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.

We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.

The influence of iodoacetamide and N-ethylmaleinimide on the activity of the acetyl transferase component of fatty acid synthetase from baker's yeast was studied using [Wlacetyl-CoA and pantetheine as substrates. No inhibition by either of the blocking agents was observed. This, together with previous information, suggests, that a non-sulfhydryl acceptor group is involved in the catalytic process of acetyl transfer from acetyl-CoA to the acyl carrier protein component of the multienzyme complex.To identify this carrier group in the active site of its acetyl transferase, fatty acid synthetase was treated with [14C]acetyl-CoA after first blocking the free SH-groups of the complex with Ellman's reagent. I n this way a radioactive acetyl-enzyme was formed, which By comparing the amino acid composition of [14C]malonyl-peptides, stable towards performic acid, with the partial sequence of the [14C]acetyl-enzyme it was concluded that acetyl and malonyl transfer reactions are catalyzed by enzyme components of the multienzyme complex, which are distinctly different. I n both cases however, the catalytic mechanisms probably involve the participation of serine residues as acyl carrier groups. 19 Eur.

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