Definitive identification of the compounds revealed in brain extracts in the above two ion SIM and multiple ion screens was attempted by monitoring co-elution of three ions of these MO-TMS-derivatives (SIM methods 6-9 for free steroids, SIM methods 10-13 for steroid sulphates and 14-15 after adjustments, see below, p. 176). Additional confirmation was sought by using a second derivatisation method on these brain extracts using HFBA and two ion SEM (SIM methods 16-17 for free steroids, SIM methods 18- 19 for steroid sulphates and 20-21 after adjustments). The additional ions for SEM of the MO-TMS derivatives were chosen after the same criteria as for the two ion SIM methods (see 3.2.5). Similarly, specific ions for two ion SEM of EEFBA-derivatives were chosen (see Appendix 1).
The criteria for identification were both RRT and qualifier to target ion ratios (Q/T). The first criterion used was for the RRT of a potential endogenous compound to be within ± 0.5% of the RRT of a standard compound run alongside the tissue sample. The second criterion was for the endogenous compound to be within ± 20% relative abundance of
both T and Q ions. The lower and upper limits of the respective Q/T ratios are therefore 0.67 and 1.5 x the standard value.
Examples of RRTs and Q/Ts for unconjugated and sulphate conjugated endogenous rat brain steroids for which all ions monitored were above the detection limit are shown in Table 5-4 and Table 5-5. Table 5-4 shows the RRT results for derivatives from several extracts, alongside the values given by standard compounds. These identification limits were developed as pragmatic limits in this study by analysis of numerous samples of reference compounds. In order to test and finther strengthen the basis of those pragmatic limits, confidence limits (at the 99.9% level) for RRTs using parametric statistics were also calculated from a number of standard samples. These are shown alongside the above pragmatic limits. As the examples show, the former are in good agreement with those confidence limits. The identification results were in agreement when either the pragmatic 0.5% or the 99.9% parametric confidence limits were applied in 82 out of 87 cases. For the number of analyses carried out, it can be expected for practical reasons that some standard compounds will show a narrower distribution of relative retention times than given by the above pragmatic limits. Generally, this was taken as a good backup for the pragmatic 0.5% limits and these can be justifiedly applied.
Table 5-5 shows the Q/T values of several derivatives from brain extracts alongside values from standard compounds. Again, pragmatic limits are used and compared with parametric 99.9% confidence limits calculated from various standard runs. The identification results were the same with both the pragmatic 20% and the 99.9% parametric confidence limits except in 4 cases out of 102, thus providing further confidence for the use of these identification criteria.
Overall definitive identification was then assumed, if either co-elution of three ions of the MO-TMS-derivative within the RRT- and Q/T- limits or co-elution of two ions within the RRT- and Q/T-limits from both of two different derivatisation methods (MO-TMSI and HFBA) was observed. Tentative identification of compounds was suggested where RRT- and Q/T-criteria were fulfilled in two ion SIM of HFB-derivatives. The identification procedures of both RRT and Q/T analyses of brain steroids and the final decision on identification are summarised in Table 5-6. Examples of the co-eluting ion peaks o f both MO-TMS- and HFB- derivatives of endogenous free and sulphate conjugated steroids are shown in Figure 5-4 and Figure 5-5 respectively.
Among the free steroids, 5a-DHPR0G showed co-elution with the Q/T-ratios somewhat outside the limits. However, this compound did not derivatise with HFBA, thus could not be confirmed in this way and was presented as tentatively identified. The free steroids unequivocally confirmed are DHEA, 3a,5a-THPROG, PREG, PROG, 3a,5a-THDOC,
CORT, 20P-DHPREG, 20a-DHPREG, TESTO, 5a-pregnan-3a,17-diol-20-one.
Tentative identification is suggested for 5a-DHPR0G, 20a-DHPROG, 5a,20a- THPROG, 5a-pregnane-3a,20a-diol, 3p,5a-THPROG, 3p,5a-THDOC, 5a-pregnan- 3a, 11 p-diol-20-one.
In the sulphate fraction only DHEA was unequivocally identified. Analysis of steroid sulphate conjugates gave interferences for several compounds in some ion responses. In subsequent analyses different ions were chosen in those cases as described below. For PREGS after MO-TMSI derivatisation, ions 402, 386, 312 were detected, but large interferences seen for ion 402. Thereafter ion 296 was monitored for the compound instead. However, Q/T ratios did not fulfil the diagnostic criteria. Furthermore, signals for the compound in brain extracts after HFB-derivatisation were below detection limit. The MO-TMS derivative of 3a-DHPR0GS ion 417 was detected in extract 5, but interferences were observed for ions 244 and 386. In analyses of subsequent extracts, the ions were thus changed to 417, 312, 326. However, no signal above detection limit was detected in those extracts for those ions HFB-derivatives of the compound were not analysed as they could not be resolved from the 3P-DHPR0G derivative. Interferences were also observed for ions 386 and 364 of MO-TMS- 5a-pregnan-3a,17-diol-20-one- S. Other ions monitored were 476, 188 and 296 in subsequent analyses, but in those were below detection limit. The HFB derivative of this steroid gave a signal in analysis of a brain extract for only the target ion (442). The qualifier ion (487) was below detection limit. For the MO-TMS derivatives of 5a-pregnane-3a,20a-diol-S and 5p-pregnane- 3a,20a-diol-S two ions (269, 284) were detected, but a third ion was below detection limit (449). However, this ion has an expected relative abundance o f approximately 10- 15% and thus might be easily missed. There are no other unique ions with higher relative abundance for those compounds and with 269, 284 and 346 as diagnostic ions the compound could not be further confirmed. No detectable signals were observed for the HFB-derivative of 5a-pregnane-3a,20a-diol-S. For the HFB-derivative of 5p-pregnane- 3a,20a-diol-S only ion 712 was above detection limit. Ion 469 was observed for 5a- pregnan-3a,l lp-diol-20-one-S as HFB-derivative, but the compound could not be
confirmed as MO-TMS-derivative. Various ions (386, 244, 417, 296, 312) of MO-TMS- 3p-DHPR0GS were monitored in different brain extracts. However, there were either interferences or no detectable signals in the three ion monitoring of the compound. This compound was not determined in two ion monitoring after HFB-derivatisation, because it could not be resolved from 3a-DHPR0G as mentioned earlier. For compounds where all ion peaks were above detection limit and peaks were not distorted by interference, the RRTs and Q/Ts are shown in Table 5-4 and Table 5-5.
Both confirmed and tentatively identified compounds were quantified in either two or three ion SIM. Quantitation was done using area ratios of the target ions of MO-TMS- derivatives to the internal standards and calibration curve functions shown in Table 3-5. For quantitation in two ion SIM of HFB derivatives and certain compounds in three ion SIM of MO-TMS-derivatives single point calibrations were used. All extracts had been prepared as described in Methods (2.3.7), except extract 8 which was twice subjected to Lipidex chromatography after HFBA derivatisation. The concentrations of endogenous steroids in male adult rat whole brain from several extracts are shown in Table 5-7. Recoveries of standard steroids added to brain hoinogenates and carried through the extraction, fractionation and assay procedure are also shown in this Table.
Detection limits for the two ion SIM methods of all the compounds listed in Table A-1 and carried in pure solution reagent blanks alongside extracts of rat brain were determined for both the free steroid and the steroid sulphate fractions. Compounds were not considered to be detected, unless their ion areas were at least three times the equivalent signals at the same relative retention time in reagent blanks analysed alongside the tissue samples (for consideration of detection limits see 3.1). The concentrations at those levels were determined from calibrations performed using linear regression (with the exception of 17-OH-PREG which gave a quadratic curve, calibration functions see Table 3-5) and the minimal values from four free steroid and three steroid sulphate extractions carried out are also shown in Table 5-7.
Blood contamination of brain samples was estimated by the spectrophotometric measurement o f haemoglobin and found to be less than 0.6% (v/v). Other estimates of blood contamination of rat brain samples [202] from assays of peroxidase activity have yielded similar values (0.3-0.7 %). The blood contamination is for most steroids thus unlikely to contribute significantly to the brain concentrations measured (it was estimated
for PREG, DHEA, TESTO and found to be maximally about 1%, using plasma values reported in [12,103,190,213]). An exception is CORT. The CORT concentration reaches values of up to approximately 115 ng/ml at the peak of the daily cycle at 20.00 h in regimens of lights on from 7.00 - 19.00 h [103]. At this level the blood content of the brain samples of this study would amount to 12% of the mean concentration determined. The samples in this study were collected around about the nadir of the plasma CORT diurnal cycle (11.00 h), where its concentration is typically about 13 ng/ml [103], however it cannot be definitely excluded that higher plasma concentrations were present in the animals used.
As can be seen from Table 5-7, the most abundant steroid found in the present study of male rat brain was CORT. The second most abundant compound was PREG, followed by TESTO, PROG, 5a-DHPR0G and 3(3,5a-THDOC. Somewhat lower were then 20a- DHPREG, 20P-DHPREG, 3a,5a-THPR0G, 3p,5a-THPROG, 5a,20a-THPROG and 3a,5a-THDOC. The lowest levels were found for DHEA, 20a-DHPROG, 5a-pregnane- 3a,20a-diol, 5a-pregnan-3a,17-diol-20-one and 5a-pregnan-3a, 11 p-diol-20-one. DHEAS was somewhat higher than DHEA. The concentrations found were varying notably between analyses of extracts (see Table 5-7), even though animals were kept strictly under the same controlled conditions. Variabilities of steroid concentrations in brain extracts from animals kept under the same conditions were previously reported, however. Shimada et al. [176] found PREG levels to vary between animals under the same treatment by a factor o f ~10. Variations can also be seen in Table 5-1 summarising previous reported identifications of steroids. Consistent with previously reported findings the levels of PREG and DHEAS were much higher compared to DHEA [48,106]. However, the previously reported high concentrations of PREGS (see 5.1, for example [50,119]) could not be confirmed here and no further sulphate esters could be formally described, with exception of DHEAS. The recovery of PREGS in the extraction procedure here was relatively low, but even after correction for procedural losses levels of 0.25 ng/g would have been detected, which is much lower than in some previous reports. It is possible that the lack of detection of this steroid here is due to natural variation. Another possible reason for the discrepancy is the difference in the methodologies used. The major difference in the sample preparation between the present and other studies on steroids in brain is the separation of free steroids and their conjugates. Here Oasis MAX® ion exchange chromatography was used for the complete
separation o f free steroids from sulphate esters as opposed to the partial separation of organic solvent extraction from aqueous phases or polarity based solid phase extraction used for this task in previous reports (see Chapter 4). Complete separation of free steroids from the sulphate esters on the MAX cartridges used in the present study has also been confirmed by analysis of urine samples (see Appendix 2).
Criteria for unequivocal identification in SIM are a subject of wide debate (see for instance [19]) and there is a wide variability of criteria used throughout the literature. For mass spectrometric identification of steroids in brain, several methods have been employed. Whereas PREG, DHEA, their sulphate and fatty acid esters have been subject to many studies and have been well characterised including by 5 ion SIM, other compounds have only been measured with single ion monitoring. The more ions are used in SIM the higher the specificity achieved. However, there is a limit to the practicability of this approach, as there is a decline in sensitivity as more ions are monitored. Furthermore, it is in most cases not possible to find several specific ions that are without interference from neighbouring compounds when a large number o f close-by eluting compounds are monitored simultaneously. In the above mentioned analyses using 5 ion SIM a single compound was characterised using pooled tissue from several animals. An investigation into specificity of low-resolution MS has shown that three ion monitoring with ion response ratios within ± 20% of those of standards allows unique identification (see 3.1.4). Additional specificity is introduced if MS three ion monitoring is used in conjunction with LC or GC and selective sample preparation steps. Three (or more) ion monitoring has not been applied so far for identification of brain steroids, except in the case of PREG, DHEA, 3a,5a-THPROG, 3p,5a-THPROG and EpiA. In those cases relative ion variabilities were not shown, however. In the past few years several methods for measurement of steroids in brain tissue based on GC-MS or LC-MS have been developed (see for example [38,66,106,130,175,177,194,196]). These methods, however, are all limited to the quantitation of the already identified compounds PREG, DHEA, PROG, 3a,5a-THPROG or PREGS and DHEAS. In this study it was for the first time attempted to identify a wider range o f compounds with unequivocal identification methods.
Previously identified compounds in rat brain could be confirmed and several further compounds were identified. Further evidence was presented for a wide range of analytes for further confirmation.
The source of the identified steroids in the present study could not be assigned to entry via the BBB after synthesis by peripheral steroidogenic glands or to synthesis within the brain. However, knowledge of steroid metabolising enzymes found in brain (see Section 1.3.1.5) allows some speculation as to the possible pathways generating the compounds reported here (see Figure 5-6).
Based on the present results, the main metabolites of PREG would be PROG, 20a- DHPREG and 20p-DHPREG. 20P-HOR activity has not yet been discovered in rat brain, but the possibility for the presence of the enzyme remains, especially as expression of the enzyme was described in mouse and neonatal pig brain (see Chapter 1). PROG is possibly metabolised further to 20a-DHPROG and 5a-DHPR0G. Furthermore, two reduced metabolites of 5a-DHPR0G were identified, 3a,5a-THPROG and 3P,5a- THPROG (with the latter to be confirmed). Another PROG metabolite, DOC, as well its 5a-reduced form, 5a-DHD0C could not be identified. This is consistent with lack of brain P450c21 expression. The downstream metabolite 3a,5a-THDOC was confirmed, however and potentially also 3p,5a-THDOC. The latter two compounds would thus seem likely to enter the brain from the circulation after peripheral synthesis rather than in situ synthesis.
17-OH-PROG and another compound arising by the action of P450cl7, 17-OH-PREG could not be detected. This is consistent with the lack of detection of P450cl7 in the adult rat CNS so far despite many attempts. However, the downstream product of 17- OH-PREG, DHEA was found in the brain. DHEA was the first documented neurosteroid [48], that remains in brain long after removal o f peripheral glands. Thus the findings here are consistent with the hypothesis that DHEA arises via a different pathway than CNS P450cl7 (see 1.3.1.5.1). Nevertheless, a different 17-hydroxylated compound has been found, 5a-pregnan-3a,17-diol-20-one. This finding could be explained by the compound entering the brain via the BBB or by the existence of another mechanism of 17- hydroxylation than via P450cl7. Formation o f the compound in peripheral glands and secretion has not been reported. Furthermore, a precursor o f the compound, 3a,5a- THPROG is present in brain, thus it is possible that the compound is formed in brain.
The significance of 3a,5a-THPROG and 3a,5a-THDOC is well established with their potent GABAa-R modulatory properties, that are the basis for their anxiolytic, sedative and hypnotic actions (see 1.3.2.1, 1.3.4). The findings here point to the possibility of the origins of the two steroids, at least in part to be different, with 3a,5a-THPROG being partially or completely produced within the brain and 3a,5a-THDOC most likely not, as described above. This leads to the questions of the functions of those compounds. One possible reason could be that 3a,5a-THDOC is produced by the adrenal as part of the stress response and 3a,5a-THPROG at least partially within the brain as a localised signalling function. The 3 p-isomers of the above compounds are inactive at the GABAa-
R. They can, however possibly act as inhibitors o f the action of the respective 3a- isomers [148]. Furthermore, 3p,5a-THPROG was shown to be a substrate for 3p,5a- diol-hydroxylase, the most abundant P450 steroid hydroxylase in the brain [204]. Hydroxylation by this enzyme at positions C-6 or C-7 is a major catabolic pathway in prostate and possible could serve this function in brain, acting to reduce the levels of 3a,5a-THPROG [IBS]. This function could be fulfilled in the following way: by up- regulation o f 3p-H 0R activity, 3p,5a-THPROG (and possibly 3p,5a-TH D 0C) is produced and acts as antagonist of the 3a,5a-reduced isomer to reduce GABAa-R
potentiation. To remove the 3a,5a-isomers, the levels of their precursors 5a-DHPR0G and/or 5a-DHD0C are reduced by increased production of 3P,5a-metabolites, which in turn are removed by action of 3p,5a-diol-hydroxylase. The 3 a-H 0R reaction is then tilted in the oxidative direction due to reduced levels of the products of the reaction in this direction, thus reducing levels o f 3a,5a-isomers.
The significance of 5a-pregnane-3a,20a-diol (the 20-reduced metabolite of 3a,5a- THPROG) found here (to be confirmed), is not known. Pregnanes reduced at C-20 have a reduced efficacy compared to the 20-ketones in enhancing the current induced by GAB A at recombinant GABAa-R expressed in Xenopus oocytes [15]. The maximum potentiation of GABA-stimulated ^^Cl'-uptake and currents is also significantly lower than for 3a,5a-THPROG, suggesting it to be a partial agonist [14,15]. As expected from their effects on GABAa-R modulation, 3a,5a/p,20a/p-reduced pregnane-diols were