Seymour Lieberman

Reflections on sterol sidechain cleavage process catalyzed by cytochrome P450scc

The essay examines the evidence upon which the presently accepted version of the mechanism of the cytochrome P450(scc)-catalyzed-cleavage of the sidechain of cholesterol is based. This analysis indicates that the generally held view of the process (two consecutive hydroxylations, followed by cleavage of the resulting glycol) most likely does not describe the true mechanism. The available evidence can not be used to support this traditional notion. Two alternative hypotheses are proposed.

17-Hydroxylase: an evaluation of the present view of its catalytic role in steroidogenesis

This survey analyses the evidence that has led to the belief that the catalytic role of 17-hydroxylase in the biosynthesis of cortisol, estradiol, testosterone and dehydroepiandrosterone is confined to two chemical reactions: pregnenolone-->17-hydroxypregnenolone-->dehydroepiandrosterone. This analysis suggests that the evidence supporting this view is not compelling enough to accept it unquestioningly. Different interpretations of the data can suggest other catalytic roles for 17-hydroxylase that are worthy of consideration. One such alternative is proposed.

An Abbreviated Account of Some Aspects of the Biochemistry of DHEA, 1934–1995

I propose to discuss some features of the biochemistry of dehydroepiandrosterone (DHEA) which I believe are incomplete. Attention needs to be given to these issues particularly in this volume because they may be relevant to some of the subjects that will be presented. DHEA was first isolated in 1934 from urine by Butenandt and Dannenbaum.’ Even at the very beginning the haze that envelops our understanding of the significance of this compound was evident. In this study DHEA itself was not isolated; the substance isolated was its 3-chloro derivative. The chlorine-containing substance was immediately recognized as an artifact produced from DHEA by boiling the urine with HCI. What was not known until 1944 was that the most probable precursor of the chloro compound was the conjugate, dehydroepiandrosterone-3-sulfate (DHEAS) which was isolated from urine by Munson, Gallagher, and Koch2 in 1944. In 1954 Migeon and Plager3 showed that complete extraction of DHEA from human plasma could be achieved only after acid solvolysis, and later in 1959 Baulieu4 showed that DHEAS was also the form that was most abundant in plasma. In the early years, confusion reigned even about the name of this compound. Butenandt and Dannenbaum’ first named the compound dehydro-androsterone. Ruzicka et al.,5 who synthesized the compound by degradation of cholesterol, called it trans-dehydroandrosterone. Fieser,h in the first edition of his famous 1936 book, “Chemistry of Natural Products Related to Phenanthracene,” named it “dehydroisoandrosterone.” Between the years 1936 and 1949, the biochemical and endocrine communities almost always used this designation. In 1949 Fieser, in the third edition of his book, declared that the compound should be dubbed dehydroepiandrosterone. Although both names, dehydroisoandrosterone and dehydroepiandrosterone, were trivial and the distinction between the two was minimal, the influence of Fieser at that time was dominant and the community generally accepted the new name. Ten years later, Fieser and Fieser’ recommended still another name for this compound. In their classic book, “Steroids,” published in 1959 they suggested the name “androstenolone.” This time the new name did not prevail, and dehydroepiandrosterone retained acceptance. To return to the conjugates of DHEA, its sulfate, DHEAS, is indicated in FIGURE 1 by the structure at the bottom left. R stands for the steroid moiety. At the top are shown the structures of the glucuronidate and the N-acetyl glucosaminidate. At the bottom right is the structure of the hypothetical and contentious sulfatide proposed by OerteL8 In the middle 1960s Ocrtel claimed that DHEA exists in tissue as a lipophilic derivative composed of the steroid sulfate esterified to a diacyl glycerol residue. The steroid moiety of one of Oertel’s sulfatides was DHEA. During the

The detection of 20(S)-hydroxycholesterol in extracts of rat brains and human placenta by a gas chromatograph/mass spectrometry technique

The presence of 20(S)-hydroxycholesterol in rat brains and human placenta has been established using the gas chromatography/mass spectrometry (GC/MS) select ion monitoring (SIM) technique. Identification was ensured by three criteria: the specific retention time when the compound emerges from the gas chromatogram and the two m/z ions (201 and 461amu) which are characteristic of its mass spectrum. The possible role of 20(S)-hydroxycholesterol in steroid hormone biosynthesis and in other biological processes is discussed.

Other conceivable renditions of some of the oxidative processes used in the biosynthesis of steroid hormones

The generally accepted version (GAV) of the chemical processes by which the steroid hormones are biosynthesized cannot be considered to be an inerrant description of in vivo processes. Customarily this version is derived by piecing together the results obtained from several independent artificial in vitro incubation experiments. Extrapolation of such results from in vitro to in vivo requires untested assumptions which introduce varying degrees of uncertainty. In vitro incubation experiments reveal only what is possible; not what actually prevails in situ. Presented here are hypothetical alternative renditions of some of the oxidative processes involved in steroidogenesis. These versions suggest that some cytochrome P-450s catalyze the introduction of both oxygen atoms of dioxygen into an appropriate sterol precursor. The products are conceived as oxygen free radicals (peroxy or 1,2-cyclic peroxy) which serve as the reactive intermediates (the precursors) for the hormones. The true intermediates are not stable, isolable, hydroxylated compounds as they are customarily portrayed in the GAV. Central to these new renditions is the hypothesis that the appropriate P-450 introduces dioxygen into the precursor yielding either: A, a 20 peroxy sterol species or B, a species oxygenated at both C-17 and C-20 or C, a species oxygenated at both C-20 and C-21. In this hypothesis, A would serve as the precursor for progesterone, B, for the C19-androgens and C18-estrogens and C, for the mineralocorticoids (corticosterone and aldosterone) and the glucocorticoid (cortisol). How this version of steroidogenesis can be used to understand the etiologies of various genetically derived enzyme deficiency diseases of the adrenal and ovaries will be discussed. If as proposed here, the various polyfunctional cytochromes (P-450(scc), P-450(c17,) P-45011B1 (P-450(cortisol)), P-45011B2 (P-450(aldo)), etc.) catalyze conversions that are different from simple hydroxylations, the labels usually given these deficiency diseases may not be appropriate. More importantly, these new conceptions may clarify the etiology of some of the characteristic symptoms of these diseases that are not now adequately explained by the GAV.

New assumptions about oxidative processes involved in steroid hormone biosynthesis: is the role of cytochrome P-450-activated dioxygen limited to hydroxylation reactions or are dioxygen insertion reactions also possible?

The traditional conception of the chemical pathways leading to the formation of the steroid hormones is derived by piecing together the results of several independent in vitro incubation experiments. The results of these experiments have led to the assumption that some relevant cytochrome P-450s (P-450scc, P-450arom, P-450aldo, etc.) are polyfunctional and catalyze several successive hydroxylation reactions, which lead to the formation of the hormonal products. This essay offers an alternative view. It advances the suggestion that the oxygenated intermediates in the relevant biosynthetic conversions are reactive species that are formed by addition of both atoms of dioxygen onto two neighboring carbon atoms of steroidal precursors. Space-filled Stuart molecular models, generated by a computer program, suggest that the oxidized intermediates resemble hydroperoxides or cyclic peroxides (1,2-dioxanes). For the aromatization process required for estrogen biosynthesis, the atoms of dioxygen are bonded to C-2 and C-19 of the C19-precursor. For aldosterone formation, dioxygen is bonded to C-11 and C-18 of an appropriate precursor. Moreover, the results obtained from a computer program that provides information about molecular mechanics (bond angles and bond distances as well as total potential energies for each conformation of a molecule) suggest that consideration be given to the possibility that cortisol also can be biosynthesized by P-450-activated dioxygen addition to C-11 and C-17 of an appropriate precursor. Neither the traditional view of steroidogenic pathways nor the suggestions advanced here have been established by compelling experimental findings. Both hypotheses are saddled with untested assumptions, which are necessary because the dynamic processes can only be discerned by indirect means. The origins of some naturally occurring steroids hydroxylated at C-17, C-18 and C-19 are examined in the light of the suggestions made in this essay.