Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

This chapter contains section titled: The Relationship of Urinary Steroid Metabolites to Their Hormonal Precursors References Discussion

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-450's 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-450scc, P-450c17, P-45011B1 (P-450cortisol), P-45011B2 (P-450aldo), 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.

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-450's (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.

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 461 amu) 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.

The steroid hormones in addition to their function in sex physiology have important roles in the regulation of carbohydrate, protein, and electrolyte metabolisms. Although abnormalities involving the steroids are known to be present in certain diseases, the exact role of these disturbances in the etiology and manifestations of these diseases has not been established, because, hitherto, the methods which have been available have not been satisfactory for exploring the details of steroid metabolism. In the course of a comprehensive study which has been in progress in the laboratories of the Memorial Hospital since 1940 (l-5), more adequate procedures have been developed and have been utilized in the investigation of the steroids excreted in the urine of human beings in health and in sickness. The results will be reported in a series of communications, of which this, the first, describes the methods developed for the isolation of the neutral steroids from urine and for their quantitative measurement. Subsequent reports will include the characterization of the ketosteroids isolated (6), the application of infra-red spectroscopy to their analysis and identification (7), and the descriptions of the qualitative and quantitative patterns of t.he steroids excreted by normal individuals and by patier1t.s with various disorders including neoplastic growth. Such comparisons have been made possible by the application of standardized procedures involving the methods herein described to the urine specimens of a large number of individuals.

Desmosine (D) and isodesmosine (I), the intramolecular crosslinking amino acids that occur in chains of elastin, have now been found in free form in human urine. Until now, these amino acids (Mr = 526) were found to occur in urine only as higher molecular weight (Mr = 1,000–1,500) peptides. Thus, the previously used analytical methods required, as the first step, acid hydrolysis of the urine at elevated temperature to liberate D and I from their peptides. The analytical method described here uses HPLC followed by electrospray ionization MS for the detection and quantitation of free D and I in unhydrolyzed urine. Identities of both D and I were established by their retention times on LC and by their mass ion at 526 atomic mass units, characteristic of each compound. The sensitivity of the method is 0.10 ng. The average values of free D and I in the urine of seven healthy subjects were 1.42 ± 1.16 and 1.39 ± 1.04 μg/g of creatinine, respectively. After acid hydrolysis of the urine, the amounts of D and I were 8.67 ± 3.75 and 6.28 ± 2.87 μg/g of creatinine, respectively. The method was also successfully used to measure peptide-bound D and I levels in the sputum of patients with chronic obstructive pulmonary disease.

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 chamical 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.

The isolation from human urine of Ag-etiocholenolone (III) (Fig. 1) and Ag-androstenolone (IV), both derived by dehydration of the adrenocortical metabolites 11-hydroxyetiocholanolone (I) and 11-hydroxyandrosterone (II), is described in this report. Since the presence of ll-hydroxyetiocholanolone has been established in a very high proportion of patients with neoplastic disease and is very rarely found in normal subjects (5-8), this investigation has a very significant bearing on cancer. The presence of this unusual steroid could easily have been overlooked, were it not for the systematic application of the powerful tool afforded by infra-red spectrometry. The investigation, therefore, is reported as an application of a methodical procedure to the elucidation of the chemical structure of the compounds. The more purely clinical aspects of the problem have been published elsewhere (5-8). Infra-red spectrometry permits the positive identification of a compound by comparison of the spectrum with that of a known, pure substance (9, 10). For this purpose, it is not necessary that the substance be obtained in crystalline form, since, indeed, non-crystalline eluates from a chromatogram often show spectra identical with those obtaiqed from authentic samples. Prior to the use of infra-red spectrometry, it was necessary to depend upon the more usual criteria of identity, such as the constancy of properties after repeated chromatography, melting point of a pure sample and of mixtures, rotation, and the like, together with the preparation of suitable derivatives. By these means we, as well as others, have isolated and characterized a number of urinary steroids, including androsterone and etiocholanolone, two of the more abundant ketosteroids.

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.

Dehydroepiandrosterone (D) is biosynthesized in the brain by a pathway different from that existing in the adrenal cortex. C6 rat glioma tumor cells in culture biosynthesize both pregnenolone (P) and D. They possess the mRNA, protein, and side-chain cleavage activity of P450scc. On the other hand, P450c17 was not detected. Adding FeSO4 to C6 cells increased the synthesis of both P and D. Even in the presence of aminoglutethimide, an inhibitor of P450scc, FeSO4 increased the synthesis of both steroids, indicating that the Fe2+-sensitive process does not involve P450scc. Likewise, the FeSO4-induced formation of D was not blocked by the P450c17 inhibitor, SU-10603. These results suggest that the FeSO4-induced synthesis of D as well as of P in C6 cells may be due to the fragmentation of in situ-formed tertiary hydroperoxides. It is likely, however, that the effect of the Fe2+ is not limited to this one reaction. When exogenous P was added to C6 microsomes, along with FeSO4, the amount of D formed was greater than control values, indicating that Fe2+ facilitated the conversion of P to D. Unlike the constituents that are converted by Fe2+ to P, the precursor of D in C6 cells is not soluble in a 1:1 mixture of ether and ethylacetate. Treatment of C6 cells with KI, NaBH4, or HIO4 resulted in an increase in D synthesis. From this it seems clear that a precursor of the D produced in C6 cells is a steroid where both C-17 and C-20 are oxygenated.

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 ketosteroids. Among the chemicals producing these increments are triethylamine, HCl, FeCl3, and Pb(OAc)4. Most revealing is the action of FeSO4 on these extracts. This reducing agent also converts components of the extract into the two neurosteroids, suggesting the presence of sterol hydroperoxides or peroxides in brain. The clues provided by this study indicate that the chemical nature of the steroidal constituents existing in extracts of mammalian brains remains to be determined. It is likely that their association with neurological functions will be. better understood when the structures of these substances are established.

Five steroids--3 beta-hydroxypregn-5-en-20-one (pregnenolone; P), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-AP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-AP), 3 beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone; D), and 3 beta-hydroxy-5 alpha-androstan-17-one (EpiA)--were extracted from the brains of adult male rats, rabbits, and dogs. The steroids exist in this organ as unconjugated compounds and as sulfates, lipoidal esters, and sulfolipids. The techniques for separating these four classes of steroids 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 (Rt) on HPLC, their Rt 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 conjugates 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.

When incubated with [3H]cholesterol, a bovine adrenocortical mitochondrial pellet obtained by centrifugation at 12,000 x g yielded, as expected, only the C21O2 metabolites progesterone and pregnenolone. However, the steroidogenic potential of the 12,000 x g pellet fraction was augmented significantly by lyophilization. After lyophilization, the 12,000 x g pellet converted the sterol into C19 androgens and corticosteroids, in addition to C21O2 pregnane derivatives. Leaching the lyophilized mitochondrial fraction with either hexane or acetone increased substantially the yields of the metabolites. It did not change qualitatively the array of metabolites formed during in vitro incubation, but 5 alpha-reductase activity was unmasked by the washings, particularly with acetone. Thus, the fraction sedimented at 12,000 x g contains the complete complement of steroidogenic enzymes required for the biosynthesis of the aforementioned adrenal hormones. These results cast doubt upon the widely held belief that the various enzymes required for adrenocortical steroidogenesis are distributed between two different subcellular compartments, the mitochondrion and the endoplasmic reticulum.

Annals of the New York Academy of SciencesVolume 595, Issue 1 p. 1-16 Reconsidering Some of the Biosynthetic Pathways Leading to Formation of C19-Steroids V. V. K. Prasad, V. V. K. Prasad Department of Obstetrics and Gynecology The St. Luke's-Roosevelt Institute for Health Sciences New York, New York 10019 and The College of Physicians and Surgeons Columbia University New York, New York 10032Search for more papers by this authorS. Lieberman, S. Lieberman Department of Obstetrics and Gynecology The St. Luke's-Roosevelt Institute for Health Sciences New York, New York 10019 and The College of Physicians and Surgeons Columbia University New York, New York 10032Search for more papers by this author V. V. K. Prasad, V. V. K. Prasad Department of Obstetrics and Gynecology The St. Luke's-Roosevelt Institute for Health Sciences New York, New York 10019 and The College of Physicians and Surgeons Columbia University New York, New York 10032Search for more papers by this authorS. Lieberman, S. Lieberman Department of Obstetrics and Gynecology The St. Luke's-Roosevelt Institute for Health Sciences New York, New York 10019 and The College of Physicians and Surgeons Columbia University New York, New York 10032Search for more papers by this author First published: June 1990 https://doi.org/10.1111/j.1749-6632.1990.tb34278.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume595, Issue1Steroid Formation, Degradation, and Action in Peripheral TissuesJune 1990Pages 1-16 RelatedInformation

WIDELY accepted dogmas in the life sciences often require revision. Some of these revisions are made necessary not by the subsequent correction of faulty data but rather by the fact that the dogmas are founded on data that, although accurate, do not allow for a single, unequivocal interpretation. In many such instances, the dogma arises when experimental data are considered in the light of contemporary conceptions. These evoke the most obvious explanations which, in turn, with the passage of time become cherished beliefs. This is true even when an alternative and sometimes equally logical explanation of the experimental data provides a different view of the natural situation, one that is no less tenable than the original proposal. Several years ago we examined in some detail the proposition that the true intermediates in the biosynthetic processes leading to the formation of the steroid hormones are enzyme-bound, transient species that proceed from precursor to hormonal products by means of concerted reactions (1).