Johnny Silva Cancer Research

Ldh Test Methodology

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all cytoplasmic membranes of all cells of the body, especially in osteoblasts (bone-forming cells), intestinal mucosa, placenta, and renal tubules. Table 7-3 summarizes tissue origins of key liver enzymes. Methods of analysis for transaminases are presented in Test Methodology 7-4, and for GGT in Test Methodology 7-5.

TEST METHODOLOGY 7-4. TRANSAMINASES1 Aspartate Transaminase

The Reaction

Analysis of aspartate transaminase (AST) can be achieved by a coupled reaction involving pyridoxal-5'-phosphate (P-5'-P) and malate dehydrogenase (MDH) at 37°C:

Aspartate + alpha-oxoglutarate AST P-5 -P ) oxaloacetate + glutamate Oxaloacetate + NADH + H+ MDH ) malate + NAD+

Decrease in absorbance at 340 nm is determined by continuous monitoring.

The Calculation

The AST level (U/L) is calculated using the following formula: AA/min X 1 X total volume (mL)

If 3.0 mL total volume and 0.2 mL sample volume are used, the activity factor is 2381.

The Specimen

Serum or heparinized plasma free from hemolysis may be used. Interferences are minimal since the sample is measured at 340 nm, but preanalytical variation can occur due to hemolysis (erythrocyte source of AST), and there may be slight variation due to gender and fasting state. Alcohol and drugs such as opiates, salicylates, or ampicillin may increase AST activity. If temperature is not maintained at a constant 37°C, the rate of the product formation will be inaccurate.

Reference Ranges

Adult male <35 U/L Adult female <31 U/L

0.0063 ^mol X sample volume (mL)

For example: AA/min X 2381 = 0.020 X 2381 = 48D U/L

TEST METHODOLOGY 7-4. TRANSAMINASES1 (continued)

Alanine Transaminase

The Reaction

Analysis of alanine transaminase (ALT) can be achieved by this coupled reaction involving pyridoxal-5'-phosphate (P-5'-P) and lactate dehydrogenase (LDH) at 37°C:

Alanine + alpha-oxoglutarate ALT. P-5'-P ) pyruvate + glutamate Pyruvate + NADH + H+-LD^. lactate + NAD+

Decrease in absorbance at 340 nm is determined by continuous monitoring.

The Calculations

The ALT level (U/L) is calculated using the following formula: AA/min X 1 X total volume (mL) 0.0063 ^mol X sample volume (mL) If 3.0 mL total volume and 0.2 mL sample volume are used, the activity factor is 2381.

For example: AA/min X 2381 = 0.020 X 2381 = 48D U/L The Specimen

Serum or heparinized plasma free from hemolysis may be used. Interferences are minimal since the sample is measured at 340 nm, but preanalytical variation may occur due to moderate hemolysis (erythrocyte source of ALT), loss of stability over time, and slight variation due to gender. Certain drugs and alcohol may also increase ALT activity. Maintaining substrate concentration in excess, as well as stable temperature, are also critical for accurate enzyme analysis. Stability of the enzyme activity can be maintained by refrigeration of the sample for up to 3 days and freezing the sample for up to 30 days.

Reference Ranges

Adult male <45 U/L

Adult female <34 U/L

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