LACTATE DEHYDROGENASE

L-lactate: NAD Oxidoreductase

Other name of LDH

1.1.1.27

E.C of LDH

oxidoreductase

Belonging to the class ?

hydrogen acceptor or as coenzyme

Hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate with the mediation of NAD as ?

LDH

the reaction is reversible and strongly favors as the reduction of pyruvate to lactate

NAD+

Uses ? as cofactor

Embden-Meyerhof pathway

In the body, pyruvate is formed as end product of the ? which converts glucose to energy

heart and liver

RBCs

Skeletal muscle and kidney

WBCs

TISSUE DISTRIBUTION

High concentrations are found in the ?

Significant amounts are present in ?

? also contain considerable concentration

Also found in ?

lungs

smooth muscles

brain

Lesser amount in:

100-150x

Hemolysis produces an artefactual increase in serum levels; ? greater activity in RBC than serum

Serum

What is the sample choice?

Hemolysis

? must be avoided

Freezing

? may distort the isoenzymes distribution markedly

CSF and urine

Other body fluids as sample:

Activators

SH-

Inhibitors

Mercuric chloride

N-ethymaleimide (NEM)

Chloromercuribenzoate

Borate or oxolate

Oxomate

Urea

Sulfite

Iodate, EDTA

NADH

Most determination are based on the detection of ? in the reaction.

direct spectrophotometric measurement

NADH has strong absorbance at 340 nm which permits ?

redox indicator

NADH can be coupled with a ?

p-nitrophenylhydrazine

One other approach utilizes ? to form a color complex.

This method is imprecise and is not employed by many laboratory

Forward reaction

Reverse reaction

METHOD OF ESTIMATION

methylethylketone (MEK)

In reverse reaction of fluorometry; NAD is measured by its fluorescence following the addition of ?

Colorimetry in reverse reaction

Pyruvate depletion is monitored by 2,4 dinitrophenylhydrazone

European System and American System

What are the methods/system used in measuring the isoenzymes?

Heart and Muscle

5

LD is basically made of 4 polypeptides comprised of two types of polypeptide chains designated as ?

This combined in ? arrangements to yield ? major isoenzyme fractions.

European System

based on how fast isoenzyme moves towards an anode

LD1

being the fastest moving

LD5

? being the slowest

American System

Numbers the isoenzymes in exactly the reverse direction so that LD1 of the American System is LD5 of the European system.

Enumerate and memorize the two tables under the American System

nonspecific finding

Because of many conditions that contribute to increase activity, an elevated total LD value is rather ?

separated into isoenzyme fractions

LD assays assume more clinical significance when ?

Electrophoresis

Column Chromatography

Immunoinhibition

Enumerate the assay used in studying isoenzymes:

Fluorometrically or colorimetrically

After electric separation, the isoenzyme can be detected either ?

Wacker Method (colorimetric)

Solution of buffer, lactate, NAD is gently rolled onto the surface of the film

Incubated at 30-37°C for 30 mins or more

Allows ID and quantitation of each of the 5 fractions through time consuming

Provides more useful information than any other methods for separation and quantitation of LD isoenzymes

Column Chromatography

Workers developed a system using a column that could isolate LD1 and LD2 along with CK-MB fraction

ion exchange column

Sample is applied to an ?

Tris buffer

Unwanted fractions are eluted with ? (containing a low concentration of NaCl)

increasing the amount of NaCl in the buffer

the desired CK and LD fractions are removed from the column by ?

Column Chromatography

Disadvantages:

Sample volume required is quite large with that for electrophoresis

Useful information is lost regarding damage to other tissues

Difficult to process a large number of samples efficiently

Immunoinhibition

Useful in detection of LD1

Antibody to M subunit of LD + sample

Any isoenzyme containing an M subunit binds Ab it rendering the CHON inactive

Since only LD1 does not contain the M subunit, it will be the only one showing activity when assayed for total LD

relative % of LD1

By comparing total activity, with or without antibody present in the sample, the ? can be determined

Immunoinhibition

Procedures are reasonably quick to perform and cost is in the same range as electrophoresis

Some data suggest that LD isoenzyme changes associated with MI measuring the relative amount of LD1 and LD2 by electrophoresis

Megaloblastic anemia

Widespread carcinomatosis, esp. hepatic metastases

Septic shock and hypoxia

Hepatitis

Renal infarction

Thrombotic Thrombocytopenic purpura

Enumerate the pronounced elevation (5 or more times Normal)

MI

Pulmonary infarction

Hemolytic conditions

Leukemia

IM

Delerium tremens

Muscular dystrophy

Enumerate the moderate elevation (3-5 times normal)

Most liver diseases

nephrotic syndrome

Hypothryoidism

Cholangitis

Enumerate slight elevation (up to 3 times normal)

100 - 150 x

approximately False High

Hemolysis

Erythrocytes contain an LDH concentration ? that found in serum ?

25°C and analyze 48 hours

If the sample cannot be analyzed immediately, it should be stored at ?

LDH 5

4°C than at 25°C

? is the most labile (loss of activity at ? )

Approximately 35-90 U/L

95-200 U/L

Reference Ranges:

Forward Reaction: ?

Reverse Reaction: ?