2.5.

Lactate Dehydrogenase

LD
Learning Objectives

After listening to the lectures and completing the

exercises, the student will be able to:
Describe the biochemical theory & metabolic pathways,
and physicochemical properties of Lactate
dehydrogenase (LD)
Discuss the normal & abnormal states affecting levels
of LD and LD isoenzymes
Describe the principles of LD and LD isoenzyme
analysis in terms of key reagents and their role.
Learning Objectives
After listening to the lectures and completing the exercises,
the student will be able to:
 Differentiate causes of common preanalytical, analytical and
postanalytical errors in LD and LD isoenzyme analysis.
 Interpret results of LD and LD isoenzymes compared to
reference ranges.
Outline of Lecture: LD and LD
Isoenzymes
 Introduction
 Source
 Clinical Significance
 Methods of Analysis
 Specimen
 Interpretation of Results
 Quality Control
 Sources of Errors
 Reporting and Documentation
 Summary
Introduction to Lactate Dehydrogenase
L-lactate: NAD+ oxidoreductase, LD
Oxidase
(pH 8.8-9.8)
L-Lactate + NAD+ → Pyruvate + NADH + H+
( pH 7.4-7.8)

Enzyme specificity includes other  hydroxy acids

LD is inhibited by mercuric ions
Sources of LD
All cells
Heart
Liver
Kidney
Erythrocytes
Skeletal Muscle
Brain
Clinical Significance of LD
Myocardial infarction
Liver disease (hepatic inflammation)
Hemolytic conditions
Malignancies
Skeletal muscle disease
Renal disease
Pulmonary embolisms
Clinical Significance of LD
Time Frame of LD following Acute Myocardial
Infarction (AMI)
 Elevated 2-3 days after AMI
 Returns to normal by 7-10 days after AMI
Analytical Methodology of Total
Lactate Dehydrogenase Activity
Reverse method (P L)
NADH + H+ + Pyruvate -- LDH  Lactate + NAD+
Absorbance of NAD can be measured with photometer at
340 nm
The molar absorptivity (epsilon) of NAD at 340 nm is
6220 cm. L/ mole
Analytical Methodology of Total Lactate
Dehydrogenase Activity
End-point colorimetric method
Test principle: Pyruvate released by LDH is reacted
with 2, 4-dinitrophenyl hydrazine to form the
corresponding golden-brown colored hydrazone at an
alkaline pH. The intensity of the color is proportional
to enzyme activity and is measured at 410 nm.
Specimen for LD
Nonhemolyzed serum or plasma.
Heparinized plasma
< 2 day old samples
Stored at room temperature
Reference Ranges and Interpretation of LD
Results
Age Specific Reference Ranges
Dependent on methods
Serum for Adult: 100-190 U/L
CSF for Adult: 10% of serum value

Compare patient result to reference range to assess for

cardiac, liver, skeletal muscle or other diseases.
Quality Control
A normal & abnormal quality control sample should
be analyzed along with patient samples, using
Westgard or other quality control rules for acceptance
or rejection of the analytical run.
Assayed known samples
Commercially manufactured (Humastar)

Validate patient results

 Detects analytical errors.
Sources of Error in LD
Nonlinear results from side reactions can be repeated
with diluted sample
Hemolyzed samples cause false positive
Loss of activity if frozen or stored more than 3 days at
room temperature
Use of anticoagulant is source of error
Documentation of LD
Enzyme Analysis
Record patient results in result logbook
Record QC results in QC logbook
Retain records for recommended time
Introduction to Lactate Dehydrogenase
Isoenzymes
Isoenzymes
Multiple forms of one type of enzyme
React with the same substrate
Composed of slightly different polypeptide chains
Have some unique characteristics such as temperature
inactivation or clinical significance
LD1 or HHHH
LD 5 or MMMM
5 LD Isoenzymes

HHHH, LD1: cardiac muscle, erythrocytes, brain, and

renal cortex
HHHM LD2: cardiac muscle, erythrocytes, brain and
renal cortex
HHMM LD3: lung, spleen and the platelets
HMMM LD4: liver and skeletal muscle
MMMM LD5: liver and skeletal muscle
Clinical Significance of LD Isoenzymes
LDH-1 and LDH-2 :cardiac muscle, erythrocytes, and
renal cortex
LDH-3: lung, spleen and the platelets
LDH-4 and LDH-5: liver and skeletal muscle.

Diseases affecting these organs and tissues will cause

elevation of individual isoenzyme % compared to
reference ranges.
Method of Separation of LD Isoenzymes:
Electrophoresis
pH 8.0 buffer
migrated with electrical current
agarose or cellulose membrane.
d,l- lactate + NAD + Substrate is placed on separated
fractions, incubated at 37C to develop colored
formazen bands.
Method of Separation of LD
Isoenzymes: Electrophoresis
Densitometric Scan of Normal Serum
Note the anode view is on the right side.
Calculation of Results of LD
isoenzyme electrophoresis
Densitometer is used to determine isoenzyme %
% OD increases with larger, darker bands.
Total % must add up to 100%

The electrophoretic pattern is also significant.

Interpretation of LD Isoenzyme
Electrophoresis Results

Line A: myocardial
infarction
Line B: normal
Line C: liver disease
1 = LD1 etc.
Other Methods for Isoenzyme of LD
Selective Chemical Inhibition
Ion exchange chromatography
Immunoprecipitation
Selective Substrate to measure 2 hydrobutryase
activity
Reference Ranges and Interpretation of LD
Isoenzyme Results
LD1: 14- 26%
LD2: 29-39%
LD3: 22-26%
LD4: 8-16%
LD5: 6-16%
Compare patient results to reference ranges to indicate
if diseases of heart, liver or others may be present.
Isoenzyme patterns provide additional information.
Specimen for LD isoenzymes
Nonhemolyzed serum or plasma.
Heparinized plasma
< 2 day old samples
Stored at room temperature
Quality Control
A normal & abnormal quality control sample should
be analyzed along with patient samples, using
Westgard or other quality control rules for acceptance
or rejection of the analytical run.
Assayed known samples
Commercially manufactured

Validate patient results

 Detects analytical errors.
Sources of Error in LD Isoenzymes
Hemolyzed samples cause false positive
LD 1 and LD 2
Loss of activity if frozen or stored more than 3 days at
room temperature
LD 4 and LD5
Use of anticoagulant is source of error
Documentation of LD Isoenzyme
Enzyme Analysis

Record patient results in result logbook

Record QC results in QC logbook
Retain records for recommended time
Summary LD and LD Isoenzymes
 Discussion of source and clinical Significance of LD and
LD isoenzymes
 Description of methods of analysis, specimen,
interpretation of results, QC, sources of error and reporting
and documentation procedures for LD and LD isoenzymes
References
 Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. Philadelphia, 2001
 Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis
 Bekele T., Lecture notes on clinical chemistryII, Carter
centre.