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Using isotopic dilution techniques, certain metabolic properties of cholesteryl 3/3-sulfate [cholesterol sulfate (CS)], a substance that has recently been isolated from human blood and adrenal tissue, were studied in two normal human subjects. Thus, the concentration (c) of circulating CS" in the plasma was found to be 328 and 924 jug/100 ml. Following the injection of [14C]CS, the metabolic clearance rate (MCR) of this compound was determined from the disappearance curve of the tracer from plasma. From these data, the half-life was calculated to be 14-15 hr in these subjects. The production rate of cholesterol c X^>holesterol sulfate (CS)* 1 *has recently been shown by Drayer et al. (1964) to be a naturally occurring entity. This conjugate has been found in bovine adrenal tissue, human blood (Drayer et al., 1964), and adrenal tumor tissue (N. M. Drayer, unpublished data). Recently, Moser et al. (1966) also isolated this sulfate from natural sources. That CS may serve, too, as a substrate for the mitochondrial side-chain cleaving enzyme of bovine adrenal tissue has been described by Raggatt et al. (1965). Moreover, Roberts et al. (1964) perfused CS through a human adrenal adenoma, in situ, and the results suggest that CS was converted to a variety of A5-3/3-sulfated steroids via intermediates which retain the sulfate grouping. The purpose of the present study was to determine the plasma concentration of CS, the rate at which blood-borne CS is produced (PRcs), and the quantitative importance of circulating CS as a precursor of steroids. Values for the metabolic clearance rate, MCR, were obtained by injecting 14C-labeled CS intravenously and measuring the concentration of radioactivity present as CS in plasma samples withdrawn at various intervals fol-

Bovine adrenal cortical tissue contains a lipoidal derivative of pregnenolone (3#-hydroxypregn-en-20-one) from which.the free steroid can be liberated by treatment with alkali. Evidence for the presence of such an entity comes from examination of a nonpolar extract of tissue from which pregnenolone and its sulfate had been removed by chromatography. Treatment of the nonpolar fraction with alkali followed by exhaustive chromatographic analysis led to the detection of pregnenolone. The steroid was identified by both gas chromatography/mass spectrometry and double isotope procedures. Quantitative analysis indicated that the three forms of pregnenolone are present in bovine adrenal cortical tissue in the following amounts (jtg/kg): lipoidal derivative, 290; free steroid, 435; and sulfate, 65. Because the only known metabolic function of pregnenolone is to serve as a precursor of the steroid hormones, these findings have far-'reaching implications for steroid hormone biochemistry. This paper describes experiments that indicate that there is present'in bovine adrenal cortices a lipoidal substance that yields pregnenolone (3fl-hydroxy-pregn-5-en-20-one) upon treatment with alkali. Adrenals have long been known to contain pregnenolone and pregnenolone sulfate, but we now present conclusive evidence for the existence of a nonpolar steroidal derivative which, upon alkaline treatment, leads to the liberation of pregnenolone. This C21-steroid, as is well known, serves as a common precursor for all the steroid hormones. Although it is not now possible to assess what significance the lipoidal derivative of pregnenolone has, its existence is noteworthy and accounts for the fact that this report is being made before its exact structure has been determined.We were led to search for such a lipoidal form of pregnenolone by the recent observation (1) that acyl esters of cholesterol, as well as some inorganic esters of that sterol, such as the nitrate or phosphate, can serve as substrates for the cholesterol sidechain cleavage enzyme present in adrenal mitochondria. When cholesterol nitrate, cholesterol acetate, or cholesterol caproate was used as substrate for this enzyme, the corresponding pregnenolone ester was found to be the product. These results suggested that the side-chain cleavage enzyme was unable to distinguish between cholesterol derivatives that differ from each other by the presence of various carboxylic and inorganic acid esters at C-3.These findings recalled some observations that were made almost a decade ago and whose full significance still remains unexplained. During a search for intermediates that might be involved in the conversion of cholesterol into pregnenolone, we reported the isolation of (20S)-20-hydroxycholesterol from adrenal extracts (2). In that study, fractions that were assumed to contain the fatty acid esters of cholesterol were processed in a manner that would permit us to explore the possibility that an esterified form of (20S)-20-hydroxycholesterol was also present in these extracts....

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