G. Phillipou

Formation of some bicyclic systems by radical ring-closure

Abstract The rates and stereochemistry of ring closure of the radicals ( 2 ), ( 9 ), ( 10 ), and ( 16 ) have been determined and rationalised.

On the presence of melatonin in pineal glands and plasma of foetal sheep

Abstract Melatonin is shown to accumulate within the foetal sheep pineal gland in the last few days of gestation. The mean ± S.E.M. foetal content was 79 ± 25 pg per gland for 30 foetuses obtained from day 124–145 gestation and 836 ± 286 pg per gland for 21 foetuses judged to be within 5 days of birth. In 5 neonates, the mean pineal content on day 1 was 899 ± 280 pg per gland. This accumulation correlates to a rise in tissue hydroxyindole-O-methyltransferase activity and foetal plasma melatonin. The possible relationship between these parameters and the endocrine changes preceding parturition is discussed. Significant levels of monoamine oxidase activity were found in foetal sheep pineal and the possibility is raised of other indoles (e.g. 5-methoxy tryptophol) being of importance in foetal pineal function.

Ovarian steroidogenesis studied by mass fragmentography.

A practical mass fragmentographic procedure is described which is suited to in vitro studies of gonadal endocrine function and which allows reliable determination of nanogram amounts of a wide range of steroids, including those formed from deuterium-labelled precursors. Steroids were isolated by chromatography using Dextran gel (Lipidex®-5000) and determined by mass fragmentography as the 3-enol trifluoroacetates or t-butyldimethylsilyl ethers. Application of the technique is illustrated by experiments on steroid hormones produced in organ culture by isolated sheep ovarian follicles with and without gonadotrophic (HCG) stimulation, and an analysis of steroids produced by entire ovine and human ovaries maintained in vitro in a perfusion apparatus. Large ovarian follicles from sheep on Days 4–14 of the oestrous cycle produced in organ culture predominantly oestradiol-17β and testosterone, as well as small amounts of pregnenolone, androstenedione, oestrone and DHA. Addition of HCG transiently enhanced the release of oestradiol-17β, caused a marked and sustained increase in the production of pregnenolone, and initiated the synthesis of progesterone and 20α-hydroxy-4-pregnen-3-one. When [16-2H1]-pregnenolone (200–500 ng ml−) was added to the culture medium, it was transformed into oestrogens and androgens, albeit in yields which suggested a clear preference for endogenous precursors. At least nine steroids were produced by sheep and human ovaries maintained in the perfusion apparatus

Deuterium labelled steroid hormones: Syntheses and applications in quantitation and endocrinology

Abstract Methods for the syntheses of various multi-deuterated steroid hormones have been summarized and evaluated. Applications of these latter compounds in combination with the technique of gas chromatography/mass spectrometry are described in the general areas of quantitation and human endocrinology, including their potential for the establishment of definitive reference methods or measuring steroid production rates.

Melatonin in Man

Considerable advances in the understanding of melatonin in man are now possible. The advent of melatonin measurement has allowed previous conjectural possibilities for melatonin and pineal gland function in man to be tested. The impressive feature of melatonin physiology in the human is the stability of secretion, which fits with an enviromental sensing function of the pineal gland and with melatonin as having some coordinating role in human endocrinological function. The possibilities for the use of melatonin in human and veterinary clinical medicine is raised.

Metabolism of the steroid anaesthetic alphaxalone by the isolated perfused rat lung.

Abstract We have investigated the metabolism of the steroid anaesthetic, alphaxalone (3α-hydroxy-5α-pregnane-11, 20-dione), in the isolated rat lung perfused at 10ml min−1 with the plasma substitute, Haemaccel. When [14C]-alphaxalone was introduced into the perfusate during a 90 min recirculating perfusion, four metabolites appeared in the reservoir. We have identified two of these by gas chromatographic-mass spectrometric (GC-MS) methods as 3α,11-dihydroxy-5α-pregnane-20-one (metabolite 2), the major metabolite and 5α-pregnane-3α, 11,20-triol (metabolite 3); precise assignment of the stereochemistry at C11 and C20 was not possible. Following perfusion of up to 30 min with concentrations of alphaxalone ranging from 0.08 to 8.9 μM, we have measured metabolite 2 in both the lung tissue and in the reservoir. From this data we have obtained a Km of 0.25 μ and a Vmax of 7.8 nmol min−1 g dry lung−1. Extrapolating from reported plasma concentrations in humans, it would seem that this ability of the lung to metabolize alphaxalone would have tittle influence during induction of anaesthesia. However, it could significantly influence the concentration of alphaxalone in the cerebral circulation during administration of the considerably lower doses of alphaxalone used for sedation during regional anaesthesia.

Deuterium labelled steroid hormones: Tracers for the measurement of androgen plasma clearance rates in women

A method employing stable isotope-labelled tracers and gas chromatograph-mass spectrometry (GC-MS) analysis has been used to measure the plasma clearance rates (PCRs) of androstenedione (A) and testosterone (T) in normal women and women with androgen abnormalities including hirsutism and polycystic ovary syndrome. A solution of deuterium-labelled A and T is infused at a constant rate and blood samples taken at 2 and 2.25 h. Solvent extracts of the derived plasma samples, to which an internal standard has been added, are derivatized with pentafluoropropionic anhydride and the endogenous steroid and deuterated steroid are quantitated after an injection of the derivatization mixture into a capillary column GC-MS. The concentration of the deuterated steroid in the infusion mixture is measured and the PCR is calculated. In premenopausal normal women the PCRA is 1950 +/- 184 1/24 h (n = 5) and the PCRT is 484 +/- 82 1/24 h (n = 7).

SPECIAL USES OF A GAS CHROMATOGRAPH–MASS SPECTROMETER IN ENDOCRINOLOGY

Based on the isotope dilution principle, and using deuterium-labelled steroids as tracers combined with GC-MS analysis, clinically acceptable procedures for the measurement of daily urinary production rates (UPRs) and metabolic clearance rates (MCRs), of hormonal steroids, have been developed. In clinical tests, using progesterone and androstenedione as models the UPRs and MCRs obtained compared favourably with values obtained using radioisotope-labelled tracers. The heavy isotope procedure proved, however, to be superior to the radioisotope methods in accuracy, speed and patient safety.

Capillary Gas Chromatographic Measurement of Neutral Urinary Steroids: Evaluation and Comparison with Alternative Methodology

The major neutral urinary steroids have been quantitated by capillary gas chromatography and the reproducibility and accuracy of the method have been fully evaluated. Typical intra- and inter-assay coefficients of variation for individual steroids ranged from 4·9 to 10·1% and from 15·6 to 21·5%, respectively. Comparison of steroid values with alternate and commonly employed procedures for measuring urinary 17-oxosteroids, total 17-oxogenic steroids, and cortisol and plasma dehydroepiandrosterone-sulphate yielded correlation coefficients between 0·71 and 0·79. A definite relationship between serum cortisol and urinary trihydroxy-pregnanedione was also shown. Urinary androstenedione metabolites, however, could not be significantly (P>0·05) correlated with the normalised androgen ratio in plasma.

Urinary 5α-androstane-3α,17β-diol levels in normal and hirsute women: Discriminating power and relation to other urinary steroids

The urinary levels of seven steroids, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, androsterone, etiocholanolone, tetrahydrocortisone, tetrahydrocortisol and allotetrahydrocortisol were measured in both normal (n = 18) and hirsute (n = 24) women. The results confirmed 5α-androstane-3α,17β-diol as the most significant steroid with respect to discrimination between hirsute and normal subjects. Investigation of the inter-steroid relationships, using multivariate techniques established that the mode of steroid metabolism was different between the two groups. Whereas in normal women the strong correlation amongst all the androgen metabolites inferred a predominant hepatic route to 5α-androstane-3α,17β-diol formation, the same analogy was not applicable to the hirsute subjects. Excellent agreement was found for the predicted vs actual excretion of 5α-androstane-3α,17β-diol in normal women, based on a regression model involving the six other steroids as independent variables. When the same model was used for estimation of 5α-androstane-3α,17β-diol levels in thirteen hirsute subjects, misclassified as “normal”, 50% gave values which were considerably less than actually measured. It is suggested that this discrepancy, with respect to these hirsute subjects is a reflection of extrahepatic production of 5α-androstane3α, 17β-diol due to increased 5α-reductase activity.