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Interferences with assay of creatine kinase activity in vitro.

The release of intracellular enzymes, e.g. creatine kinase and lactate dehydrogenase, is frequently used as an indicator of skeletal muscle damage in vivo and in vitro. The biochemical events leading to enzyme release from skeletal muscle fibres are not completely characterized. A useful approach to define these events is to examine the effects of various enzyme inhibitors on muscle damage and enzyme release. For example, Jackson and his associates (Duncan & Jackson, 1987; Jackson et al., 1987) recently showed that chlorpromazine (a phospholipase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) reduced creatine kinase efflux in vitro induced by 2,4-dinitrophenol or A23187 from isolated soleus muscles of rats and mice. Earlier work (Jackson et al., 1984) demonstrated that dibucaine, another phospholipase inhibitor, also reduced lactate dehydrogenase efflux in vitro induced by 2,4-dinitrophenol from the isolated mouse soleus muscle. Other compounds, such as mepacrine (a phospholipase inhibitor) and phenidone (a joint cyclo-oxygenase/lipoxygenase inhibitor) have also been shown to have similar effects (Jackson et al., 1984, 1987). These studies implicated possible roles for phospholipase and lipoxygenase in mediating intracellular enzyme release during muscle damage. A necessary prerequisite in using this approach is that the enzyme inhibitors added would not interfere with the determination of enzyme activity (e.g. that of creatine kinase) used as an index of muscle damage. Jackson et al. (1987) have reported that this is the case for phenidone and nordihydroguaiaretic acid. In a recent series of studies on the mechanisms of muscle damage caused by organic cosolvents, we also used these inhibitors for probing the biochemical mechanism. However, in initial experiments, we found that significant interferences occurred between several agents/solvents with the determination of creatine kinase activity. In documenting these interference results, we used three different experimental methods involving two different sources of muscle creatine kinase at two initial concentrations of creatine kinase. The first method, method A, utilized rabbit muscle creatine kinase Type I obtained from the Sigma Chemical Co. A spiking solution of creatine kinase was prepared by dissolving approx. 1 mg of the enzyme in 10 ml of a balanced salt solution (BSS, pH 7.4). A given aliquot of this solution was spiked into two incubation vessels containing the same BSS at 37 °C and bubbled with 02/C02 (19: 1). The test compound was added to one incubation vessel while the second vessel served as the control. These solutions were incubated for 30 min and creatine kinase activity was determined immediately following incubation. In method B, creatine kinase was generated from slicing a rat muscle [the extensor digitorum longus (EDL) muscle] and allowing the enzyme to be released into the BSS. An aliquot of this solution was added to two incubation vessels and processed subsequently according to method A. Method C also utilized creatine kinase that was generated from the sliced rat EDL muscle. However, the BSS containing the enzyme was divided equally between the test and control incubation vessels. The appropriate amount of test agent or BSS was then added, the solutions incubated for 30 min, and assayed for creatine kinase. This method was designed to prevent dilution of the enzyme activity as well as the concentrations of other simultaneously released substances that might affect creatine kinase activity in vitro. Creatine kinase activity was determined spectrophotometrically at 340 nm using a commercially available kit (Sigma Diagnostics no. 47-UV). This assay is based upon the rate ofchange in the absorbance caused by a reduction of NAD+ to NADH. The degree of interference with creatine kinase activity was evaluated by the ratio of creatine kinase activity in the presence of the test compound to the activity measured in the absence of the test compound. Statistical significance was performed when appropriate using Student's t-test or one-way analysis of variance. The results of the interference studies are shown in Table 1. The degree of enzyme interference did not exhibit a dependency upon the initial creatine kinase activity for chlorpromazine, dibucaine, propylene glycol, poly(ethylene glycol) 400, verapamil, nordihydroguaiaretic acid, 2,4-dinitrophenol and deoxycholic acid. In contrast, the degree of creatine kinase inhibition caused by ethanol and the calcium ionophore A23187 was shown to be dependent upon the initial concentration of the enzyme. Chlorpromazine caused a 65-950 inhibition of creatine kinase activity, with significant differences in the degree of inactivation among the three methods. Dibucaine, on the other hand, did not apparently interfere with the determination of creatine kinase activity and there were no statistical differences among the three methods. Mepacrine and phenidone were found to inhibit enzyme activity to about 700, while nordihydroguaiaretic acid completely abolished creatine kinase activity in the system in vitro. Verapamil, deoxycholic acid, 2,4-dinitrophenol, and A23187 (at high initial creatine kinase concentrations) did not interfere with the determination of creatine kinase activity in vitro. The organic cosolvents propylene glycol and ethanol did not appear to interfere with creatine kinase activity while poly(ethylene glycol) 400 appeared to inhibit enzyme activity substantially (60-70 %). With the exception of chlorpromazine and phenidone, there did not appear to be a difference in the degree of assay interference with the source of the creatine kinase, i.e. rabbit versus rat.