Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

LactateþNADþ ⇄

LDH

pyruvateþNADHþHþ

32.4 Reagents........................................

1. Buffer substrate solution (consisting of 50 mM phosphate buffer, pH 7.5 and
   0.6 mM pyruvate, and 0.095% sodium azide)


2. NADH 2 : 0.18 mmol/L

32.5 Procedure

Take 2 ml of NADH solution and add 50μl of serum sample. Incubate for 2 min, and
then add 2 ml buffer substrate. Measure the decrease in absorbance at 340 nm at
1 min interval for 3 min. If absorbance change exceeds 0.1/min, dilute sample
accordingly and include dilution factor in calculation.

32.6 Calculation

Lactate dehydrogenase activity/liter of serum

¼

ΔOD=mintotal volume in cuvette 1000

ðÞmolar extinction coefficient volume of serum used mlðÞ

¼U=L

Molar absorption coefficient of NADH at 340 nm¼6.22 103  10 ^6 /μmole/
cm.

32.7 Clinical Significance...............................

Normal total LDH activity is 100–190 U/L at 37C. The isoenzymes of LDH have
diagnostic value in heart and liver diseases. In a healthy individual, LDH2 value is
higher than LDH1 in serum. In myocardial infarction, the peak levels of LDH1
activity is much higher than LDH2 (although both LDH1 and LDH2 may be
elevated, but LDH1 is more elevated), and that occurs within 12–24 h after onset
of infarction and elevated levels persist for 10–14 days. LDH5 levels increase
predominantly in muscular dystrophy, while in liver infarction or liver malignancy
both LDH4 and LDH5 are increased. It should be noted that LDH activity is 80– 100
times more in red blood cells (RBC) than in serum. Hence for estimation of enzyme
activity, serum should be free of hemolysis.

130 32 Determination of Total Lactate Dehydrogenase Activity in Serum Sample