Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes.

We report the effect of serum pH on the storage stability of the human creatine kinase isoenzymes and on the creatine kinase assay lag phase (Szasz et al., Clin. Chem. 22: 650, 1976). We also investigated the effect of including mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, or ethylene glycol bis(betaaminoethyl ether)-N,N,N',N'-tetraacetate at 20, 4, and --20 degrees C. Storage stability of the isoenzymes is profoundly affected by pH. For patients' samples and semi-purified human creatine kinase isoenzymes added to heat-inactivated sera, increasing diluent pH above 7.0 decreases creatine kinase stability. The thiol agents or chelators generally give little or no protection above pH 7.5; at pH 8.5 they contribute significantly to isoenzyme instability. Storage at 4 degrees C provides greater stability than storage at 20 degrees C, particularly in the case of creatine kinase isoenzyme BB. The lag phase was minimum at a serum pH of 6.5, in the presence of 10 mmol of monothioglycerol per liter. Increasing serum pH to 8.5 prolongs the reaction lag phase by about 1 min over the minimum. We recommend that, before they are stored at 4 degrees C, the pH of patients' samples be adjusted to 6.5 and oxidation of SH-groups be minimized by adding monothioglycerol to the sample.