Uptake and metabolism of sulphated steroids by the blood–brain barrier in the adult male rat

Abstract Little is known about the origin of the neuroactive steroids dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PregS) in the brain or of their subsequent metabolism. Using rat brain perfusion in situ, we have found 3H‐PregS to enter more rapidly than 3H‐DHEAS and both to undergo extensive (> 50%) desulphation within 0.5 min of uptake. Enzyme activity for the steroid sulphatase catalysing this deconjugation was enriched in the capillary fraction of the blood–brain barrier and its mRNA expressed in cultures of rat brain endothelial cells and astrocytes. Although permeability measurements suggested a net efflux, addition of the efflux inhibitors GF120918 and/or MK571 to the perfusate reduced rather than enhanced the uptake of 3H‐DHEAS and 3H‐PregS; a further reduction was seen upon the addition of unlabelled steroid sulphate, suggesting a saturable uptake transporter. Analysis of brain fractions after 0.5 min perfusion with the 3H‐steroid sulphates showed no further metabolism of PregS beyond the liberation of free steroid pregnenolone. By contrast, DHEAS underwent 17‐hydroxylation to form androstenediol in both the steroid sulphate and the free steroid fractions, with some additional formation of androstenedione in the latter. Our results indicate a gain of free steroid from circulating steroid sulphates as hormone precursors at the blood–brain barrier, with implications for ageing, neurogenesis, neuronal survival, learning and memory.


(TLC) plates showing purity of 3 H-dehydroepiandrosterone sulphate (DHEAS) and 3 H-pregnenolone sulphate (PregS) used in the present study.
As shown in the legend to Fig. 4 of the main text, control experiments (n = 4) in which standard 3 H-DHEAS, 3 H-DHEA, 3 H-PregS or 3 H-Preg were added to rat brain homogenates followed by extraction and separation into free steroid and steroid sulphate fractions, as for the perfused rat brain samples, showed negligible sulphation or desulphation of the labels during these procedures. Thus the rapid desulphation of 3 H-DHEAS or 3 H-PregS seen on their uptake into the brain was not an artefact of the subsequent extraction and fractionation of steroids. Further TLC showed that there was no other detectable metabolism of these labels during the extraction and fractionation procedure.

Section 3. Distributions of radioactivity following TLC of steroid sulphate and free steroid fractions from the brain parenchyma of rats perfused with either 3 H-DHEAS or 3 H-PregS.
Positions of these steroids on TLC are given below relative to the solvent front (Rf) as mean ± SEM. Typical profiles of radioactivity as detected by phosphorimaging are also shown, with intensity in arbitrary units (au). The positions of standards are indicated by horizontal bars above the profiles. Non-radioactive steroid standards were visualised by exposure to iodine vapour whereas the 3 H-steroids were detected by the phosphorimager.

TLC of steroid sulphate fractions from the parenchyma of 3 H-DHEASperfused and from 3 H-PregS-perfused rat brains
Upon TLC in solvent system A, the steroid sulphate fractions from the parenchyma of 3 H-DHEAS-perfused (n = 5) and from 3 H-PregS-perfused (n = 5) rat brains gave single peaks corresponding to their appropriate standards, although that from the 3 H-DHEAS-perfused rats Chromatography was in solvent system A and the positions of steroid standards are indicated by horizontal bars above the profiles.

PregS-perfused rat brains in comparison with the free steroid fraction from the same parenchyma samples
Upon TLC in solvent system B, both the 3 H-label from the steroid sulphate fractions and the standard 3 H-PregS gave peaks on desulphation corresponding (Rf = 0.45 ± 0.01) to standard Preg (Rf = 0.44). Likewise, TLC of the free steroid fraction from these 3 H-PregS-perfused rat brains in the same solvent system showed no evidence of metabolism other than desulphation, with peaks (Rf = 0.45 ± 0.01) corresponding to standard Preg and not to other possible Preg metabolites (see Fig. S3).

TLC of the putative Preg isolated from the desulphated steroid sulphate fraction and the free steroid fraction from the parenchyma of 3 H-PregSperfused rat brains, following acetylation alongside known steroid standards
Upon TLC

DHEAS-perfused rat brains in comparison with the free steroid fraction from the same parenchyma samples
Upon TLC in solvent system B, both the desulphated steroid sulphate fraction and the free steroid fraction from the parenchyma of 3 H-DHEAS-perfused rat brains gave two peaks: from the desulphated steroid sulphate fraction at Rf 0.29 ± 0.01 and Rf 0.38 ± 0.01 and from the free steroid fraction at Rf 0.28 ± 0.01 and Rf 0.38 ± 0.01 (see Fig. S5). The earlier peaks from both fractions corresponded with androstenediol (Rf = 0.25) and the later peaks with 3 H-labelled and non-radioactive DHEA at Rf = 0.36. There were no detectable 7hydroxymetabolites of DHEA (Rf < 0.05). The two peaks which arose from the steroid sulphate fraction were not an artefact of the deconjugation procedure because desulphation of the 3 H-DHEAS standard gave only one peak at 0.37.

TLC of the putative DHEA and androstenediol isolated from the desulphated steroid sulphate fraction and the free steroid fraction from the parenchyma of 3 H-DHEAS-perfused rat brains, following acetylation alongside known steroid standards
Following acetylation and TLC in solvent system C, the putative DHEA from both the desulphated steroid sulphate fractions and the free steroid fractions gave single peaks at Rf = 0.38 ± 0.01 and Rf = 0.36 ± 0.01, respectively. These peaks corresponded with acetylated standard 3 H-labelled (Rf = 0.38) and non-radioactive DHEA (Rf = 0.37). Likewise, acetylation of the putative androstenediol gave peaks from the desulphated steroid sulphate fractions at Rf = 0.47 ± 0.01 and from the free steroid fractions at 0.45 ± 0.01, both corresponding with acetylated standard androstenediol at Rf = 0.46 (see Fig. S6). However, acetylation of the 3 H-label eluted from the putative androstenediol peak in the free steroid fraction also gave two additional peaks, one of which Rf = 0.11 ± 0.01 corresponded to standard androstenedione carried through the acetylation procedure (Rf = 0.14; although this would not be acetylated) and another Rf = 0.34 ± 0.01 which could not be identified. There was no 3 H-peak corresponding with standard acetylated testosterone (Rf = 0.24).