BY DR BRIJESH MUKHERJEE

INTRODUCTIONy LDH is an enzyme (EC

1.1.1.27) ubiquitous in tissue y LDH catalyzes the conversion of pyruvate to lactate in the glycolytic pathway y It has five isoenzymes, each with a different composition

SYNONYMS OF LDHy LD; y Lactate dehydrogenase; y Lactic dehydrogenase; y Total LDH; y LDH isoenzymes y Formal name: y Lactate dehydrogenase, Total and Isoenzymes

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

STRUCTURE OF LDH ISOENZYMEThe multiple form of an enzyme catalyzing the same reaction are called isoenzyme or isozyme They differ in physical, chemical and electrophoretic properties y As discussed LDH is an tetrameric enzyme made up of 4 polypeptide subunits y Two types of subunits namely M(for muscles) and H(for heart) are produced in different genes y M-subunit is basic and H-subunit is acidic in nature y Characteristic feature of LDH isoenzyme are given in next slide

LDH ISOENZYME AND THEIR CHARATERISTICSISOENZYME SUBUNIT ELECTROP HORETIC MOBILITY Fastest Faster Fast Slow Slowest WHETHER DESTROYE D BY HEAT(60 C) No No Partially Yes Yes % OF NORMAL SERUM IN HUMANS 25% 35% 27% 08% 05% LDHI LDH2 LDH3 LDH4 LDH5 H4 H3M H2M2 HM3 M4

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

ISOZYMESy y y y y

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[citationneeded]

y LDH-X(also called LD-C) composed of 4X subunits is

present in post pubertal human testis y Seventh LD called LD-6 identified in severely ill individuals

INHIBITIONy Mercuric ions y P-chloromercuribenzoate y y y y y

(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 LDHLevels 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 y Liver 145U/g y Heart 124U/g y Kidney 106U/g y Skeletal muscle 147U/g y Erythrocyte 36U/g

CLINICAL SIGNIFICANCEIncreased levels can be divided into 2 groups y CARDIAC CAUSES y NON CARDIAC CAUSES CARDIAC y Myocardial infarction(LD-1) y Myocarditis(LD-1) y CCF with hepatic congestion

CLINICAL SIGNIFICANCEy Total LDH will begin to rise 2 to 5 days after an MI;

the elevation can last 10 days. y LDH-1 and LDH-2 LDH isoenzymes - Compare LDH 1 and LDH 2 levels. Normally, the LDH-1 value will be less than the LDH-2. In the acute MI, however, the LDH 2 remains constant, while LDH 1 rises. When the LDH 1 is higher than LDH 2, the LDH is said to be flipped, which is highly suggestive of an MI. A flipped pattern appears 12-24 hours post MI and persists for 48 hours.

CLINICAL SIGNIFICANCENON CARDIAC CAUSES y Severe shock y Anoxia y Haemolysis y Megaloblastic anaemia(50 times elevated) y Liver disease( toxic hepatitis with jaundice, VH, infectious mononucleosis y Renal diseases like tubular necrosis and pyelonephritis y Progressive muscular dystrophy(LD-5) y Pulmonary embolism(LD-3)

CLINICAL SIGNIFICANCE(cont.)a. b. c. d. e. f. g.

Malignant diseases Liver metastasis Hodgkins lymphoma(relevant in predicting the survival rate) Abdominal cancers Lung cancer Teratomas Seminomas of testis(LD-1 is useful in staging and predicting the outcome of disease) Dysgerminoma of ovaries

LDH IN URINE AND CSFy LDH levels are 6 times increased in chronic glomerulonephritis, SLE, diabetic nephrosclerosis, bladder and kidney malignancies y 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 LEVELS IN TRANSUDATE AND EXUDATEy Measuring LDH in fluid aspirated from a pleural

effusion (or pericardial effusion) can help in the distinction between 1. Exudates (actively secreted fluid, e.g. due to inflammation) 2. Transudates (passively secreted fluid, due to a high hydrostatic pressure or a low oncotic pressure). y The usual criterion is that a ratio of fluid LDH versus upper limit of normal serum LDH of more than 0.6 or indicates an exudate, while a ratio of less indicates a transudate. y Different laboratories have different values for the upper limit of serum LDH, but examples include 200 and 300 IU/L. y In empyema, the LDH levels, in general, will exceed 1000 IU/L.

PHYSIOLOGICAL RISE IN LDHy Strenuous exercise can cause temporary elevations in

LDH. y Hemolysis of the blood specimen can cause falsely elevated results. This may happen if the specimen is handled roughly, stored in extreme temperatures, or if the sample was difficult to collect. y If your platelet count is increased, serum LDH will be artificially high and not reflective of the LDH actually present.

DRUGS AFFECTING LDH LEVELSy Ascorbic acid (low level of LDH) y Anesthetics y Aspirin y Narcotics y Procainamide y Alcohol

LDH ASSAYy Hemolyzed serum should not be used as erythrocyte y

y y y y y y

contain 150 times more activity than serum 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 25C 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 A/min. in Tris buffer and keep cold.

PROCEDUREy Set spectrophotometer at

340 nm and 25C. y Pipette into cuvette as follows:Tris HCl, 0.2 M pH 7.3 6.6 mM NADH

2.8 ml

0.1 ml

30 mM Sodium pyruvate

0.1 ml

y Incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration and establish a blank rate, if any. y Add 0.1 ml of appropriately diluted enzyme and record A340/min from initial linear portion

y CALCULATIONy Units/mg =

A 340/min 6.22xmg enzyme/ml reaction mixture

NORMAL LAB VALUESy 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. y Normal Adult Range: 0 - 250 U/L Optimal Adult Reading: 125

ELECTROPHORESIS OF ISOZYMES OF LDHy 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

y Lactate + NAD+ -------------- Pyruvate + NADH + H+

y In order to detect the LDH isozyme in an agarose gel after electrophoresis, the above enzymatic reaction is coupled to a color producing reaction: y 1) Lactate + NAD+ --------Pyruvate + NADH + H+ y 2) NADH + PMS -------NAD+ + PMS-H (PMS - Phenazine methosulfate) y 3) PMS-H + TNBT ----------PMS + TNBT-Formazan. (TNBT- Tetranitroblue tetrazolium) y 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.

y Prepare a 1.2% agarose gel in 50 mM Tris HCl buffer pH 8.2. Load 20 l 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 y Turn off electricity, and place the gel into the developing chamber which already contains the developer solution ( H2O 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 oC to develop color reaction for 30 minutes.

Data Analysis y 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. y The gels can be stored in standard destain solution containing 10% methanol and 5% acetic acid.

GRAPH OF LDH (ELECTROPHORESIS)

ELECTROPHORESIS OF LDH

CARDIAC MARKERSy Cardiac markers are biomarkers measured to

evaluate heart function. y They are often discussed in the context of myocardial infarction, but other conditions can lead to an elevation in cardiac marker level. y Most of the early markers identified were enzymes, and as a result, the term "cardiac enzymes" is sometimes used y Depending on the marker, it can take between 2 to 24 hours for the level to increase in the blood.

Test

Sensitivity and specificity The most sensitive and specific test for myocardial damage. Because it has increased specificity compared with CK-MB, troponin is a superior marker for myocardial injury.

Approximate peak

Troponin test

12 hours

Creatine Kinase (CKMB) test Lactate dehydrogen ase(LDH) Myoglobin( Mb)

It is relatively specific when skeletal muscle damage 10 24 hours is not present.

LDH is not as specific as troponin.

72 hours

low specificity for myocardial infarction

2 hours

Ischemiamodified low specificity albumin (IM A)

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