Five steroids-38-hydroxypregn-S-en-20-one (pregnenolone; P), 3fi-hydroxy-5a-pregnan-20-one (3.3-AP), 3a-hydroxy-5a-pregnan-20-one (3a-AP), 3.-hydroxyandrost-5-en-17-one (dehydroepiandrosterone; D), and 3fi-hydroxy-5a-androstan-17-one (EpiA)-were extracted from the brains of adult male rats, rabbits, and dogs. The steroids exist in this organ as uncoajugated compounds and as sulfates, lipoidal esters, and sulfolipids. The techniques for separating these four classes ofsteroids from each other and for separating the five steroids from each other are described. In all cases, the steroids were identified by their retention time (Re) on HPLC, theirRt by gas chromatography, and by selected ion monitoring of their mass spectra. The latter were also used for quantification. In their reaction toward organic bases, the sulfolipid conjugates resemble previously described sulfolipids of cholesterol and sitosterol. These conijugates are relatively abundant in brain, particularly those of P and D, and this suggests that, in the search for the physiological significance of these brain constituents, these conjugates warrant attention.also seem to indicate that the above-mentioned steroids may be present in brain in amounts somewhat larger than those previously reported. Furthermore, until now only the sulfate and lipoidal esters of both P and D have been detected in the brain. Our findings reveal that another steroidal conjugate(s), still to be characterized, is also present. In particular, evidence is given here that indicates that steroidal conjugates comparable in nature to those that we previously described (11) for cholesterol and sitosterol are also present in substantial amounts in brain. We have designated this conjugated form a sulfolipid, with the possible structure StO-SO2-OL (where St is a steroid and L is a lipid moiety), because this nonpolar conjugate, when treated with an organic base such as triethylamine, can cleave to give either a product that behaves like the sulfuric acid ester of a steroid or an unconjugated steroid, like P. Although their exact structures have yet to be determined, the abundance of these conjugates in brain suggests that their relationship to brain function warrants corsideration.The most recent facet of steroid hormone biochemistry to evoke intense interest, astonishing activity, and discoveries that presage previously unforeseen scientific advances is that which deals with the relationship between steroids and neuronal-especially brain-function. One ofthe factors that has been responsible for stimulating the interest in this chapter of biochemistry has been the detection of steroids in brain (neurosteroids) by Baulieu and his colleagues (1). Another impetus for the development was the finding of Majewska et al. (2) that certain steroid derivatives are potent modulators of the Cl-channel associated with the type A receptor for y-aminobutyric acid. More recent findings have shown that other ion channels, particularly those associated with N-methyl-D-aspartate receptors, may also ...

In addition to the neurosteroids pregnenolone and dehydroepiandrosterone, organic solvent extracts of rat brains contain related compounds that react with various reagents to yield additional amounts of these ketosterolds. Among the chemicals producing these increments are triethylamine, HCI, FeCl3, and Pb(OAc) In a recent paper (1), estimates of the concentrations of five steroids and their conjugates in mammalian brains were recorded. Also described were the techniques used for the separation, identification, and quantification of these socalled neurosteroids. An unusual feature of the reported findings was the relatively high concentrations of steroidal conjugates, which were designated "sulfolipids." Following from our previous experience with analogous conjugates of cholesterol (2), we found that there appear to exist in mammalian brain nonpolar conjugates of neurosteroids, particularly pregnenolone (3f3hydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3f-hydroxy-5-androsten-17-one).These, when treated with an organic base such as triethylamine or pyridine, were converted to a steroid sulfate that could be identified and quantified (after acid hydrolysis) as the free steroid. Some of these conjugates appeared to be present in relatively high concentrations in brain, suggesting that these conjugates play a physiological role in the nervous system.The experiments described in this paper bear on these substances. More The second part was separated into its ketonic and nonketonic (part B) components by treatment with polystyrenebenzyloxime resin to remove carbonyl compounds (8), including the endogenous ketosteroids and their derivatives. Sample B was dissolved in 20 ml of benzene containing 2 ml of acetic acid and a bolus of 200,000 dpm of tritiated pregnenolone. The oxime resin (100 mg) was added, and the suspension was stirred for 2 hr at room temperature. A check of the tritium content of the supernatant revealed that >90% of the tritiated steroid had bound to the resin. The reaction was allowed to proceed to Abbreviation: P450scc, side chain-cleaving P450.

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A simple and precise assay is described for measurement of the enzymatic conversion of cholesterol into the C21 steroid, pregnenolone. This procedure determines quantitatively the amount of the isotopically labeled C6 fragment (isocaproic acid) formed from cholesterol, when the sterol substrate bears a radioisotope, 14C or 3H, in the side chain. The method is based upon the fact that the substrate is separated from the C6 product by percolation through a T Ahe biosynthetic processes, by which steroid hormones are made from cholesterol, all begin with cleavage of a C6 fragment from the side chain of the sterol. Most of the other