Simultaneous separation of creatine kinase and lactate dehydrogenase isoenzymes using an electrophoresis method]

A specific and sensitive rapid method for simultaneous electrophoretic separation of the blood serum creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes is described, fit for rapid diagnosis. A common electrophoresis scheme was used for the separation of both enzymes on the same cellulose acetate plate. Then the isoenzymes were separately detected by their enzymic activities. To detect the enzymic activities at the sites of isoenzymes' localization, the samples were incubated with substrate gel. Optimal conditions for the detection of CK and LDH activities were defined. To illustrate the diagnostic value of the method, the blood serum CK and LDH isoenzymic activities were measured in the patients with acute myocardial infarction and in those operated on for aortocoronary shunting.     

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.     

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.     

Chromatographic and electrophoretic separation of creatine kinase isoenzymes compared.

We compared two techniques for separating and evaluating serum creatine kinase isoenzymes--fluorometric agarose electrophoresis and Sephadex chromatography--in 50 patients, 25 of whom had confirmed acute myocardial infarction. In every case isoenzyme MB (heart isoenzyme) was detected with equal sensitivity by either procedure. Evidently, only the presence or absence of MB is clinically significant; none of the 25 patients without infarction had detectable MB activity in their serum. Columns connected to a continuous-flow sample line for analyses of the eluting stream without further modification produced satisfactory results.     

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。     

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.     

Evaluation of isoenzymes of creatine kinase and lactate dehydrogenase in cases of suspected acute myocardial infarct

In 176 patients who were hospitalized with a suspected anterior myocardial infarction, the total enzymatic activity of CPK and LDH in addition to the percentage of various isoenzymes have been determined through electrophoretic separation. The blood samples were taken upon the patients admittance to the hospital and repeated every 6 hours until the 24th hour after admittance. We have considered significant an increase of CPK-MB of more than 6% of the total, along with the LDH1/LDH2 ratio greater than 1. We have also taken into consideration the LDH1/LDH-total ratio. The patients were then subdivided into four groups and the results obtained were compared to the electrocardiographic data. Our results confirmed the need to examine both isoenzymatic groups for a correct laboratory diagnosis of anterior 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.     

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.     

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.     

Creatine kinase and lactate dehydrogenase isoenzymes in serum and tissues of patients with stomach adenocarcinoma

Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.     

Improved column method for separating lactate dehydrogenase isoenzymes 1 and 2

Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was evaluated by electrophoresis on polyacrylamide gel. Results for column-fractionated LD-1 and LD-2 were expressed in two ways: LD-1/LD-2 ratios and total LD-1 + LD-2 activities. The former is a more specific indicator of myocardial infarction than the latter. Sera from 10 patients with acute myocardial infarction (increased creatine kinease isoenzyme MB activity) exhibited ratios in the range of 0.92 to 1.56, ratios for 10 patients without heart disease (normal creatine kinase MB) ranged from 0.33 to 0.69.     

Distribution of serum concentrations of creatine kinase MM and BB isoenzymes measured by radioimmunoassay.

Because of the recent interest in measuring serum enzyme concentration as opposed to catalytic activity, we measured the serum concentration of creatine kinase isoenzymes BB and MM by radioimmunoassay and the total creatine kinase enzymatic activity in healthy adults. For sex-race subgroups we report mean values and the 2.5, 5, 25, 50, 75, 90, 95 and 97.5 percentiles. Differences among average values of the subgroups were highly significant. Results within subgroups frequently departed from a gaussian distribution.     

Creatine kinase and lactate dehydrogenase isoenzymes in stage D prostatic carcinoma

Serum creatine kinase (CK) and lactate dehydrogenase (LD) isoenzymes were determined electrophoretically, along with various other biochemical markers of malignancy, in 19 patients with metastatic carcinoma of the prostate. Mitochondrial CK appeared in 15 patients, the CK-BB isoenzyme in 6. As a result, CK activity not inhibited by anti-M-subunit antibodies, CK non-M, was above the reference value in altogether 17 patients. There was a cathodic shift among the LD isoenzymes, significantly more prominent with increasing total LD, and a positive correlation between elevations of CK non-M and LD-5, suggesting a relation to tumour burden for both. An LD 'flip' (LD-1 greater than LD-2) was present in 10/15 patients. The frequency of CK non-M elevations was similar to--but not quantitatively correlated with--elevations of prostatic acid phosphatase and alkaline phosphatase. Thus, changes in CK and LD patterns are frequent in patients with prostatic cancer and must be taken into consideration when acute cardiac symptoms are evaluated in such patients.