Download 1 APPENDIX 1 TEST PRINCIPLES USED IN THE BIOCHEMICAL

APPENDIX 1
TEST PRINCIPLES USED IN THE BIOCHEMICAL ANALYSIS
Glucose
Glucose is the most important monosaccharide in the blood, an indispensable
energy supplier which supports cellular function. Glucose degradation occurs in glycolysis.
Glucose measurement is used in the diagnosis and monitoring of carbohydrate metabolism
disorders including diabetes, neonatal hypoglycemia, idiopathic hypoglycemia and
pancreatic islet cell carcinoma.
Test method: Enzymatic colorimetric assay

Plasma sample and addition of R1(phosphate buffer,GOD [microorganisms], POD
[horseradish],4-aminophenazone,phenol) and start of the reaction

Glucose is oxidized by glucose oxidase (GOD) to gluconolactone in the presence of
atmospheric oxygen. The resultant hydrogen peroxide oxidizes 4-aminophenazone and
phenol to 4-(p-benzoquinone-monoimino)-phenazone in the presence of peroxidase
(POD). The colour intensity of the red dye is directly proportional to the glucose
concentration and measured photometrically.
Glucose + O2 + H2O
GOD
Gluconolactone + H2O2
2H2O2 + 4-aminophenazone + phenol
POD
4-(p-benzoquinonemonoimino)phenazone + 4H2O
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Cholesterol
Cholesterol is a steroid with a secondary hydroxyl group in the C3 position. It
is synthesized in many types of tissue, but particularly in the liver and intestinal wall.
Approximately three quarters of cholesterol is newly synthesized and one quarter originates
from the dietary intake. Cholesterol assays are used for screening for atherosclerotic risk
and in the diagnosis and treatment of disorders involving elevated cholesterol levels as well
as lipid and lipoprotein metabolic disorders.
Test method: Enzymatic colorimetric assay

Plasma sample and addition of R1(cholesterol reagent) and start of the reaction

Cholesterol is determined enzymatically using cholesterol esterase and cholesterol
oxidase.
Cholesterol esters + H2O
cholesterol esterase
cholesterol + RCOOH
Cholesterol esters are cleaved by the action of cholesterol esterase to yield free
cholesterol and fatty acids.
cholesterol
+ O2
cholesterol oxidase
cholest-4-en-3-one + H2O2
Cholesterol is converted by oxygen with the aid of cholesterol oxidase to cholest-4-en3-one and hydrogen peroxide.
2H2O2 + 4-aminophenazone + phenol
peroxidase
4-(p-benzoquinonemonoimino)-phenazone
+ 4H2O
The hydrogen peroxide created forms a red dyestuff by reacting with 4aminophenazone and phenol under the catalytic action of peroxidase. The colour
intensity is directly proportional to the cholesterol concentration and measured
photometrically.
cholesterol
+ O2
cholesterol
oxidase
cholest-4-en-3-one + H2O2
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Albumin
Albumin is a carbohydrate –free protein, which constitutes 55-65% of total
plasma protein. It maintains oncotic plasma pressure, and is also involved in the transport
and storage of a wide variety of ligands and a source of endogenous amino acids.
Test method: Colorimetric assay with endpoint method

Plasma sample and addition of R1(Citrate buffer)

Addition of R2(substrate-Citrate buffer and bromocresol green) and start of reaction

At a pH value of 4.1 albumin displays s sufficiently cationic character to be able to bind
to bromocresol green (BCG), an anionic dyestuff, to form a blue-green complex.
Albumin+BCG
AlbuminBCG complex
The colour intensity of the blue-green colour is directly proportional to the albumin
concentration and determined photometrically.
Alkaline phosphatase (ALP)
ALP consists of four structural genotypes: the liver-bone-kidney type, the
intestinal type, the placenta type and the variants from the germ cells. I t occurs in
osteoblasts, hepatocytes, leucocytes, the kidneys, spleen, placenta, prostate and the small
intestine. The liver-bone-kidney is particularly important. A rise in ALP occurs in all
forms of cholestatis, particularly with obstructive jaundice. It is also elevated in diseases of
skeletal system, such as Paget’s disease, hyperparathyroidism, rickets and osteomalacia, as
well as with fractures and malignant tumours. A considerable rise is sometimes seen in
young and activity juveniles. It is caused by increased osteoblast following accelerated
bone growth.
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Test method: Colorimetric assay in accordance with a standardized method

Plasma sample and addition of R1(2-Amino-2-methyl-1-propanol, mage=nesium
acetate, zinc sulfate, N-(2-hydroxyethyl)-ethylenediamine triacetic acid)

Addition of R2 (p-Nitrophenylphosphate) and start of reaction
p-nitrophenylphosphate + H2O
ALP
phosphate + p-nitrophenol
In the presence of magnesium and zinc ions, p-nitrophenyl phosphate is cleaved by
phosphatases into phosphate and p-nitrophenol. The p-nitrophenol released is proportional
to the ALP activity and is measured photometrically.
Blood Urea Nitrogen (BUN)
Urea is the final degradation product of protein and amino acid metabolism. In
protein metabolism the proteins are broken down to amino acids and deaminated. The
ammonia thus formed is synthesized to urea in the liver. This is the most important
catabolic pathway for eliminating excess nitrogen in the mammalian body.
The determination of BUN is the most widely used test for the evaluation of
kidney function.
This test is frequently used in conjunction with creatinine for the
differential diagnosis of prerenal hyperuremia ( cardiac decompensation, water depletion,
increased protein catabolism), renal hyperuremia ( glomerulonephritis, chronic nephritis,
polycystic kidney, nephrosclerosis, tubular necrosis) and post renal hyperuremia
(obstructions of urinary tract).
Test method: Kinetic UV assay in accordance with a standardized method


Plasma sample and addition of R1(buffer,NADH)
Addition of R2 (buffer,urease, GLDH, ά-ketoglutarate) and start of reaction
Urea is hydrolysed by urease to form CO2 and ammonia
Urea + H2O
urease
2NH4 + CO2
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The ammonia thus formed then reacts with ά-ketoglutarate and NADH in presence of
GLDH to yield glutamate and NAD+
ά-ketoglutarate + N H4+ + NADH GLDH
L-glutamate + NAD+ + H2O
The decrease in the absorbance due to consumption of NADH is measured kinetically.
Alanine aminotransferase (ALT)
Alanine aminotransferase (glutamate pyruvate transaminase) belongs to the
group of transaminases which catalyse the conversion of amino acids to the corresponding
α-keto acids via the transfer of amino groups;they also catalyse the reverse process.
Test method: UV test according to standardized method

Plasma sample and addition of R1(Tris buffer,L-alanine and NADH)

Addition of R2(α-ketoglutarate) and start of reaction:
α-ketoglutarate + D-alanine
ALT
L-glutamate + pyruvate
ALT is the enzyme which catalyzes this equilibrium reaction. The pyruvate
increase is measured in a subsequent indicator reaction which is catalyzed by lactate
dehydrogenase (LDH)
Pyruvate + NADH+H+
LDH
L-lactate +NAD+
In the second reaction , NADH is oxidized to NAD. The rate of decrease in
NADH (measured photometrically) is directly proportional to the rate of formation of
pyruvate, and thus the ALT activity.
Aspartate aminotransferase (AST)
Aspartate aminotransferase (glutamate oxaloacetate transaminase) belongs to
the group of transaminases which catalyse the interconversion of amino acids and α-keto
acids by transfer of amino groups.
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Test method: UV test according to standardized method


Plasma sample and addition of R1(Tris buffer,L-aspartate,NADH, MDH and LDH)
Addition of R2(α-ketoglutarate) and start of reaction:
α-ketoglutarate + L-aspartate
AST catalyzes this equilibrium reaction.
AST
L-glutamate + oxaloacetate
The oxaloacetate increase is measured in a
subsequent indicator reaction which is catalyzed by malate dehydrogenase (MDH)
Oxaloacetate + NADH+H+
MDH
L-malate +NAD+
NADH is oxidized to NAD+. The rate of decrease in NADH ( measured photometrically) is
directly proportional to the rate of formation of oxaloacetate, and thus the AST activity.
Total Bilirubin
Bilirubin is produced during normal and abnormal degradation of erythrocytes
in the reticuloendothelial system. Bilirubin determinations are made use of in the diagnosis
of liver diseases, detection of hemolytic anemia and the assessment of the degree of
severity of jaundice.
Test method: Colorimetric assay

Plasma sample and addition of R1(detergent, hydrochloric acid)

Addition of R2( diazoreagent) and start of reaction
Indirect bilirubin is liberated by the detergent.
In strongly acidic solution
containing 2,5-dichlorophenyl diazonium salt, total bilirubin couples to form azobilirubin.
The colour intensity of the red azo-dye formed is directly proportional to the total bilirubin
and determined photometrically.
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Gamma-glutamyltransferase (GGT)
Gamma-glutamyltransferase is used in the diagnosis and monitoring of
hepatobiliary diseases.
Test method: Enzymatic colorimetric assay

Plasma sample and addition of R1(TRIS buffer,glycylglycine)

Addition of R2(acetate buffer, L-y-glutamyl-3-carboxy-4-nitroanilide) and
start of reaction
L-y-glutamyl-3-carboxy-4-nitroanilide + glycylglycine
L-y-glutamyl-glycylglycine + 5-amino-2-nitrobenzoate
γ-GT
Gamma-glutamyltransferase transfers the γ-glutamyl group of L-y-glutamyl-3carboxy-4 nitroanilide to glycylglycine. The amount of 5-amino-2-nitrobenzoate liberated
is proportional to the GGT activity and measured photometrically.
Creatinine
Creatinine is synthesized endogenously from creatine and creatine phosphate.
Creatining determination is performed for the diagnosis and monitoring of acute and
chronic renal disease as well as for monitoring of renal dialysis.
Test method: Kinetic colorimetric assay (Jaffe method)


Plasma sample and addition of R1(sodium hydroxide)
Addition of R2(picric acid) and start of reaction
creatinine + picric acid
creatinine- picric acid complex
In alkaline solution, creatinine forms a yellow-orange complex with picrate.
The colour intensity is directly proportional to the creatinine concentration and measured
photometrically.
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Triglycerides
Triglycerides are esters of the trihydric glycerol with 3 long chain-fatty acids.
They are partly synthesized in the liver and partly ingested in food. The determination of
triglycerides is used in the diagnosis and treatment of patients having diabetes mellitus,
nephrosis, liver obstruction, lipid metabolism disorders and numerous endocrine disorders.
Test method: Enzymatic colorimetric assay

Plasma sample and addition of buffer, 4-chlorophenol, lipoprotein lipase,
glycerokinase and glycerol phosphate oxidase and start of reaction.
LPL
Triglycerides + 3 H2O
Glycerol + ATP
glycerol + 3 RCOOH
GK
Mg2+
Glycerol-3-phosphate
GPO
glycerol-3-phosphate + ADP
dihydroxyacetone phosphate + H2O2
peroxidase
H2O2 + 4-aminophenozone + 4-chlorophenol
4-( pbenzoquinone-monoimino)-phenazone + 2 H2O + HCl
Triglycerides are hydrolysed by lipoprotein lipase to glycerol followed by
oxidation to dihydroxyacetone phosphate and hydrogen peroxide. The hydrogen oxide
produced then reacts with 4-aminophenazone and 4-4-chlorophenol under the catalytic
action of peroxidase to form a red dyestuff (Trinder end point reaction).
Total Protein
Plasma proteins are synthesized predominantly in the liver, plasma cells, lymph
nodes, the spleen and in bone marrow.
In the course of disease the total protein
concentration and also the percentage represented by individual fractions can significantly
deviate from normal values.
Hypoproteinemia may be observed in cases of severe
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dehydration and illnesses such as multiple myeloma. Change in the relative percentage of
one plasma protein fraction.
Total protein measurements are used in the diagnosis and
treatment of a variety of diseases involving the liver, kidney or bone marrow, as well as
other metabolic or nutritional disorders.
Test method: Colorimetric assay


Plasma sample and addition of R1(blank reagent)
Addition of R2(biuret reagent) and start of reaction
Protein + Cu2+
alkaline solution
Cu- protein complex
The colour intensity is directly proportional to the protein concentration and
measured photo metrically.