Clinical Biochemistry

Urea can be estimated in the serum sample by colorimeter using diacetyl monoxime. The sample is treated with diacetyl monoxime and thiosemicarbazide under acidic condition. Urea condenses with diacetyl monoxime and forms a pink colored complex (a diamine derivative). The absorbance can be measured at 520 nm. The absorbance or intensity of the complex is directly proportional to the concentration of urea in the serum. First, a standard curve is prepared using standard urea solution and the concentration of the urea in the unknown serum sample can be calculated from it. The normal level of urea in the serum is 10–40 mg/dL. Uric acid in serum can be estimated by colorimeter using 4-aminoantipyrene and hydrogen peroxide. Uric acid is estimated in blood along with urea and creatinine as a parameter to check kidney function. Uricase enzyme acts upon uric acid and forms allantoin and hydrogen peroxide. The by-product H2O2 in the presence of catalytic enzyme peroxidase reacts with 4-amino antipyrine and dihydroxybenzene sulfonic acid (DHBS) to form a red-colored quinone-imine dye complex. The absorbance can be measured at 505 nm. The amount of uric acid in serum is directly proportional to the absorbance of the final product. Uric acid in serum can be estimated by colorimeter using picric acid. This method is known as Jaffe's method. The sample is treated with picric acid in alkaline condition. Creatinine forms a red-orange complex with picric acid. The absorbance can be measured at 520 nm. The intensity of this colored complex (creatinine-picrate) is directly proportional to the amount of creatinine present in the sample. Estimation of transaminase activity in the serum is the important parameter to check the liver function. ALT (alanine aminotransferase; formerly GPT—glutamate pyruvate transaminase) and AST (aspartate-aminotransferase; formerly GOT—glutamate-oxaloacetate-transaminase) enzymes are mainly present in liver and kidney cells. Glutamate pyruvate transaminase (SGPT) activity in serum can be estimated using 2,4-dinitrophenylhydrazine. The sample is treated with ALT substrate (alanine, α-ketoglutarate, phosphate buffer, and NaOH). ALT enzyme catalyzes the reaction between l-alanine and α-ketoglutarate to form pyruvate and l-glutamate. The pyruvate reacts with 2,4-dinitrophenylhydrazine in the alkaline condition to form a red-brown complex. The intensity of the color of the complex is directly proportional to the amount of ALT enzyme in the serum. Glutamate oxaloacetate transaminase (SGOT) activity in serum can be estimated using 2,4-dinitrophenylhydrazine. The sample is treated with AST substrate (aspartate, α-ketoglutarate, phosphate buffer, and NaOH). AST enzyme catalyzes the reaction between l-aspartic acid and α-ketoglutarate to form glutamate and oxaloacetate. The oxaloacetate reacts with 2,4-dinitrophenylhydrazine in the alkaline condition to form a red-brown complex. The intensity of the color of the complex is directly proportional to the amount of AST enzyme in the serum. The absorbance can be measured at 505 nm. Alkaline phosphatase (ALP) enzymes activity estimation is another important parameter to assess the liver function.Any damage or injury in the liver leads to the outflow of ALP from the liver to blood. Thus, its high level of blood indicates liver disease. The sample is treated with disodium phenyl phosphate. Alkaline phosphatase in the serum breaks it into phenol and phosphate radical at alkaline pH. The phenol then forms a red-orange complex with 4-amino antipyrine in the presence of potassium ferricyanide. The absorbance can be measured by colorimeter at 510 nm. The intensity of the color is proportional to the phenol formed which in turn related to the activity of ALP enzyme. Serum albumin can be estimated by colorimeter using bromocresol. The sample is treated with bromocresol. Albumin in the serum reacts with bromocresol green (BCG) at acidic pH to form a green-blue color complex. The absorbance can be measured at 630 nm. The intensity of the color is directly proportional to the albumin level. Blood glucose estimation is the primary and most used method to check hyperglycemia or diabetes. The normal glucose level is 80–100 mg/dL under fasting condition and 120–140 mg/dL 2 h after eating. The blood glucose above these levels is termed as hyperglycemia or diabetes. This is enzymatic method where blood glucose is oxidized to gluconic acid by glucose oxidase. Hydrogen peroxide which formed as a by-product coupled with 4-amino antipyrine or 4-aminophenazone and phenol to form a red dye quinonimine in the presence of peroxidase enzyme. The absorbance of the colored product can be measured at 505 nm and its intensity is directly proportional to the amount of glucose. For diabetes, urine glucose estimation is another parameter. Normally the detectable level of glucose in the urine is below 100 mg/dL (0–0.8 mmol/L). A higher level of glucose in the urine is the indication of higher glucose in the blood (diabetes mellitus). Urine glucose can be estimated by the Benedict test. The urine sample is treated with Benedict reagent (copper sulfate). Glucose present in the sample reduces the copper sulfate (Cu2 +) to red color cuprous oxide (Cu+) and oxidizes itself to gluconic acid. More amount of glucose in the sample results in the reduction of copper sulfate and disappearance of the blue color. Thus, the glucose amount can be checked by the final color development. Urine glucose percentage can be estimated by Benedict titration method. The sample is titrated with Benedict solution (copper sulfate and potassium thiocyanate). In the reduction reaction of Cu2 + by glucose, a white precipitate of cuprous thiocyanate (CuCNS) is formed instead of a red precipitate of cuprous oxide. The potassium ferricyanide of the benedict reagent keeps the cuprous oxide in the solution. Copper sulfate and sodium carbonate (Na2CO3) reacts to form CuCO2 which remains dissolved by the sodium citrate. The formation of white precipitate with complete disappearance of the blue color indicates the complete reduction of copper. Thus, glucose in the sample can be calculated titration. In these tests’ patients are given 75 mL of glucose in 250 mL water after 12 h fasting. The blood glucose level is estimated after 2 h. The normal level is below 140 mg/dL. The level above 140 mg/dL is an indication of diabetes. Cholesterol estimation in serum is a parameter to check lipid profile. This method is also known as the Liebermann-Burchard test. This method was described by Watson. The normal cholesterol level is below 200 mg/dL. A sample is treated with acetic anhydride in acidic condition (glacial acetic acid and concentrated sulfuric acid). Cholesterol reacts with acetic anhydride and forms a green color complex which absorption maxima at 575 nm. The intensity of the color complex is the direct measurement of the amount of cholesterol present in the sample. Cholesterol can be estimated by enzymatic method as well. The sample is treated with cholesterol esterase which hydrolyze cholesterol ester into cholesterol and fatty acids. The free cholesterol oxidized to cholest-4-en-3-one and hydrogen peroxide by the enzyme cholesterol oxidase (CHO). The hydrogen peroxide forms a red color by reacting with phenol and 4-amino antipyrine in the presence of peroxidase. The intensity of the color dye or solution is directly proportional to the amount of cholesterol in the sample. The absorbance can be measured at 505 nm. The level of triglycerides is checked for lipid profile. The normal blood triglyceride level is less than 150 mg/dL. High triglyceride level is associated with heart disease. The sample is treated with the enzyme lipase which hydrolyzes the triglyceride into glycerol and three molecules of fatty acid. Glycerol is phosphorylated to glycerol-3-phosphate by the enzyme glycerol kinase. The glycerol-3-phosphate is oxidized to dihydroxyacetone phosphate by the enzyme glycerol-3-phosphate oxidase (GPO). The by-product hydrogen peroxide forms a red color complex by reacting with 4-amino antipyrine (4-AAP)/4-aminophenazone (chromogenic substrate) and 4-chlorophenol. The absorbance can be measured at 505 nm. Its intensity is directly proportional to the content of triglyceride in the sample. The hemoglobin(Hb) level in the blood can be estimated by Sahli's method. The Sahli's hemoglobinometer consists of a comparator with color standard, Hb tube, Hb pipette, dropper, and stirrer. Hemoglobinis first converted to acid hematin by treating with N/10 HCl. Acid hematin is brown. The color intensity of the solution depends on the concentration of Hb in the blood. The resultant color solution is diluted with water and matched against the color standard on the glass. The reading is in g/dL (g in 100 mL of blood) or g%. The normal range of Hb in the blood is 14–18 g/dL in males and 12–16 g/dL in females. The low level of Hb is termed as anemic condition.