Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity.

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarcation patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.     

Simultaneous separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography.

Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.     

Alkaline phosphatase. II. Conditions affecting determination of total activity in serum.

Results of previous experiments on isolated purified isoenzymes of alkaline phosphatase from humans have been confirmed on sera containing relatively large activities of the different isoenzymes. The most remarkable finding is that activation by N-ethylaminoethanol is much more pronounced, in the case of the intestinal and placental isoenzymes, than is activation by diethanolamine. For several reasons, it is suggested that N-ethylaminoethanol is the buffer of choice, 0.1 mol/liter concentration for routine measurements and 1 mol/liter in those cases where the determination of the intestinal or placental isoenzymes is important. Mg2+ could be omitted because its addition increases the activity only marginally. Normal values for adults with use of 0.1 mol/liter N-ethylaminoethanol are 59 +/- 36 (2 SD) U/liter (n = 126).     

An immunochemical procedure for determination of mitochondrial aspartate aminotransferase in human serum

An immunochemical procedure is described for quantitation of mitochondrial aspartate aminotransferase (m-AspAT; EC 2.6.1.1) activity in human serum specimens. Antibodies directed against purified soluble aspartate aminotransferase (s-AspAT) from human erythrocytes were produced in rabbits and partly purified. Antibody sufficient for analyses of > 6000 specimens could be obtained from 15 mL of rabbit antiserum; contaminant AspAT activity of the antibody preparation was < 0.4 U/L. Addition of antibody directly to purified AspAT isoenzymes resulted in inhibition of s-AspAT but had no measurable effect upon m-AspAT. Antibody is incubated with serum in the presence of polyethylene glycol for 60 min at room temperature, then 60 min at 4 degrees C, and centrifuged (7000 x g, 4 degrees C, 15 min). No detectable s-AspAT activity remains in the supernatant fluid; thus m-AspAT activity can be measured directly. Precision, both within-day and day-to-day, was < 1 U/L, or 3.0% of residual m-AspAT activity. The method completely removed 1200 U of purified s-AspAT activity per liter; addition of s-AspAT to serum in increasing concentrations of about 500 U/L had no effect upon the measurement of residual m-AspAT activity. Results of the procedure described showed excellent correlation with those by an alternative procedure involving antibodies directed against m-AspAT. Addition of both anti-s- and anti-m-AspAT antibodies resulted in complete removal of serum AspAT activity. Univalent Fab fragments prepared anti-s-AspAT antibodies were capable of directly inhibiting s-AspAT activity without precipitation. Although a homogeneous immunoinhibition assay was possible, the greater precision of the precipitation assay made it preferable.     

Serum creatine kinase isoenzyme MB activity: Evaluation of a kit employing agarose-gel electrophoresis with overlay paper fluorescence scanning

A commercial kit for determining serum creatine kinase isoenzyme MB activity was evaluated. The kit employed agarose-gel electrophoresis followed by incubation of overlay paper on the agarose and then fluorescence scanning of the paper. Within-day coefficients of variation ranged from 24.9% for a specimen with no elevation of MB activity to 6.6% for a specimen with moderately elevated MB activity. The kit appeared to demonstrate MB in all sera and showed higher than expected values in recovery studies. The kit performed in a relatively linear fashion from 50 to 500 I.U./1 total creatine kinase activity. Hemolysis appeared to lower measured MB. For comparison with another method, specimens were also analyzed by microcolumn chromatography, which was found to incompletely separate isoenzymes. The kit produced lower values than microchromatography for specimens with low MB activities and higher values for specimens with elevated MB activities. Patients without corroborative evidence of myocardial injury showed a somewhat hyperbolic relationship between per cent MB and total creatine kinase activity, but MB activity was generally 4 I.U./1 or less. Although the kit had serious laboratory shortcomings, it may be as clinically useful as other methodologies.     

Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase

A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions.     

Preparation and stability of a liquid creatine kinase isoenzyme control from rabbit serum.

The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.     

New manual and automated method for determining activity of creatine kinase isoenzyme MB, by use of dithiothreitol: clinical applications.

The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.     

Sensitive, rapid assay of subforms of creatine kinase MB in plasma

The subforms of creatine kinase (CK; EC 2.7.3.2) in plasma have received recent attention as potential markers for the early diagnosis of acute myocardial infarction. Because changes in CK-MM subforms are not specific for myocardial injury, we developed an assay, based on high-voltage electrophoresis, that is sufficiently sensitive to detect the CK-MB subforms at concentrations substantially below the upper limit of normal (14 U/L). The assay can detect 1.25 U of either MB subform per liter with a precision of 0.20 U/L and gives responses that vary linearly with activity concentration from 0.0 through 30.0 U/L, with an identical signal response for both subforms. When both subforms are present in a serum sample, the assay accurately measures both the relative percentage and the absolute quantity of each: assay activity/known activity was 1.03 for each subform at a total MB subform activity of 5.0 U/L (r = 0.98). Assay time is 25 min, and there is no loss of CK during electrophoresis. Thus, this system can be used to rapidly, sensitively, and precisely quantify the two CK-MB subforms at activities well within the normal reference interval.     

Preservation of acid phosphatase activity in medico-legal specimens

Vaginal acid phosphatase has been preserved with a protective broth containing, per liter, 50 of bovine albumin, 0.2 g of sodium azide, 10 mmol of phosphate (pH 7.4), and 9.0 g of NaCl. Samples may be maintained at ambient temperature for one month without loss of activity. Several other commonly used preservative methods are compared and are shown to be inadequate. With a constant 2.5 ml volume of the support medium, and use of a sodium thymolphthalein monophosphate method (Worthington Diagnostics), vaginal acid phosphatase activity in non-coital women is less than 10 U/liter of broth, and in recently post-coital women is more than 50 U/liter (242 +/- 104 U/liter). In vivo degradation of vaginal activity follows a nearly logarithmic course until four days after intercourse, when it reaches nearly normal values.     

Development and clinical evaluation of a microcentrifugal analyzer method for determining creatine kinase MB isoenzyme

We have adapted to a microcentrifugal analyzer an immunoinhibition assay for measuring the activity of creatine kinase MB by using an inhibitory antibody for the M monomer. The method actually measures half the MB activity, but results are not multiplied by two because atypical isoenzymes of creatine kinase, including BB, IgG-BB, and the isoenzyme derived from mitochondria, are also detected, if they are present. Results correlated well with an electrophoresis method for 36 serum samples. Myocardial infarction was assessed in 175 patients admitted to our coronary-care unit, with respect to sensitivity (100%) and specificity (98%) when a decision point of 100 U/L (30 degrees C) was chosen for total creatine kinase activity (dithiothreitol-activated) and 6 U/L (30 degrees C) for the isoenzyme (by immunoinhibition). Atypical isoenzymes are easily recognized and confirmed by electrophoresis when the MB activity (by immunoinhibition) exceeds 6 U/L and 20% of the total creatine kinase activity.     

自制超薄型琼脂糖凝胶板电泳分离检测血清LDH同工酶及成年人参考范围的建立

目的 自制超薄型琼脂糖凝胶板,建立电泳分离检测血清乳酸脱氢酶(LDH)同工酶的方法并建立成人血清的参考区间。方法 用自制琼脂糖凝胶板分离检测LDH同工酶,对方法学性能包括精密度、正确度、线性范围、参考区间进行确认和建立。结果 5种LDH同工酶图谱分离清晰,批内CV值均小于8.77%,批间CV值均小于13.12%,胶板间CV值均小于7.89%。LDH1在13.48～287.65U/L,LDH2在18.19～575.67U/L,LDH3在19.42～504.62U/L,LDH4在8.00～342.27U/L和LDH5在13.88～199.79U/L的范围内呈线性,斜率趋近于1。与Sebia配套电泳试剂盒的结果比较,5种同工酶在两方法间的差异无统计学意义(t=0.028 1～0.567 4,均P>0.05),相关系数r=0.949 4～0.985 5。建立的成人参考区间为LDH1:15.7%～34.3%,LDH2:26.8%～38.9%,LDH3:18.8%～28.1%,LDH4:5.7%～13.7%和LDH5:2.7%～15.3%。结论 自制超薄型琼脂糖凝胶板电泳分离检测血清LDH同工酶的方法性能良好,可用于临床检测。     

Use of the Du Pont aca to measure cholesterol in high-density lipoprotein fractions prepared by the heparin/Mn2+ precipitation method.

The enzymatic cholesterol method used with the Du Pont aca has been modified to provide a reliable measurement of high-density lipoprotein cholesterol in serum after heparin/Mn2+ precipitation of the low- and very-low-density lipoproteins. Interference by Mn2+, equivalent to about 90 mg of cholesterol per liter, is decreased to less than 40 mg of cholesterol per liter by the presence of ethylenediaminetetraacetate (8 mmol/liter) in the diluent; the residual effect of Mn2+ is compensated by calibrating the aca with standards containing Mn2+ and heparin. With an 80-microliter sample, the sensitivity is 236 muA/mg per liter and linearity ranges from 50 to 1500 mg/liter. Average analytical recovery of cholesterol added to the high-density lipoprotein fraction was 103%. Diluted fractions give the expected results. Between-run reproducibility (CV) is 1.3 and 1.6% at 463 and 554 mg/liter. Correlation with the Lipid Research Clinics' procedure (25 samples) gave a regression line of y(aca) = 1.039x- 15, and a correlation coefficient of 0.997.     

A "column-batch" method for separating MB and LD1 in a single fraction

In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.     

Determination of phosphodiesterase I activity in human blood serum.

Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis.     

Simple, highly selective screening method for barbiturates in urine.

I present a new, simple colorimetric method for detecting and estimating barbiturates in urine. After the barbiturates are extracted with ether, an aliquot of the washed ether phase is added to the color reagent (a bivalent mercury/dithizone chelate in chloroform). On addition of diluted pyridine and shaking, a pinkish-violet color appears if a barbiturate is present. The overall sensitivity of the above method was evaluated by probit analysis in the case of sodium phenobarbital. The concentration of sodium phenobarbital in urine detectable at least 99% of the time was 6.72 mg/liter, with 95% confidence limits of 5.37 to 10.36 mg/liter. Sodium phenobarbital, 10 mg/liter, can be detected in the presence of phenytoin (50 mg/liter), glutethimide (100 mg/liter), or bemegride (100 mg/liter). The whole procedure requires less than 10 min. An excretion study illustrates application of the procedure.     

Rapid estimation of hemoglobin A2 without chromatography or electrophoresis.

Hemoglobin A2 is batch fractionated with diethylaminoethyl Bio-Gel A (Bio-Rad Laboratories) equilibrated with a tris(hydroxymethyl)aminomethane HCl buffer (pH 7.68, 8.75 mmol/liter, 6.36 mmol of CL- per liter). Hemoglobin A1 and F become bound to the resin, allowing the separation and quantitation of A1. Hemoglobin S alters the equilibrium condition, but adjustments are easily made so that A2 can be separated in the presence of S. The procedure is simpler than electrophoretic or chromatographic methods, requires 5 min, is accurate (A2 fraction is at least 94% A2, less than 6% A1), precise (SD +/-0.24 for duplicates), and has a normal limit of 2.6 +/-1.02%.     

Manual and semi-automated procedures for measurements of triglycerides in serum.

We describe manual and semi-automated procedures for serum triglyceride determinations, in which lipids are partitioned between a water/isopropanol phase and a nonane phase. More than 99% of the triglyceride is extracted into the nonane phase, as determined by recovery of 3H-labeled triolein. Studies with 14C-labeled lecithin demonstrate that less than 1.3% is extracted into the nonane phase at concentrations up to 2.5 g/liter. A novel feature of the method is that glycerol can be liberated from triglyceride by sodium hydroxide at room temperature in less than 5 min. Glycerol is oxidized by periodate in 1-2 min at 25 degrees C; the formaldehyde produced is reacted with 2,4-pentanedione to yield 3,5-diacetyl-1,3-dihydrolutidine. The manual procedure requires less than 20 min; the semi-automated method requires 7 min from sampling to readout. The procedure may be run at 30-40 samples/h, with stable baseline and less than 2.0% carryover. Both methods are linear to 0.50 g (5.6 mol) of triolein per liter. Analytical recoveries at several concentrations ranged from 97-101% (mean, 100%).     

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.     

Colorimetric measurement of ornithine carbamoyl transferase activity in plasma, and results for a supposedly healthy population.

Determination of ornithine carbamoyl transferase (EC 2.1.3.3) activity in plasma is important for detection of liver diseases. The assay established in this paper has been made optimum. A blank is needed containing both substrates, carbamoyl phosphate and ornithine. We used a new colorimetric assay, based on a complex with a phosphoferric-antipyrine reagent and diacetyl monoxime, to measure the citrulline formed. The highly sensitive assay permits low activities to be determined accurately. Values for blood plasma from 425 supposedly healthy people, varied from 0 to 16 U/liter (95th percentile), and 27% of this population showed an activity of less than 2 mumol of citrulline formed per minute per liter, 2 U/liter being the limit of the method's sensitivity.