Creatine Kinase Assay Research Articles

Creatine kinase in serum: 2. Interference of adenylate kinase with the assay.

Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.

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.

Creatine kinase reactivation by thiol compounds

It is now commonly accepted that thiol activated creatine kinase (CK) assay systems measure CK activity more accurately and more reproducibly than non-activated systems. However, some differences have arisen in the literature in regards to the nature and course of CK activation. Some thiol compounds have been reported to yield higher enzyme activities than others. Dalal et al. (1) reported that mercaptoethanol at 6.5 mM is suboptimal in the Siegel and Cohen assay (2); whereas, dithiothreitol (DTT) at 4 mM yields maximum activity. Warren has shown that DTT and mercaptoethanol produce significantly greater CK activities than cysteine, dithioerythritol (DTE), glutathione, or mercaptoacetate (3). Bishop et al. (4) and Kar and Pearson (5) reported that CK was equally activated independent of the thiol activator. We report here our findings on the relative effectiveness of mercaptoethanol, cysteine, glutathione, and DTT in the reactivation of serum CK using the Oliver-Rosalki method (6,7) and some characteristics of the reaction process.

Evaluation of Direct and Indirect Coupled Enzyme Assay Systems for Measurement of Creatine Kinase Activity

Abstract Direct assay of creatine kinase by the method of Siegel and Cohen with the Technicon "SMA 12/60" is compared with two indirect coupled-enzyme assay methods. The direct method was linear to greater than 2000 U/liter, whereas the indirect methods were linear to 250 and 1500 U/liter, with use of serial dilutions of a creatine kinase preparation. Increasing the activity of the auxiliary enzymes, hexokinase and glucose-6-phosphate dehydrogenase, in one of the coupled enzyme assay methods increased its linear range from 250 to 700 U/liter. The observed findings are explained on the basis of the fundamental differences between direct and indirect coupled-enzyme assay systems and, within the latter system, the effect of the activities of auxiliary enzymes.

A "Reagentless" Fluorometric Method for Creatine Kinase Activity in Serum

Abstract A "reagentless" fluorometric method is described for the analysis of serum creatine kinase (CK) activity. The method is based on the use of silicone rubber pads, upon which are placed all the reagents for assay of CK. The rate of formation of NADH fluorescence at 460 nm is measured and equated to CK activity. The method is simple, rapid, inexpensive, and as little as 3 µl of serum is needed.

Single Stable Reagent for Creatine Kinase Assay

Abstract A single stable reagent for spectrophotometric assay of creatine kinase in one step is described. Reagent cost has been decreased by running the reaction at Km conditions for creatine phosphate and relating the activity to maximum velocity.