The document discusses the lactate dehydrogenase (LDH) blood test used for heart attack victims. LDH is an enzyme found in most body cells that is released when cells are damaged or die. An LDH blood test can detect elevated levels after a heart attack, indicating heart muscle cell death. The test is most useful within 12-24 hours of a heart attack. Higher-than-normal total LDH combined with higher LDH-1 than LDH-2 isoenzymes suggests a heart attack. The LDH level is measured using a UV-Vis spectrophotometer to detect the decrease in absorbance of NADH at 340nm as LDH converts pyruvate to lactate.

1. PRESENTER: DUNG TRONG TRAN
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LACTATE DEHYDROGENASE (LDH)
BLOOD TEST OF HEART ATTACK VICTIMS

2. Contents
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 What it is
 When it’s used
 How it’s used

3. What it is
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 Lactate dehydrogenase (LDH) is an enzyme
catalysing the reaction:
 LDH is in almost all of the body's cells and is
released into the blood when cells are damaged
or destroyed
 the blood level of LDH is a general indicator
of tissue and cellular damage
LDH +
Pyruvate+NADH Lactate+NAD

4. What it is
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 LDH exists in five forms, or isoenzymes.
 Each isoenzyme has a slightly different structure
and is found in different tissues.
 The results of each LDH isoenzyme
concentration indicates which tissue may be
damaged or injured.

5. Heart attack
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 Heart muscle cells die
 Release LDH into the bloodstream

6. When it’s done
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 The assay is used within 12-24 hours after the
infarction.
 The increase in enzyme activity tends to be
proportional to the extent of the myocardial lesion
 If
Total LDH increases
LDH-1 > LDH-2 (normally LDH1<LDH2)
 Heart attack
 Rarely used now

7. How it’s used
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 Enzyme activity = moles of substrate converted per
unit time = rate × reaction volume
 1 enzyme unit (U) = 1 μmol/min, where μmol refers to
the amount of substrate converted
 The LDH activity in serum is measured by the rate of
NADH disappearance
LDH +
Pyruvate+NADH Lactate+NAD

8. How it’s used
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 Using UV-Vis spectrometer to measure enzyme
activity
o Easy handling.
o Low susceptibility against disturbances.
o Cover UV range, where NADH shows absorption.

9. NADH/NAD+ Absorbance
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 The rate of NADH disappearance can be
measured as an decrease in absorbance at 340nm

10. How it’s used
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 Reagents:
 NADH solution
 Sodium Pyruvate solution
 Buffer solution: pH ~ 8.0
 Sample:
 Centrifuge the blood samples
 Serum can be separated and assayed directly

11. Procedures
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1. Determining e of NADH
A = C x e
where
C is the concentration of NADH in moles/L
e is extinction coefficient
 Obtain e from calibration curves of A vs known C
 e = 6220

12. Procedures
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2. Measuring blood LDH:
i. Set up the LDH reaction using the mixture
without an enzyme as a blank
ii. Put the full mixture in the spectrophotometer
and read the absorptions

13. Procedures
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iii. Plot of the absorption vs time of the LDH
reaction. The slope we got for our graph
was ΔA/min

14. Calculations
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1. The change in concentration of NADH was
calculated by using the equation :
∆CNADH/min = ∆A/min/e = slope / 6220 (εNADH)
2. Concentration of enzyme in unit (U):
∆CNADH/min x V(L) x 106 (μmole/mole)= μmol/min= U

15. Summary
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 Lactate dehydrogenase assay is often used as a
diagnostic tool in monitoring patients after heart
attack
 UV-Vis photometric assay is applied
 Temperature and pH must be maintain in the
assay

16. References
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 http://www.labtestsonline.org.uk/understanding/an
alytes/ldh/
 http://www.biopacific.net/pointe-package-
inserts/Lactate_dehydrogenase.pdf
 http://www.biotek.com/resources/docs/NADH_Ap
p_Note.pdf
 Jack E.Dixon (1975), “Determining blood enzyme
concentrations: A Unified laboratory experiment”,
Biochemical education, 3(2), pp.31-33.
 Hans Bisswanger (2014), “Enzyme assays”,
Perspectives in Science, Volume 1, pp.41-55.

17. Thanks for listening
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