Evaluation of the thermal stability of clinically relevant enzymes at 37 degrees C.

The thermal stability at 37 degrees C of several clinically relevant enzymes and isoenzymes was assessed by measuring changes in enzyme activity as a function of time under incubation and reaction conditions. Selwyn plots were used in the reaction-condition assessments. Except for CK-1 (BB), all the enzymes investigated are stable enough at 37 degrees C to permit assay. These enzymes were LDH-1, LDH-5, s-AspAT, m-AspAT, apo-s-AspAT, apo-m-AspAT, ALP-liver, ALP-bone, ALP-intestine, ALT, apo-ALT, CK-2, and CK-3. CK-1 is stable at 37 degrees C under assay conditions but not under incubation conditions. We specifically avoided using Arrhenius plots to evaluate thermal stability and point out pitfalls inherent in their indiscriminate use.

Selective inactivation of lactate dehydrogenase isoenzymes with ionic surfactants.

Ionic surfactants selectively inactivate porcine lactate dehydrogenase (LDH) isoenzymes in 30 mM phosphate buffer, pH 7.4. The cationic surfactants hexadecylpyridinium bromide and hexadecyltrimethylammonium bromide rapidly inactivate LDH isoenzymes containing the B subunit; inactivation of LDH-A4 is slower and also retarded by the cofactor reduced nicotinamide adenine dinucleotide. The anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate rapidly inactivate LDH isoenzymes containing the A subunit; inactivation of LDH-B4 is much slower and retarded by the cofactor. The selectivity of the inactivation process correlates with electrostatic interactions: positively charged surfactants preferentially inactivate isoenzymes containing a subunit of net negative charge, and negatively charged surfactants preferentially inactivate isoenzymes containing a subunit of net positive charge. Inactivation takes place near the critical micelle concentration for the cationic surfactants. Inactivation with anionic surfactants occurs above the critical micelle concentration. The cationic surfactants show little discrimination among LDH-B4 and the hybrid isoenzymes, AB3, A2B2, and A3B, inactivating all at approximately the same surfactant concentration. The anionic surfactants, however, show a more graded inactivation-concentration profile with discrete differences in threshold surfactant concentrations required for complete inactivation of the four A subunit containing isoenzymes. At a particular surfactant concentration, loss in activity can be correlated with the percent A- or B-subunit composition of the isoenzyme.

Multilayer film elements for clinical analysis: general concepts.

Dry, thin films containing all necessary reagents for clinical analysis by colorimetry have been designed. Reagents in a matrix of hydrophilic polymer are coated on top of a transparent plastic base. A white isotropically porous polymer spreading layer, 80% void volume, is coated over the reagent layer(s). In the analysis, a drop (typically 10 microliter) of undiluted serum or other fluid is touched to the spreading layer. The fluid spreads rapidly and uniformly through the pore structure, filling a void volume corresponding to the drop volume. Water and low-molecular-weight components diffuse from the spreading layer into the reagent layer(s), initiating the reaction sequence. The spreading layer acts also as a white optical diffuser for reflection densitometry. Optical reflection density is linearized through use of the function developed by Williams and Clapper [J. Opt. Soc. Am. 43, 595 (1953)] to convert reflection to transmission density. A wide variety of chemical assays are compatible with this format. As an example, for the glucose film we found coefficients of variation of 1.5% in predicting glucose concentrations in control sera during 20 days. Results for glucose concentrations in several hundred patients' sera by the present method were very cose to those obtained with the Center for Disease Control's hexokinase reference method.