“34 BRIEF NO’iX5

Spectrophotometric estimation of serum lactate dehydrogenase with

semicarbazide in the reaction medium

The enzyme lactate dehydrogenase converts lactic acid to pyxvic acid in

presence of the coenzyme ?\‘AD at an alkaline pH, KAD itself being reduced to
KADH in the process. In the spectrophotometric estimation of the cnz!.me, the rate
of accumulation of SADI- determined by measuring the rate of increase of absorbance
at 340 nm is utilized for the enzyme assay.
Xmador 1-8 nl.l emplo!-ed the above reaction for the estin~atj~~n of lactate
dehydrogenasc by using lactic acid (77.5 m&l) a~ the substrate in sodium pyre-
phosphate buffer (0.05 JI; pH 8.8). This method was tested by us with the object
of finding out the limit up to which the linearity between the increase of the absorbanw
and the reaction time is maintained. In the present investigaticxi the reagents used
were: (I) lactic acid (M K- K, England) ; (2) sodium p~rophosphate (Kiedel, Germany) :
(3) NAT> (R.D.H., IZngland) ; (4) senli~arbazide l~~dr~)~hloride (R.T>.H., I’ngland).
.U~sorbancr increase at 340 nm was measured every 30 WC for a total period
of IO min in a EIilger spectrophotometer. The temperature in the wvette was noted.
NXD was not added in the buffered substrate; instead, solutions of NAD made b!.
dissolving 20 mg per ml of distilled water were prepared fresll ever\. da!. and used.
The increase of absorbance at 340 nm was plotted against time to construct the
reaction rate curx’r. This experiment xvas repeated with different ~iamples of serum.
A typical cm-x-e for the ass;a- carried out at 20’ is slwan in l-is. I. It is evident from

0.15r

the curve that linearity between the increase in absorbance and the reaction time
is maintained only up to an increase of absorbance of 0.04, beyond which the curve
starts flattening out. This deviation from linearity. after only a small increase in the