Determination of Lactate Dehydrogenase Activity

In this determination of lactate dehydrogenase activity post we have briefly explained about lactate dehydrogenase activity assay principle, requirements, lactate dehydrogenase activity assay procedure, and calculation.

Lactate Dehydrogenase Activity Assay

A large number of enzymes are known to catalyze reactions in which NAD+ accepts a hydride ion from a reduced substrate or NADH donates a hydride ion to an oxidized substrate.

They are commonly called dehydrogenases e.g., alcohol dehydrogenase, lactate dehydrogenase. The reduced form of nicotinamide adenine dinucleotide (NADH) has an absorption peak at 340 nm whereas the oxidized form (NAD+ ) does not have it.

The progress of an enzyme catalyzed reaction involving nicotinamide adenine dinucleotide coenzyme can easily be monitored by following the rate of appearance or disappearance of NADH by measurement of absorbance at 340 nm. 

Pyruvate + NADH + H+   ↔ lactate + NAD+

lactate dehydrogenase activity assay can be determined spectropotometrically by measuring the increase or decrease in extinction at 440 nm due to the change in levels of NADH.

Lactate dehydrogenase activity assay has a wide variety of clinical uses. It is extensively expressed in body tissues but is especially plentiful in cardiac and skeletal muscle, liver, kidney and red blood cells.

Lactate dehydrogenase is cytoplasmic in its cellular location. It has been widely used to evaluate the presence of damage and toxicity of tissues & cells. An increase in serum lactate dehydrogenase is found in myocardial infarction.

Principle

The lactate is acted upon by lactate dehydrogenase to form pyruvate in the presence of NAD. The pyruvate forms pyruvate phenyl hydrazone with 2, 4 dinitrophenyl hydrazine. The color developed is read in a spectrophotometer at 440nm in lactate dehydrogenase activity assay.

Requirements

Equipment’s

1. Incubator

2. Spectrophotometer

3. Test tube

4. Test tube stand

Chemicals

1. 2, 4 Dinitrophenyl Hydrazine

2. Glycine

3. Hydrochloric acid

4. Lithium Lactate

5. Nicotainamide Adenine Dinucleotide

6. Sodium Chloride

7. Sodium Pyruvate

Reagents

1. Glycine buffer, 0.1M, pH 10: 7.505 g of glycine and 5.85 g of sodium chloride were dissolved in 1 litre of water.

2. Buffered substrate: 125 ml of glycine buffer and 75 ml of 0.1 N NaOH were added to 4 g of lithium lactate and mixed well.

3. Nicotainamide Adenine Dinucleotide: 10 mg of Nicotainamide Adenine Dinucleotide (NAD) was dissolved in 2.0 ml of water.

4. 2, 4 Dinitrophenyl hydrazine: 20 mg of 2, 4 Dinitrophenyl hydrazine (DNPH) was dissolved in 100 ml of 1 N HCl.

5. Standard, 1μmol/ml: 11 mg of sodium pyruvate was dissolved in 100 ml of buffered substrate (1 μmole of pyruvate/ml).

6. NADH solution, 1 μmol/ml: Dissolve 8.5 mg NADH solution, 1 μmol/ml in 10 ml buffered substrate (8.5 mg/10 ml buffered substrate).

7. 0.4 N NaOH: For a 0.4 N solution for lactate dehydrogenase analysis 16.00 g of NaOH per liter is needed.

Procedure

1. Placed 1.0 ml of buffered substrate and 0.1 ml of sample into each of two tubes. Added 0.2 ml of water to the blank.

2. Then to the test added 0.2 ml of NAD. Mixed and incubated at 37ºC for 15 min. Exactly after 15 min, 1.0 ml of dinitrophenyl hydrazine was added to each (test and control). Left for further 15 min.

3. Then added 10 ml of 0.4N sodium hydroxide and the color developed was read immediately at 440 nm.

4. A standard curve with sodium pyruvate solution with the concentration range 0.02 – 0.10μmole was taken at lactate dehydrogenase activity assay.

Result

Lactate dehydrogenase activity assay in serum was expressed as μmoles of pyruvate liberated/L and in tissue homogenate as nmoles of pyruvate liberated/min/mg protein.

Further Readings

Reference