Enzymatic determination of total cholesterol in serumA simple, enzymatic method for the determination of total cholesterol in serum is described. All cholesterol esters in the serum are split quantitatively into free cholesterol and fatty acids by cholesterol esterase. In the presence of oxygen, the free cholesterol is converted by cholesterol oxidasc into A 4 -cholestcnone. The hydrogen peroxide produced in this reaction oxidizes methanol to formaldehyde in the presence of catalase. The formaldehyde reacts with ammonium ions and acetylacetone to form 3,5-diacetyl-l,4-dihydrolutidinc, which is determined colorimetrically at 405 nm. The accuracy and precision of this method are very good. Proportionality is achieved up to 25.86 mmol cholesterol/1 serum. Serum proteins, bilirubin, creatinine, hemoglobin and drugs do not interfere.
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.
To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.
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