Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.

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.

724 CLINICAL CHEMISTRY, Vol. 20, No. 6, 1974 Enzymatic Determination of Total Cholesterol in Serum To the Editor: For many years, dissatisfaction has been expressed with direct methods for cholesterol (1). Because the Liebermann-Burchard color reaction may yield entities with extinction coefficients for cholesterol that differ from those for its esters, extraction and saponification steps are necessary in the procedure (2, 3). These steps are particularly cumbersome when large numbers of samples are to be analyzed, and make development of new cholesterol methodology highly desirable. We have been developing an enzymatic method fortotalcholesterol in serum that would eliminate the extraction steps as well as provide overall enhanced conveniences and rapidity in the analysis. A recent publication by Richmond (4) describing a cholesterol assay in which cholesterol oxidase is used prompted us to communicate our approach, which we believe to be significantly better. The Richmond technique (4) requires incubation at two different temperatures and the ordered addition of five separate reagents, one of which involves saponification of the serum cholesterol esters with alcoholic potassium hydroxide. After saponification, cholesterol oxidase (EC 1.1.3.6) is used to produce hydrogen peroxide which, in turn, is developed into a colored product. Our approach, in contrast, is completely enzymatic and is based on the following sequence of reactions:

This chapter highlights some methods previously used for determining l-aspartate and l-asparagine and the difficulties found in these methods. The chapter discusses a method for the enzymatic determination of L-aspartate and L-asparagine that can be applied in biochemistry, microbiology, clinical chemistry, and foodstuff chemistry. In the method, the decrease of the extinction due to NADH at 340, 334, or 365 nm is proportional to the aspartate concentration. The addition of asparaginase to the assay system leads to a further decrease of the NADH concentration and this gives the asparagine content. The chapter discusses the optimum conditions for measurements, the equipment, and the reagents used in the method. It also presents a list of the solutions used in the method and their preparation procedure. The chapter further reviews the specificity, accuracy, and precision of the method.

Eine einfache enzymatische Methode zur Bestimmung des Gesamt-Cholesterins im Serum wird beschrieben. Die im Serum vorhandenen Cholcsterinester werden mittels Cholcstcrinesterase quantitativ in freies Cholestcrin und Fettsauren gespalten. Das freie Cholesterin wird in Gegenwart von Sauerstoff und Cholesterinoxydase in A-Cholestenon umgewandelt. Das bei dieser Reaktion entstehende Wasserstoffperoxyd oxydiert bei Anwesenheit von Katalase Methanol zu Formaldchyd. Dieses reagiert mit Ammoniumionen und Acetylaceton unter Bildung von 3,5-Diacctyl-l,4-dihydrolutidin, dessen Farbintcnsitat bei 405 nm gemessen wird. Richtigkeit und Prazision der Methode sind sehr gut. Die Proportionalitat ist bis 25,85 mmol Cholcsterin/1 Serum gegeben. Serumprotcine, Bilirubin, Creatinin, Hamoglobin und Pharmaka storen nicht.

Es werden enzymatische Methoden zur quantitativen Bestimmung von Acetacetat und D-(-)-3-Hydroxybutyrat im Blut beschrieben, die sich für Routine-Analysen eignen. Vorläufige Normal-Werte und das Verhältnis 3- Hydroxybutyrat : Acetacetat wurden für Gesunde und eingestellte Diabetiker ermittelt.