67 Relationships of circulating pregnanolone isomers and their polar conjugates to the status of sex, menstrual cycle, and pregnancy Radmila Kancheva, Martin Hill, David Cibula1, Helena Vcˇela´kova´, Lyudmila Kancheva, Jana Vrbı´kova´, Toma´sˇ Fait1, Antonı´nParˇ´ızek1 and Luboslav Sta´rka Institute of Endocrinology, Na´rodnı´trˇı´da 8, CZ 116 94 Prague 1, Czech Republic 1Department of Gynecology and Obstetrics, 1st Medical Faculty, Charles University, Apolina´rˇska´ 18, CZ 128 51 Prague 2, Czech Republic (Requests for offprints should be addressed to M Hill; Email: [email protected]) Abstract Pregnanolone isomers (PIs) and their polar conjugates (PICs) found in P indicate the more intensive conjugation of 5a-PI modulate ionotropic receptors such as g-aminobutyric acid during pregnancy. This mechanism probably provides for the or pregnane X receptors. Besides, brain synthesis, PI elimination of neuroinhibitory P3a5a in the maternal penetrates the blood–brain barrier. We evaluated the compartment. Additionally, our result points to a limited physiological importance of PI respecting the status of sex, sulfation capacity for neuroinhibitory P3a5b in P. In contrast menstrual cycle, and pregnancy. Accordingly, circulating to the situation in M, F, and L where the P3a5bC is the most levels of allopregnanolone (P3a5a), isopregnanolone abundant PIC, and P3a5b is present in minor quantities (P3b5a), pregnanolone (P3a5b), epipregnanolone (P3b5b), compared with the P3a5a,P3a5b may acquire physiological their polar conjugates, and related steroids were measured in importance during pregnancy, contributing to the sustaining 15 men (M), 15 women in the follicular phase (F), 16 women thereof. On the other hand, the declining formation of in the luteal phase (L), and 30 women in the 36th week of P3a5b may participate in the initiation of parturition, given gestation (P) using GC–MS. The steroid levels were similar in the relative abundance of the steroid, its potency to suppress M and F, increased about thrice in L and escalated in P (38– the activity of oxytocin-producing cells and its effectiveness in 410 times compared with F). The PICs were prevalent over uterine relaxation. the PIs (16–150 times). Higher ratios of 5a-PIC to 5a-PI Journal of Endocrinology (2007) 195, 67–78 Introduction 1997, Corpechot et al. 1997). Although several studies have brought information to light regarding the levels of PI in Reduced progesterone metabolites, including pregnanolone humans, only a small number have included data regarding isomers (PIs) and their polar conjugates (PICs), are efficient the respective polar conjugates (Mickan & Zander 1979, Hill neuromodulators. They are effective on neurotransmitter et al. 2000, Havlikova et al. 2006). receptors influencing the permeability of ion channels In addition to the action on GABAA-R, the 5b-PI block (Majewska 1990). PIs with a hydroxy group in the 3a- type T calcium channels in rat peripheral neurons; these position shorten the period of paradoxical sleeping, attenuate channels play a significant role in pain perception (Todorovic the release of acetylcholine in the neocortex and hippo- et al. 2004). The 5b-PI also binds to nuclear progesterone campus, suppress neurogenesis, and deteriorate spatial receptors (Putnam et al. 1991). It has recently been reported memory. These effects are provided via modulation of the that 5b-PI may act chronically through a mechanism receptors of type A g-aminobutyric acid (GABAA-R; mediated by pregnane X receptors to regulate uterine Darnaudery et al. 1999). The 3b-PI competes with 3a-PI contractility (Mitchell et al. 2005). The 5a- and 5b-PIC are for the binding sites on GABAA-R (Prince & Simmonds effective as positive and negative modulators of N-methyl- 1992, Wang et al. 2002, Lundgren et al. 2003). Conjugation D-aspartate receptors (NMDA-R) respectively (Park-Chung counteracts the effect of 3a-PI, and further amplifies the et al. 1997, Weaver et al. 2000). The activation of NMDA-R antagonistic effect of the 3b-PI on GABAA-R. For instance, in hypothalamic magnocellular neuroendocrine cells of the the GABAA-R inhibiting efficiency of 3b-hydroxy-5a- supraoptic nucleus may induce oxytocin production (Pak & pregnane-20-one sulfate is comparable with the GABAA-R Curras-Collazo 2002), resulting in onset of parturition. activating effectiveness of allopregnanolone (P3a5a). Some PIs originate from progesterone (Prog) through the action reports indicate that in addition to the brain’s in situ synthesis, of the ubiquitous 5a- and 5b-reductase, which is most active circulating PIs penetrate the blood–brain barrier (Bixo et al. in the liver (Ingelman-Sundberg 1976). Both enzymes show Journal of Endocrinology (2007) 195, 67–78 DOI: 10.1677/JOE-06-0192 0022–0795/07/0195–067 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 09:36:04PM via free access 68 R KANCHEVA and others . Importance of pregnanolone isomers significant activity in the tissues associated with pregnancy transported to the maternal compartment via the placenta – (Lisboa & Holtermann 1976, Milewich et al. 1979, Sheehan where they are again converted into PI (Dombroski et al. et al. 2005); they catalyze the formation of 5a- and 5b- 1997). The levels of all PI in pregnancy are strikingly higher dihydroprogesterone (P5a and P5b)respectively.The in comparison with the situation in non-pregnant women subsequent metabolism of dihydroprogesterone epimers to (Genazzani et al. 1998, Hill et al. 2000, Luisi et al. 2000, individual PI is catalyzed by stereospecific 3a-or3b- Pearson Murphy et al. 2001, Havlikova et al. 2006). hydroxysteroid oxidoreductases (Okuda & Okuda 1984, The aim of this study was to evaluate the physiological Melcangi et al. 1994, Corpechot et al. 1997, Ottander et al. impact of endogenous neuroactive PI and PIC in humans, 2005). The latter enzyme may be identical to the 3b- respecting the status of sex, menstrual cycle, and pregnancy. hydroxysteroid dehydrogenase, as indicated in the human The evaluation was based on the steroid circulating levels and placenta study (Dombroski et al. 1997). steroid neuromodulating activities reported in the literature. PIs, and particularly their polar conjugates, are synthesized A further objective was to assess inter-group differences in the in large quantities in the human fetoplacental unit from Prog biosynthesis and metabolism of PI. For these reasons, we (Fig. 1; Milewich et al. 1979, Dombroski et al. 1997). As has measured the circulating levels of all PIs, such as P3a5a, been documented for 5a-PI, the steroids are metabolized to isopregnanolone (P3b5a, epiallopregnanolone, isoallopreg- respective 3-oxo-derivatives (P5a/b), which are then nanolone), P3a5b, epipregnanolone (P3b5b), and PIC as Figure 1 The biosynthesis of pregnanolone isomers. Journal of Endocrinology (2007) 195, 67–78 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 09:36:04PM via free access Importance of pregnanolone isomers . R KANCHEVA and others 69 conjugates of P3a5a (P3a5aC), P3b5a (P3b5aC), P3a5b after centrifugation for 5 min at 2000 g at 0 8C. The plasma (P3a5bC), and P3b5b (P3b5bC) in adult men (M), in samples were stored at K20 8C until analysis. women in the follicular (F) and luteal phases (L) of the menstrual cycle (MC), and in women in the 36th week of Steroids and chemicals gestation (P), using GC–MS. In addition, the levels of the respective precursors, free pregnenolone (Preg) and The steroids were purchased from Steraloids (Wilton, NH, conjugated pregnenolone (PregC), Prog, P5a, and P5b USA). The solvents for the extraction and high performance were also quantified. liquid chromatography (HPLC) were of analytical grade, sourced from Merck. The derivatization agent Sylon BFT (bis(trimethylsilyl)-trifluoroacetamide 99% and trimethylchlor- osilane) was purchased from Supelco (Bellefonte, PA, USA). Materials and Methods Instruments The circulating levels of all PICs, Preg and PregC were measured in all groups. The levels of all PIs and Prog were The GC–MS system was supplied by Shimadzu (Kyoto, measured in F, L, and P. The quantification of P5a and P5b Japan). The system consisted of a GC 17A gas chromatograph was limited to P. equipped with automatic flow control, AOC-20 autosampler and for the MS a QP 5050A quadrupole electron-impact detector with a fixed electron voltage of 70eV.A Zebron ZB- Subjects 50 medium polarity capillary column (50% phenyl/50% The subjects in all groups were Caucasian. The M group methylpolysiloxane) from Phenomenex (St Torrance, CA, consisted of 15 healthy men (16–62 years of age) participating USA) was used for the analysis. The length of the column was in a study on iodine deficiency in the Czech Republic. The 15 m, the internal diameter was 0.25 mm, and the film blood from non-pregnant women was collected on the 5th thickness was 15 mm. and 22nd days of the MC for the F (nZ15) and L (nZ16) groups respectively. Eleven women were followed in both Preparation of the plasma samples for GC–MS free steroids phases. The 30 pregnant volunteers in the P group entered the analysis study protocol according to the inclusion and exclusion criteria. The volunteers were enrolled into the study at a Frozen samples were thawed and 1 ml sample was spiked with single institution between January 2004 and September 2005, 17a-estradiol as an internal standard to attain a concentration of according to the following inclusion criteria: age between 18 1 mg/ml. The spiked sample was extracted with 3 ml diethyl and 30 years, physiological pregnancy, cephalic presentation ether. The water phase was kept frozen in a mixture of solid of the fetus. The exclusion criteria were as follows: carbon dioxide and ethanol, and the organic phase was decanted manifestation of pre-eclampsia, chronic medication, anemia, intoglass tubes and evaporated to dryness. The dryorganic phase any chronic disease or medication that might influence steroid residue was used for the determination of free steroids.