Evaluation of the optimized bioluminescent assay for the determination of total creatine kinase and creatine kinase MB activities

A very sensitive, optimized bioluminescent assay for certain kinase and creatine kinase MB activities is tested. We evaluated reagent blanks, sensitivity, precision and compared the results with those of the spectrophotometric immunoinhibition test. The main advantage of the new method is a detection limit of less than 1 U/l which, together with a high precision (s = 0.1 at detection limit), allows determinations of the creatine kinase MB activity even in normal sera in about 20 minutes. A disadvantage of the manual procedure is that it may be necessary to include up to five pipetting steps.     

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.     

A sensitive bioluminescent immunoinhibition test for CK-B subunit activity and a CK-MB specific ELISA compared: correlation with agarose electrophoresis and influence of CK-isoenzyme profile on results

Searching for alternatives to the imprecise spectrophotometric tests for low-concentration creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB), we investigated the analytical performance of two potentially superior approaches--a bioluminescent immunoinhibition assay (I, LKB-Wallac) and an ELISA (enzyme-labeled immunosorbent assay) technique (II, Hybritech)--in comparison with an electrophoretic method (III, Beckman). Only I showed good between-day precision (CV 8.3%) at the upper reference limit, allowing reproducible assay of CK-B subunit activity down to at least 3 U/L. In conditions where CK isoenzyme assays remained unaffected by CK-MM concentrations, test results were proportional to the amount of CK-MB in the sample up to at least 50 U/L for I, 120 micrograms/L for II, and 100 U/L for III (r greater than 0.998 by linear regression analysis). For CK-MB-positive samples, the data by I correlated more closely with values by III (n = 24; r = 0.994) than did results by II (n = 15; r = 0.909), but both methods were equally effective in discriminating between samples with or without electrophoretically supranormal CK-MB activity (93% sensitivity). II was entirely CK-MB specific, whereas CK-B activity by I was consistently (18/18) increased in CK-MB-negative samples containing CK-BB (n = 6; r = 0.996) or macro CK, types 1 or 2 (n = 12; r = 0.930). I is highly sensitive for screening for increased non-MM CK activity, the nature of which should be subsequently clarified by electrophoresis.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Serum guanase activities after myocardial infarction.

Serum guanase, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase and hydroxybutyrate dehydrogenase activities were measured in 290 blood samples from 96 consecutive patients admitted to a Coronary Care Unit. Elevated serum guanase activities (greater than 2 U/l) were found in 19 patients (20%). The magnitude and frequency of these elevations did not negate the value of guanase as a "liver function test", since all cases with raised guanase also had abnormal serum alanine aminotransferase activities. This fact, together with other information in the literature, indicated that elevated serum guanase activity following myocardial infarction was consequent upon some degree of sub-clinical hepatic necrosis. Caution must be exercised when serum asparate aminotransferase is used as an index of heart muscle necrosis unless guanase or some other "liver specific" enzyme is known to be normal, or unless creatine phosphokinase or hydroxybutyrate dehydrogenase activities are elevated.     

Creatine kinase in serum: 3. Further study of adenylate kinase inhibitors.

In search of an appropriate inhibitor to suppress the interference of adenylate kinase with the creatine kinase assay, we found that the combination diadenosine pentaphosphate (10 mumol/liter) and AMP (5 mmol/liter) is a better inhibitor than is fluoride (25 mmol/liter). The latter inhibits adenylate kinase uncompetitively and weakly (Ki = 2.5 mmol/liter), and must be incorporated in the starting reagent, and at 30 degrees C it becoms fully effective only after a lag phase of 6 min. In this concentration, fluoride inhibits adenylate kinase from erythrocytes, muscle, liver or platelets by 94, 92, 88, and 87%, respectively, and creatine kinase by 8%. Bromide and chloride also inhibit creatine kinase. Attempts to replace AMP by a specific inhibitor of liver adenylate kinase failed. Homologs of diadenosine pentaphosphate with either fewer or more phosphoryl groups in the polyphosphate bridge inhibited even more weakly than did the pentaphosphate. Platelets can significantly contribute to adenylate kinase activity in serum. The inhibitor combination inhibited adenylate kinase from platelets by 90%.     

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies

Abstract

Objective: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. Material and methods: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. Results: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. Conclusion: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.     

Automatic bioluminescent glucose determination using commercially available reagent kits coupled to the bacterial NAD(P)H-linked luciferase system

A sensitive and specific automatic bioluminescent method is described for glucose determination in serum samples using commercially available reagent-kits. The Boehringer Gluco-quant kit, originally designed for spectrophotometric measurement, was successfully coupled to the bacterial luciferase NAD(P)H-linked system. The method's validity was proven by comparison with a spectrophotometric method. Correlation was excellent (r = 0.98, n = 50). Precision attained by 30 assays was good (CV 1.19%). The assay was verified by determining the glucose concentrations of more than 1000 serum samples. Using a microcomputer-controlled automatic luminescence analyser (Berthold LB 950 T) and reagent kits for luminometry (Boehringer Mannheim, LKB Wallac, Lumac/3M), the complete assay can be performed fully automatically with commercially available reagent kits. More than 200 samples can be assayed by one individual per day. The bioluminescence method is at least 100 times more sensitive than spectrophotometric measurements. Other reagent kits tested (Behring, Merck, Sigma) are also suitable for coupling to the NAD(P)H-linked luciferase system.     

Myoglobin concentration,creatine kinase,and creatine kinase sub-unit B activity in serum after myocardial ischaemia

The myoglobin concentration. creatine kinase and creatine kinase sub-unit B activity were estimated in fourteen patients with ischaetmic heart disease before and after exercise induced angina pectoris. No changes in these parameters were found.     

Use of creatine kinase MB isoenzyme for diagnosing myocardial infarction when total creatine kinase activity is high

The usefulness of measuring creatine kinase MB isoenzyme for diagnosing myocardial infarction when activities of total creatine kinase are very high is unclear. We conducted a retrospective study in an urban hospital that serves a largely indigent population. We concentrated on 146 patients whose creatine kinase activity was greater than 1000 U/L (upper limit of normal: 165 U/L for women and 225 U/L for men), with MB isoenzyme greater than 10 U/L and less than 5% of total creatine kinase. The positive predictive value of MB isoenzyme (isoimmune method) values greater than 10 U/L was between 11.6% and 56.8% when the value for total creatine kinase exceeded 1000 U/L. Using different values (MB greater than 4% of total creatine kinase) as positive for myocardial infarction would have resulted in far fewer false-positives, but 10 cases of myocardial infarction would have been missed. The most appropriate cutoff value for MB isoenzyme in this population (total creatine kinase greater than 1000 U/L) was found to be greater than 2% of total creatine kinase.     

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.     

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.     

Bioluminescence assay of creatine kinase and its isoenzymes in serum and cerebrospinal fluid.

We examined the sensitivity of bioluminescence for the determination of very low concentrations of creatine kinase brain-type subunit (CK-BB) in serum and in cerebrospinal fluid. To optimize the sensitivity of CK-isoenzyme assays and eliminate possible sources of error, we separated the isoenzyme fractions by using inhibiting anti-MM and precipitating anti-MM and anti-BB antibodies. The results with the bioluminescence assay correlated with spectrophotometric values such that r = 0.97 for the total CK activity and r = 0.98 for the CK-B activity. The reproducibility of the present method was comparable with the spectrophotometric method and was even better at low enzyme activities. The within-series precision for assay of total CK activity at 2 U/L corresponded to a CV of 9%; at 13 U/L the CV was 5.8%. All the assays were carried out at 25 degrees C. Even at this low temperature, CK activities as low as 0.2 U/L could be determined. In eight patients without any evidence of cerebral cell damage, total CK activity in cerebrospinal fluid was x = 1.05 +/- 0.6 U/L, and CK-BB activity was x = 0.7 +/- 0.4 U/L. In sera of these patients CK-BB activity was x = 0.6 +/- 0.5 U/L. Differences in CK and CK-BB activities in four patients with transient or progressive brain-cell damage are discussed.     

Enzymatic pattern of glucose metabolic pathways in pyruvate kinase-deficient erythrocytes.

It has been shown that in some cases of congenital non-spherocytic haemolytic anaemia (CNSHA) with pyruvate kinase deficiency, the primary defect may be related to diminished magnesium-stimulated ATPase activity, followed by elevation of the erythrocyte ATP level. ATP as the end product of glycolysis inhibits by negative feedback control the activities of key glycolytic enzymes involved in energy production, i.e. pyruvate kinase (PK) and phosphofructokinase (PFK). Erythrocyte-deficient PK, however, is insensitive to the stimulating effect of fructose 1,6-diphosphate (FDP), which is normally a positive effector of PK. Both competing effectors, i.e. ATP and FDP, seem to show specific interaction. PK, inactive in the presence of high concentrations of ATP, seems to lose its sensitivity to FDP. This effect persists until ATP molecules are present in excess. In vitro incubation of deficient PK with ATPase resulted in increased PK activity as well as in recovery of its sensitivity to the stimulating effect of FDP. The same effects were obtained in vivo by administering magnesium levulinate intravenously to CNSHA patients with PK deficiency. This may indicate that magnesium ions stimulate deficient ATPase activity and lead to diminution of ATP as a negative effector for other regulatory enzymes.     

重组人肌酸激酶MM同工酶的酶动力学研究

目的:对纯化后重组肌酸激酶MM同工酶(CK-MM)的米氏常数(Km)进行研究,为其作为参考品或校准品应用于临床奠定基础。方法:将重组酶置于自制类人血清基质中,按IFCC参考方法配制试剂,采用HITACHI全自动生化分析仪测定重组酶与天然酶[底物分别是磷酸肌酸(PCr)和腺苷二磷酸(ADP)]的Km常数。结果:重组酶KmPCr值高于天然酶1.4倍,Km ADP重组酶与天然酶近似。结论:重组CK-MM的酶动力学常数基本符合参考方法的测定要求,初步具备作为基因工程人源酶校准品的基本特点。     

低血糖对肌肉损伤影响的实验研究

目的;研究胰岛素剂量和低血糖持续时间对血清酶活性影响,确定低血糖损伤的脏器。方法：根据胰岛素剂量和低血糖持续时间的不同,将30只家兔分为5组：A2组,胰岛素2U／kg低血糖持续30min;A10组,胰岛素10U/kg低血糖持续30min;B2组,胰岛素2U／kg低血糖持续60min;B10组：胰岛素10U/kg低血糖持续60min;C对照组：胰岛素10U/kg加50％葡萄糖注射不诱发低血糖。胰岛素注射前后对血清酶的活性及肌酸激酶（CK）同功酶的分布进行观察。结果：所有低血糖组其血清CK的活性较对照组明显增高,且在A10和B10组CK活性的升高持续24h,而血清ALT、AST、LDH的升高仅见于B10组。此外,主要存在于心肌和骨骼肌中的CK－Band4在B10组可见显著升高。结论：低血糖所致血清酶活性及CK－Band4的升高是由于肌肉损伤的结果而非肝脏的损伤,低血糖持续的时间和胰岛素剂量可以影响脏器损伤的程度。     

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.     

肌酸激酶受精神分裂症症状及药物的影响

目的探讨以氯丙嗪、氯氮平、利培酮为代表的精神病药物对精神分裂症患者治疗前后肌酸激酶(CK)的影响差异。方法将2010年入住该院的150例精神分裂症患者的血清CK进行检测,并按CK正常与否、服用药物的种类及精神病症状进行分组对照。结果 150例患者服用抗精神病药物后,其中有105例(70%)患者CK有不同程度的升高,平均值为(450.0±385.0)U/L;有45例(30%)患者CK在正常范围内,平均值为(150.0±45.5)U/L;服用不同药物后,CK均明显升高,但不同药物间差异无统计学意义(P>0.05);精神分裂症首发兴奋状态或躁狂症服药后CK分别为(502.0±266.0)U/L和(478.0±293.0)U/L,较癔症及抑郁症组显著升高,差异有统计学意义(P<0.01)。结论精神病患者服用药物后CK明显升高,与性别、年龄和药物的种类关系不大,与精神病患者的症状关系密切。     

Enzymatic determination of acetate in serum or plasma using a centrifugal fast analyser

A simple enzymatic spectrophotometric micromethod is described for direct kinetic assay of acetate in serum or plasma using the Eni-Gemsaec centrifugal fast analyser. The method is based on the transformation of acetate and ATP into acetylphosphate and ADP by acetate kinase (EC 2.7.2.1). ADP is further measured by two coupling reactions involving pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) with measurement of NADH consumption at 340 nm.The method involves a reagent blank for compensation of reagent deterioration, a preincubation of 3 min without acetate kinase to eliminate any interference due to endogenous pyruvate, and a two-point kinetic protocol with measurements of absorbance at 95 s and 395 s. The analytical performances of the proposed method were investigated using an evaluation scheme proposed by the French Society of Clinical Biology.     

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.