Evaluation of a commercial immunoenzymometric assay kit for creatine kinase MB isoenzyme determination using monoclonal antibodies

A simultaneous two-site immunoenzymometric assay for creatine kinase MB determination (Hybritech Tandem-E CK-MB) using monoclonal antibodies was evaluated and compared with cellulose acetate electrophoresis using fluorometric scanning densitometry. The assay has satisfactory precision (between-day analysis gives a coefficient of variation between 2.1 and 9.4%) and is not susceptible to interference by concentrations of creatine kinase MM up to 5000 micrograms/l (3400 U/l) and creatine kinase BB up to 1000 micrograms/l (1085 U/l). The upper limit of MB isoenzyme concentration in 250 apparently healthy people was 5.5 micrograms/l. Comparison between the immunoenzymometric assay (y) and electrophoresis (x) yielded the following linear regression equation: y = 0.37x + 1.9, with a correlation coefficient of 0.828. The characteristics of the temporal kinetics of MB isoenzyme, calculated by two methods, in 49 patients with acute myocardial infarction, were nearly identical in terms of the rate of creatine kinase MB release and the time at which the peak value is obtained, but not in terms of the rate of elimination of the isoenzyme. The fractional disappearance rate of MB isoenzyme from the circulation was significantly higher if calculated with Tandem-E results rather than with electrophoresis results (-0.035 vs -0.028, p less than 0.001). Whereas in the first day after infarction immunoenzymometric assay and electrophoresis had the same clinical sensitivity for identifying patients with acute myocardial infarction, in specimens collected more than 24 hours after the onset of the chest pain, the clinical sensitivity of the immunoenzymometric method was lower. Our results show that it is still premature to draw definitive clinical conclusions from the immunoassay results.     

Clinical evaluation of an automated chemical inhibition assay for lactate dehydrogenase isoenzyme 1

We evaluated an automated assay for lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes, supplied by Boehringer Mannheim Diagnostics (BMD) and based on selective chemical inhibition of non-LD-1 isoenzymes by guanidine thiocyanate. Results were compared with the Roche Isomune LD-1 method. The Hitachi 717 analyzer was used to measure enzyme activity for both procedures in 229 serum samples. One hundred specimens were also analyzed by the Helena rapid electrophoresis (REP) method. We determined the limit of linearity of the BMD method to be about 1200 U of LD-1 per liter. The analytical correlation of BMD (y) with Isomune (x) yielded y = 1.0x + 0.5 U/L, r = 0.997, Sy/x = 16.9 (range 20-1397 U/L). The regression equation for BMD vs REP was y = 1.1x + 7.2% (r = 0.800, Sy/x = 7.4, range 14-83%). Average values for within-run precision for low (38 U/L), medium (180 U/L), and high (865 U/L) controls were 4.1%, 1.0%, and 0.5%, respectively (16 trials of six each). The average values for run-to-run precision were 4.1%, 1.7%, and 1.1%, respectively, for these controls (n = 16). We used receiver-operating characteristic curves to determine optimum decision limits. Using an LD-1 cutoff of 40% of total LD, we obtained a clinical sensitivity of 97-100% and a specificity of 95% when blood was collected during the optimum interval, 24-48 h after the onset of chest pain. We conclude that the BMD LD-1 assay is equivalent to the immunochemical and electrophoretic assays for measuring the LD-1 isoenzyme.     

Specific assay of serum lactate dehydrogenase isoenzyme 1 by proteolysis with alpha-chymotrypsin and protein denaturation.

We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.     

The quality control of serum lactate dehydrogenase isoenzyme separations using commercial quality control sera

The lactate dehydrogenase (l-lactate:NAD oxidoreductase, EC 1.1. 1.27; LDH) isoenzyme content of 31 commercially available quality control sera was determined using thin layer agarose and fluorimetry. Only one of these sera was specifically intended for isoenzyme quality control purposes. The group of human sera, with no additives, proved to be the only one having an LDH isoenzyme distribution suitable for quality control use. Within-batch and between-batch precision has been determined over a 2–3 month period for LDH isoenzyme separations using several human quality control sera. The control of quality of this procedure has been shown to be feasible.     

A colorimetric assay for serum lactate dehydrogenase

A simple, rapid, colorimetric assay for serum lactate dehydrogenase is described. The method requires 0.05 ml serum and a 12-min incubation at 37° and utilizes a tetrazolium salt in conjunction with a stable intermediate electron carrier (Meldola Blue). Good correlation between the colorimetric assay and a reference spectrophotometric assay was observed.     

Analytical quality specifications for serum lactate dehydrogenase isoenzyme 1 based on clinical goals.

The aim of the study was to deduce analytical quality specifications for the determination of catalytic concentration of serum lactate dehydrogenase isoenzyme 1 (S-LD-1) according to clinical goals (the clinical utility model). We defined clinical goals for false positive and false negative S-LD-1 measurements in the monitoring of patients with testicular germ cell tumors (TGCT), clinical stage I, on a surveillance only program. The absolute S-LD-1 catalytic concentrations were routinely corrected for contamination from preanalytical hemolysis. A reference group of 37 men had a near In-Gaussian distribution for the absolute S-LD-1 catalytic concentration. The geometric mean was 76 U/l and an S-LD-1 >128 U/l (99.72 percentile, the decision limit) indicated a high risk of a relapse of TGCT. We have previously shown that an S-LD-1 >160 U/l (treatment limit) was associated with a suboptimal outcome from the treatment of metastatic TGCT. The maximum allowable analytical positive bias was 5 U/l, and the maximum allowable analytical negative bias was -32 U/l. The maximum allowable analytical coefficient of variation, CV(A), was 11% (approximately 14 U/l) at a bias = -5 U/l. For S-LD-1 measurements not corrected for hemolysis, the decision limit was 145 U/l, the maximum allowable negative bias -19 U/l, and CV(A) 8%(approximately 12 U/l). A routine correction for hemolysis had a large impact on the analytical quality specifications.     

Selective determination of lactate dehydrogenase isoenzyme 1 in serum after inhibition by 1,6-hexanediol

A rapid selective method for measuring the activity of lactate dehydrogenase isoenzyme LD-1 in serum by using 1,6-hexanediol as an inhibitor of the M-subunit was developed. Hexanediol was added to serum at a final concentration of 0.7 mol/l. After incubation at 30 degrees C for 15 min, the activity was measured with an automatic analyser. The inter-assay coefficient of variation was 6.9% for the lactate dehydrogenase isoenzyme LD-1 measurement. The results obtained from the sera of 100 patients analysed by the proposed selective method and by the conventional electrophoretic method, respectively, showed an excellent correlation. This selective method was used to determine the lactate dehydrogenase isoenzyme LD-1 activity of sera from patients with acute myocardial infarction, and the results were correlated well with those obtained by the immunological, Roch Isomune method. Addition of 1,6-hexanediol did not affect the measurement of activities of other enzymes such as alkaline phosphatase, gamma-glutamyltransferase, aspartate aminotransferase and alanine aminotransferase.     

Increased lactate dehydrogenase isoenzyme 1 in serum and tumor tissue of a patient with small-cell carcinoma

Histological examination of supraclavicular lymph node tissue obtained at biopsy from a 63-year-old man disclosed metastatic small-cell carcinoma. On admission and for four days subsequently, total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum was 6.5 times normal; studies of LD isoenzyme showed persistently increased LD-1, with LD-1 greater than LD-2. Isoenzyme electrophoresis of tissue homogenates prepared from the patient's tumor also showed the LD-1 greater than LD-2 pattern. Isoenzyme studies for supraclavicular lymph node tissue from five control subjects showed contrasting isoenzyme patterns as compared with the patients in whom LD-2, LD-3, and LD-4 predominated. Because these abnormalities were persistent, they differ from the temporal sequence for LD usually seen in myocardial infarction. This emphasizes the importance of repetitive sampling for clinical interpretation of data on this enzyme.     

Abnormal lactate dehydrogenase isoenzyme in serum and tumor tissue of a patient with neuroblastoma

Serum and tumor tissue of a patient with neuroblastoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), which, on agarose gel electrophoresis, migrated between LDH-2 and LDH-3 with a mobility the same as that of the extra LDH isoenzyme found in normal human erythrocytes. On surgical removal of the tumor, the high total LDH activity (775 U/L) in the serum of the patient rapidly decreased to normal (70-220 U/L), and the abnormal LDH isoenzyme was no longer detected. The total LDH activity of the abnormal LDH isoenzyme per gram of hemoglobin in the tumor tissue was 26 times that of erythrocytes, suggesting that the abnormal isoenzyme originated mainly from the tumor cells themselves rather than the erythrocytes contained in the tumor tissue. This first report on the appearance of the abnormal LDH isoenzyme in a patient with neuroblastoma suggests that this abnormal LDH isoenzyme may have some significance as a marker enzyme for neurogenic tumors.     

Abnormal serum lactate dehydrogenase isoenzyme in a case of laryngeal carcinoma and thyrotoxicosis.

An abnormal lactate dehydrogenase (LDH) isoenzyme was found in the serum of a patient with laryngeal carcinoma and hyperthyroidism. On electrophoresis it migrated as an additional band between LDH-1 and LDH-2. Follow-up studies suggested that this higher molecular weight, rather thermostable LDH isoenzyme, might have originated from the cancer tissue, though a possible relationship with the thyrotoxic state cannot be excluded.     

Increased serum lactate dehydrogenase isoenzyme 1 and macro creatine kinase type 2 in a patient with lung cancer

A 72-yr-old man, having a lung tumor with metastatic spread to liver and bone, was hospitalized with a lactate dehydrogenase (LD; EC 1.1.1.27) activity and a creatine kinase (CK; EC 2.7.3.2) activity in serum of 30 and 2 times the upper reference limit (URL), respectively. The LD isoenzyme-1/LD activity ratio was 62% (2 times URL). This ratio was maintained throughout hospitalization, during which LD activity increased up to 90 times URL. Macro CK type 2 activity represented almost all of the CK activity, which increased up to 4 times URL during hospitalization. The patient died the 29th day after admission. The enzyme abnormalities were thought to stem from tumor tissue.     

Evaluation of a new enzymatic kit for serum triglycerides.

We evaluated a new enzymatic kit for serum triglycerides (triacylglycerols) in two laboratories. The procedure involved both enzymatic hydrolysis of the triglycerides and conversion of the glycerol, which was linked to an indicator reaction. There was excellent agreement with the mechanized procedure of Kessler and Lederer and with a manual method. The test requires 50 mul of serum and can be completed in about 50 min. Inter- and intra-laboratory variation, linearity, accuracy, and specimen storage were studied.     

Use of purified lyophilized human lactate dehydrogenase isoenzyme 5 in a study of measuring lactate dehydrogenase activity

We examined the stability of human lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5--purified to a specific activity of about 400 kU/g--when lyophilized in a buffered, stabilized matrix of bovine albumin. This isoenzyme was prepared with a final activity of about 500 U/L and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. This isoenzyme decayed with approximate first-order kinetics, with an estimated half-life at -20 degrees C of about 475 years. Stability of reconstituted samples stored at 20 or 4 degrees C was poor, suggesting that the reconstituted material should be used without delay; material stored at -20 degrees C showed excellent stability for 15 days. We propose that such preparations might be further investigated as standards for use in electrophoresis of lactate dehydrogenase isoenzymes.