Automated kinetic determination of lactate dehydrogenase isoenzymes in serum.

A steady-state kinetic method has been revised for measuring lactate dehydrogenase isoenzyme activities, which relates the inhibition of heart-type isoenzyme activity to the overall isoenzyme composition of the enzyme subunits. The method depends on the pH-dependent formation of an inhibitory ternary complex by the heart-type isoenzyme with NAD+ and pyruvate (if the reaction is measured by NADH oxidation). A preincubation step in the previous method is eliminated. The isoenzymes are measured by measuring the reduction of pyruvate in two different concentrations, which favor either the total or fractional activity, depending on the concentrations of pyruvate and the percentage of heart-type subunits. The method has been adapted to a centrifugal analyzer, which has speeded automated isoenzyme determinations, with an accuracy comparable to that for electrophoretic methods.     

High-pressure liquid-chromatographic separation of lactate dehydrogenase isoenzymes.

The isoenzymes of lactate dehydrogenase (EC 1.1.1.27) were separated with excellent resolution in less than 1 h by high-pressure anion-exchange chromatography. These isoenzymes were determined in some tissue extracts and high-activity serum samples by this technique.     

Automated separation and measurement of lactate dehydrogenase isoenzymes.

We describe a totally automated, computer-controlled system for separating and measuring the activity of lactate dehydrogenase (EC 1.1.1.27) isoenzymes. These isoenzymes in tissue extracts were separated on disposable DEAE-cellulose mini-columns. Resolution was complete except that LD-1 was not resolved from LD-2.     

A transient variant of serum lactate dehydrogenase isoenzymes.

An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.     

An appraisal of routine methods for the determination of the anodal isoenzymes of lactate dehydrogenase

The relative value of four tests currently in common use for assaying the lactate dehydrogenase (LDH) isoenzymes liberated following myocardial infarction hs been assessed. Kinetic spectrophotometric methods, using an assay temperature of 37° have been employed for the determination of total LDH, hydroxybutyrate dehydrogenase (HBD), heat-stable LDH and urea-stable LDH activity in the serum of patients with myocardial infarction and with liver disease. Heat-stable LDH was the most sensitive of the four tests for the diagnosis of myocardial infarction, although urea-stable LDH was only slightly less sensitive. Heat-stable LDH and urea-stable LDH were also found to be more specific for the detection of anodal LDH isoenzymes than either HBD or total LDH. Methodological problems encountered with the assays are discussed. It is concluded that, using an assay temperature of 37°, heat-stable LDH and urea-stable LDH are superior to both HBD and total LDH for the detection of myocardial infarction.     

Reversible loss of lactate dehydrogenase isoenzymes and lactate dehydrogenase activity in patient's serum

An abnormal lactate dehydrogenase (LD; EC 1.1.1.27) electrophoretogram (only one band, at the application site) and a low LD activity (7 U/L) was seen for a patient's serum during storage at 22 and 4 degrees C. Both reverted to normal when the serum was incubated at 37 degrees C.     

Studies on alkaline phosphatase isoenzymes. Relation to gamma-glutamyltransferase and lactate dehydrogenase isoenzymes.

Gamma-Glutamyltransferase (GT) and isoenzymes of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) have been studied in 282 cases with increased S-ALP and in 18 chronic alcoholics with normal routine liver tests. There was a high degree of correlation between S-GT and the bile (alpha 1) and liver (alpha 2) fractions of S-ALP. Fractionation of alkaline phosphatases sometimes yielded clinical information, which could not be obtained by determinations of S-ALP and S-GT only. The presence of alpha 1-ALP and increased S-GT appeared to be more sensitive indicators of ethanol-induced liver involvement than other liver tests, including LDH-5/LDH-4 ratios.     

Serum lactate dehydrogenase isoenzymes linked to immunoglobulin A

Electrophoretically abnormal serum LDH isoenzyme patterns due to the presence of LDH-IgA complexes are described. Evidence was obtained indicating that the complex formation is caused by the presence of an anomalous immunoglobulin A.     

Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.     

毛细管电泳法分离血清中乳酸脱氢酶同工酶

目的利用还原型辅酶I（NADH）在340nm波长处特异性吸收峰，建立用毛细管区带电泳在线检测人血清中乳酸脱氢酶（LDH）同工酶的方法。方法实验中采用未涂层毛细管，长60cm，内径75μm，pH9．4（23℃）二氨基二甲基1，3丙二醇（AM2P）缓冲液含20mmol/L氧化型辅酶I（NAD^＋）、51，6mmol/L乳酸锂。结果在25min内完成电泳分离，乳酸脱氢酶同工酶1（LDH1）、乳酸脱氢酶同工酶5（LDH5）纯品各自均可得到较好峰形，而且LDH1、LDH2纯品的混合物亦可得到完全分离，在分析正常人血清与肝癌患者血清时，其结果与美国Helena公司REP全自动琼脂糖电泳结果一致，取得满意效果。结论该法快速、低耗，具有较好的推广应用价值。     

Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated

We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.     

Diagnostic value of lactate dehydrogenase isoenzymes in cerebrospinal fluid

To evaluate the diagnostic value of lactate dehydrogenase (LD) isoenzymes in cerebrospinal fluid (CSF), 93 consecutive CSF specimens were analyzed. These specimens were from patients of four categories: tumors, infections, hemorrhages, and others. It was found that the isoenzyme patterns overlapped among different categories, but they differed within each category and were thus helpful in differential diagnosis. For instance, metastatic tumors showed prominent LD-5, whereas a primary brain tumor demonstrated an increase in all fractions. Viral encephalitis revealed an increase in the first three isoenzymes and bacterial meningitis, the last two. In acquired immune deficiency syndrome (AIDS) cases, however, LD isoenzyme changes were demonstrated in CSF when only cryptococcal meningitis and not when encephalitis was present. Both subdural and subarachnoid hemorrhages showed elevation of all fractions in our study. Elevation of the first three fractions was usually due to brain tissue damage or hemorrhage, as proven by our isoenzyme study of hemolysate mixed with CSF. The prominence of the last two fractions was related to anaerobic metabolism in the central nervous system or to granulocytic infiltration. In conclusion, LD isoenzyme analysis in CSF is helpful in differential diagnosis of various CNS disorders, although its sensitivity awaits further improvement.     

Increased activities of creatine kinase and lactate dehydrogenase isoenzymes in a patient with metastatic ovarian tumor

A 50-year-old woman with metastatic rhabdomyosarcoma of the ovary had increased activities of creatine kinase (CK; EC 2.7.3.2), CK-MB isoenzyme, lactate dehydrogenase (LD; EC 1.1.1.27), and LD-2 isoenzyme in her serum. The isoenzyme activities did not show a pattern of increasing, then decreasing. Clinical findings, including electrocardiograms, did not support the diagnosis of myocardial infarction. We suggest that high activities of CK-MB and LD-2 in serum may serve as a marker of rhabdomyosarcoma.