Estimation of serum biochemical parameters by using a semi-autoanalyzer
α ketoglutarate reacts with L- almandine in the presence of ALT to form L- glutamate plus pyruvate. The pyruvate is used in the indicator reaction for a kinetic determination of the reduced form of nicotinamide adenine dinucleotide (NADH) consumption.
The International Federation of Clinical Chemistry (IFCC) has to know recommended standardized procedures for ALT determination, including
1) Optimization of substrate concentrations,
2) the use of tries buffers,
3) Preincubation of a combined buffer and serum solution to allow side reactions with NADH to color,
4) Substrate start (α- ketoglutarate), and
5) Optimal pyridoxal phosphate activation.
As a group, the transaminases catalyze the conversion of amino acids and α-Kato acids by transferring the amino groups. The enzyme ALT has been found to be in the highest concentration in the liver, with decreasing concentrations found in the kidney, heart, skeletal muscle, pancreas, spleen, and lung tissues. Almandine amino transferees measurements are used in the diagnosis and treatment of certain liver diseases (e.g. Viral hepatitis and cirrhosis) and heart disease. Evaluated levels of the transaminases can indicate myocardial infarction, hepatic disease, muscular dystrophy, or organ damage. Serum elevations of ALT activity are rarely observed except in parenchyma liver disease since ALT is a some-more liver- specific enzyme than aspirate amino transfers(AST).
- Reagent 1 (R1) working solution :
(Bottles 1 and 1 a) Tries buffer: 125mmol/1, pH 7.3; L-almandine: 625mmol/1; NADH: 0.23mmol/1(Yeast); LDHS 1.5 U/ml (microorganisms); preservative connect bottle 1to Bottle 1a and dissolve the granule into the buffer.
- Reagent 2 (R2) Working solution:
Ketoglutarate: 94mmol/1; preservative C until the use α-catalogue solution, supplied “ready to use”. Store capped at 2-8 expiration date on the package.
At the reaction pH, the bromcresol purple(BCP) in the Roche Diagnostics (RD) albumin system reagent binds selectivity with albumin. This reaction is based on a modification of a method described y Dumas (4). Although BCP is structurally similar to the conventional bromcresol green (BCG), its pH color change interval is higher (5.2 – 6.8)than the color change interval for BCG (3.8-5.4), thus reducing the number of weak electrostatic dye/protein interactions. The BCP system eliminates many of the nonspecific other serum proteins as a result of the increased pH. In addition, the use of a sample blank eliminates background spectral interferences not completely removed by dichromatic analysis. Albumin constitutes abut 60% of the total serum protein in normal, healthy individuals. Unlike most of the other serum proteins, albumin serves a number of functions which include transporting large insoluble organic (e.g., long0 chain fatty acids and bilirubin), binding toxic metal ions, transporting excess quantities of poor’s soluble hormones (e.g., cortical, aldosterone, and thyroxin), maintaining serum osmotic pressure, and provisioning a reserve store of protein. Albumin measurements are used in the diagnosis and treatment of numerous diseases primarily involving the liver or kidneys.
- Reagent 1(R1) working solution:
Citrate buffer: 95mmol/1, pH 4.1; preservative
- Reagent 2 (R2) working solution:
Citrate buffer: 95mmol/1, pH 4.1; bromcresol green :0.66mmol/1; Use supplied ready to use. Transfer the contents of BCP chromate to an analyzer bottle.
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