Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis
Biological and Pharmaceutical Bulletin
In the 1980s, Baulieu and co-workers demonstrated that some steroids, such as pregnenolone (PREG), dehydroepiandrosterone and their sulfates, are present in higher concentrations in the brain than in blood and are synthesized de novo in the central nervous system. 1) Such steroids are now universally referred to as neurosteroids. They act as modulators of several neurotransmitter receptors (g-aminobutyric acid A , N-methyl-D-aspartate and s 1 receptors) either as stimulators or inhibitors 1,2)
... nd are involved in learning and memory performance. 3) For these reasons, there is a considerable interest in determining the location and levels of different neurosteroids in the brain. The levels of PREG sulfate (PREGS) have conventionally been evaluated by measuring its genin formed after solvolysis by 5) or radioimmunoassay (RIA). 6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid. To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue). 4-6) One potential explanation for this discrepancy is that the LC-MS-MS method is more specific for brain samples than the GC-MS or RIA assay combined with solvolysis; the GC-MS may be detecting PREG derived from PREGS as well as that from other conjugated forms, such as sulfolipid conjugate, by solvolysis, and the RIA employs an antibody that reacts with other steroids having structures closely related to that of PREG. Although our LC-MS-MS method was specific and did not need a solvolysis procedure, it did not have sufficient sensitivity for the analysis of PREGS in the brain (limit of quantitation: ca. 160 pg/injection), and the obtained data using ca. 400 mg of tissue were not so reliable. 7) Based on the results, we developed an enzyme-linked immunosorbent assay (ELISA) for PREGS using an antibody raised against 11ahemiglutaryloxy-PREGS-bovine serum albumin conjugate. 8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS. MATERIALS AND METHODS Materials and Reagents Standard PREGS, PREG and all other reagents were those used in the previous study. 8) Oasis HLB cartridges (60 mg: Waters Assoc., Milford, MA, U.S.A.) were successively washed with AcOEt (1 ml), EtOH (2 ml) and H 2 O (2 ml) prior to use. Rats and Treatment Wistar strain rats (7 weeks old, male, 190-200 g) obtained from Japan S.L.C. (Hamamatsu, Japan) were used and euthanized by decapitation. Brain samples were collected from the rats assigned either to a control group (without injection, nϭ10), a group of animals receiving an intrapertioneal (i.p.) vehicle injection (5% EtOH/ saline, 5 ml/kg i.p., nϭ3) and a group receiving an injection of PREGS (dissolved in 5% EtOH/saline, 2 mg/5 ml/kg i.p., nϭ5). In the second and third groups, one hour following injection of vehicle or PREGS solution into the rat, the brain was collected. Blood from the control rats was collected from the cut end, left at 4°C for 2 h and then centrifuged at 1500 g (4°C, 10 min). The serum was separated and stored at Ϫ20°C. Pretreatment Procedure The whole brain (1.6-1.8 g) was homogenized in 1% AcOH/MeOH (ca. 10 ml) using an ultrasonic homogenizer. The concentration of the homogenate was adjusted to 100 mg brain tissue/ml with 1% AcOH/MeOH, and 0.5 or 2 ml of homogenate (corresponding to 50 or 200 mg of brain tissue) was pipetted into tube. Pregnenolone sulfate (PREGS) is reported to be present in higher concentration in the brain (more than 5 ng/g tissue in the rat) than in blood and is considered to be a neurosteroid. However, there are some doubts on its brain levels, because they were determined by indirect methods (e.g., GC-MS or radioimmunoassay after solvolysis). In the present study, PREGS in rat brains was determined by an enzyme-linked immunosorbent assay, which did not require solvolysis, after pretreatment with an Oasis HLB cartridge. The absolute recovery rate of PREGS through the pretreatment was 60.8%, and the quantitation limit was 33 pg/g tissue for a 200-mg of brain aliquot. Intra-and inter-assay coefficients of variation were less than 15.1 and 9.2%, respectively. The brain PREGS levels in the control rats (n)01؍ were less than 0.15 ng/g tissue except for one sample (0.42 ng/g tissue) and were lower than the serum levels (n,5؍ 0.25-0.47 ng/ml). On the contrary, the brain PREGS levels were sufficiently increased after intrapertioneal injection of 2 mg/kg body of PREGS (n,5؍ 0.37-1.29 ng/g tissue). These results demonstrate that, in rats, the brain PREGS may be derived from peripheral sources, and its actual levels are much lower than those previously measured by indirect methods.