advanced clinical chemistry for medical lab2

chemistry

1. LD
2.5. Lactate Dehydrogenase
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2. 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.
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3. 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.
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4. 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
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5. 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
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6. Sources of LD
All cells
Heart
Liver
Kidney
Erythrocytes
Skeletal Muscle
Brain
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7. Clinical Significance of LD
Myocardial infarction
Liver disease (hepatic inflammation)
Hemolytic conditions
Malignancies
Skeletal muscle disease
Renal disease
Pulmonary embolisms
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8. 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
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9. 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
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10. 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.
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11. Specimen for LD
Nonhemolyzed serum or plasma.
Heparinized plasma
< 2 day old samples
Stored at room temperature
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12. 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.
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13. 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.
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14. 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
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15. Documentation of LD
Enzyme Analysis
Record patient results in result logbook
Record QC results in QC logbook
Retain records for recommended time
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16. Introduction to Lactate
Dehydrogenase Isoenzymes
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17. 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
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18. 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
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19. 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.
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20. 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.
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21. Method of Separation of LD
Isoenzymes: Electrophoresis
Densitometric Scan of Normal Serum
Note the anode view is on the right side.
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22. 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.
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23. Interpretation of LD Isoenzyme
Electrophoresis Results
Line A: myocardial
infarction
Line B: normal
Line C: liver disease
1 = LD1 etc.
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24. Other Methods for Isoenzyme of LD
Selective Chemical Inhibition
Ion exchange chromatography
Immunoprecipitation
Selective Substrate to measure 2 hydrobutryase activity
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25. 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.
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26. Specimen for LD isoenzymes
Nonhemolyzed serum or plasma.
Heparinized plasma
< 2 day old samples
Stored at room temperature
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27. 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.
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28. 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
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29. Documentation of LD Isoenzyme
Enzyme Analysis
Record patient results in result logbook
Record QC results in QC logbook
Retain records for recommended time
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30. 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
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31. Reference
1. Bishop ML, Fody EP, Schoeff LE. Clinical Chemistry:
Principles, Procedures, Correlations. 6th edition.
2. Burtis C, Ashwood E, Bruns D. Tietz Textbook of Clinical
Chemistry and Molecular Diagnostics. 7th edition,
3. Arneson W and Brickell J .Clinical Chemistry: A Laboratory
Perspective. 6thedition. Jan 25, 2007
4. Bekele T. Clinical chemistry lecture note for medical
laboratory technology
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