An Oxygraphie Method for the Determination of Cholesterol and Measurement of the Slow Oxygen-consuming Reactions of Hepatic Microsomes

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Abstract
Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphie method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5nmoles. An oxygraphie method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed β-NADH-dependent oxygen consumption and cyanide-insensitive α-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1 mg protein/ml). Furthermore, the temporary cessation of α-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with α-NADH is due to electron transfer from α-NADH via αNADH-cytochrome b5 reductase and cytochrome b5 in which the rate-determining step lies at some reaction after the reduction of cytochrome b5

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