Neurosteroids: pregnenolone in human sciatic nerves.

Abstract

The characterization and quantification of pregnenolone in human sciatic nerves were undertaken, following previous demonstration of the synthesis of this steroid in rat brain oligodendrocytes, to explore the hypothesis that Schwann cells may demonstrate the same biosynthetic activity. Pregnenolone was definitively identified by mass spectrometry and quantified by specific radioinmunoassay. Its concentration (mean ± SD, 63.9 ± 45.9 ng/g of wet tissue, n = 12) was 2100 times the plasma level and concentration found in tendons and muscle. No correlation was found with sex or age. Free dehydroepiandrosterone as well as sulfate and fatty acid esters of pregnenolone and dehydroepiandrosterone were also measured. Results are discussed in terms of the concept that these "neurosteroids" may be synthesized in the peripheral nervous system. Pregnenolone (3(3-hydroxy-A5-pregnen-20-one) accumulates in the rat brain independently of endocrine gland function, and its synthesis from cholesterol in oligodendrocytes has been demonstrated (1, 2), justifying the term "neurosteroid" (3). Measurements by radioimmunoassay have indicated that pregnenolone is present also in human brain and cranial nerves at levels similar to those in the rat (i.e., -0.1 ,umol/g of wet tissue) (4-6). Since (wt/vol) KOH for 1 hr at 80°C followed by 12 hr at room temperature. To measure the sulfated steroids present in the aqueous phase after isooctane/ethyl acetate extraction, solvolysis was performed in three cases as follows: the aqueous phase, once brought to pH 1 with sulfuric acid and made 20%o-NaCl, was extracted three times with ethyl acetate and the pooled extracts were incubated at 37°C for 16 hr. Further processing was carried out according to Burstein and Lieberman (9). Free steroids recovered in each of the free, saponified, and solvolyzed fractions were separated by partition chromatography on celite microcolumns before radioimmunoassay (4, 10). The addition of 3H-labeled steroids allowed appropriate corrections to be made for losses during the isolation procedures.Radioimmunoassays. Pregnenolone and DHEA and their respective sulfate and fatty acid esters were measured by radioimmunoassays using specific antibodies (5, 11). Measurements were made in triplicate on three dilutions of the fractions, thus allowing the construction of regression lines (measured picograms versus dilution) whose slopes gave the concentrations of steroids in the samples. Results were expressed as nanograms of steroid (free or released from conjugates) per gram of tissue, and standard deviations were calculated.Gas Chromatography-Mass Spectrometry (GC-MS). Unconjugated pregnenolone from sciatic nerves was identified by GC-MS. After individual measurements by radioimmunoassay of the fractions from the celite partition chromatography, the remaining unconjugated pregnenolone from three The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C...

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

“…Its mean concentration (64 ± 46 ng/g) was about two orders of magnitude higher than levels in blood, tendons and muscle. No correlation was found with sex or age (Morfin et al, 1992).…”

Mass spectrometric assay and physiological–pharmacological activity of androgenic neurosteroids

“…Similarly, de novo synthesis of neurosteroids in the human brain is supported by the recent reports showing the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) type 1, which catalyzes conversion of pregnenolone into progesterone (Lanthier and Patwardham, 1986;Morfin et al, 1992;Bixo et al, 1997;Beyenburg et al, 1999;Stoffel-Wagner, 2003). Moreover, multiple isoforms of 3β-HSD are capable of exhibiting the same activity but differ by their affinity to the substrates, their optimal pH and temperature as well as by their tissue specific expression Inoue et al, 2002;Yu et al, 2002).…”

Immunohistochemical Study of 3.BETA.-Hydroxysteroid Dehydrogenase /.DELTA.5-.DELTA.4 Isomerase in the Rat Cardiovascular System.

“…Threef-HSD activity has been found not only in steroidogenic but also in various other tissues including nervous and cardiovascular organs (LE GOAS-COGNE et al, 1987;JUNG-TESTAS et al, 1989;ZHAO et al, 1991;MORFIN et al, 1992;AKWA et al, 1993;MARTEL et al, 1994;KOENIG et al, 1995;SANNE and KRUEGER, 1995). There have been many papers reporting that a major adrenal steroid hormone DHEA and sex steroid levels are strongly associated with cardiovascular diseases (ELDRUP et al, 1987;LACROIX et al, 1992;EICH et al, 1993;SLOWINSKA-SKZEDNICKA et al,1995).…”

“…Pregnenolone converts into progesterone in the brain and sciatic nerve (JUNG-TESTAS et al, 1989;MORFIN et al, 1992;AKWA et al, 1993;KOENIG et al, 1995). The progesterone synthesized within both central and peripheral nervous systems has been suggested to regulate neurotransmission and to be related to myelination (KOENIG et al, 1995;BAULIEU et al, 1996).…”