Automated methods for optimal rapid quantitation of the levels of 29 enzymes in the liver and brain of CS7BU6J mice using a miniature fast analyzer (MFA) are described. Enzyme assays were developed by varying reagent concentrations and pH until we obtained the highest activities with the lowest coefficient of variation for the 29 selected enzymes while maintaining linearity with protein concentration and time. Our techniques should be readily adaptable to the commercial prototypes of the MFA and other automated assay systems, for other strains or species, and a variety of applications such as the study of dietary effects or other types of perturbations or experimental manipulations on certain enzyme pathways. Using the described methods, we define the mean normal activity of 29 enzymes as well as the associated statistical parameters used to define these ranges of activity in C57BL16J populations. These values are in turn useful in the identification ofabnormal activities during routine analysis. The methods described are presently being incorporated into a mutagenesis test system to detect chemically induced mutations expressed as altered enzyme activity.
' Abbreviations used: MFA, miniature fast analyzer; DlT, dithiothreitol; C.V., coefficient of variation. zyme activity coupled with the lowest coefficient of variation was determined by varying the reagent concentrations and the pH while maintaining a linear response of rate versus time for a variety of protein concentrations.
Methods
Animals. C57BL/6J mice, obtained from Jackson Laboratory (Bar Harbor, Me.), were established as a breeding colony at the National Center for Toxicological Research (Jefferson, Ark.). Mice were maintained on 5010-C Mouse Chow (Ralston Purina Co., St. Louis, MO.) and hyperacidified water (pH 2.5) ad libitum (7). Mice (10 weeks of age) were fasted 4 h and killed by cervical dislocation after light ether anesthesia. Individual brains and livers were excised, rinsed in distilled HzO, quickly frozen in liquid Nz, and stored at -60°C.