Separation of five isoenzymes of serum lactate dehydrogenase by discontinuous gradient elution from a miniature ion-exchange column.

We describe a simple, fast chromatographic technique for quantitatively separating the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) in serum. A 250-microL serum sample is applied to a 6.0 x 0.7 cm column of QAE-Sephadex A-50 and eluted stepwise with five different buffers. The isoenzyme fractions, assayed by the method of Wroblewski and LaDue [Proc. Soc. Exp. Biol, Med. 90, 210 (1955)], are stable at room temperature for 24 h. For all five isoenzymes the average within-day coefficient of variation is 4.3%; the day-to-day CV for 20 days is 6.3%. Of some common potentially interfering substances tested, only sodium fluoride (238 mmol/L) was found to do so, by slowing the elution from the column and making the fractions turbid. The expected range in international (IUB) united and percent for each isoenzyme was determined from data on 73 men and 70 women. From these data we calculate, by a percentile estimate of a nongaussian distribution, a normal range of LD-1/LD-2 ratio of 0.55--0.87 for men and 0.52--0.91 for women. The LD isoenzyme patterns in both normal and above-normal samples of 147 sera, as evaluated by the present column method and by electrophoresis, correlated well.