and fatty acid
REAGENT: alcoholic potassium hydroxide (KOH
STEP 3: Purification:
PURPOSE: to precipitate free cholesterol; removes error of nonspecific
chromogen interference
REAGENT: digitonin measure cholesterol before and after digitonin treatment to
-

determine cholesterol fraction

STEP 4: Colorimetry
PURPOSE: color development, measured spectrophotometrically
REAGENT: color reagent

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CHECKPOINT! Clinical Chemistry by Jude

cOLORIMETRY Liebermann-Burchard reaction Salkowski reaction

Reagent Acetic anhydride + Sulfuric acid Sulfuric acid + Ferric iron
Endproduct Cholestedienyl monosulfonic acid Cholestedienyl disulfonic acid
End color Green (not stable; add sodium sulfate Red (stable)
to stabilize) measured at 410nm
(Calbreath) or 620nm (Tietz)

CDC Reference Method for Cholesterol (Abell, et. al.)

Hydrolysis with alcoholic potassium hydroxide
Extraction with hexane
Extract is dried in vacuo
Dried extract is then treated with Liebermann-Burchard regent (Acetic acid + acetic
anhydride+ sulfuric acid)
Read at 620nm after 30 minutes

2. Enzymatic Method
a. Spectrophotometric
Absorbance of quinoneimine dye is measured at 500nm
4-aminoantipyrine is also known as 4-aminophenazone
Some methods use methanol on the third step instead of 4-aminoantipyrine.
Methanol, in the presence of peroxidase is oxidized into formaldehyde. Formaldehyde
is then detected using acetylacetone (similar to colorimetric step of triglyceride
chemical method)
Some methods measure the absorbance of cholest-4-en-3-one at 240nm

Cholesteryl ester+ H0 Cholesterol +Freefatty acid

hydrolase

Cholesterol+O -Cholestero Cholest-4-en-3-one + H,O

oxidase

Peroxidase
H,O+Phenol +4-aminoantipyrine
Quinoneimine dye + 2H,O
b. Oxygen consumption
Uses cholesteryl ester hydrolase and cholesterol oxidase
Hydrogen peroxide that forms is mixed with peroxidase, which causes the formation
of water and oxygen
oxygen electrode measures the oxygen released after the decomposition ofhydrogen
peroxide
not easily automated and generally require a lot of cholesterol oxidase

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 CHECKPOINT! Clinical Chemistry by Jude

C. Standing Plasma Test

Chylomicrons, if present in appreciable quantities are detected using the standing
plasma test
2mL of plasma is placed in 10 x 75mm test tube and allowed to stand in the refrigerator
at 4°C undisturbed overnight
Chylomicrons appear as "loating cream layer" visually
Chylomicrons in fasting plasma is considered abnormal
Plasma sample that remains turbid after standing overnight indicates excessive
amounts of VLDL

D. Methods for HDL cholesterol quantitati on:

1. Precipitation
VLDL and LDL is precipitated by adding precipitating agents (polyanion-divalent cation)
such as:
Heparan sulfate-Mn** (Heparin-Manganese chloride)
Dextran sulfate-magnesium chloride
Phosphotungstate-magnesium chloride (sodium phosphotungstate-Mg)
Heparin-Car
CDC reference method
(1) ultracentrifugation to remove VLDL
-

(2) precipitation with Heparin-Manganese chloride to remove LDL-

(3) measurement of the supernate using Abell-Kendall method

2. Immunoassays
ELISA
Immunonephelometry

E. Methods for LDL cholesterol quantitation:

1. Calculation
- ( tiglycerides
LDL = total cholesterol HDI. -

TAG/5 = VLDL" is applicable only if TAG is <400mg/dL

=
"TAGx 0.16 VLDL" is applicable if TAG is >400mg/dL.
Friedewald
(TAG divided by 5) is used if the unit used is mg/dL
(TAG divided by 2.175) is used if the unit use is mmol/L
DeLong
(TAG divided by 6.5) is used if the unit used is mg/dL
(TAG divided by 2.825) is used if the unit use is mmol/L

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 CHECKPOINT! Clinical Chemistry by Jude

F. Assays for Phospholipids

Chemical Method
Extraction
Oxidation: converts phospholipid phosphorus into inorganic phosphorus
Reagent: concentrated acid (wet-ash oxidation)
Colorimetry
Phosphorus is mixed with molybdate reagent to form phosphomolybdate ion
Phosphomolybdate ion is then subjected to reduction to form molybdenum blue
Total phosphorus x 25 = Phospholipid mass
Enzymatic Method
Phospholipase D: Phospholipid is hydrolyzed using phospholipase D into choline
Choline oxidase: Choline is then oxidized into betaine and hydrogen peroxide
Peroxidase: Hydrogen peroxide is then mixed with phenol and 4-aminoantipyrine
to form quinoneimine dye, which is measured at 500nm

G. Assays for Fatty acids

Perform lipid extraction
Wash with dilute sulfuric acid to remove contaminants
Titrate purified extract with dilute NaOH with thymolphthalein as indicator

IV. Specimen Considerations and Patient Preparation:

Fasting requirement of at least 12 hours - patient can drink water, but no food should
be consumed. Non fasting state can affect triglyceride but cholesterol is not significantly
affected
Ethanol consumption must be restricted at least 2 days prior to the test since it can
cause an increase in triglyceride
Serum or EDTA-plasma may be used (citrate and fluoride can alter osmolarity: heparin
interferes with electrophoresis); EDTA is preferred for lipoprotein assays
No to hemolyzed sample; no to icteric specimens (presence of bilirubin levels greater
than 5mg/dL can interfere)
Avoid water contamination, especially for cholesterol assays (Lieberman Burchard)
Acceptable CV for Lipid analysis:
Total Cholesterol 3% HDL-cholesterol = 49%
LDL-cholesterol = 4% TAG = 5%

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 CHECKPOINT! Clinical Chemistry by Jude

V. Clinical Significance
Arteriosclerosis vs Atherosclerosis
A.
Arteriosclerosis
is a general term for the thickening and hardening of arteries
Atherosclerosis
is a type of arteriosclerosis
hardening of the arteries, caused by plaques that build up inside the arteries
Plaque is made of cholesterol, fatty substances, cellular waste products, calcium and
fibrin
B. Coronary Heart Disease
Refers to the broad spectrum of heart disease resulting from impaired coronary blood
flow
Clinical (Non-Laboratory) Risk factors for CHD
Cigarette smoking (any smoking in the past month)
Hypertension (blood pressure >140/90 mm Hg or on antihypertensive
medication)
Family history of premature CHD (CHD in male first-degree relative
<55 years, or in female first-degree relative <65 years)
Age (men >45 years; women>55 years)
Obesity
Diabetes mellitus
Sedentary lifestyle
C. Analphalipoproteinemia - also known as Tangier disease; deficiency of HDL
D. Abetalipoproteinemia~ also known as Bassen-Kornzweig syndrome; deficiency of apoB
(B48 and B100); notable acanthocytes in peripheral blood smear
E. Fredrickson and Levy's Hyperlipoproteinemia Phenotypes
Blood Lipoprotein Patterns in Patients with Hyperlipoproteinemia
TYPE LIPOPROTEIN PATTERN
Extremely elevated TG due to the presence of chylomicrons
lla Elevated LDL
Ilb Elevated LDL and VILDL
Elevated cholesterol, TG; presence of B-VLDL
IV Elevated VLDL
| Elevated VLDL and presence of chylomicrons

TYPE APPEARANCE OF PLASMA AFTER OVERNIGHT STORAGE AT

4°C
Creamy layer on clear plasma
Tla Clear
Clear or slightly turbid
Clear or slightly turbid
Clear or turbid
Creamy layer on turbid plasma

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 CHECKPOINT! Clinical Chemistry by Jude

F. Lipid Storage Diseases

LIPID STORAGE DISEASE ENZYME DEFICIENTT
Fabry's disease_ alpha galactosidase
GM-1gangliosidosis beta galactosidase
Gaucher beta glucosidase
Krabbe cerebroside beta galactosidase
Niemann Pick sphingomyelinase
Metachromatic leukodystrophy_ arylsulfatase A
Sandhoff total hexosaminidase (hexosaminidase A and B)
LTay Sach hexosaminidase A

VI. Values to Remember

A. ATP I1 Classification for LDL, Total and HDL Cholesterol, and Triglyceride values
(Henry 21st ed)
LDL Cholesterol Triglycerides
<100 mg/dL = Optimal <150mg/dL Normal
100-129mg/dL =Near optimal/above optimal 150-199mg/dLBorderline high
130-159mg/dL Borderline high 200-499mg/dL High
160-189mg/dL High
=
500 mg/dL Very high
2190mg/dL = Very high

Total Cholesterol HDL Cholesterol

200 mg/dL = desirable <40 mg/dL = low
200 to 239 mg/dL = borderline high 260 mg/dL = high
2 240 mg/dL = high

B. Reference rang
Total cholesterol = 140-200mg/dL
HDL cholesterol = (Male) 29 to 60mg/dL (Female) 38-75mg/dL
LDL cholesterol = 57 to 130mg/dL
Triglycerides 67 to 157mg/dL

C. Conversion factors
Cholesterol (mg/dL. to mmol/L) = 0.026
Triglycerides (mg/dL to mmol/L) = 0.0113

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 PROTEINS
LWhat are Proteins?
A. Characteristics of Proteins
Most abundant macromolecule in the body
The only macromolecule expressed in grams per deciliter, unlike lipids and
carbohydrates, which are both expressed in milligrams per deciliter
Contains carbon, hydrogen, oxygen and nitrogen
Nitrogen is the element that differentiates protein from other macromolecules -

lipids and carbohydrates do not contain nitrogen

Mostly synthesized in the liver, except for some proteins (i.e. immunoglobulins which
are produced by plasma cells)
Water soluble; Amphoteric can be positively charged or negatively charged
Alkaline pH (predominance of OH ions) proteins are negatively-charged
Acidic pH (predominance of H* ions) proteins are positively-charged
Amino acids are the building blocks are proteins. Amino acids have an amino group (-
NH) and a carboxylic acid group (-coOH)
Peptide bonds (also known as amide linkages) hold amino acids together
Essential amino acids are amino acids that are important for protein synthesis. They
are not produced in the body and are therefore "essential" constituent of the diet
Essential amino acids include:
Isoleucine Leucine Lysine Methionine
Phenylalanine Threonine Tryptophan Valine
Structure of proteins:
Primary structure - refers to the sequence of amino acids in a peptide or
protein
Secondary structure may inchude the alpha-helix.beta-sheet.beta-turn or
random structure of proteins
Tertiary structure - folding of proteins into a three-dimensional shape;
denaturation of proteins refers to unfolding that occurs when temperature
change or in the presence of organic solvents, detergents or reagents that may
change the tertiary structure
Quaternary structure -
incorporation of two or more polypeptide chains or
subunits into a larger unit; example include creatine kinase with two subunits as
well as LDH and hemoglobin having four subunits
 CHECKPOINT! Clinical Chemistry by Jude

Proteins can be differentiated based on:

Differential solubility - proteins are affected by pH, ionic strength, temperature
and dielectric constant. Isoelectric point is the pH where proteins have no net
charge. Zwitterion is an ion that has two differing charges but the net charge on
the molecule is zero.
Molecular size - can be accomplished through ultracentrifugation or dialysis
Molecular mass can be accomplished through mass spectrometry
Electrical charge can be accomplished using ion-exchange chromatography
-

and electrophoresis,
Surface adsorption can be done using chromatography
-

Plasma vs Serum protein - plasma contains fibrinogen whereas serum does not; an
approximate 4% decrease in total protein content in serum due to absence of
fibrinogen
B. Protein Metabolism
Protein digestion begins in the stomach and completed in the smal intestines
High acidity denatures the protein making them susceptible to enzyme digestion
Pepsin is an enzyme in the stomach responsible for digesting proteins into amino
acids
Amino acids are absorbed from the small intestines into the blood and then
transported to the liver
C. Functions of Protein
Regulate colloidal oncotic pressure
Act as carrier for transport of different substances
Coagulation cascade
Complement fixation
Serves as biocatalysts or enzymes
Maintenance of acid-base balance (proteins act as buffer)
Immunologic functions (antibodies and complement proteins)
Form many important intracellular and extracellular structures
Important for tissue repair (collagen)
Generate energy through catalysis and electron transfer
Produce motility through contractile elements
Assemble molecules
Serve as ion channels and pumps
Serve as receptors, hormones, and cytokines for intercellular regulation
Constitute signaling networks for intracellular regulation

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 CHECKPOINT! Clinical Chemistry by Jude

D. Major Plasma Protein

FRACTION SPECIFIC PROTEINS
Prealbumin Prealbumin
Albumin Albumin
Alpha1 globulin Alphai antitrypsin, alpha-fetoprotein, alpha-lipoprotein, alpha-1-acid
glycoprotein, alpha-1-anti-chymotrypsin, inter-alpha-trypsin
inhibitor, Gc-globulin
Alphaz globulin Ceruloplasmin, haptoglobin, alpha2 macroglobulin,
Beta globulin Transferrin, hemopexin, beta-2 microglobulin, complement system,
fibrinogen, LDL, VLDL, C-reactive protein
Gamma globulin Immunoglobulins, C-reactive protein (in some books)

E. Proteins and their Specific Functions

PREALBUMIN
also known as transthyretin, migrates anodal (faster) than albumin
2 most predominant protein in the CSF
md

functions as a carrier protein for thyroid hormones and vitamin A

ALBUMIN
protein that is present in highest concentration in the plasma
synthesized by the liver
derived from the Latin name "albus" which means white, originated from the
white precipitate formed during the boiling of acidic urine in patients with
proteinuria
normal life span of albumin in the circulation is 15-19 days
serves the transport protein carrier for most substances
maintains oncotic pressure (or colloidal osmotic pressure) which is defined as
the pressure exerted by proteins in blood plasma that tends to pull water into the
circulation, thereby preventing its extravasation into the tissues
a negative acute phase reactant, since it decreases during inflammation
high serum albumin levels are often associated with dehydration or prolonged
tourniquet application or specimen evaporation
low serum albumin levels can be related to:
inflammation
hepatic disease
urinary loss due to kidney problems
gastrointestinal loss
protein-calorie malnutrition
burn injury
edema and ascites

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CHECKPOINT! Clinical Chemistry by Jude

Terms: Analbuminemia - absence of albumin

Bisalbuminemia presence of 2 bands in the albumin region
-

Hypoalbuminemia decreased levels of albumin

-

GLOBULINS
Alpha-1-antitrypsin accounts to majority of the alpha-1 globulins
Alpha-1-acid glycoprotein serves as carrier for steroid hormones
Alpha-2-macroglobulin accounts to majority of the alpha-2 globulins
Transferrin accounts to 90% of the beta globulins
Transferrin is also known as siderophilin; transferrin is the transport protein for
iron. It is capable of transporting 2 ferric ions; ferritin serves as the soluble
storage form of iron
Haptoglobin transports free hemoglobin; hemopexin transports heme
Haptoglobin and Hemopexin decreases in patients with hemolytic anemia
Ceruloplasmin is an oxidase. It is a blue alpha-2 globulin that transports copper.
90-95% of copper in plasma is bound to ceruloplasmin, the rest to albumin.
Wilson's disease is associated with low ceruloplasmin levels, leading to low
serum copper and copper overload (deposits) in tissues
C-reactive protein serves as a non-specific indicator of inflammation
MISCELLANEOUS PROTEINS
Bence-Jones protein are proteins identical light chains excreted in the urine of
patients with MULTIPLE MYELOMA. Bence Jones Protein characteristically
precipitates at 40-60°C but dissolves at 100°C. Immunofixation is the method
used when to identify Bence Jones Protein.
Myoglobin is a heme containing protein found in the striated skeletal and
cardiac muscles. It can be used as marker of acute myocardial infarction.
Rises at 1-3 hours
Peak at 5-12 hours
Returns to normal in 18-30 hours
Myoglobin is not specific for AMI since it can also be elevated in muscle-related
disorders.
Troponin is a complex of three proteins: Tnl (inhibitory subunit), TnT
(tropomyosin-binding subunit) and TnC (calcium-binding subunit). Cardiac
isoforms of Tnl and TnT are specific for heart muscles.
Cardiac troponin T rises within 3-4 hours
Peak in 10-24 hours
Remains elevated for up to 10-14 days
I
Cardiac Troponin rises within 3-6 hours
Peak in 14-20 hours
Returns to normal in 5-10 days

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 CHECKPOINT! Clinical Chemistry by Jude

II. Specimen Considerations and Patient Preparation:

Serum is preferred; 24-hour urine and serous fluids can also be used
Protein in cSF is LESS THAN 1% compared to PLASMA PROTEIN
Can be used to determine if a certain body fluid is a transudate (low protein) or an
exudate (high protein)
No to lipemia and hemolysis

II. Methodologies of Protein Determination:

Direct Optical Method
Absorbance of UV light at 200-225nm (or 210) and 270-290nm (or 280) have been
used to measure protein concentrations and is applied to monitor chromatographic
separations of peptides and proteins
Kjeldahl method
is based on the digestion of protein with consequent measurement of nitrogen
content
Steps:
1. Initial precipitation: serum proteins are precipitated using organic acids such as
trichloroacetic acid or tungstic acid
aciddigestion to release ammonium ions from nitrogen-containing compounds
by heating with sulfuric acid in the presence of a catalyst (such as cupric sulfate;
potassium sulfate; or H202+ mercury or copper), at 340-360°C
Ammonium ions that form are quantitated by alkalinization, distillation and acid
titration, or by Nesslerization
Alkalinization-Distillation-Titration is done using alkali, boric acid and HCI
Nesslerization is done using Hglz and KI
Considered as the reference method; assumes average nitrogen content of proteins is
16% (multiply N by 6.25). Actual nitrogen content of serum protein ranges from
15.1-16.8%. Some references prefer the use 6.54, which is the average nitrogen
content of different proteins (albumin 6.53, alpha 6.63, beta 6.78, gamma globulins
6.52, etc).
Interferences: NPNs like urea and amino acids can cause an increase therefore
precipitation test is required
Biuret reaction
is based on the formation of violet colored chelate between Cu2 ions and requires at
least 2 peptide bonds, measured at 540nm
Biuret is a molecule that forms when urea, the end product of protein metabolism is
heated at 180°C. Two molecules of urea form one molecule of biuret
Kinetic Biuret method is prefered
amino acids and dipeptides will not be detected by Biuret reaction

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 CHECKPOINT! Clinical Chemistry by Jude

reagents include:
a. Alkaline copper sulfate
b. Sodium hydroxide
C. Rochelle salt (also known as sodium potassium tartrate -~used to complex cupric
ion and prevent their precipitation)
d. Potassium iodide (keeps copper in cupric form)
Interferences in Biuret Reaction
a. High bilirubin, Elevated lipids, Hemolyzed sample false increased
-

b. High blood ammonia level false decreased

-

Lowry (Folin-Ciocalteu) Method

Specimen are mixed with an alkaline copper solution followed by addition of Folin-
Ciocalteu reagent
Phosphotungstomolybdic acid (Phosphotungstic acid + phosphomolybdic acid) are
reduced to tungsten blue by copper complex with peptide and by tyrosine and
tryptophan
Absorbance products are measured at 650-70Onm; dependent on phenolic groups
Dyes:
a. Bromcresol purple ~ most sensitive, specific and precise among the dye-binding assays
b. Bromcresol green (BCG)~ most commonly used;, measure absorbance
eTetrabromphenol blue~ used in urine reagent strip, sensitive to albumin
d Ninhydrin ~ for amino acids
e. Methyl orange
E Hydroxybenzeneazobenzoic acid (HABA)
g Coomassie brilliant blue
h. Pyrogallol red~ used for analysis of fluids with lower protein concentrations such as
urine and CSF
Refractometry
Rapidly estimates protein at high concentrations
Often used in urine specimen
Salt precipitation (turbidity) Test~used for urine and cSF
a. sulfosalicylic acid often used as 3% SSA, precipitates all forms of protein
-

b. trichloroacetic acid preferentially precipitates globulins than albumin

-

C. sulfosalicylic acid +sodium sulfate

d. benzethonium chloride under alkaline condition
e. benzalkonium salts under alkaline condition
Electrophoresis
Movement of proteins during alkaline electrophoresis, from origin towards the
anode:
GammaBeta Alpha 2 Alpha 1 Albumin Prealbumin
Gamma globulins tend to migrate towards the cathode instead of moving towards
the anode during electrophoresis. This is known as electroendosmosis.

"Never put off til tomorrow the book you can read today"- Holbrook Jackson 34
 CHECKPOINT! Clinical Chemistry by Jude

a. First, serum is electrophoresed, at an alkaline pH (8.6). Since the pH is alkaline,

proteins in serum become negatively charged, causing them to be attracted towards
the positive pole or electrode known as the anode
b. After separation, regions are stained using Coomassie Brilliant blue, Amido black and
Ponceau S
C. After staining, stained bands are quantified using densitometer
d. Electrophoretic patterns:
beta-gamma bridging effect-seen in patients with liver cirrhosis caused by an
increase in lgA which is found between the beta and gamma region during
electrophoresis
gamma spike - seen in cases of monoclonal gammopathy (i.e. multiple myeloma
and Waldenstrom's macroglobulinemia)
alpha-2-macroglobulin elevation with albumin decrease seen in patients
with nephrotic syndrome
alpha-1-antitrypsin deficiency- seen in patients with emphysema
increased beta region - use of plasma instead of serum during electrophoresis
low albumin, high alpha-1 and alpha-2 region - acute inflammation

V. Values to Remember:
Reference Range: Total Protein: 6.5 to 8.3 g/dL
Albumin: 3.5 to 5.5 g/dL
Conversion Factor: g/dL to g/L = 10

"Never put off til tomorrow the bookyou can read today"- Holbrook Jackson 35
 ENZYMES c
I. What are Enzymes?
A. Characteristics of Enzymes
Biocatalysts, mostly protein in nature, that enable the necessary metabolic reactions to
occur at body temperature, speeding up reaction rates
Act by decreasing activation energy required for the biochemical reaction to push through
Affects only the rate of the reaction; affects the rate equally on both directions (for
reversible reactions)}; they are not used up or consumed in the reaction
Most chemical reactions in the body occur at a pH 7.0 and temperature of 37°C
B. Enzynmology - Definition of Terms
Holoenzyme
Whole enzyme molecule; apoenzyme + cofactor
Apoenzyme
Protein portion of the enzyme
Cofactor
Non-protein portion of the enzyme; can be organic or inorganic
Coenzymes - organic (carbon-containing) cofactors
Examples: NAD/NADH or NADP/NADPH used in dehydrogenase reactions
Cysteine used in creatine kinase reaction
Pyridoxal (vitamin B6) phosphate used in transaminase reactions
Activators- inorganic (non carbon-containing) cofactors; can be metallic or non
metallic
Metallic Calcium for amylase
Magnesium for creatine kinase
Zinc for lactate dehydrogenase
Others: iron, manganese, copper
Non-metallic Chloride for amylase
Zymogens or Proenzymes
inactive forms of enzyme
Pepsinogen - inactive form of enzyme pepsin, which promotes protein digestion
- produced by the chief/zymogenic cells of the stomach
converted into pepsin upon contact with gastric acid
Plasminogen inactive form of enzyme plasmin, which promotes fibrinolysis
produced by the liver
-
activated by urokinase or tissue plasminogen activator (tPA)
 CHECKPOINT! Clinical Chemistry by Jude

Isoenzymes
enzymes that catalyze the same reaction but differ in terms of physical or chemical
characteristics, tissue distribution as well as electrophoretic mobility
Example:
Lactate dehydrogenase - LD1, LD2, LD3, LD4, LD5
Creatine kinase CKMM, CKMB, CKBB

Alkaline phosphatase -
intestinal ALP, placental ALP, liver ALP, bone ALP
Acid phosphatase - prostatic and erythrocytic ACP
Macroenzymes
are high-molecular-mass forms of the serum enzymes; large enzymes
two types: macroenzyme type 1 = enzyme bound to an immunoglobulin
macroenzyme type 2 = enzyme bound to non-immunoglobulin substance
such as drugs or plasma membrane fragments
usually found in patients who have an unexplained persistent increase of enzyme
concentrations in serum
larger enzymes leads to decreased clearance of enzymes in the circulation
enzyme bound to immunoglobulin especially, have longer half-life (since IgG have
lifespan of approximately 3 weeks)
Example: macro-CK, macroamylase
Substrate
precursor of a product in an enzymatic reaction
binds to the active site of the enzyme
C. Enzymatic Reaction

ENZYME+ SUBSTRATE > ES cOMPLEX ENZYME + PRODUCT

mmmmmm.mme
D. Factors that affect enzymatic reaction
pH
excessively alkaline or acidic pH can increasing
en2yme
affect the enzymes and can denature activity
enzymesS
optimum
some enzymes however, perform well in pH

alkaline (eg. ALP) and acidic (eg. ACP

10
and pepsin) environment pH
hitte-//www.bbc.co.uk/staticarchive/db 7bs8 i3 5d8ct5ce20bs8387bf6778443 6ce279,git
wwww

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 37
CHECKPOINT! Clinical Chemistry by Jude

Temperature
Reaction velocities of most chemical reactions
increasing
increase with temperature and approximately enzyme
activity
double for each 10°C rise
most enzymes become denatured and optimum
temperature
insoluble within minutes of being subjected to
10 20 30 40 50 60 70
temperatures of 60°C and above temperature (°c
Most body enzymes have temperature s/chesesnerpersad.ties wordpress.com/a2015/04/gcsechem 18par

optimum close to 37-38°C and have progressively less activity as temperature rises
above 42-45°C
Low temperatures decrease enzyme activity and they may be completely inactive at
temperatures of 0°C and below
Inactivity at low temperatures is reversible, most enzymes may be preserved by
storage at -20 to -70°C
Enzyme concentration
Velocity of reaction is directly More enzyme nolecules
can react with more
substrate molecules, so
the reactuon rate
proportional to enzyme ncreases.

concentration in the presence of

excess substrate
Reaction proceeds more rapidly
when more enzyme molecules are
Enzyme Concentration -
present to bind substrate
This scenario assumes that there is
Substrate concentration a targe excesS of substrate.
httprsabeerahmohammed.fiies, wordpress.com/2045/04/enzyme.gif

Michaelis-Menten hypothesis
The higher the substrate concentration, the more substrate bound to enzyme and
the greater the rate/velocity of the
reaction
When all enzyme is bound to
substrate, there will be no further
increase in velocity- this is the
maximum velocity
When the substrate is present in an
adequate amount (all enzymes are
bound and saturated), the rate of
reaction depends only in enzyme Substrate concentration
http://classes.midlandstech.edu/carterp/courses/bio225/chapo5/slide7.GIF
concentration

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
- Vivian Greene 38
CHECKPOINT! Clinical Chemistry by Jude

First order reaction

substrate readily binds to free enzyme at a low-substrate concentration
with the amount of enzyme exceeding the amount of substrate, the reaction rate
steadily increases as more substrate is added
ratedependsonsubstrate concentration
reaction rate is directly proportional to substrate concentration
Zero order reaction
high substrate concentration saturates all available enzymes; fewer binding sites
are available for attachment
reaction velocity reaches maximum; rate of increase of the velocity falls to zero
velocity is independent of substrate concentration when binding sites are
saturated
rate does not depend on substrate concentration; reaction rate depends only on
enzyme concentration
when product is formed, the resultant free enzyme immediately combines with
excess free substrate
Inhibitors
Substances that inhibit enzymatic reaction
Competitive Inhibition
inhibitor binds the active site of enzyme
substrate and inhibitor compete for the same binding site
addition of more substrate results in the displacement of inhibitor molecules by
substrate molecules, with a consequent increase in enzymatic activity
can be reversed by the addition of substrate molecules
Non Competitive Inhibition
inhibitor binds enzyme at a place other than the active site (allosteric site) of
enzyme
alter the configuration of the enzyme in such a way as to reduce or abolish excess
of the substrate to the active site, causing a decrease in enzyme activity
may be reversible (if binding is reversible or may be separated) or irreversible (if
inhibitor destroys a part of enzyme)
addition of substrate has NO effect since it cannot reverse the alteration caused by
inhibitor

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
- Vivian Greene 39
 CHECKPOINT! Clinical Chemistry by Jude

Uncompetitive Inhibition
inhibitor binds the ES complexX

the resulting enzyme-substrate-inhibitor complex does not yield product

increasing substrate concentration results in more ES complexes to which the
inhibitor binds and, thereby, increases the inhibition

The Lineweaver-Burk plots for inhibition

inhibitor -inhibitor inhibitor

1/ no inbibator 1/ 10 inhibitor 1/ noinhibitou

stope-K/
1/sl 1/s 1/s)
Competitive Uncompetitive Noncompetitive
inhibitio inhibitio inhibitio
Ky increased K reduced Kt unaffected
may unaffected max reduced max reduced
Taken fram: https://biochem eek.wordprezs.com/page/3/

lonic strength or Electrolyte environment

Electrolytes such as calcium, magnesium, zinc and chloride are activators and may
be required by some enzymatic reactions
if ionic strength is too high, enzyme activity drops
in protein-free solutions, enzymes lose activity rapidly
E. Importance of Enzyme Detection
To detect tissue injury or damage
To identify abnormalities or deficiency of enzymes
F. Measuring Enzymatic activity
Most enzyme tests used in clinical chemistry laboratory follow Michaelis-Menten kinetics
and are performed under conditions of zero-order reaction -a large excess of substrate is
present so that the amount of enzyme activity is the only rate-limiting factor in the assay
Enzymes are measured based on their activity rather than their mass or molar
concentration
Enzymatic activity = can be measured as a rate of product produced or as a rate of
substrate consumed per unit of time
Units used in expressing enzymatic activity
Enzyme activity can be expressed either in IU or KU.
International Unit - micromole ofsubstrate per minute; also known as U/L
Katal Unit mole of substrate per second
-

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
Vivian Greene
-
40
 CHECKPOINT! Clinical Chemistry by Jude

Approaches to measurement of enzymatic activity:

Fixed-Time (static or two-point) assay
the reactants are combined, the reaction proceeds for a designated time, the
reaction is stopped (usually by inactivating the enzyme with a weak acid)
measurement of a product or substance produced over a given amount of
time
the reaction is assumed to be linear over the reaction time
the larger the reaction, the more enzyme is present.
Multipoint Continuous Monitoring (Kinetic assays)
Multiple measurements, usually of absorbance change, are made during the
reaction, either at specific time intervals (usually every 30 or 60 seconds) or
continuously by a continuous-recording spectrophotometer.
Measurement of a substrate product per minute (or hour) produced
constantly over a period of time
advantageous over fixed-time methods because the linearity of the reactioni
may be more adequately verified; usually preferred
if absorbance is measured at intervals, several data points are necessary to
increase the accuracy of linearity assessment
Continuous measurements are preferred because any deviation from
linearity is readily observable.
Immunoassays nowadays measure concentration of some isoenzymes (eg. prostatic-
specificisoenzyme, CKMB mass,)
Immunoassays may overestimate active enzyme as a result of possible cross-reactivity
with inactive enzymes, such as zymogens, inactive isoenzymes, macroenzymes, or
partially digested enzyme.
Most enzyme measurements require the use of serum since anticoagulants may inhibit
activators (like calcium and magnesium) which are necessary for enzyme reactions
G. Methods for Measuring Enzymatic activity
Spectrophotometric (colorimetric)
More cumbersome and less sensitive
Requires large amount of substrate (which may later on act as enzyme inhibitor)
and longer incubation time
Manometry (measures pressure of gases and vapor)
Evolution of gas or disappearance of gas as the reaction proceeds
Fluorometry
Coupled-enzymatic reactions

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 41
 CHECKPOINT! Clinical Chemistry by Jude

H. Principles of Enzyme Data Interpretation

1. There is no truly "organ-specific" enzyme - since all enzymes are found in more than one
tissue
2. Serial measurements provide most useful data; a single measurement can be misleading
i.e. serial measurements over the course of several days checking for patterns of
increase or decrease]
3. Negative (normal) results are useful especially when ruling out presence of tissue
damage
4. Enzyme date must be integrated with other information -
other pertinent data must be
used in conjunction with enzyme result before a diagnosis can be made
I. Classification of Enzymes
1. Oxidoreductases- oxidize one substrate and reduce the other
2. Transferases -
transfer a group (other than hydrogen) from one substrate to another
3. Hydrolases - catalyze hydrolysis or splitting up of a bond in a substrate, resulting in the
formation of two or more molecules
4. Lyases-Catalyze removal of groups from substrates without hydrolysis; the product
contains double bonds
Isomerases -
catalyze the interconversion of geometric, optical, or positional isomers
6. Ligases - Catalyze the joining of two substrate molecules, coupled with breaking of the
pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound
(1) Oxidoreductase (2) Transferase (3) Hydrolases
(dehydrogenase) kinase/ transaminase)
Lactate creatine kinase acid & alkaline
dehydrogenase aspartate phosphatase
Glucose-6-phosphate aminotransferase (SGOT) cholinesterase
dehydrogenase alanine aminotransferase lipase
-Malate (SGPT) trypsin
dehydrogenase gamma glutamyl pepsin
Isocitrate transferase (GGT) leucine
dehydrogenase hexokinase aminopeptidase
Cytochrome oxidase pyruvate kinase amylase
Glutamate glycogen phosphorylase glucosidase
dehydrogenase glutathione-S-transferase galactosidase
Beta-hydroxybutyric 5 nucleotidase
dehydrogenase chymotrypsin
elastase
(4) Lyase (5) Isomerase (6) Ligase
(decarboxylase)
aldolase glucose phosphate isomerase glutathione
pyruvate decarboxylase ribose phosphate isomerase synthetase
glutamate decarboxylase triosephosphate isomerase
tryptophan decarboxylase

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 42
 CHECKPOINT! Clinical Chemistry by Jude

I. Enzymes Detected in the Laboratory

A. Aldolase
1. Characteristics of Aldolase
catalyzes an early step in glycolysis for glucose which is the conversion of:

Ald dihydryoxyacetone phosphate

D-fructose-1, 6-diphosphate
D-glyceraldehyde-3 phosphate
2. Clinical Significance of Aldolase
Highest level in progressive muscular dystrophy
Elevated in skeletal muscle disease or injury, metastatic carcinoma of the liver,
granulocytic leukemia, megaloblastic anemia, hemolytic anemia and tissue infarction in
general
In myocardial infarction: rises 6-8 hours stays elevated up to 3-4 days
3. Specimen Considerations
No to hemolyzed sample (RBC aldolase level is 150 times as high as the serum level)
Plasma is preferred over serum because of the possible release of platelet enzyme
during clotting
4. Methodologies
Sibley-Lehninger Method
Substrate: fructose-1, 6-diphosphate
Product measured: dihydroxyacetone-phosphate
Coupled enzymatic reaction
Reagent Enzymes: Triosephosphate isomerase and glycerol-3-phosphate
dehydrogenase
Measure the decrease in NADH concentration
5. Reference Range: 2.5 to 10.0U/L at 37°C

B. Lactate Dehydrogenase (LDH)

1. Characteristics of LDH
catalyzes the reversible conversion of lactate and NAD into pyruvate and NADH
LDH
LACTATE NAD PYRUVATENADH
has zinc as one of its component
present in almost all types of tissue; tissue non-specific

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-
Vivian Greene 43
CHECKPOINT! Clinical Chemistry by Jude

2. Isoenzymes of LDH
Electrophoresis
ORIGIN ANODE
LDS >>> LD4 LD3 >>> LD2 >>> LD1
Concentration in serum: LD 2>1>3>4>5
LD4 and LD5 are cold labile fractions
LD1 is the most negatively charged isoenzyme
LD1 isthe fastest toward the anode; LD5 is the slowest
Alpha-hydroxylbutryic dehydrogenase (HBD) has the same kinetic properties as LD1 but
does not act on the substrate lactate
HBD can be measured using Rosallki-Wilkinsonmethod, which uses ketobutyrate as a
substrate with subsequent measurement of change in NADH absorbance
APPROXIMATE % OF
CHAIN TISSUE RICH IN THE
TOTAL NORMALLY
COMPOSITION IsOENZYME
PRESENT IN SERUM
LD HHHH 29-37 Heart, brain, erythrocytes
LD2 HHAM 42-48 Heart, brain, erythrocytes
LD3 HHMM 16-20 Brain, kidney
LD4 HMMM 2-4 Liver,skeletal muscle, kidney
LDs MMMM 0.5-1.55 Liver, skeletal muscle, ileum
3. Clinical Significance
used as a non-specific enzyme marker for acute myocardial infarction (AMI)
Pattern of increase during AMI: Increases 12 to 24 hours after the onset of AMI
Peaks at 48-72 hours after the onset of AMI
Normalizes within 10 days
LDflip (LD1>LD2) is oftentimes associated with AMI
Highest elevation of LDH is found in megaloblastic.anemia [pernicious anemial
Conditions affecting total lactate dehydrogenase activity
PRONOUNCED ELEVATION MODERATE ELEVATION SLIGHT ELEVATION
(5 OR MORE TIMES (3-5 TIMES NORMAL) (UP TO 3 TIMES
NORMAL) NORMAL)
Megaloblastic anemia Myocardial infarction Most liver diseases
Widespread carcinomatosis, Pulmonary infarction Nephrotic syndrome
especially hepatic Hemolytic conditions Hypothyroidism
metastases Leukemias Cholangitis
Systemic shock and hypoxia Infectious mononucleosis
Hepatitis Delirium tremens
Renalinfarction Muscular dystrophy

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
- Vivian Greene 44
 CHECKPOINT! Clinical Chemistry by Jude

4. Specimen Considerations and Patient Preparation

Serum is preferred, anticoagulants can inhibit LDH
No to hemolysis; LDH is a lot more concentrated in RBCs than in plasma
Do not store in freezing temperature; LD5 is cold labile, specimens for LDH should be
stored at room temperature
5. Methodologies:
Wacker (forward reaction)
measures enzymatic activity as lactate is converted to pyruvate
measurement of activity can be through kinetic (uses UV) or colorimetric (addition of
color reagents)
KINETIC: measures the increase in absorbance at 340nm as NAD is converted to
NADH

COLORIMETRIC: addition of phenazine methosulfate and nitroblue tetrazolium

which reacts with NADH to produce a positive blue-purple color
COLORIMETRIC: addition of p-nitrophenylhydrazine (or 2,4

dinitrophenylhydrazine) which reacts with pyruvate producing phenylhydrazone

-a golden brown color at alkaline pH measured at 440 or 525nm
Wrobleuski La Due (reverse reaction)
measures enzymatic activity as pyruvate is converted to lactate
measures the decrease in absorbance at 340nm as NADH is converted to NAD
3x faster than the forward reaction

C. Creatine Kinase (Creatine phosphokinase)

1. Characteristics of Creatine kinase
catalyzes the transfer of phosphate to creatine

creatine+ ATP creatine phosphate+ ADP

CK requires magnesium and thiol source (cysteine)
inhibited by zinc and manganese; excess Mg can also inhibit CK
2. Isoenzymes of Creatine Kinase
95% of total CK activity is derived from CK-MM
Half-life of CK isoenzymes: CK-BB = 2-3 hours CK-MB 12 hours
CK-MM = 15 hours
Most CK are found in cytoplasm
mitochondrial CK is the CK found within the mitochondria of the cell. It does not
appear in the circulation unless more severe cellular damage takes place

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-Vivian Greene 45
CHECKPOINT! Clinical Chemistry by Jude

3. Clinical Significance of Creatine Kinase

CK-MB in AMI: rises after 4to 8 hours
peaks at 12-24 hours
returns to normal within 48 to 72 hours (2-3 days)
Highest elevation of creatine kinase is found in Duchenne's muscular dystrophy
NOT present in liver
CONDITIONS AFFECTING CREATINE KINASE
PRONOUNCED ELEVATION MILD OR MODERATE ELEVATION
(5 ORMORE TIMES NORMAI (2-4 TIMES NORMAL)
Duchenne's muscular dystrophy Severe exercise, trauma, surgical
Polymyositis procedure, intramuscular injections
Dermatomyositis Delirium tremens, alcoholic myopathy
Myocardial infarction severe ischemic injury
Pulmonary infarction
Pulmonary edema (some patients)
Hypothyroidism
Acute agitated psychoses
4. Specimen Considerations and Patient Preparation:
No to hemolysis. CK is NOT present in RBCs. However, adenylate kinase, which
catalyze a similar reaction as that of CK is present, causing a false increase in CK
when hemolysis is present
Stored in the dark since it has been found that CK is inactivated by light
5. Methodologies
Sulfhydryl activator (e-g. cysteine, N-acetyl cysteine, glutathione, dithiothreitol or
Cleland's reagent, monothiolgycerol) is required
Methods:
Tanzer-Gilvarg (forward reaction)
ATP +creatine ADP +creatine phosphate
ADP formed from the forward CK reaction of CK is reacted with PKand LDH
Pyruvatekinase: ADP + phosphoenol pyruvate ATP + pyruvate
LDH: pyruvate + NADH lactate + NAD

*NADH absorbs light at 340nm; NAD does not; pH is maintained at 9.0

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-
Vivian Greene 46
 CHECKPOINT! Clinical Chemistry by Jude

Oliver-Rosalki (reverse reaction)

ADP +creatine phosphate > ATP + creatine
ATP formed from the reverse CK reaction is reacted with HKand G6PD
Hexokinase: ATP + glucose ADP + glucose-6-phosphate
+
G6PD: G6P NADP* 6-phosphogluconate + NADPH
*NADPH absorbs light at 340nm; NADP does not
**pH is maintained at 6.7
Reverse reaction is 6X faster compared to forward reaction
CK-MB mass vs CK-MB
CK-MB mass assay uses monoclonal antibody technology it does not
-

depend on protein's enzymatic activity unlike the usual CK-MB assays

D. Aspartate Aminotransferase/ Transaminase (AST)

1. Characteristics of AST
previously known as the serum glutamic-oxaloacetic transaminase (SGOT)
catalyzes a reversible reaction between:
ASPARTATE + a-KETOGLUTARATE OXALOACETATE+ GLUTAMATE
requires pyridoxal phosphate as coenzyme
widely distributed in tissue; highest in cardiac tissue, liver, muscle
2. Clinical Significance of AST
often used in conjunction with ALT for hepatocellular disorders
AST during AMI: rises after 6-8 hours
peaks at 24 hours
returns to normal within 5 days
conditions affecting AST
PRONOUNCED ELEVATION MODERATE ELEVATION SLIGHT ELEVATION
OR MORExN)
(5 (3-5 x N) (UP TO3x N)
Acute hepatocellular damage Biliary tract obstruction Pericarditis
Myocardial infarction Cardiac arrhythmias Cirrhosis
Circulatory collapse (shock) Congestive heart failure Pulmonary infarction
Acute pancreatitis Metastatic or primary Delirium tremens
Infectious mononucleosis tumor in liver Cerebrovascular
Muscular dystrophy accident
3. Specimen Considerations
Hemolysis should be avoided because it can dramatically increase serum AST
concentration.
AST activity is stable in serum for 3 to 4 days at refrigerated temperatures

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 47
 CHECKPOINT! Clinical Chemistry by Jude

4. Methodologies
Karmen method: coupled enzymatic reaction
Enzymatic reaction for AST:
aspartate + alpha ketoglutarate + Oxaloacetate +glutamate
Add malate dehydrogenase:
oxaloacetate + NADHmalate + NAD+

Measure loss of absorbance at 34Onm due to formation of NAD

Optimal pH is 7.3-7.8
Diazonium salt
formation of diazonium derivative
Colorimetric method: Reitman-Frankel
reagent: dinitrophenylhydrazone
formation of blue color, measured at 505nm
lack of specificity, reacts with any keto-compound

E. Alanine Aminotransferase/ Transaminase (ALT)

1. Characteristics of ALT
previously known as serum glutamic-pyruvic transaminase (SGPT)
catalyzes:
ALANINE+ a-KETOGLUTARATE GLUTAMATE+PYRUVATE
2. Clinical Significance
Highest elevation is found in acute liver hepatitis
specific for liver disease compared to AST
3. Specimen Considerations
ALT is stable for 3 to 4 days at 4°C
relatively unaffected by hemolysis.
4. Methodologies
Enzymatic method
Enzymatic reaction for ALT:
alanine + alpha ketoglutarate pyruvate + glutamate
Add lactate dehydrogenase:
pyruvate +NADHlactate + NAD

Measure loss of absorbance at 340Onm due to formation of NAD

Optimal pH is 7.3-7.8
Diazonium salt
formation of diazonium derivative

"Life isn't about waiting for the storm to pass. t's about learning to dance in the rain"
-
Vivian Greene 48
 CHECKPOINT! Clinical Chemistry by Jude

Colorimetric method: Reitman-Frankel

reagent: dinitrophenylhydrazone
formation of blue color, measured at 505nm
lack of specificity, reacts with any keto-compound

F. Alkaline phosphatase
1. Characteristics of ALP
Catalyzes hydrolysis of phosphomonoesters (or organic phosphate esters) into alcohol
and phosphate at an alkaline pH (9.0-10.0)
Requires activator zinc
2. Isoenzymes
Normal isoenzymes: intestinal, placental, bone and liver
Liver and bone ALP are the most predominant fractions
can be differentiated using electrophoresis, heat denaturation and chemical inhibition
Carcinoplacental isoenzymes include Regan, Nagao and Kasahara. They are usually
found in patients with malignancy and their characteristics resemble that of placental
ALP
Electrophoresis (origin towards anode)
INTESTINAL PLACENTAL BONE > LIVER
Liver and bone fractions are difficult to resolve during electrophoresis
To improve separation of bone and liver forms, use:
neuraminidase (to remove sialic acid)
wheat germ lectin (to bind other isoenzymes)
Heat denaturation (most heat stable to most heat labile)
PLACENTAL INTESTINAL > LIVER > BONE
Heat stability is determined by heating serum at 56°C for 10-15 minutes
Placental ALP~ most heat stable of all the normal ALP isoenzymes (up to 60°C for
10 mins)
Regan ALP ~ most heat stable among all the types of ALP
Chemical Inhibition
L-phenylalanine inhibits placental, intestinal, Regan and Nagao
~

Levamisol~ inhibits liver and bone isoenzymes

L-homoarginine ~ inhibits liver and bone
isoenzymes
2M Urea~ inhibits bone isoenzyme
L-leucine~inhibits Nagaoisoenzyme
20% ethanol denatures liver ALP rapidly than bone

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 49
CHECKPOINT! Clinical Chemistry by Jude

3. Specimen Considerations
No hemolysis since AlLP is 6x more concentrated in RBCs than serum
ALP assays should be run ASAP after collection since activity in serum increases
approximately 3% to 10% on standing at 25°C or 4°C for several hours
Diet may induce elevations in ALP activity of blood group B and O individuals who are
secretors.
Values maybe 25% higher following ingestion of a high-fat meal due to increase
intestinal fraction
4. Methodologies for Total ALP Activity
REACTION NAME sUBSTRATE COMMENTS
USED
Shinowara-Jones- Beta- Long incubation time
Reinhart glycerophosphate high blank values
King-Armstrong Phenylphosphate Endpoint, requires protein removal
Bessey-Lowry- p-Nitrophenyl Endpoint or kinetic, rapid
Brock phosphate p-nitrophenol (colorless end product)
at alkaline pH forms quinoid structure
with intense yellow color
reaction time is 30 minutes; reaction is
stopped using NaOH (inactivates
enzyme and acts as color developer)
absorbance is measured at 400-410nm
original BLB method uses glycine
buffer
Bowers-McComb p-Nitrophenyl Uses phosphate-accepting buffer (a
(Comb) phosphate transphosphorylating buffer, i.e. AMP
buffer also known as 2-amino-2-
methyl-1-propanolol) at pH 10.5 at
30°C
Other modifications include use of
diethanolamine as buffer
reference method
measures p-nitrophenol (yellow)
characterized by increased absorbance
at 405nm
4-nitrophenyl Kinetic; automated
phosphate Measurement of 4-nitrophenoxide
(yellow)

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Vivian Greene 50
 CHECKPOINT! Clinical Chemistry by Jude

5. Clinical Significance
Often used in evaluation of hepatobiliary (obstructive conditions) and bone disorders
(osteoblast involvement)
Highest elevation of ALP is seen in: Paget's disease (osteitis deformans)
Hepatocellular disorders (cirrhosis and hepatitis) produces only slight elevations (less
than 3 x ULN)
Biliary obstruction (cholestasis)~3-10 times elevation
Physiologic elevation of ALP can be seen in growing children due to osteoblastic
activity
Conditions affecting alkaline phosphatase
PRONOUNCED ELEVATION MODERATE ELEVATION SLIGHT ELEVATION
(5 OR MORE x N) (3-5 x N (UP TO 3 N) x
Bile duct obstruction Granulomatous or Viral hepatitis
(intrahepatic or extrahepatic) infiltrative diseases of Cirrhosis
Biliary cirrhosis liver Healing fractures
Osteitis deformans (Paget's Infectious mononucleosis Pregnancy (t
disease) Metastatic tumors in bone placental ALP)
Osteogenic sarcoma Metabolic bone diseases Normal growth
Hyperparathyroidism rickets, osteomalacia) patterns in children

G. Acid Phosphatase
1. Characteristics of Acid Phosphatase
Catalyzes hydrolysis of phosphomonoesters (or organic phosphate esters) into alcohol
and phosphate at an acid pH (5.0-6.0)
Greatest concentrations occur in prostate, liver, spleen, RBCS, platelets and bone
Highest concentrations in prostate gland secretions and erythrocytes
Lysosomal, prostatic, erythrocyte, macrophage, and osteoclastic ACPs are five
important types found in humans
Tartrate-resistant acid phosphatase can be found in certain leukemia (hairy cell
leukemia)
2. Isoenzymes
Electrophoretic separation
Erythrocytic ACP remains in origin Prostatic ACP migrates with great mobility
Chemical Inhibition:
Erythrocytic acid phosphatase Prostatic acid phosphatase
~inhibited by 2% formaldehyde solution inhibited by L-tartrate
and 1mM cupric sulfate solution specific acid phosphatase
non specific acid phosphatase

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CHECKPOINT! Clinical Chemistry by Jude

3. Specimen Considerations
Specimen must be acidified to prevent loss of ACP. Citrate is the preferred
anticoagulant, buffered to a pH of 6.2-6.6
Plasma is the sample of choice to minimize platelet contamination
Serum should be separated from clot to prevent leakage of ACP from RBCs and
platelets
Serum activity decreases within 1to 2 hours at room temperature without the
addition of a preservative, due to loss of carbon dioxide in the sample which results in
an increase in pH
if not assayed immediately, serum should be frozen or acidified to a pH lower than 6.5
Hemolysis should be avoided because of contamination from erythrocyte ACP, causing
false elevation.
Fluoride inhibit ACP; oxalate and heparin causes a false decrease
4. Methodologies for Total ACP Activity
REACTION NAME SUBSTRATE USED COMMENTS
| Bodansky Beta-glycerophosphate | Lengthy assay, nonspecific
Gutman, King- Phenylphosphate Nonspecific
Armstrong
Hudson Rapid, nonspecific
nitrophenylphosphate Substrate is converted to p-
nitrophenol, addition of a base
produces p-nitrophenolate (yellow)
which is measured at 410nm
Babson and Reed Alpha- Complicated, less sensitive; preferred
naphthylphosphate forcontinuous monitoring reactions
Roy Thymolphthalein more specific for prostatic form;
monophosphate method of choice for quantitative
endpoint reactions
measurement of product
thymolphthaleinat 590nm
Rietz, Guilbault 4-Methylumbelliferone Fluorescent, some improved
phosphate sensitivity
5. Clinical Significance
Elevated in patients with prostatic carcinoma, however it is not specific since it can
also be elevated in prostatic hyperplasia and prostatic surgery
Useful in forensic clinical chemistry- especially in medico legal evaluation of rape; ACP
can be detected in vaginal washings for up to 4 days; it can also be detected in
beddings, undergarments or clothes stained with seminal fluid in the crime scene
ACP may also be elevated in bone diseases due to osteoclastic activity, as wel as in
Gaucher disease

"Life isn't about waiting for the storm to pass. t's about learning to dance in the rain
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Vivian Greene 52
 CHECKPOINT! Clinical Chemistry by Jude

H. Gamma Glutamyl Transferase

1. Characteristics of GGT
involved in the transfer of the glutamyl residue from gamma glutamyl peptides to
amino acids
regulates synthesis of glutathione and certain components of the movement of amino
acids into cells
widely distributed; highest in kidneys
found on outside surface of the cell membrane
2. Clinical Significance of GGT
used for evaluation of obstructive jaundice, biliary obstruction and liver cancer
it is only slightly increased in hepatitis and liver disorder
most sensitive enzymatic indicator of hepatobiliary disease
first abnormal liver function test in heavy drinkers; elevated levels may indicate
chronic alcoholism and alcoholic cirrhosis
can be used as a means to differentiate the cause of elevation of ALP (as to whether
it is due to liver or bone disorder)
Patients on anticonvulsants manifest marked increase in GGT (dilantin or
phenobarbital)
Newborns have higher values compared to adults but decline by about 6 months
Gross obesity results in some increase in GGT
3. Specimen Considerations
Serum is preferred
Citrate, oxalate and fluoride inhibit the enzyme; heparin causes turbidity
GGT activity is stable, with no loss of activity for 1 week at 4°PC

4. Methodologies
Substrate: gamma-L-glutamyl-p-nitroanilide
Product to be measured: p-nitroanilide (405-420nm); kinetic method
Substrate: gamma-L-glutamyl-2-carboxy-4-nitroanilide
Product to be measured: 5-amino-2 nitrobenzoate (410nm)
Also known as the Szasz method
5. Reference Values
Males: up to 40U/L Females: up to 25U/L

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
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Vivian Greene 53
 CHECKPOINT! Clinical Chemistry by Jude

I. 5-nucelotidase
1. Characteristics of 5-NT
also known as 5-ribonucleotide phosphohydrolase
a metalloprotein with zinc as it's integral component
acts only on nucleotides phosphorylated on the 5th ribose C-atom to give nucleotide
and phosphate
5'ribonucleotide + H20> ribonucleoside + phosphate
2. Specimen Considerations
Do not used EDTA
3. Clinical Significance of 5'-NT
Elevated in liver-related conditions
Similar to GGT, 5-NT is most commonly used to determine whether the source of an
elevated ALP is liver or bone.

Glucose-6-phosphate dehydrogenase
1. Characteristics of G6PD
Converts glucose-6-phosphate and NADP into 6-phosphogluconate and NADPH
Sources of G-6-PD include the adrenal cortex, spleen, thymus, lymph nodes, lactating
mammary gland, and erythrocytes. Little activity is found in normal serum
2. Clinical Significance of G6PD
For assessment of the X-linked disorder G6PD deficiency
3. Specimen Considerations
Red cell hemolysate is used to assay enzyme deficiency; serum is used to assay
enzyme elevation
EDTA whole blood; must be shipped chilled at 4°C
4. Methodology
Measurement of increase in NADPH concentration by measuring increase in
absorbance at 340nm
5. Reference Values
Normal value: 8-14U/g of hemoglobin in RBC hemolysate

K. Leucine Aminopeptidase
Catalyzes hydrolysis of N-terminal residue from certain peptides and amides
(especially leucine) containing a free amino group
Elevated in disease of the liver, biliary tract and pancreas

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
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Vivian Greene 54
 CHECKPOINT! Clinical Chemistry by Jude

L.Angiotensin-ConvertingEnzyme (ACE)
1. Characteristics of ACE
also known as kininase lI and peptidyl-dipeptidase
Converts the decapeptide angiotensin I into a vasoconstricting-octapeptide
angiotensin 2

Conversion occurs in the lungs

Macrophages and epithelioid cells are the main tissue sources
2. Clinical Significance
Elevated in patients with active sarcoidosis and in disorders involving macrophages
like Gaucher's disease and leprosy
3. Specimen Considerations
Serum samples should be frozen for transport

M. Cholinesterase
1. Characteristics of Cholinesterase
Catalyzes: Choline ester acid + choline
Occurs in two forms:
Pseudocholinesterase
present in plasma and liver
acts on wider variety of choline esters compared to true cholinesterase
active at both high and low substrate concentrations
True cholinesterase (Acetylcholinesterase)
present in nerve endings and erythrocytes
optimally active only at low concentrations of acetylcholine and is inhibited
in higher ones
important for transmission of nerve impulses
2. Clinical Significance
organophosphorus insecticides cause a reduction on both cholinesterases
acetylcholinesterase can be detected in amniotic fluid for assessment of neural tube
defects (spina bifida and anencephaly)
3. Specimen Considerations
Serum or heparinized plasma can be used
4. Methodologies
UV method

Change in UV absorption; hydrolysis of benzoylcholine

Needs special equipment

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
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Vivian Greene 55
 CHECKPOINT! Clinical Chemistry by Jude

Michel method
Substrate: acetylcholine
Measurement of change in pH
Temperature sensitive; much variability among different laboratories
Ellman method
Colorimetric: uses thiol derivative as substrate; formation of thiocholine
derivatives
Common reaction:
Propionylthiocholine propionic acid + thiocholine
Thiocholine + 5,5-dithiobis-(2-nitrobenzoic acid) colored compound
measured at 410nm
Sensitive, rapid and recommended method

N. Amylase (diastase)
1. Characteristics of Amylase
catalyzes the breakdown of starch and glycogen into smaller sugars
requires calcium and chloride for activation
Small in size, normally fltered or cleared from the circulation immediately
originates from the pancreas, liver and salivary glands
2 Isoenzymes
salivary amylase is inhibited by wheat germ lectin
Salivary amylase = Ptyalin migrates fastest to the anode
Pancreatic amylase = Amylopsin migrates slowest to the anode (cathodal)
3. Clinical Significance
early acute pancreatitis marker rises within 2-12 hours
peaks at 24 hours
returns to normal within 3-5 days
non-specific for pancreas since it is also produced by the salivary glands
Conditions affecting serum amylase
PRONOUNCED ELEVATION MODERATE ELEVATION
(5 OR MORExN) (3-5 xN)
Acute pancreatitis Pancreatic carcinoma affecting head of
Pancreatic pseudocyst pancreas (late manifestation)
Morphine administration Mumps
Salivary gland inflammation
Perforated peptic ulcer
lonizing radiation

Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
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Vivian Greene 56
CHECKPOINT! Clinical Chemistry by Jude

4. Specimen Considerations
Amylase can be detected in serum, urine and even in peritoneal fluid
AMS in serum and urine is stable at room temperature for 1 week or at 4°C for 2
months.
Do not use calcium-binding anticoagulants such as citrate, oxalate and EDTA since
amylase requires calcium
Specimen should not be contaminated with saliva since saliva contains 700 times
more amylase compared to serum
Red cells do not have amylase; hemolysis will not interfere except for coupled-
enzymatic reactions
triglycerides suppress or inhibit serum AMS activity - AMS may be normal in acute
pancreatitis with hyperlipemia
morphine and opiates lead to falsely elevated levels due to cause constriction of the
sphincter of Oddi and of the pancreatic ducts, increases inarticulate pressure leading
to regurgitation of AMS into serum
5. Methodologies
Saccharogenic (sugar-generating)
includes classical methods of Folin-Wu or Somogyi- Nelson
measures reducing sugars produced by hydrolysis of starch
Amyloclastic (starch-cutting or iodometric method)
includes method of Caraway
measures decrease in substrate (starch) concentration due to amylase activity
if starch reactions with elemental iodine, blue color forms the intensity of which
-

is proportional to the amount of starch present in the mixture

decrease in blue color means breakdown of substrate (starch), and indicates
increased activity of amylase
uses dye-labeled substrate materials from which amylase releases colored dye
Coupled enzyme reaction or Continuous monitoring
coupling of several enzyme systems to monitor amylase activity
often used for automated procedures
maltopentose or maltotetrose are used as substrate
optimal pH is 6.9
coupled enzymes used include:
a. Amylase glucosidase glucose oxidase
-

b. Amylase -
glucosidase- hexokinase G6PD (change in absorbance at 340nm
as NAD is converted to NADH)

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-Vivian Greene 57
 CHECKPOINT! Clinical Chemistry by Jude

Chromogenic (color breakdown)

measures the increasing color from production of product coupled with a
chromogenic dye
6. Reference Values
serum, 25-130 U/L urine, 1-15 U/h
unit used for amylase activity vary according to each procedure.
Somogyi units are frequently used
conversion factor between Somogyi units and international units is 1.85.

0. Lipase
1. Characteristics of Lipase
hydrolyzes the ester linkages of fats to produce alcohol and fatty acids
2. Clinical Significance of Lipase
found primarily in the pancreas
more specific for acute pancreatitis compared to amylase
rises more slowly compared to amylase
serum lipase level gradually increase 2-4 days after the onset of acute pancreatitis
and remains elevated for 5 days up to 2 weeks
3. Specimen Considerations
No to hemolysis. Hemolysis can cause a false decrease in lipase activity
Serum is preferred
False elevation can occur if specimen is contaminated with bacteria
4. Methodologies:
Cherry and Crandali
uses olive oil as substrate; measurement of the liberated fatty acids by titration
with NaOH after 24-hour incubation
triolein is now used as a substrate for a more pure form of triglycerides
copper salts can be used to determine the amount of acid released; copper ion
forms complex with the fatty acids generated by hydrolysis this fatty acid-
copper complex dissolves in water while the original substrate does not
colorimetric measurement of copper indicates the amount of fatty acids present -

showing the level of enzyme activity

Turbidimetric method- simpler and more rapid; measurement of rate of clearing
as an estimate for lipase activity
Colorimetric method - coupled reactions with peroxidase or glycerol kinase

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
-
Vivian Greene 58
 CHECKPOINT! Clinical Chemistry by Jude

II. Clinical Significance of Enzymes

A. Cardiac Profile
Time Course of Enzyme Activity in Myocardial Infarction (Bishop)
Onset of Elevation Peak Activity Duration of Elevation
() (h) (days)
CK 4-8 12-24 3-4
CK-MB 4-8 24-38 2-3
AST 6-8 24 5
LD 12-24 10
B. Pancreatic profile -
Amylase and Lipase
C.Hepatic and Hepatobiliary Profile
Hepatic Disorders- AST and ALT
Hepatobiliary Disorders - ALP and GGT
LIVER DISEASES BONEDISEASES
GGT
Leucine aminopeptidase INCREASED NORMAL
5-nucleotidase

"Life isn't about waiting for the storm to pass. It's about learning to dance in the rain"
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Vivian Greene 59
 co
ACID-BASE BALANCE
What are Acids and Bases?
Arrhenius' Definition
An acid is a substance that increases the concentration of hydrogen ion (H+) when
dissolved in water
A base is a substance that increases the concentration of hydroxyl ion (OH-) when

dissolved in water
Bronsted and Lowry's Definition
An acid is a substance that donates a proton in a reaction
A base is a substance that accepts a proton in a reaction
Lewis' Definition
An acid is a molecule or ion that accepts a pair of electrons to form a covalent bond
A base is a molecule that donates a pair of electrons for a covalent bond.
II. What is Acid-Base Balance?
Important in order to maintain the pH within the normal range (normal range of pH = 7.35
7.45)
Important to maintain homeostasis, important in enzyme function
L. Buffer Systems
Buffers are substances that resist change(s) in pH
A Buffer system is composed of a "weak base and its conjugate acid" or a "weak acid and its
conjugate base"
Buffer Systems in the body
a. Bicarbonate-carbonic acid buffer systenm
The most important buffer system in the body
Bicarbonate-carbonic acid ratio must be 20:1 in order to maintain normal pH
Over 90% of blood carbon dioxide exists in the form of bicarbonate ion
b. Biphosphate-dihydrogen phosphate buffer system
must be maintained at a ratio of 4:1
C. Hemoglobin since it transports gases (oxygen and carbon dioxide)
d. Plasma proteins - since proteins are amphoteric (have positive and negative charge)
IV. Organs associated with the maintenance of acid-base balance
Lungs
Lungs help maintain acid-base balance through gas exchange or respiration
Rapid and short term compensation
Analyte(s) controlled: 02 and COz
Kidneys
Kidneys help maintain acid-base balance through reabsorption or excretion of bicarbonate
Slow but long term compensation; Analyte controlled: bicarbonate (HcOs)
 CHECKPOINT! Clinical Chemistry by Jude

V. Henderson-Hasselbach Equation
States the relationship between lungs, kidneys and pH
Bicarbonate = Total Carbon dioxide minus Carbonic Acid
Carbonic acid = partial pressure of carbon dioxide x 0.0307
Formula:
pH
HCO3
6.1+ l08 CO2 x0.03007
pH is directly proportional to bicarbonate - an increase in bicarbonate causes and increase
in pH and vice versa
pH is indirectly (inversely) proportional to partial pressure of carbon dioxide - an increase
in pCOz causes a decrease in pH and vice versa

VI. Clinical Significance

Acidosis refers to a decrease in blood pH
Alkalosis refers to an increase in blood pH
Changes in pH can be caused by either defect in the lungs (respiratory) or defect in the
kidneys (metabolic)
When one organ has a problem, the other organ will compensate. That means when
lungs have problem, the kidneys will compensate. When kidneys have problem, the
lungs will compensate
ACID BASE Organ Organ to Primary
Primary Cause
DISTURBANCES Defective Compensate Compensation

Respiratory Lungs Hypoventilation Kidneys Bicarbonate

reabsorption
Metabolic Kidneys Bicarbonate Hyperventilation
Lungs
excretion
Bicarbonate
Respiratory Lungs Hyperventilation Kidneys
excretion

Metabolic Kidneys
Bicarbonate
Lungs Hypoventilation
reabsorption

VIl. Specimen Collection

Specimen: Arterial whole blood using heparin as anticoagulant
Venous blood is usually 0.03 pH units lower than arterial blood
Arterialized venous blood may be obtained by heating the hand and forearm in water at
45degC for 5 minutes and then drawing blood from the dilated veins on the back of the
hand
Capillary blood is arterialized by warming the ear, finger or heel at 45degC before taking
the sample
Syringe with rubber stopper; specimen should be sealed

"fegg is broken by outside force, life ends. Ifbroken by inside force, life begins. Great things
aways begin from inside" - Jim Kwik 61
 CHECKPOINT! Clinical Chemistry by Jude

Anaerobic collection
not use vacutainer tube
Do
Place specimen in ice water or ice bath
No to clots, no to hemolysis, no to bubbles
Measurements are done at 37 + 0.05°C (Kaplan)
For each degree of fever in the patient, pO2 will fall 7% and pCO2 will rise 3%.
pH and blood gases are to be done within 20 minutes, no refrigeration is necessary
If
VII. Cases related to Blood Gas Analysis
Specimen was exposed to room air
Increase in oxygen, Decrease in carbon dioxide, Increase in pH
Room air is composed of 20-22% oxygen. This atmospheric air may enter the
specimen causing an increase in oxygen while displacing carbon dioxide in the
process -leading to a decrease in carbon dioxide in the specimen
Sealed specimen was left at room temperature
Decrease in oxygen, Increase in carbon dioxide, Decrease in pH
Changes are due to the presence of blood cells utilizing glucose and oxygen at room
temperature, causing the formation of acid products and carbon dioxide
Excess heparin
Heparin is an acid mucopolysaccharide
It is often used at a concentration of 0.2mg/mL of blood
Excess of acid mucopolysaccharide leads to acidic pH of blood specimen
IX. Methodology
pH- glasselectrodeconnected toa reference electrode (calomel electrode, mercury-
mercuric chloride)
pCO2-Severinghaus electrode -a modified pH electrode; glass electrode with weak
bicarbonate solution enclosed in silicone membrane
pOz-amperometric/polarographic; Clark electrode- composed of oxygen permeable
membrane (i.e. Tefion, polyethylene) with electrode composed of a platinum cathode
and silver-ilver chloride anode
Bicarbonate and Carbon dioxide content may be obtained by nomogram from blood
gas analyzers.
CO2 content - consists of bicarbonate, undissociated carbonic acid, dissolved carbon
dioxide and carbamino-bound carbon dioxide
Methods for COz content
Automated Enzymatic method
Allforms of COz are converted to bicarbonate by addition of base
Bicarbonate is converted to oxaloacetic acid using phosphoenol pyruvate
carboxylase
Add malate dehydrogenase and measure consumption of NADH at 340nm, as
Oxaloacetic acid is converted to malate

"fegg is broken by outside force, life ends. Ifbroken by insideforce, life begins. Great things
always begin from inside" - Jim Kwik 62
CHECKPOINT! Clinical Chemistry by Jude

Automated Colorimetric method

Bicarbonate, carbonic acid and carbamino-bound carbon dioxide is released by
addition of acid
Gaseous carbon dioxide is dialyzed through a silicone-rubber gas-dialysis
membrane into a buffer solution of cresolred at pH 9.2
Decrease in color intensity is proportional to the carbon dioxide content
Decrease in color intensity is measured at 430nm
Other continuous flow methods used phenolphthalein as indicator
Gasometric (Van Slyke or Kopp-Natelson)
Determines the amount of physically dissolved carbon dioxide and amount of
carbon dioxide released from bicarbonate and carbonic acid
Gaseous carbon dioxide present can be measured using:
AVolumetrically volume of gas at atmospheric pressure
-

Manometrically - pressure of gas at a fixed volume

Reagents:
ALactic acid = releases carbon dioxide
Caprylic alcohol = prevents foaming
Sodium hydroxide = absorbs carbon dioxide
Autoanalyzer (Autotechnicon)
Reagents:
A Sulfuric acid = releases carbon dioxide

Buffered phenolphthalein = absorbs carbon dioxide

Oxygen content and Percent Oxygen Saturation
Methods for O2 content
Spectrophotometric measurement of absorbance of a hemolyzed blood sample
-

at two wavelengths (650 ad 805nm)

Oximetric- uses Pulse Oximetry; noninvasive; light shines through a finger or the
bridge of the nose to a detector and absorbance is measured at 650 and 805nm
Gasometric manometric method devised by Van Slyke
-

Transcutaneous pO; and pCO2 - for continuous monitoring of partial pressures of

oxygen and carbon dioxide on a noninvasive basis

fegg is broken by outside force, life ends. If broken by inside force, life begins. Great things
always begin from inside" - Jim Kwik 63
 ELECTROLYTES AND CO
MINERALS
L.
What are Electrolytes?
Electrically charged substances; can be positively-(cation) or negatively-(anion) charged
Electrolyte panel usually includes: sodium, potassium, chloride and bicarbonate measurements
Heparin is the anticoagulant of choice for electrolyte analysis
II. Sodium
A. Characteristics of Sodium
major extracellular cation
solute that contributes most to total serum osmolarity
blood levels of sodium are mainly controlled by aldosterone
B. Functions of Sodium
regulates osmolarity and blood volume
C. Clinical Considerations
CAUSES OF HYPERNATREMIA CAUSES OF HYPONATREMIA
Diabetes insipidus Diuretics, Potassium depletion
Osmotic dieresis Aldosterone deficiency, Ketonuria
Loss of thirst Salt-losing nephropathy, Vomiting
Insensible loss of water Diarrhea, Excess fluid loss as with burns, excess sweating or
Gastrointestinal loss of trauma
hypotonic fluid SIADH, Excess water intake
Excess intake of sodium Adrenal insufficiency
Reset osmostat
Acute or chronic renal failure
Nephrotic syndrome, Hepatic cirrhosis, Congestive heart
failure
Pseudohyponatremia (hyperglycemia, hyperlipidemia,
hyperproteinemia)
D. Specimen Considerations and Patient Preparation:
Serum, plasma, 24-hour urine sample, as well as sweat and CSF can be used as sample
If plasma is used, lithium & ammonium salts of heparin, as well as lithium oxalate may be used
Heparin is the anticoagulant of choice
Hemolysis does not cause significant changes but marked hemolysis should be avoided
The equation below can be used to check on accuracy of electrolyte determination:
Na COz+ Cl+ 10
or
=
Na CO2+Cl+12
 CHECKPOINT! Clinical Chemistry by Jude

E. Methodologies of Sodium Measurement

Flame emission photometry
sodium produces yellow color when exposed to flame
sodium emits light at a wavelength of 590nm
dilute sample first to prevent interferences, to prevent atomizer plugging and to
acquire increased sensitivity
dilute sample using high purity water (deionized water with electrolyte content of
<0.5ppm)
serum is usually diluted 1:100 or 1:200
lithium or cesium may act as internal standard
Atomic absorption spectrophotometry
lon-selective electrode (glass aluminum silicate)
F. Values to Remember:
Reference Values: Serum: 135 to 145mmol/L
24-hour urine: 40 to 220mmol/day
CSF: 138 to 150mmol/L
Conversion factor: mEq/L to mmol/L = 1.0

III. Potassium
A. Characteristics of Potassium
major intracellular cation
B. Functions of Potassium
Involved in proper transmission of nerve impulses
Important for contraction of the heart abnormal levels of potassium can lead to
-

altered electrocardiographic patterns

C. Clinical Considerations
CAUSES OF HYPERKALEMIA CAUSES OF HYPOKALEMIA
Decreased Renal Excretion GI Loss
Acute or chronic renal failure Vomiting Diarrhea, Gastric suction,
Hypoaldosteronism; Addison's disease Intestinal tumor, Malabsorption
Diuretics Cancer therapy chemotherapy, radiation
Increased Intake therapy
Oral or IV potassium replacement Large doses of laxatives
therapy Decreased Intake
Cellular Shift Renal Loss
Acidosis, Muscle/cellular injury Diuretics- thiazides, mineralocorticoids
Chemotherapy Nephritis, Renal tubular acidosis
Leukemia (increased WBC) Hyperaldosteronism; Cushing's syndrome
Hemolysis Hypomagnesemia
Increased Intake Acute leukemia
Sample hemolysis, Thrombocytosis Cellular Shift
Prolonged tourniquet use or excessive Alkalosis, Insulin overdose
fistclenching

"Destroy negative thoughts when they first appear. This is when they're the weakest"
-
Songide Makwa 65
 CHECKPOINT! Clinical Chemistry by Jude

D. Specimen Considerations and Patient Preparation:

Serum or plasma can be used; plasma/serum must be separated from the cells quickly to
prevent potassium from shifting from RBCs to serum
Heparin is the anticoagulant of choice
Whole blood samples for potassium determination should be stored at room temperature
NO to prolonged tourniquet application, excessive fist clenching and hemolysis
E. Methodologies of Potassium Measurement:
Flame emission photometry
Potassium produces violet color when exposed to flame
Potassium emits light at a wavelength of 768nm
dilute sample first to prevent interferences, to prevent atomizer plugging and to
acquire increased sensitivity
dilute sample using high purity water
serum is usually diluted 1:100 or 1:200
lithium or cesium may act as internal standard
Atomic absorption spectrophotometry
lon-selective electrode (valinomycin membrane)
F. Values to Remember:
Reference Values: Serum: 3.4 to 5.0mmol/L
24-hour urine: 25 to 125mmol/day
Conversion factor: mEq/L to mmol/L = 1.0

IV.Chloride
A. Characteristics of Chloride
major extracellular anion
chloride is the counterion of sodium - a counterion is an ion that accompanies an ionic
species in order to maintain electric neutrality
B. Functions of Chloride
Maintains water balance, osmotic pressure ad anion-cation balance in the extracellular
fluid
Responsible for chloride shift an exchange mechanism between chloride and
-

bicarbonate across the membrane of RBCs

C. Clinical Considerations
HYPERCHLOREMIA HYPOCHLOREMIA
Excess loss of bicarbonate Prolonged vomiting, Diabetic ketoacidosis, Aldosterone
Renal tubular acidosis deficiency
Metabolic acidosis Salt-losing renal diseases (i.e. pyelonephritis)
High serum bicarbonate (ie. compensated respiratory
acidosis or metabolic alkalosis)

"Destroy negative thoughts when they first appear. This is when they're the weakest"
-Songide Makwa 66
 CHECKPOINT! Clinical Chemistry by Jude

D. Specimen Considerations and Patient Preparation:

Serum, plasma, whole blood, 24-hour urine and sweat can be used as sample
Lithium heparin is the anticoagulant of choice
marked hemolysis should be avoided
E. Methodologies of Chloride Measurement:
Ion-selective electrode
Most commonly used
using an ion exchange membrane selective for chloride ions. Membrane used is a
combination of silver wire coated with AgCl.
Amperometric-Coulometric titration
nciple of Cotlove chloridometer
Uses coulometric generation of silver ions which combine with chloride to
quantitate chloride concentration.
Excess silver ions, which were not bound to chiloride is used to indicate endpoint.

Ag +Cl> AgCI
Sample diluted in acid with small amount of gelatin
Nitric acid provides good conductivity
Acetic acid provides sharper endpoint by reducing solubility of silver chloride
by decreasing polarity
Gelatin makes a smoother titration curve by equalizing the reaction rate over
the entire electrode
Mercuric (Mercurimetric) titration
principle of Schales and Schales method
based on the reaction of chloride ions to mercuric ions to form mercuric chloride
blood containing bromide leads to positive error
Excess mercuric ions are then made to react with diphenylcarbazone in order to
form violet blue color.
2CI +Hg HgC
excess Hg + diphenylcarbazone violet blue color
Colorimetric method
uses mercuric thiocyanate and ferric nitrate to form ferric thiocyanate, which is a
reddish colored complex with a peak absorbance at 480nm
Used in Autoanalyzer (Technicon)
2C1 +Hg(SCN), >HgCl, +2SCN
3SCN+Fe Fe(SCN),
F. Values to Remember:
Reference Values: Serum: 98 to 107mmol/L 24-hour urine: 110-250mmol/day
Conversion factor: mEq/L to mmol/L = 1.0

"Destroy negative thoughts when they first appear. This is when they're the weakest"
Songide Makwa 67
 CHECKPOINT! Clinical Chemistry by Jude

V. Calcium
A. Characteristics of Calcium
99% of calcium is found in bones and teeth; 1% is found in the blood
Calcium in the blood exists in three forms
Free calcium = 50% Complexed calcium = 10% Protein Bound = 40%%
1g/dl. decrease in albumin causes 0.8mg/dL decreased in total calcium
Calcium levels are altered by blood pH - alkalosis lowers calcium, acidosis increases
calcium
Calcium is absorbed in ileum at acid pH
Calcium level in the blood is controlled by parathyroid hormone, calcitonin and vitamin D
Low levels of parathyroid hormone leads to lovw serum calcium and high serum
phosphorus level
Low vitamin D leads to low serum calcium and increased calcium and phosphorus in feces
Has reciprocal or inverse relationship with phosphate
B. Functions of Calcium
Contributor to structure of bone and teeth
Coagulation (calcium is coagulation factor IV)
For proper contraction of heart muscles
Activator to enzymes
Neurotransmission regulator
C. Clinical Considerations Associated with Calcium

HYPERCALCEMIA HYPOCALCEMIA
Primary hyperparathyroidism (most Primary hypoparathyroidism
common PTH mediated hypercalcemia) Severe hypomagnesemia
Familial hypocalciuric hypercalcemia Longstanding hypercalcemia
Ectopic secretion of PTH by neoplasms Pseudohypoparathyroidism
Malignancy associated (most common non Vitamin D deficiency, chronic renal ailure
PTH mediated hypercalcemia) renal tubulopathies, Fanconi's syndrome
Vitamin D intoxication, Thyrotoxicosis Mutations of vitamin D receptor
Hypoadrenalism, Immobilization with Hypoalbuminemia, acute pancreatitis
increased bone turnover, Milk-alkali rhabdomyolysis
syndrome, Sarcoidosis, Multiple myeloma
D. Specimen Considerations and Patient Preparation:
Serum, plasma or 24-hour urine sample may be used; Lithium heparin is the preferred
No to EDTA, oxalate and citrate
Samples should be collected anaerobically
For 24-hour urine calcium, sample should be acidified using 6M HCl (1mL of HCl per
100ml of urine)
For assays that are subject to interference with magnesium, magnesium can be removed
by adding 8-hydroxyquinoline

"Destroy negative thoughts when they first appear. This is when they're the weakest"
Songide Makwa 68
 CHECKPOINT! Clinical Chemistry by Jude

E. Methodologies of Calcium Measurement:

Orthocresolphthalein complexone (Hitachi and Dimension)
a calcium chelator; produces reddish complex with absorbance at 570-578nm
8-hydroxyquinoline binds magnesium which may interfere
~urea can be used to decrease the turbidity of lipemic serum and increase intensity of the
calcium-dye complex
ethanol can be used to decrease the absorbance of the blank
Arsenazo IlI (Vitros and Synchron)
Alizarin
Methylthymol blue
Atomic absorption spectrophotometry
~calcium compounds in a flame dissociate into free calcium atoms
~free atoms absorb light of a characteristic wavelength
lanthanum is used to bind phosphate that might instead bind the calcium and cause
falsely low result
lon-selective electrode
Clark Collip Precipitation method
~classic method that measures oxalic acid as the end product
critical points: washing the calcium oxalate precipitate
Titration of oxalic acid with KMnO4 at 70°C
Ferro Ham Chloroanilic acid Precipitation method ~ precipitation of calcium with
chloroanilic acid
Compleximetric~ removal of calcium by EDTA
F. Values to Remember:
Reference Values:
Total Calcium (child) 2.20-2.70mmol/L (adult) 2.15-2.50mmol/L
Tonized Calcium (child) 1.20-1.38mmol/L (adult) 1.16-1.32mmol/L
24-hour urine: 2.50-7.50mmol/day
Conversion factor: mg/dL to mmol/L = 0.25

VI. Magnesium
A. Characteristics of Magnesium
Second most abundant intracellular cation (after potassium)
Fourth most abundant cation in the body
50% of magnesium in the body is found in the bone; around 25% is in the muscle
Exists in the blood in three forms:
Free magnesium = 55% Complexed magnesium = 15%
Protein Bound = 30%

"Destroy negative thoughts when they first appear. This is when they're the weakest"
Songide Makwa 69
 CHECKPOINT! Clinical Chemistry by Jude

B. Functions of Magnesium
Contributor to bone structure
For muscle contraction and heart rhythm
Activator to enzymatic reactions
C. Clinical Considerations
HYPERMAGNESEMIA HYPOMAGNESEMIA
Acute or chronic renal failure Poor diet; prolonged magnesium-deficient IV
Hypothyroidism therapy, Chronic alcoholism
Hypoaldosteronism Pancreatitis, Vomiting, Diarrhea
Hypopituitarism Laxative abuse, Hyperparathyroidism,
Antacids Hyperaldosteronism, Hyperthyroidism
Enemas Hypercalcemia, Diabetic ketoacidosis
Cathartics Diuretics, Excess lactation; pregmancy
Dehydration Tubular disorders, glomerulonephritits,
Bone carcinoma/ bone metastasis pyelonephritis
D. Specimen Considerations and Patient Preparation:
Serum, lithium heparinized-plasma or 24-hour urine may be used
Oxalate, EDTA and citrate should not be used
No to hemolysis
24-hour urine should be acidified with HCl to prevent precipitation
E. Methodologies of Magnesium Measurement:

Colorimetric method:
a. Calmagite (Hitachi and Synchron)
~a naphthol sulfonic acid derivative
use of polyvinylpyrrolidone minimizes the effects of serum protein
~Mg+ calmagite = reddish violet (or violet) complex read at 520-532nm
strontium chelate = masks the effect of calcium
~triethanolamine = to mask the effect of iron
b. Formazen dye (Vitros)~ colored complex at 66Onm
C. Methyl-thymol blue (Dimension, DuPont aca)
Dye-lake method Titan yellow dye (Clayton yellow/ thiazole yellow); titan yellow
forms a red lake with magnesium; polyvinyl alcohol increases the sensitivity of the
method
Fluorometry - magnesium reacts with the reagent 8-hydroxy-5-quinoline sulfonic acid
or calcein to form a fluorescent compound (390-410nm)D
Atomic Absorption Spectrophotometry~ reference method
lon-selective electrode
F. Values to Remember:
Reference Value: 0.63-1.0 mmol/L
Conversion factor: mEq/L to mmol/L = 0.5

"Destroy negative thoughts when they first appear. This is when they're the weakest"
- Songide Makwa 70
 CHECKPOINT! Clinical Chemistry by Jude

VI. Phosphate
A. Characteristics of Phosphate
Intracellular anion; most phosphorus is in the form of phosphate
Most serum phosphate are inorganic; most phosphorus inside the cell is organic
Phosphate metabolism is controlled by parathyroid hormone, calcitonin and vitamin D
Exists in the blood in three forms:
Free phosphate = 55% Complexed phosphate = 35%
Protein Bound 10%
=
B. Functions of Phosphate
Serves as a buffer (biphosphate-dihydrogen phosphate buffer system)
Serves as part of energy molecules likes ATP
C. Clinical Considerations Associated with Phosphate:

HYPERPHOSPHATEMIA HYPOPHoSPHATEMIA
Renal failure Infusion of dextrose solution
Increased breakdown of cells (i.e. Use of antacids
intravascular hemolysis) Alcohol withdrawal
Neoplastic disorders (i.e. Lymphoblastic Poor diet
leukemia) Vomiting
Intensive exercise, Severe infections ketoacidosis
D. Specimen Considerations and Patient Preparation:
Serum, lithium heparin-plasma or 24-hour urine sample may be used
Oxalate, EDTA and citrate should not be used
Noto hemolysis
Diurnal variation = highest levels are found in late morning: lowest in the evening
E. Methodologies of Inorganic Phosphorous Measurement
Fiske-Subbarow method
Uses molybdate reagent; products that can be measured include:
measurement of ammonium molybdate complex at 340nm (Hitachi, Dimension,
Synchron)
reduction to form molybdenum blue, which is read at 660nm (or 600 to 700nm in
some references; used in Vitros); reducing agents that can be used in this
reduction process include: ANSA, stannous chloride, ascorbic acid and N-phenyl-p
phenylenediamine
serum proteins are precipitated by trichloroacetic acid and phosphate is
converted into phosphomolybdate complex (Mov) by the addition of sodium
molybdate. The addition of p-methylaminophenol reduces the MoW into Mov. The
absorbance of the solution at 700nm is proportional to the serum phosphate
concentration

"Destroy negative thoughts when they first appear. This is when they're the weakest"
-Songide Makwa 71
 CHECKPOINT! Clinical Chemistry by Jude

F. Values to Remember:
Reference Values: Serum/Plasma (neonate) 1.45-2.91mmol/L
(child) 1.45-1.78mmol/L
(adult) 0.87-145mmol/L
24-hour urine: 13-42mmol/day
Conversion factor for Phosphorous: mg/dL to mmol/L = 0.323

VII. Anion Gap

Difference between unmeasured anions and unmeasured cations
Useful in indicating an increase in one or more of the unmeasured anions in the serum
Serves as a form of quality control for the analyzer used to measure these electrolytes
A.Formula:
AG Na- (Cl + HCOa) or AG (Na + K) - (CI+ HCOs)
B. Reference values:
7 to 16 mmol/L or 10 to 20mmol/L
C. Clinical Significance of Anion Gap
Increased AG = uremia/ renal failure, ketoacidosis, poisoning due to ingestion of toxic
substances like methanol, ethanol, ethylene glycol poisoning or salicylate; lactic
acidosis; severe dehydration; instrument error
"MUDILES" common causes of increased anion gap
-

Methanol
Uremia
Diabetic ketoacidosis
Iron, inhalants (i.e. carbon monoxide, cyanide, toluene), isoniazid, ibuprofen
Lactic acidosis
Ethylene glycol poisoning, ethanol ketoacidosis
Salicylates, Starvation ketoacidosis, Sympathomimetics
Decreased AG = rare; hypoalbuminemia (decreased in unmeasured anions); severe
hypercalcemia (increase in unmeasured cations); patients with multiple myeloma;
instrument error

"Destroy negative thoughts when they first appear. This is when they're the weakest"
Songide Makwa
-
72
 NON-PROTEIN NITROGENS
. What are Non-Protein Nitrogens?
A. Characteristics of Non-Protein Nitrogens
Substances that are not considered as proteins, but contains nitrogen
Mostly considered as waste products
Include urea, uric acid, creatinine, creatine and ammonia and amino acids
Urea, uric acid and creatinine has been often used to assess renal function
Ammonia is used assess liver function

I. Urea
A. Characteristics of urea
End product of protein metabolism
Molecular formula of H;N-CO-NH2
Major NPN of the body, accounts to almost 45% of the total NPNs
Produced by the liver through the Krebs-Henseleit Cycle; excreted by the kidneys but
partially reabsorbed (approximately 40% is reabsorbed)
Major organic component of the urine
Urea concentration vs BUN
Blood urea nitrogen pertains to the nitrogen content only of urea - it is not the
same as urea, it more or less a part of urea. This value is often obtain using indirect
methods
Urea concentration refers to the concentration of urea as a whole molecule, not
just the nitrogen portion. This values is often obtained using the direct methods.
However, it can be calculated from BUN.
BUN can be converted into urea concentration using the following equation:
BUN x 2.14 = urea concentration
2.14 isa constant. It is derived from the molecular weight of the two nitrogen atoms
in urea (2x 14 = 28) and molecular weight of the urea itself (urea MW = 60).
Molecular weight of urea is divided by the molecular weight of the two nitrogen
atoms, which means 60 divided by 28 2.14
B. Specimen Considerations and Patient Preparations:
Plasma, serum or urine can be used
Ammonium containing anticoagulants should not be used
Sodium fluoride and Sodium citrate should not be used
Non hemolyzed sample is recommended
Refrigerate if cannot be analyzed immediately
 CHECKPOINT! Clinical Chemistry by Jude

C. Methodologies for Urea Measurement

1. Direct Method - measures urea directly as urea
a. Diacetyl Monoxime Condensation/Method
Also known as Fearon reaction
Reactions: Urea+ diketone (DAM) + strong acid + heat

unstable, photosensitive yellow complex (known as diazine)

measured at 540nm
b. O-phthaldehyde
Reactions: urea +0-phthaldehyde isoindoline
isoindoline + N-(l-naphthyl) ethylene diamine colored product
C. Isotope Dilution-Mass Spectrometry~ reference method

2. Indirect Method -measures urea by converting it first into ammonium ions using
urease; ammonium ions formed from urea are then measured
INITIAL REACTION: urease converts urea into ammonium ions (or ammonia) and
carbonate ions (or carbon dioxide)
Urease is often derived from jack bean meal
SECONDARY REACTIONS: quantifies ammonium ions that form after the initial reaction
Secondary Reactions that can be used include:
Nesslerization Reaction
Reagents: potassium iodide, mercuric iodide
Product measured: yellow-orange colloid (NH:Hg2l) known as ammonium
dimercuric iodide
Berthelot Reaction
Reagents: sodium hypochlorite (NaoCl), phenol, sodium nitroprusside
Na0Cl chlorinates ammonia into monochloramine; monochloramine reacts with
phenol to form indophenol (blue or green)
Reaction is maintained at alkaline pH (>10.0) with sodium nitroprusside acting as
catalyst
Product measured: indophenol at 630-660nm
GLDH-coupled (glutamate dehydrogenase)
ammonia +2-0xoglutarate + NADH + GLDH> NAD+ glutamate + water
used in automated instruments
measurement of decrease in absorbance at 340nm
Conductimetric
conversion of unionized urea into ammonium and bicarbonate ion; measure
increase in conductivity rate
Indicator dye
color change; used in dry reagent strips and multilayer film reagents

"Faith is seeing light with your heart when allyour eyes see is darkness"- Anonymous 74
 CHECKPOINT! Clinical Chemistry by Jude

D. Clinical Conditions Associated with Urea:

1. DECREASED BUN LEVELS
Decreased protein intake Vomiting and diarrhea
Liver disease Pregnancy
2. UREMIA (UREMIC SYNDROME) vS AZOTEMIA
Azotemia~refers to increase NPNs, particularly urea in blood
Uremia increase in nitrogen-containing compounds in the blood such as urea,
creatinine and uric acid; defined as increase in NPNs with symptoms of organ
involvement such as renal failure
3. PRE-RENAL AZOTEMIA vs RENAL AZOTEMIA vs POST RENAL AZOTEMIA
Pre-renal azotemia
Reduced blood flow to the kidneys less urea is excreted, more urea is retained
-

in the blood
Diet increased protein intake
-

Increased protein catabolism or degradation

Renal azotemia
Decreased kidney function
Post renal azotemia
Urinary tract obstruction
E. Values to Remember:
ReferenceValue: 7 to 18mg/dL
Conversion factor: mg/dL to mmol/L = 0.357
Urea Concentration = BUN x 2.14
III. Uric Acid
A. Characteristics of Uric acid
End product of purine metabolism in humans
Produced in the liver, excreted through the kidneys
Filtered by the glomerulus but 40% of uric acid is reabsorbed in the kidneys
A strong reducing
agent
Exists as monosodium urate in the plasma; in an acidic urine, uric acid is the most
predominant form
B. Specimen Considerations and Patient Preparations:
Heparinized plasma, serum or urine can be used
Serum should be separated immediately to prevent dilution
EDTA &fluoride containing anticoagulants should be avoided if uricase is to be used
No fasting required; gross lipemia should be avoided
High bilirubin levels can cause false decrease by peroxidase method
Hemolysis causes low values

"Faith is seeing light with your heart when all your eyes see is darkness" Anonymous
-
CHECKPOINT! Clinical Chemistry by Jude

C. Methodologies for Uric Acid Measurement:

Chemical Method (Caraway method)
Reagent: phosphotungstic acid (PTA)
Reaction: uric acid + PTA tungsten blue + allantoin
Sodium cyanide (NaCN) is used to increase color and inhibit fading but considered
to be hazardous
Other compounds such as sodium carbonate (Na2C0s) may be used instead of
NaCN to adjust pH to 10.0-10.4 (for full color development and decreased
turbidity)
Positive result: tungsten blue measured at 660nm or 710nm (in some references;
usual range is 650-700nm)
Aspirin, acetaminophen, caffeine and theophylline leads to falsely elevated results
Enzymatic method (uricase)
Step 1: Uricase converts uric acid into allantoin and hydrogen peroxide
Step 2:
UV method:
A uric acid absorbs light at 285nm (or 293nm in some books; usual range is
290-295nm)
allantoin does not absorb light at 285 or 293nm
decreased in absorbance is proportional to concentration ofuric acid
originally present before addition of uricase
Colorimetric method:
Phosphotungstic acid chromophores; method of Bittner
Coupled Enzymatic:
Uricase is coupled with peroxidase
A Hydrogen peroxide that forms from uricase step is reacted with

peroxidase and an indicator/chromogen

AChange in color is measured
Indicators/chromogen include: 4-aminophenazone + phenol;
methylbenzothiazoline hydrazone; o-dianisidine; 3, 5-dichloro-2-
hydroxybenzenesulfonic acid and 4-aminophenazone (formatiorn of red
dye)
Reducing substances cause a false decreased result
D. Clinical Conditions Associated with Uric Acid:
1. Increased Blood Uric Acid
Increased production increased synthesis of purine precursors
-

Increased nucleic acid turnover chemotherapy in leukemia patients

Enzyme defect- Lesch-Nyhan syndrome~an X-linked condition affecting mostly
males, characterized by deficiency of the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT)
Uric Acid excretion competition organic acids, thiazides, salicylates
-

"Faith is seeing light with your heart when all your eyes see is darkness" Anonymous
-
76
 CHECKPOINT! Clinical Chemistry by Jude

Decreased uric acid excretion - kidney disease

Diet-rich in purine - beans, oatmeal, kidney, red meat, sardines
Gout-occurs when monosodium urate precipitates from supersaturated body
fluids. Gouty arthritis maybe associated with urate crystals in synovial
fluid and with deposition of crystals known as tophi in other tissues as
well.
Tophi are deposits of uric acid crystals as sodium urates in great toe, ear lobe,
elbow and in other tissues etc.
2. Decreased Blood Uric Acid
Liver disease, defective reabsorption of uric acid by kidneys (i.e. Fanconi
Syndrome)
E. Values to Remember:
Reference Value: Male: 3.5 to 7.2mg/dL Female: 2.6 to 6.0mg/dL
Conversion Factor: mg/dL to umol/L = 0.0595

IV. Creatinine
A. Characteristics of Creatinine
Anhydride of creatine creatinine is produced when creatine loses water
Can also be produced when creatine phosphate loses phosphoric acid
(dephosphorylation)
End product of muscle metabolism; more muscles, mean more creatinine
Not reabsorbed by the renal tubules in significant amounts
Can be used to assess the completeness of a 24-hr urine specimen since creatinine is
excreted at a constant rate
Normal BUN to creatinine ratio =10:1 to 20:1
B. Specimen Considerations and Patient Preparation:
Serum, plasma and urine may be used
Icteric samples should be avoided
Hemolyzed samples should be avoided
Lipemic samples can cause errors
no fasting required
No strenuous exercise, no fist clenching
C. CREATINE Analysis:
Apply Jaffe reaction to trichloroacetic acid filtrate of serum (for the value of
creatinine)
Treat the specimen with heat or dilute sulfuric acid to convert creatine to creatinine
The difference between the two values is creatine

"Faith is seeing light with your heart when all your eyes see is darkness" Anonymous
-
77
CHECKPOINT! Clinical Chemistry by Jude

D. Methodologies for CreatinineMeasurement:

Chemical Method (Jafe Reaction)
Reagent: alkaline picrate solution (picric acid + sodium hydroxide)
Product measured: red-orange complex knownm as Janovski complex measured at
490-505nm
Kinetic Jaffe reaction is preferred than endpoint since most of interfering substances
require longer time before contributing significantly to the absorbance
Modifications of Jaffe uses adsorbents to remove interferences
Modification of Jaffe Reaction:
Fuller's earth reagent adsorbent is aluminum magnesium silicate
Lloyd's n
reagent adsorbent is sodium aluminum silicate
Enzymatic Method
=
Creatinine aminohydrolase creatine kinase method measure change in
NADH to NAD+

Creatininase-CK Creatininase
Creatinine + H20 creatine
CK
Creatine+ ATP creatine phosphate + ADP
PK
Phosphoenolpyruvate +ADP-pyruvate + ATP
LD
Pyruvate + NADH + H lactate +NAD
ACreatininase hydrogen peroxide method
- = measure formation of color as
benzoquinonemine dye forms
Creatininase-H202 Creatininase
Creatinine + H20 creatine
Creatininase
Creatine H20> sarcosine + urea
Sarcosine oxidase
Sarcosine + Oz + H0-glycine + CH20 + H02
Peroxidase
H202+ colorless substrate > colored product + H2O
Isotope Dilution-Mass Spectrometry~accepted reference method; detection of
characteristic fragments following ionization, quantification using isotopically-
labelled compound
E. Clinical Considerations Associated with Creatinine

Increased Creatinine
Relative to muscle mass- more muscles, higher creatinine
Renal function decreased removal of creatinine from plasma by the kidneys
-

Rate of creatine turnover increased conversion of creatine into creatinine

-

F. Values to Remember
Reference Range: Male: 0.6 to 1.2 mg/dL Female: 0.5 to 1.1mg/dL
Conversion factor: mg/dL to mmol/L)= 88.4

"Faith is seeing light with your heart when all your eyes see is darkness" - Anonymous 78
 CHECKPOINT! Clinical Chemistry by Jude

V. Ammonia
A. Characteristics of Ammonia
Immediate product of amino acid deamination
Deamination refers to the removal of the amino terminal end of amino acids
Primary site of ammonia production is the small intestine
Used as a liver function test
Most ammonia in the circulation are derived from:
Breakdown of amino acids obtained from dietary protein in the intestines
Hydrolysis of urea, an end product of metabolism
Ammonia exist in the ionized or unionized form: lower pH results in the formation of
ammonium ions, a higher pH results in the formation of unionized form of ammonia
(NHa)
Liver removes the toxic ammonia through the Krebs-Henseleit urea cycle, which
converts harmful ammonia into urea which is a lesser harmful substance
B. Specimen Considerations and Patient Preparations:
Eliminate source of contamination- detergent and cigarette
Serum is NOT used; EDTA plasma or heparinized plasma can be used
Placed immediately on ice bath and analyzed immediately
No to hemolysis
cMethodologies for Ammonia Measurement:
Conway microdiffusion ~ ammonia is released from alkaline solution and
determined by back titration with acid
lon-exchange~ ammonia is absorbed onto dowex 50 resin, eluted and quantitated
with Berthelot reaction
Coupled enzymatic ammonia reacts with alpha ketoglutarate and NADPH in the
presence of glutamate dehydrogenase to form glutamate, water and NADP+
lon selective electrode~ ammonia diffuses through selective membrane into
ammonium chloride causing a pH change which is then measured potentiometrically
D. Clinical Considerations Associated with Ammonia:
High levels of ammonia in the blood may indicate that the liver cannot effectively
convert the toxic ammonia into urea - ammonia, therefore a liver function test
For assessment of patients with hepatic coma (hepatic encephalopathy) and Reye's
syndrome
E. Values to Remember
Reference Range: 19 to 60 ug/dL
Conversion factor: ug/dL to umol/L = 0.587

"Faith is seeing light with your heart when all your eyes see is darkness"- Anonymous 79
 BILIRUBIN&LVER FUNCTION
I. Liver
A. Anatomy of the Liver
Largest and the most versatile organ in the body
Has two main lobes, separated from each other by falciform ligament; right lobe is six
times larger than the left lobe
1.2-1.5kg in weight
Smallest functional unit of the liver is known as the hepatic lobule
Blood is supplied from two sources: hepatic artery and portal vein
B. Functions
Synthetic function (i.e. protein, carbohydrate and lipid synthesis)
Detoxification function and Drug metabolism (i.e. ammonia, drugs)
Excretory function (i.e. bilirubin)
C. Tests for Liver Function
Proteins Bilirubin Ammonia Enzymes (AST, ALT, GGT, ALP)

II. Bilirubin
A. Characteristics of Bilirubin
Orange yellow pigment derived from hemoglobin degradation
Extracted and biotransformed mainly in the liver and is excreted in bile and urine
Mainly transported by albumin
Two major forms:
B1
B2
Unconjugated bilirubin Conjugated bilirubin
Non-polar bilirubin Polar bilirubin
Water-insoluble bilirubin Water-soluble bilirubin
Indirect bilirubin Direct bilirubin
Hemobilirubin Cholebilirubin

B. Bilirubin Metabolism and Excretion

When RBCs are destroyed, hemoglobin is released.
Hemoglobin in the plasma is transported by the protein carrier haptoglobin.
In cases of severe RBC destruction, hemoglobin in plasma increases which causes
utilization of the available haptoglobin. During increased RBC destructions therefore,
haptoglobin level decreases.
When hemoglobin could no longer be transported by Haptoglobin, hemoglobin
molecules are broken down into heme and globin.
Globin is the protein portion of hemoglobin and will be recycled by the liver
Heme is transported by a protein carrier known as Hemopexin.
Hemopexin becomes depleted when heme levels are increased.
When hemopexin is depleted, heme is divided into iron and protoporphyrin ring.
 CHECKPOINT! Clinical Chemistry by Jude

Iron is transported by transferrin into the bone marrow, to be used for the synthesis of
new RBCs
Protoporphyrin is broken down into biliverdin, and then further broken down into
bilirubin, particularly the unconjugated form.
Unconjugated bilirubin is transported by albumin towards the site of conjugation, the
liver.
In the liver cells, unconjugated bilirubin is converted into conjugated bilirubin through
the action of the enzyme uridyl diphosphate glucoronyl transferase (UDPGT). This
process occurs in the smooth endoplasmic reticulum of the liver cel.
Conjugated bilirubin goes out of the liver, down to the biliary tree (or bile duct) and
emptied into the intestine.
In the intestine, conjugated bilirubin is converted into the urobilinogen. Urobilinogen is
a colorless substance formed in the intestine.
Urobilinogen is converted into urobilin, an orange brown pigment responsible for the
natural brown color of the stool. It is then excreted through the stool out of the body.
Some of the urobilinogen from the intestine is reabsorbed back into the blood, only to
be excreted by the kidneys into the urine. Normal urine therefore may contain
urobilinogen.

C. Clinical Conditions Associated with Bilirubin:

Jaundice -yellowish discoloration of the skin and the sclera of the eyes
Kernicterus- deposition of bilirubin in the brain
Cirrhosis- occurs when scar tissues replaces healthy, normal liver tissues
Biliary Obstruction/Bile duct obstruction
a. Biliary atresia - failure of the common bile duct to form an opening
b. Cholecystitis -
inflammation of the gall bladder
C. Cholelithiasis - gall stones
d. Choledocholithiasis- presence of gall stones in the biliary tree

D. Inherited Bilirubin Derangements

1. Gilbert Syndrome = defective hepatic uptake of bilirubin
2. Crigler-Najjar Syndrome = defective conjugation of bilirubin due to deficiency of the
enzyme UDPGT
Type I = total UDPGT deficiency
Type II = partial UDPGT deficiency
3. Dubin-Johnson Syndrome defective hepatic excretion of bilirubin; dark pigmentation of
liver with associated abnormal gall bladder function
4. Rotor Syndrome defective hepatic excretion of bilirubin; no liver pigmentation; normal
gal bladder function
5. Lucey-Driscoll Syndrome presence of circulating antibody against UDPGT

"The strongest factor for success is self-esteem: believing you can do it,
believing you deserve it and believing you will get it" Anonymous
-
81
 CHECKPOINT! Clinical Chemistry by Jude

E. Hyperbilirubinemia
Prehepatic vs Hepatic vs Posthepatic Hyperbilirubinemia
Serum B1 Serum B2 Urine Urine
Bilirubin Urobilinogen
Prehepatic Dark
(i.e. hemolytic INCREASED NORMAL NEGATIVE INCREASED brown
anemia) stool
|
Hepatic
(i.e. liver INCREASED INCREASED POSITIVE INCREASED
disorders)
Post hepatic Clay-
INCREASED POSITIVE
DECREASED/
(i.e. bile duct NORMAL colored
NORMAL
obstruction) stool

F. Specimen Considerations and Patient Preparation

Protect specimen for light- bilirubin may be photo-oxidized when exposed to light
Hemolysis can cause interference. It can lead to falsely decreased result especially
when Jendrassik Grof method is used
Lipemia can interfere
Delta bilirubin is conjugated bilirubin that is covalently bound to albumin
Accelerator agents enhances or accelerates the unconjugated bilirubin's reaction wit
diazotized sulfanilic acid

G. Methodologies
Ehrlich reaction
diazotized sulfanilic acid for bilirubin used in urine
Van den Bergh reaction
diazotized sulfanilic acid for bilirubin used in serum
diazotized sulfanilic acid is formed by reacting sulfanilic acid with sodium nitrite
and hydrochloric acid
Jendrassik Grof method
diazotized sulfanilic acid plus accelerator/dissociating agent (caffeine-sodium
benzoate-acetate)
Advantages:
Insensitive to sample pH changes
Insensitive to variation in protein concentration of sample
Not affected by hemoglobin up to 750mg/dL
Has adequate optical sensitivity even for low bilirubin concentrations
Ha minimal turbidity and a relatively constant serum blank
Sodium acetate acts as a buffer
Addition of a strongly alkaline tartrate converts original purple color into blue
which can be measured spectrophotometrically at 600nm

"The strongest factor for success is self-esteem: believing you can do it,
believing you deserve it and believing you will get it" Anonymous
-
82
 CHECKPOINT! Clinical Chemistry by Jude

Evelyn Malloy method

diazotized sulfanilic acid plus accelerator/dissociating agent (50% methanol)
diazo product has a red to reddish purple color in acid pH with an absorption
maximum in the region of 560nm
Icterus index - subjective; test involves diluting serum with saline until it visually
matches the color of a 0.01% potassium dichromate solution; interference with
carotene, xanthophyll and hemoglobin may occur
Direct Spectrophotometric Method- absorbance of bilirubin in serum at 455nm is
proportional to the concentration; absorbance of hemoglobin at 455nm can be
corrected by subtracting absorbance at 575nm

H. Values to Remember:
(1) Reference Values:
Total Bilirubin: 0.2 to 1.Omg/dL
Direct Bilirubin: 0 to 0.2mg/dL
Indirect Bilirubin: 0.2 to 0.8mg/dL
(2) Conversion Factor of Bilirubin: mg/dL to umol/L = 17.1
(3) Critical Value for Bilirubin: >18mg/dL

"The strongest factorfor success is self-esteem: believing you can do it,

believing you deserve it and believing you will get it" -Anonymous 83
 ENDOCRINOLOGY
I. Endocrinology
A. Endocrine System
Specialized organs capable of producing hormones
Ductless organs; hormones produced by the endocrine glands are transported usually via
blood
Characteristics of the Endocrine System
Cyclicity characteristic pattern in the production of hormones (i.e. diurnal variation]
-

Pulsatility characteristic interval in the production of hormones

-

Feedback Mechanism-response of endocrine glands to stimulation or inhibition

Positive Feedback mechanism
an increase in a certain hormone leads to an increase of another hormone
a decrease of a certain hormone, leads to a decrease of another hormone
Negative Feedback mechanism
an increase in a certain hormone leads to a decrease of another hormone
a decrease ofa certain hormone, leads to an increase of another hormone
Classification of Hormones
Proteins/Polypeptides Glycoprotein Steroids Amino acid
derivative
GHRH, CRH, TRH, GnRH, TSH, LH, FSH, cortisol, melatonin,
somatostatin, PRE, ADH, hCG aldosterone, serotonin
oxytocin, erythropoietin estrogen, thyroid hormones
GH, ACTH, PRL, progesterone, epinephrine,
calcitonin testosterone norepinephrine
hPL vitamin DD

parathyroid hormone
insulin, glucagon,
-
 CHECKPOINT! Clinical Chemistry by Jude

HORMONE NATURE SITE OF MAIN ACTION(S)

ACTION
Hypothalamus
Thyrotropin-releasing Peptide Anterior Releases TSH and prolactin
hormone pituitary gland
Gonadotropin-releasing Peptide Anterior Releases LH and FSH
hormone pituitary gland
Corticotropin-releasing Peptide Anteriorr Releases ACTH and beta-
hormone pituitary gland lipotropichormone (LPH)
Growth hormone Peptide Anterior Releases growth hormone
releasing hormone pituitary gland (GH)
Somatostatin or growth Peptide Anterior Inhibits the release of different
hormone inhibiting pituitary gland hormones -GH, TSH, gastrin,
hormone (GHIH) vasoactive intestinal
polypeptide, gastric inhibitory
polypeptide, secretin, motilin,
glucagon and insulin
Prolactin-releasing Peptide Anterior Releases of prolactin (PRL)
peptide pituitary gland
Prolactin-inhibiting factor Dopamine Anterior Suppression of synthesis of
pituitary gland PRL
Anterior Pituitary Gland
Thyrotropin or Thyroid- Glycoprotein Thyroid Stimulation of thyroid
stimulating hormone hormone
(TSH)
Follicle-stimulating Glycoprotein Ovary and Testis Growth of follicles with LH,
hormone (FSH) secretion of estrogens and
ovulation
Development of seminiferous
tubules;spermatogenesis
Luteinizing hormone (LH) | Glycoprotein Ovary and Testis Ovulation; formation of copora
lutea; secretion of
progesterone
Stimulation of interstitial
tissue,secretion of androgens
Prolactin (PRL) Peptide Mammary Proliferation of mammary
glands gland, initiation of milk
secretion, antagonist of insulin
action
Growth hormone (GH) or | Peptide Liver and Production of insulin-growth
somatotropin peripheral factor-1 which promotes
tissues growth
Corticotropin or Peptide Adrenal cortex Stimulation of adrenocortical
adrenocorticotropic steroid formation and
hormone (ACTH) secretion
Beta-endorphin Peptide Brain Endogenous opiate; raising of
pain threshold and influence
on extrapyramidal motor
activity

"Those who leave everything in God's hand will eventually see God's hand in everything"
Anonymous 85
 CHECKPOINT! Clinical Chemistry by Jude

Alpha-melanocyte Peptide Skin Dispersion of pigment

stimulating hormone granules, darkening of skin
Leu-enkephalin and met Peptide Brain Same as beta-endorphin
enkephalin
Released by Posterior Pituitary Gland
Vasopressin or Anti Peptide Arterioles, renal Elevation of blood pressure
diuretic hormone tubules (distal Water reabsorption
convoluted
tubules and
collecting duct)
Oxytocin Peptide Smooth muscles Contraction, action in
of uterus and parturition and in sperm
mammary transport; ejection of milk
glands
Pineal Gland
Serotonin or 5- Indoleamine Cardiovascular, Neurotransmitter; stimulation
hydroxytryptamin respiratory and or inhibition of various smooth
gastrointestinal muscles and nerves
systems; brain
Melatonin Indoleamine Hypothalamus Suppression of gonadotropin
and growth hormone
secretion; induction of sleep
Thyroid Gland
Thyroxine (T4) and lodoamino General body Stimulation of oxygen
triiodothyronine (T3) acids tissue consumption and metabolic
rate oftissues
Calcitonin or Peptide Skeleton Uncertain; calcium and
thyrocalcitonin phosphate regulation
Parathyroid Gland
Parathyroid hormone Peptide Kidney Increased calcium
(PTH) or parathyrin reabsorption, inhibited
phosphate reabsorption
Skeleton Increased bone resorption
Adrenal cortex
Aldosterone Steroid Kidneys Salt and water balance
Androstenedione Steroid Hormone Converted to estrogens and
precursor testosterone
Cortisol Steroid Many Metabolism of carbohydrates,
proteins and fats, anti
inflammatory effect; etc.
Dehydroepiandrosterone Steroid Hormone Converted to esterogen and
(DHEA)and precursor testosterone
dehydroepiandrostenedio
nesulfate (DHEAS)
17-hydroxyprogesterone Steroid Hormone Converted to cortisol
precursor
Adrenal Medulla
Norepinephrine and Aromatic Sympathetic Stimulation of sympathetic
epinephrine amines receptorsS nervous system

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Anonymous 86
 CHECKPOINT! Clinical Chemistry by Jude

Epinephrine Aromatic Liver and Glycogenolysis, lipolysis

amines muscle, adipose
tissue
Ovary
Activin A Peptide Pituitary Stimulates release of FSH;
ovarian follicle enhances FSH action; inhibits
androgen production by theca
cells
Activin B Peptide See activin A_ See activin A
DHEA and DHEAS Steroids Hormone Converted to androstenedione
precursors
Estrogens Phenolic Female Development of secondary sex
steroids accessory sex characteristics
organs
Follistatin Peptides Pituitary Inhibits FSH synthesis and
Ovarian follicles secretion by binding activin
A4
Inhibin Peptide Hypothalamus, Inhibits FSH secretion;
ovarian follicle stimulates theca cell androgen
production
Inhibin B Peptide Seeinhibin A Seeinhibin A
Progesterone Steroid Female Preparation of the uterus for
accessory ovum implantation,
reproductive maintenance of pregnancy
structure
Relaxin Peptide Uterus Inhibition of myometrial
contraction
Testis
Inhibin B See above Anterior Control of LH and FSH
pituitary glandm | secretion
hypothalamus
Testosterone Steroid Male accessory Development of secondary sex
sex organs characteristics, maturation and
normal function
Placentaa
Estrogens See above See above See above
Progesterone Seeabove See above See above
Relaxin Seeabove See above See above
Chorionic gonadotropin Glycoprotein Same as LH Same as LH; prolongation of
or choriogonadotropin corpus luteal function
Placental growth Peptide Same as GH Same as GH
hormone
Chorionic Peptide Same as PRL Same as PRL
somatomammotropin
CS) or placental lactogen
Pancreas
Amylin Peptide Pancreas Inhibits glucagon and insulin
secretion
Glucagon Peptide Liver Glycogenolysis

"Those who leave everything in God's hand will eventually see God's hand in everything"
-
Anonymous 87
 CHECKPOINT! Clinical Chemistry by Jude

Insulin Peptide Liver, fat, Regulation of carbohydrate

muscle metabolism, lipogenesis
Pancreatic polypeptide Peptide Gastrointestinal Increased gut motility and
(PP) tract gastric emptying; inhibition of
gall bladder contraction
Somatostatin (SS) Peptide Pancreas Inhibition of secretion of
insulinand glucagon
Gastrointestinal tract
Gastrin Peptide Stomach secretion of gastric acid,
gastric mucosal growth
Ghrelin Peptide Anterior Secretion of growth hormone
pituitary gland
Secretin Peptide PancreaS Secretion of pancreatic
bicarbonate and digestive
enzymes
Cholecystokinin- Peptide Gall bladder and Stimulation of gall bladder
pancreozymin (CCK-PZ) pancreas contraction and secretion of
pancreatic enzymesS
Motilin Peptide Gastrointestinal Stimulation of gastrointestinal
tract motility
VIP Peptide Gastrointestinal Neurotransmitter; relaxation
tract of smooth muscles of gut and
of circulation; increased
release of hormones and
secretion of water and
electrolytes from pancreas to
gut
Gastric inhibitory peptide Peptide Gastrointestinal Inhibition of gastric secretion
(GIP) tract and motility; increase in
insulin secretion
Glucagon-like peptide Peptide Gastrointestinal Increase insulin and decrease
tract glucagon secretion; inhibit
gastric emptying_
Bombesin Peptide Gastrointestinal Stimulation of release of
tract various hormones and
pancreatic enzymes, smooth
muscle contractions and
hypothermia, changes in
cardiovascular and renal
function
Substance P Peptide Gastrointestinal Sensory neurotransmitter,
tract and brain analgesic; increase contraction
of gastrointestinal smooth
muscle; potent vasoactive
hormone; promotion of
salivation, increased release of
histamine

"Those who leave everything in God's hand will eventually see God's hand in everything"
-
Anonymous 88
 CHECKPOINT! Clinical Chemistry by Jude

Neurotensin Peptide Gastrointestinal Uncertain

tract and
hypothalamus
Kidneys
25 - (OH)2
1, Sterol Intestine Facilitation of absorption of
cholecalciferol Bone calcium and phosphorus;
increase in bone resorption in
conjunction with PTH
Erythropoietin Peptide Bone marrow Stimulation of red cell
formation
Renin Peptide Intravascular Converts angiotensinogen into
(Renin-angiotensin- space angiotensin I
aldosterone system) Angiotensin I is further
converted into angiotensin II
Angiotensin II increases blood
pressure
Liver
IGF-1, formerly known as Peptide Most cells Stimulation of cellular and
Somatomedin linear growth
IGF-2 Peptide Most cells Insulin-like activity
Thymus
Thymosin and Peptides Lymphocyte Maturation of T lymphocytes
thymopoietin
eart
Atrial natriuretic peptide Peptide Vascular, renal Regulation of blood volume
and adrenal and blood pressure
tissues
B-type natriuretic peptide| Peptide Vascular, renal Regulation of blood volume
and adrenal and blood pressure
tissues
Adipose tissue
Adiponectin Peptide Muscle Increases fatty acid oxidation
Suppresses glucose formation
Liver
Leptin Peptide Hypothalamus Inhibition of appetite,
stimulation of metabolism
R Resistin
Multiple Cell Types
Peptide Liver Insulin resistance
Estrogens See abovve See above Seeabove
Galanin Peptide Brain, pancreas, Regulates food intake, memory
gastrointestinal and cognition; inhibits
tract endocrine and exocrine
secretions of pancreas; delays
gastric emptying, prolongs
colonic transport times
Parathyroid hormone Peptide Kidney, bone Physiologic function
related peptide conjectural; PTH-like actions;
tumor marker

"Those who leave everything in God's hand will eventually see God's hand in everything"
-Anonymous 89
 CHECKPOINT! Clinical Chemistry by Jude

Growth factors (e.g Peptide Many Stimulation of cellular growth

epidermal, fibroblast,
transforming, platelet
derived, nerve growth
factors)
Monocytes/Lymphocytes/Macrophages
Cytokines (e.g. Peptide Many Stimulation or inhibition of
interleukins 1 to 18, cellular growth; other
tumor necrosis factor,
interferons)

Il. Hypothalamus
A. Anatomy and Physiology of the Hypothalamus
Small organ located at the base of the brain, above the pituitary gland
Connected to the pituitary gland through the infundibular stalk (infundibulum)
Most of the hormones of hypothalamus target the pituitary gland
ADH and vasopressin are produced by the hypothalamus and temporarily stored in
the posterior pituitary gland
Releasing Hormones Inhibiting Hormones
TRH, CRH, GnRH, GHRH, PRF Prolactin-inhibitory factor, GHIH

I. Pituitary Gland
AAnatomy and Physiology of the Pituitary gland
A pea-shaped organ located below the hypothalamus; Divided into two major lobes:
1. Anterior pituitary gland
Adenohypohysis
Produces six major hormones:
Thyroid-stimulating hormone Adrenocorticotropic hormone
Luteinizing hormone Follicle-stimulating hormone
Prolactin Growth hormone
2. Posterior pituitary gland
Neurohypohysis
Does not produce hormone; serves as storage site for two hormones produced
by the hypothalamus: anti-diuretic hormone and oxytocin

IV. Thyroid Gland

A. Anatomy of the Thyroid
Butterfly-shaped organ located on the posterior portion of the neck
Left and right lobes are separated by a ligament known as isthmus
Composed of two cell types:
1. Follicular cells produces metabolic hormones T3 and T4
2. Parafollicular cells also known as perifollicular cells or C-cells; produces calcitonin
-

"Those who leave everything in God's hand will eventually see God's hand in everything"
-
Anonymous 90
CHECKPOINT! Clinical Chemistry by Jude

B. Function

Produces T3 and T4- metabolic hormones

Produces calcitonin -calcium and phosphate regulator
C. Production
1. Thyroid hormone production is regulated by the HPTA (hypothalamus, pituitary
gland, thyroid gland axis)
2. Hypothalamus produces thyrotropin-releasing hormone, which stimulates the
pituitary gland to produce TSH (thyroid stimulating hormone)
3. Thyroid stimulating hormone stimulates the follicular cells ofthe thyroid to produce
T3 and T4

4 TSH binds the TSH receptors found in the thyroid follicles

5. Binding of the TSH with TSH receptor causes entry of iodine into the thyroid follicles
6. Two types of iodine may be acquired from the diet: monoatomic iodine (1) and
diatomic iodine (la). Most of the iodine in the diet are in the diatomic form. Iodine
rich foods include seafood and iodized salts.
7 Thyroid follicles are circular structures inside that thyroid that encloses a colloid
matrix known as thyroglobulin. Thyroglobulin is a protein matrix rich in the amino
acid tyrosine
8 Inside the thyroid follicles, iodine binds tyrosine. This binding is catalyzed by the
enzyme thyroid peroxidase (TP0)
9. Monoatomic iodine plus tyrosine leads to the formation of monoiodotyrosine (MIT)
10. Diatomic iodine plus tyrosine leads to the formation of diiodotyrosine (DIT)
11. The amount of DIT is greater compared to MIT
12. MIT may combine with DIT to produce triiodothyronine (T3).
13. DIT may combine with another DIT to produce tetraiodothyronine (T4 or
thyroxine)
14. In terms of concentration, there are more T4 compared to T3.
15. However, in terms of biological activity, T3 is more biologically active than T4
16. From the thyroid, T3 an T4 will move into the circulation to promote metabolism
17. In the tissues, peripheral deiodination ofT4 occurs. In peripheral deiodination, T4
is converted into the active form T3 using the enzyme thyroid deiodinase. This occurs
in the tissues.

"Those who leave everything in God's hand will eventually see God's hand in everything"
Anonymous
-
91
CHECKPOINT! Clinical Chemistry by Jude

D. Clinical Presentation of Patients with Hypothyroidism & Hyperthyroidism

HYPOTHYROIDISM HYPERTHYROIDISM
basal metabolic rate decreased increased
symphatetic response decreased increased
Weight gain loss
temperature tolerance cold intolerance heat intolerance
decreased sweating increased sweating
gastrointestinal function constipation diarrhea
decreased appetite increased appetite
cardiovascular function decreased cardiac output increased cardiac output
bradycardia tachycardia and
palpitation
respiratory function hypoventilation dyspnea
general appearance myxedemna exophthalmos
deep voice decreased blínking
impaired growth (in enlarged thyroid
children)
general behavior mental retardation (infant) restlessness
mental and physical irritability, anxiety
sluggishness hyperkinesis, wakefulness
somnolence

E. Thyroid Gland Disturbances

T3 &T4 Levels TSH levels
Hypothyroidism Primary Decreased Increased
Secondary Decreased Decreased
Hyperthyroidism Primary Increased Decreased
Secondary Increased Increased

Secondary vs. Tertiary Hypothyroidism

HYPO- T3&T4 TSH TRH TRH Stimulation/
THYROIDISM Administration Test
Secondary Decreased Decreased lncreased/ TSH before administration
Normal Low
TSH after administration
Low
Tertiary Decreased Decreased Decreased TSH before administration
Low
TSH after administration
Increased_

"Those who leave everything in God's hand will eventually see God's hand in everything"
Anonymous 92
 CHECKPOINT! Clinical Chemistry by Jude

Autoimmune Disorders of the Thyroid Hormone

HASHIMOTO'S THYRoIDITIS GRAVE'S DISEASE
Anti-microsomal antibodies (anti-thyroid Anti-TSH receptor antibodies (Thyroid
peroxidase antibodies); anti-thyroglobulin stimulating immunoglobulin)
antibodies
Primary hypothyroidism Primary hyperthyroidism

F. Protein Carriers for Thyroid Hormone

Thyroxine-binding globulin (TBG) - major protein carrier for thyroid hormones;
transports approximately 70-75% of thyroid hormones
Thyroxine-binding prealbumin (TBPA/ transthyretin)
Thyroxine-binding albumin (TBA)

G. T3 Uptake Test/ Thyroid Hormone Binding Ratio

Measures the amount of available bindings sittes in TBG
T3 Uptake test result is inversely proportional to the amount of available bindings
sites

Available Binding Sites in TBG T3U or THBR

HYPOTHYROIDISM Increased Decreased
HYPERTHYROIDISM Decreased Increased

V. Adrenal Glands
A.Anatomy and Physiology of the Adrenal Glands
Paired organs, located above the kidneys
Divided into two major parts: the outer cortex (90% of adrenal mass) and the
inner medulla (10% of the adrenal mass)
Adrenal cortex is further subdivided into three zones:
G-zone or zona glomerulosa outermost layer, salt regulation, produces
aldosterone, which is the major mineralocorticoid
F-zone or zona fasiculata middle layer, sugar regulation, produces cortisol,
-

which is the major glucocorticoid

R-zone or zona reticularis inner layer, produces weak androgens
-

Adrenal medulla produces catecholamines (epinephrine and norepinephrine) as

well as dopamine
B. Aldosterone
Major mineralocorticoid; Regulated by Renin-Angiotensin-Aldosterone System
Acts on the kidneys to promote sodium reabsorption, at the expense of potassium
Hyperaldosteronism: increased blood sodium, decreased blood potassium levels
Hypoaldosteronism: decreased blood sodium, increased blood potassium levels

"Those who leave everything in God's hand will eventually see God's hand in everything"
-
Anonymous 93
 CHECKPOINT! Clinical Chemistry by Jude

C. Cortisol
Major glucocorticoid; capable of increasing blood glucose levels
Exhibits diurnal variation: increased in the morning, low in levels during the
afternoon/evening
Hypercortisolism: Cushing's syndrome hyperglycemia and increased cortisol
-

level, buffalo hump and moon-face appearance

Hypocortisolism: Addison's disease hypoglycemia and decreased cortisol levels
-

VI. Endocrinology and Bone Metabolism

A. Calcitonin

Promotes calcium deposition; moves calcium from the blood towards the bone
Marker of medullary thyroid carcinoma
Usually decreases blood calcium levels and increases blood phosphate levels
B. Parathyroid hormone

Promotes calcium resorption; moves calcium from the bone into the blood
Usually increases blood calcium levels and decreases blood phosphate levels
C. Vitamin D
A fat soluble vitamin, can be synthesized in the skin from sunlight

Promotes calcium and phosphate reabsorption

Deficiency leads to rickets (vitamin D deficiency of children) and osteomalacia
(vitamin D deficiency of the adults)

VII. Endocrinology of Reproductive System and Pregnancy

Testosterone is the most potent male sex hormone. It is a marker of masculinity
Estrogen exists in three major forms: estrone, estradiol and estriol. The most potent
female sex hormone is estradiol. Estriol is produced by pregnant women
Estriol is produced by the placenta. During pregnancy, estrogen production shifts from
the ovaries to the placenta.
Estriol is used as a marker of stability offetoplacental unit ina pregnant woman
Estriol is part of the quadruple test for Down Syndrome (Trisomy 21)
Quadruple test:
Human chorionic gonadotropin- increased (if baby has Down syndrome)
Inhibin A increased (if baby has Down syndrome)
-

Urinary estriol decreased (if baby has Down syndrome)

-

Alpha fetoprotein - decreased (if baby has Down syndrome)

"Those who leave everything in God's hand will eventually see God's hand in everything"
Anonymous
- 94
CHECKPOINT! Clinical Chemistry by Jude

Human chorionic gonadotropin is produced by the placenta. It is a glycoprotein that has

a similar structure TSH, FSH and LH. These four hormones have similar alpha subunits
but different beta subunits. They can therefore be differentiated based on their beta
subunit.
HCG is a marker of pregnancy in women and a marker of testicular carcinoma in

males.
HCG isnormaly elevated in the first trimester of pregnancy but decreases during the
second and the third trimester.
Continuously elevated HCG levels are found related to Down syndrome,
Choriocarcinoma and Molar pregnancy.
Continuously low HCG levels in a pregnant woman may indicate ectopic pregnancy.

"Those who leave everything in God's hand will eventually see God's hand in everything"
-
Anonymous 95
 THERAPEUTICDRUGMANAGEMENT
L Therapeutic Drug Management
A. What is Therapeutic Drug Management/Monitoring?
Involves analysis, assessment and evaluation of circulating concentrations of drugs in
serum, plasma or whole blood
To ensure that a given dosage of drug produces maxinmal therapeutic benefit and
minimal toxic side effects
B. Indication of Therapeutic Drug Monitoring
Used when the consequences of overdosing and underdosing of the drug is serious
Used when there is a small difference between the therapeutic and toxic dose
Used when there is a poor relationship between the dose of drug and circulating
concentrations but good correlation between circulating concentrations and
therapeutic or toxic effects
Used when there is a change in the patient's physiologic state that may unpredictably
affect circulating drug concentrations
Used when a drug interaction is or may be occurring
Used when the physician wants to check for patient compliance

I. Pharmacology
Comprises a body of knowledge surrounding chemical agents and their effects on
living processes
Pharmacotherapeutics
Concerned with the application or administration of drugs to patients for the
purpose of prevention and treatment of disease
Requires the knowledge of pharmacodynamics and pharmacokinetics
Pharmacodynamics
Describes what the drug does to the body
Encompasses the process of interaction of pharmacologically active substances
with target sites, and the biochemical and physiologic consequences that lead
to therapeutic or adverse effects
 CHECKPOINT! Clinical Chemistry by Jude

Pharmacokinetics
Describes how drugs are received and handled by the body
Describes what the body does to the drug
Includes the processes of uptake of drugs by the body, biotransformation,
distribution, metabolism and elimination that the drugs undergo
L. Drugs and Human Body
Steady state -
the rate of administration is equal to the rates of metabolism and
excretion
Therapeutic range refers to the serum concentration of drug established to
achieve desired clinical effect
A. Drug Administration
1. Most drugs are not administered as a single bolus but instead are delivered on
a scheduled basis (multiple dosage)

2. Steady state of drugs (equilibrium) is often achieved after 4 to 7 doses

Routes of Administration
Oral through the mouth; most common route; slow-acting
Intravenous -
into the circulation; gives 100% bioavailability
Intramuscular into the muscles
-

Subcutaneous -
under the skin
Transcutaneous absorbed through the skin
Rectal -
suppository; erratic absorption
Pulmonary
Sublingual
B. Drug Absorption

Tablets and capsules require dissolution first before being absorbed

Liquid solutions have the tendency to be more rapidly absorbed
drugs can be absorbed through active transport or passive diffusion
weak acidic drugs are efficiently absorbed in the stomach
weak basic or alkaline drugs are preferentially absorbed in the intestines
changes in intestinal motility and stomach pH as well as the presence of food
or other drugs can affect drug absorption

"There will be haters. There will be doubters. There will be non-believers. And there will be
YOU, proving them wrong"- Anonymous 97
 CHECKPOINT! Clinical Chemistry by Jude

C. Drug Metabolism
First-pass metabolism
Drugs enter hepatic portal system first before entering the general circulation
Drugs are subjected to significant uptake and metabolism during the passage
through the liver
D. Drug Distribution

Drug distribution refers to the spread of drugs from its point of entry
throughout the systemic circulation and into various tissues
Drugs in blood are often bound to protein, especially to albumin, alpha-1 acid
glycoprotein and lipoproteins
Acidic drugs primarily bind with albumin
Basic drugs bind globulins and lipoproteins
Free fraction can interact with its site of action and cause a biological response
Free fraction correlates to the therapeutic or toxic effects of a drug
E. Half-life

Represents the time needed for the serum concentration of a drug to decrease
by half
F. Routes of Drug limination
Through hepatic metabolism
Through renal filtration
Combination of hepatic and renal
IV. Laboratory Assessment of Therapeutic Drugs
Sample preferred for therapeutic drug monitoring is SERUM
Do not use serum separator tubes for blood collection. Drugs might be adsorbed
into the gel.
Specimen:

Peak Specimen- maximum concentration; collected 30-60 minutes after the

drug administration but may vary depending on the type of drug
&Trough Specimen -
minimum concentration; collected before the
administration of the next dose

"There will be haters. There will be doubters. There will be non-believers. And there will be
YOU, proving them wrong" Anonymous
-
98
 CHECKPOINT! Clinical Chemistry by Jude

Laboratory results should contain:

Time of Last Dose
Time of Extraction
V. Classifications of Therapeutically monitored Drugs
CARDIOACTIVE ANTICONVULSANTS
Amiodarone Phenobarbital
Digitoxin Carbamazepine
Digoxin Phenytoin
Disopyramide Valproic Acid
A Lidocaine AEthosuximide (Zarontin)
Procainamide AFelbamate
Propanolol lamotrigine (Lactamical)
Quinidine A Neurontin (Gabapentin)
Verapamil Topiramate
ANTIDEPRESSANTS IMMUNOSUPPRESSIVE
Amitriptyline Cyclophosphamide (Cytoxan)
Desipramine Cyclosporine
Doxepin Leflunamide (LFM)
Imipramine Mycophenolate mofetil
Lithium A Prednisone

ANortriptyline Rapamycin (Sirolimus)

Tacrolimus (FK-506)
ANTI-PSYCHOTIC ANTI-NEOPLASTIC
Phenothiazines (chlorpromazine, Methotrexate (also an
thioridazine, fluphenazine) immunosuppressant)
Butyrophenones (haloperidol) Busulfan
Risperdal
Olonzapine (Zyprexa) BRONCHODILATORS
Quetiapine (Seroquel) Theophylline
A Aripirazole (Abilify)
ANTI-INFLAMMATORY/ANALGESICS ANTIBIOTICS
Acetylsalicylic acid Aminoglycosides
A Acetaminophen Vancomycin
Chloramphenicol
A Trimetophrim lactate

"There will be haters. There will be doubters. There will be non-believers. And there will be
YOU, proving them wrong" - Anonymous 99
 CHECKPOINT! Clinical Chemistry by Jude

Anticonvulsants are also known as anti-epileptic drugs

Phenobarbital is used in the treatment of generalized grand mal tonic-clonic
seizures (Henry 22nd ed)
Lithium salt (lithium carbonate) is used for treatment of bipolar disease or
manic-depression
Bronchodilators are also known as anti-asthma drugs
Chloramphenicol is often associated with aplastic anemia
Aspirin (acetylsalicylic acid) is an analgesic, anti-inflammatory and anti-pyretic
drug
Salicylate toxicity can be checked using a colorimetric method utilizing Trinder
reagent, which is composed of mercuric chloride, hydrochloric acid and ferric
nitrate. Mercuric salt precipitates proteins present in specimen. Ferric ions form
purple-complex with salicylate derivatives which can be measured at 540nm
Acetaminophen overdose causes damage to liver
Can
L 1 be detected using nitrous acid (sodium
Acetaminopheh nitritethydrochloric
acid). Addition of the reagent causes formation of 2-nitro-5-acetaminophenol
which turns into a yellow color in alkali. The yellow color is measured at 430nm

VI. Techniques of Drug Analysis

Enzyme multiplied immunoassay technique (EMIT)
Enzyme-linked immunosorbent assay (ELISA)
Enzyme immunochromatography
Fluorescence Polarization Immunoassay (FPIA)
Radioimmunoassay
Gas Chromatography-Mass Spectrometry
Thin-Layer Chromatography
Spectrophotometry
Atomic Absorption Spectrophotometry

"There will be haters. There will be doubters. There will be non-believers. And there will be
YOU, proving them wrong" - Anonymous 100
 TOXICOLOGY
What is Toxicology?
study of poisons and toxic agents
Four major disciplines in toxicology
Mechanistic Toxicology- elucidates cellular and biochemical effects of toxins
Descriptive Toxicology - uses results from animal experimentation to predict level of
exposure that will harm humans
Forensic Toxicology - concerned with medico-legal consequences of exposure to toxins
Clinical Toxicology - studies relationship between toxin exposure and disease state
any substance can cause potential harm if given at a certain dosage
ED50-effective dose; dose that would be predicted to be effective or have therapeutic benefit
in 50% of the population
TDso- toxic dose; dose that would be predicted to produce a toxic response in 50% of the
population
LDso- lethal dose; the dose that would predict death in 50% of the population
Routes of exposure to toxic agents
Ingestion, inhalation, transdermal
Toxicities are associated with suicide, accidental exposure, homicide or ocupational exposure
Acute toxicity is associated with single dose of which is sufficient to cause a toxic effect
Chronic toxicity is associated with repeated exposure for extended period of time, usually at
doses that are insufficient to cause an acute response.
Toxidromes refers to a constellation of clinical signs and symptoms that suggests a specific class
of poisoning. Toxidromes include:

d Anti-cholinergic
Agitation
Blurred vision
Cholinergic
Diarrhea
Urination
Opioid
Bradycardia
Decreased
Sedative-hypnotic Sympathomimetic
Ataxia
Blurred vision
Agitation
Diaphoresis
Decreased bowel Miosis bowel sounds Confusion Excessive motor
sounds Bradycardia Hypotension Diplopia activity
Dry skin Bronchorrhea Hypothermia Dysesthesias Excessive speech
Fever Emesis Lethargy/ Hypotension Hallucinations
Flushing Lacrimation coma Lethargy/coma Hypertension
Hallucinations Salivation Miosis Nystagmus Hyperthermia
Ileus Shallow Respiratory Insomnia
Lethargy/coma respirations depression Restlessness
Mydriasis Slow Sedation Tachycardia
Myoclonus respiratory Slurred speech Tremor
Psychosis rate
Seizures
Tachycardia
Urinary retention
 CHECKPOINT! Clinical Chemistry by Jude

I1. Toxic Agents

A. Alcohols
Toxic alcohols include: ethanol, methanol, isopropanol and ethylene glycol
Ethanol is the most commonly abused chemical substance
Methods of Analysis:
Flame ionization gas chromatography
Headspace gas chromatography
Tests for Alcohol analysis
Serum or plasma an blood ethanol levels
Breath ethanol
Oral fluid ethanol (saliva as specimen)
Urine ethanol
Differential Laboratory Findings in Alcohol Toxicity
Alcohol Serum Metabolic Acidosis Serum Urine
Osmolal Gap with anion gap acetone oxalate
Ethanol Positive Negative Negative Negative
Methanol Positive Positive Negative Negative
Isopropanol Positive Negative Positive Negative
Ethylene glycol Positive Positive Negative Positive

Stages of Acute Alcoholic Influence or Intoxication

BLOOD ALCOHOL
SIGNS AND SYMPTOMS
(% w/v)
No obvious impairment, some changes observable on
0.01-0.05
performance testing
Mild euphoria, decreased inhibitions, some impairment of
0.03-0.12
motor skills
Decreased inhibitions, loss of critical judgment, memory
0.09-0.25
impairment, diminished reaction time
Mental confusion, dizziness, strongly impaired motor skills
0.18-0.30
staggering, slurred speech)_
0.27-0.40 Unable to stand or walk, vomiting, impaired consciousness
0.35-0.50 COMA AND POSSIBLE DEATH
Presumptive Evidence Of Driving Under Influence Of
20.10
Alcohol

B. Cyanide
Characteristic odor of bitter almonds
Cyanide binds hemoglobin causing hypoxia, flushing, headache, tachypnea,
-

dizziness and respiratory depression

Methods for analysis
Photometric analysis
Headspace gas chromatography

"Working hard for something we don't care about is called stress.

Working hard for something we love is called passion" -Anonymous 102
 CHECKPOINT! Clinical Chemistry by Jude

C. Carbon Monoxide
Colorless, odorless, tasteless gas
Common sources include: car exhausts and cigarette
Has up to 250 times greater affinity for hemoglobin compared to oxygen
Produces cherry-red color of the blood
Methods for analysis:
Gas chromatography
Spot test for carbon monoxide exposure
Differential spectrophotometry
D. Arsenic
Odor of garlic
Highly keratinophilic, carcinogenic
Specimen toxicity analysis include: skin, hair or nails
E. Lead

Analyzed through measurement of blood lead levels

Can cause toxic effects in brain and can lead to anemia
F. Mercury
From Greek word "hydrargyrias" which means water silver
Metallic mercury is liquid at room temperature
Has three forms: elemental (Hg'), mercurous (Hg) and mercuric (Hg?*)
Elemental form is non toxic; methylmecury is toxic and may be acquired by
bioaccumulation in aquatic food chain especially involving fish
Previouslyused in sphygmomanometer, thermometers, barometer and manometers,
as well as in merthiolate
Toxicity: mercury can alter or denature proteins, can cause severe damage in
kidneys, lipophilic and can bind myelin - which is found in neurons
G. Organophosphates
Found in pesticides and insecticides
Toxicity is associated with decreased cholinesterase
Can cause toxicity in the liver
III. Drugs of Abuse
A. Specimen for Drugs of abuse: urine is often used
B. Methods: Screening- immunoassays
Confirmation gas chromatography-mass spectrometry
-

C. Common Drugs of Abuse

1. Barbiturates sedative hypnotics

Ultra-short acting = thiopental, methohexital, thiamylal
Short-acting and intermediate-acting = pentobarbital, secobarbital, butalbital,
aprobarbital, amobarbital, butabarbital
Long-acting = phenobarbital, methobarbital

"Working hard for something we don't care about is called stress.

Working hard for something we love is called passion" - Anonymous 103
CHECKPOINT! Clinical Chemistry by Jude

2. Benzodiazepines~ sedative hypnotics

Short-acting = midazolam, estazolam, flurazepam, temazepam, triazolam
Intermediate-acting = flunitrazepam
Long-acting = diazepam, quazepam, alprazolam, chiordiazepoxide, clonazepam,
clorazepate, lorazepam, oxazepam
3. Cannabinoids
Derived from the leaves of marijuana plant Cannabis sativa
Cannabis is the most extensively abused drug in the world
Delta-9-tetrahydrocannabinol (A-THC) is the major psychoactive component of
marihuana
Consumed by smoking the plant leaves, flower buds, and sometimes stems
THC extracted from glandular hairs of Cannabis flower produced into a resin
known as hashish
Psychotropic effects: euphoria, distorted perceptions, relaxation
4. Opiates
Opioid refers to compound involving natural or semisynthetic opiates and fully-

.
synthetic opioids
Medically used to relieve moderate to severe chronic pain
Natural opiates include morphine and codeine
Natural opiate are derived from the juice and seeds of poppy plant Papaver
somniferum
Codeine is antitussive and analgesic. It is one of the most frequently prescribed
opiates in the world
Semisynthetic opiates include: heroin, hydrocodone, hydromorphone, oxycodone
and oxymorphone
Fully synthetic opioids include: fentanyl, meperidine, methadone, propoxyphene
and tramadol
5. Cocaine
Alkaloid found in a plant, Erythroxylon coca
Medically used as a local anesthesia an vasoconstrictor in nasal surgery and to
dilate pupils in ophthalmology
Available in two forms:
1. powder (hydrochloride salt) = administered through nasal insufflation or
snorting
2. crack (free-base product) = rock crystal that is heated and smoked; term
refers to crackling sounds heard when it is heated
benzoylecgonine is the primary metabolite of cocaine
CNS stimulant

"Working hard for something we don't care about is called stress.

Working hard for something we love is called passion" -Anonymous 104
CHECKPOINT! Clinical Chemistry by Jude

6. Lysergic Acid Drugs (LSD)

Structurally similar to serotonin
Synthesized from a naturally occurring ergot alkaloid found in the fungus
Claviceps purpurea which grows in wheat and other grains
Psychedelic drug, hallucinogen
Dosage forms include: tablet, gelatin, powder, capsule
7. Amphetamines
Associated with sympathomimetic syndromes
Stimulants and hallucinogen
Include amphetamines and methamphetamines
Designer amphetamines (also known as designer drugs or club drugs) are
derivatives of amphetamine. This includes: phenylethylamines, benzylpiperazines,
phenylpiperazines and pyrrolidinophenone
Representative example is ecstasy (MDMA methylenedioxymethamphetamine)
-

D. Significance of Testing Drugs of Abuse

1. Drug-facilitated sexual assault
Includes: anti-cholinergics, antihistamines, barbiturates, benzodiazepines, chloral
hydrate, dextromethorphan, ethanol, gamma-hydroxybutyrate, ketamine, opioids,
sedative-hypnotics
2Illicit drug use during pregnancy
Includes: cocaine, opiates, cannabinoids, amphetamines, ethanol, PCP
3. "Doping" in athletic competitions
Drugs that may cause performance enhancements, but may endanger the athlete's
health
Testing for athletes include:
stimulants, narcotics, cannabinoids, glucocorticosteroids
Anabolic-androgenic steroids, hormones, hormone modulators, oxygen transfer
enhancement

"Working hard for something we don't care about is called stress

Working hard for something we love is called passion"- Anonymous 105