No abstract

We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.

Summary:A fully enzymatic assay is described for the determination of triglycerides. The coupled activities of triacylglycerol acylhydrolase and glycerol kinase result in the formation of glycerol-3-phosphate. The System also contains L-a-glycerol-phosphate oxidase, which produces hydrogen peroxide from glycerol-3-phosphate, and a sensitive chromogenic indicator System, consisting of peroxidase, 4-chlorophenol and 4-aminophenazone. We evaluated this rnethod with respect to kinetics, lineärity, blank rates, precision, accuracy, reagent stability and interfering substances.The accuracy of the triglyceride assay demands that each enzymatic reaction Step be complete and homogeneous. We therefore developed HPTLC-1 ) and HPLC-2 ) methods to monitor the course and completeness of each step. Reagenz zur enzymatischen Bestimmung von Triglyceriden im Serum mit verbesserter lipolytischer WirksamkeitZusammenfassung: Es wird ein vollenzymatischer Test zur Bestimmung Von Triglyceriden beschrieben, dessen Prinzip auf der Freisetzung von Wasserstoffperoxid aus Glycerin-3-phosphat mittels L-a-Glycerinphosphatoxidase in Kombination mit einem empfindlichen Farbindikator-System, bestehend aus Peroxidase, 4-Chlorphenol und 4-Aminöphenazon, beruht. Diese Methode wurde hinsichtlich Reaktionsgeschwindigkeit, Linearitätsbereich, Reagenzleerwert, Präzision, Richtigkeit, Reagenzstabilität und interferierender Substanzen untersucht.Da die Richtigkeit der Bestimmung von Triglyceriden einen vollständigen und eindeutigen Verlauf jedes einzelnen enzymatischen Reaktionsschrittes voraussetzt, wurden zur Verlaufskontrolle der einzelnen Teilreaktiönen HPTLC 1 )-sowie HPLC 2 )-Methpden entwickelt.

No abstract