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Download 1 APPENDIX 1 TEST PRINCIPLES USED IN THE BIOCHEMICAL
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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 2 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 3 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. 4 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 5 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. 6 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. 7 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. 8 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 9 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.