Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.

BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.

The concentrations of free Mg2+ and free Zn2+ in equine blood plasma.

The enzyme phosphoglucomutase can be used as a metal ion indicator to measure the concentrations of free Mg2+ and free Zn2+ in physiological fluids. In horse plasma, the concentration of free Mg2+ is close to 0.5 mM, whereas that of free Zn2+ is about 2 X 10(-10) M, although numerous physiological roles for Zn2+ have been postulated that would require free Zn2+ concentration orders of magnitude higher than this. A titration of plasma with Zn2+ shows that the fractional increase in free Zn2+ is essentially the same as the fractional increase in total exchangeable Zn2+, and the results are consistent with a model in which essentially all of the Zn2+ in plasma is bound to albumin. Regardless of the model, the buffering capacity of plasma for free Zn2+ is intrinsically low; however, its capacity relative to the total (exchangeable) Zn2+ present is maximal. The implications of this type of buffering for homeostasis of plasma Zn2+ are considered. Treatment of plasma with a strong reducing agent such as dithiothreitol (0.1 mM) substantially increases the apparent binding of Zn2+ and thus reduces the free Zn2+ concentration. However, the concentration of free Zn2+ appears to be insensitive to decreases in the physiological concentrations of reduced glutathione and cysteine. The concentrations of free Zn2+ and free Mg2+ in plasma are similar to those that have been reported for muscle tissue (rabbit). Their ratio is about 4 X 10(-7). The physiological implications of these concentrations are considered. In some cases, if the Zn2+ and Mg2+ complexes of an uncharacterized vertebrate protein exhibit significantly different properties, their relative importance under physiological conditions can be approximated by evaluating those of the mixed complexes present in a solution that contains the physiological concentration of free Mg2+, plus Zn2+ buffered with histidine, at the appropriate pH and ionic strength. Other metal ion/chelon systems that come close to reproducing the concentrations of free Mg2+ and free Zn2+ in horse plasma also are considered.