Lactate Dehydrogenase

LACTATE DEHYDOGENASE: 

LACTATE DEHYDOGENASE BY DR BRIJESH MUKHERJEE

INTRODUCTION: 

INTRODUCTION Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD + . It converts pyruvate , the final product of glycolysis to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver. At high concentrations of lactate, the enzyme exhibits feedback inhibition and the rate of conversion of pyruvate to lactate is decreased.

BIOCHEMISTRY: 

BIOCHEMISTRY Functional lactate dehydrogenase are homo or hetero tetramers composed of M and H protein subunits encoded by the LDHA & LDHB genes respectively Structure of LD-M & LD-H are determined by loci on human chromosome 11 & 12 respectively

The enzyme has a molecular weight of 134,000. PL optimum pH 8.8-9.8, Temp.- 37°C LP optimum pH 7.4-7.8, Temp.- 37°C: 

The enzyme has a molecular weight of 134,000. P L optimum pH 8.8-9.8, Temp.- 37°C LP optimum pH 7.4-7.8, Temp.- 37°C

ISOZYMES: 

ISOZYMES LDH-1 (4H) - in the heart and RBCs LDH-2 (3H1M) - in the reticuloendothelial system LDH-3 (2H2M) - in the lungs LDH-4 (1H3M) - in the kidneys, placenta and pancreas LDH-5 (4M) - in the liver and striated muscle [ citation needed ] LDH-X(also called LD-C) composed of 4X subunits is present in post pubertal human testis Seventh LD called LD-6 identified in severely ill individuals

INHIBITION: 

INHIBITION Mercuric ions P- chloromercuribenzoate (can be reversed by cysteine / or glutathione) Borate Oxalate Oxamate - competes with pyruvate for binding sites Excess of pyruvate and lactate EDTA inhibits the enzyme, perhaps by binding Zn ++

LEVELS OF LDH: 

LEVELS OF LDH Levels are higher in tissues than in serum LD-1, LD-2 are predominant in cardiac muscles, kidneys, erythrocytes LD-4, LD-5 are predominant in skeletal muscles, liver Liver – 145U/g Heart – 124U/g Kidney – 106U/g Skeletal muscle – 147U/g Erythrocyte – 36U/g

CLINICAL SIGNIFICANCE: 

CLINICAL SIGNIFICANCE Elevated levels are found in – Myocardial infarction(LD-1) Myocarditis (LD-1) CCF with hepatic congestion Severe shock Anoxia Haemolysis Megaloblastic anaemia (50 times elevated) Liver disease( toxic hepatitis with jaundice, VH, infectious mononucleosis

CLINICAL SIGNIFICANCE(cont.): 

CLINICAL SIGNIFICANCE(cont.) Elevated levels are also seen in: Renal diseases like tubular necrosis and pyelonephritis Malignant diseases Liver metastasis Hodgkins lymphoma Abdominal cancers Lung cancer Teratomas Seminomas of testis Dysgerminoma of ovaries Progressive muscular dystrophy(LD-5) Pulmonary embolism(LD-3)

LDH IN URINE AND CSF: 

LDH IN URINE AND CSF LDH levels are 6 times increased in chronic glomerulonephritis, SLE, diabetic nephrosclerosis, bladder and kidney malignancies LD-4 and LD-5 are undetectable in normal CSF. They are detectable in patients with bacterial meningitis. High level signifies encephalitis and poor prognosis

LDH ASSAY: 

LDH ASSAY Method : The reaction velocity is determined by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit causes the oxidation of one micromole of NADH per minute at 25°C and pH 7.3, under the specified conditions. Reagents 0.2 M Tris⋅HCl , pH 7.3 6.6 mM NADH in above 0.2 M Tris⋅HCl buffer, pH 7.3 30 mM Sodium pyruvate in above 0.2 Tris⋅HCl buffer, pH 7.3 Enzyme Dissolve at 1 mg/ml in 0.2 M Tris⋅HCl buffer. Dilute enzyme prior to use to obtain a rate of 0.02-0.04 ΔA/min. in Tris buffer and keep cold.

PROCEDURE: 

PROCEDURE Set spectrophotometer at 340 nm and 25°C. Pipette into cuvette as follows: Tris ⋅ HCl , 0.2 M pH 7. 3 2.8 ml 6.6 mM NADH 0.1 ml 30 mM Sodium pyruvate 0.1 ml

PowerPoint Presentation: 

Incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record ΔA 340 /min from initial linear portion CALCULATION Units/mg = Δ A 340/min 6.22xmg enzyme/ml reaction mixture

NORMAL LAB VALUES: 

NORMAL LAB VALUES LDH (Lactic Acid Dehydrogenase ) - Increases are usually found in cellular death and/or leakage from the cell or in some cases it can be useful in confirming myocardial or pulmonary infarction (only in relation to other tests). Decreased levels of the enzyme may be seen in cases of malnutrition, hypoglycemia, adrenal exhaustion or low tissue or organ activity. Normal Adult Range: 0 - 250 U/L Optimal Adult Reading: 125

ELECTROPHORESIS OF ISOZYMES OF LDH: 

ELECTROPHORESIS OF ISOZYMES OF LDH Tissue specific differences in LDH isoenzymes can be readily detected by the localization of LDH activity in an agarose gel after electrophoresis by an activity staining process where the product of the enzymic reaction is a water insoluble stain precipitating in the gel where the LDH proteins are located. Each of the LDH isozymes can catalyze the following reaction: LDH Lactate + NAD + --------------  Pyruvate + NADH + H+

PowerPoint Presentation: 

In order to detect the LDH isozyme in an agarose gel after electrophoresis, the above enzymatic reaction is coupled to a color producing reaction: 1) Lactate + NAD + ---------  Pyruvate + NADH + H+ 2) NADH + PMS --------  NAD+ + PMS-H (PMS - Phenazine methosulfate ) 3) PMS-H + TNBT -----------  PMS + TNBT- Formazan . (TNBT- Tetranitroblue tetrazolium ) The highly colored TNBT- Formazan product localizes in the electrophoretic zones of LDH activity and the amount of brown color formed is quantitatively related to the level of LDH isoenzyme present.

PowerPoint Presentation: 

Prepare a 1.2% agarose gel in 50 mM Tris–HCl buffer pH 8.2. Load 20 µl from serum specimens into different slots gel. Use bromophenol blue as a tracking dye Carefully cover the samples with tank buffer. Pour tank buffer into the reservoires and connect the electric cables. Electrophorese at 170 V until the bromophenol blue has migrated to within 1 mm of the positive electrode end of the gel. Detection of LDH Isoenzymes Turn off electricity, and place the gel into the developing chamber which already contains the developer solution ( H 2 O 18.4 ml, 1 M Tris 4 ml, tetrazolium-blue 12 ml, phenazine-methosulphate 4 ml, Na-lactate 4 ml and NAD 1.3 ml). Incubate at 37 o C to develop color reaction for 30 minutes.

PowerPoint Presentation: 

Data Analysis Rinse the gels with water and examine them on a light box. Locate the bands containing LDH isozymes . The amount of brown color is roughly proportional to the amount of an LDH isoenzyme present in a band. The gels can be stored in standard destain solution containing 10% methanol and 5% acetic acid.