Synthesis of Hoodigogenin A, aglycone of natural appetite suppressant glycosteroids extracted from Hoodia gordonii.

14ß-hydroxy pregnane glycosides extracted from Hoodia gordonii, a succulent plant isolated from Apocynaceae are suggested to have appetite suppressant properties in animals and humans. However, limited reports on biological studies concerning the appetite suppressant properties are available in the open literature. One reason for that is the poor availability of these glycosteroids because H. gordonii is a protected plant and the yield of extraction lies between 0.003% and 0.02%. Starting from 3α,12α-diacetoxy-pregnanone 1, we disclose in this report the synthesis of Hoodigogenin A, the aglycone of the natural 14ß-hydroxy pregnane glycosides extracted from H. Gordonii.

Purification and characterization of lysozyme-pregnanediol glucuronide conjugates: the effect of the hapten and coupling reagent on the substitution level, sites of acylation and the consequences for the development of future immunoassays.

The preparation of conjugates destined for use in homogeneous enzyme immunoassays requires careful control to maximize the yield of conjugate, limit the level of substitution and obtain highly inhibitable conjugates. Furthermore, previous studies have shown that the acylation position and orientation of haptens in enzyme conjugates is important in determining the recognition and strength of binding of the hapten by anti-hapten antibodies. In this study we have prepared and purified pregnanediol glucuronide conjugates of hen egg white lysozyme by the active ester and mixed anhydride methods and compared the resulting conjugates with those obtained when conjugating with oestrone glucuronide. Conjugation of lysozyme with pregnanediol glucuronide by the mixed anhydride procedure resulted in two major derivatives monoacylated at lysine residues 33 and 97, while acylation by the active ester procedure resulted in six major derivatives acylated at one or more of lysine residues 33, 97 and 116. Comparison of these conjugates with oestrone glucuronide conjugates suggested that when using the more reactive active ester method the protein environment of the amino groups and the acylating agent directed the site(s) of acylation and level of substitution. However, in the case of the less reactive mixed anhydride coupling procedure the chemical nature of the hapten also influenced the final conjugation product makeup. These findings have implications for the design of new, highly inhibitable enzyme conjugate immunoassay systems.

The identification of novel steroid N-acetylglucosaminides in the urine of pregnant women.

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5alpha-pregnane-3alpha,20alpha-diol. 5beta-Pregnane-3alpha,2Oalpha-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5alpha-pregnane-3beta,20alpha-diol was only found in the sulfate fraction. 5alpha-Pregnane-3alpha,20alpha,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5alpha-pregnane-3alpha,20alpha,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in beta-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 micromol/g creatinine (range 17.9-49.1 micromol) in five healthy women in the 38th-39th week of pregnancy. The mean excretion of 5beta-pregnane-3alpha,20alpha-diol glucuronide in the same women was 71 micromol/g creatinine, (range 27-127 micromol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.

The premenstrual syndrome: amelioration of symptoms after hysterectomy.

What is the role of the uterus in the aetiology of the premenstrual syndrome (PMS)? Twelve women kept a daily symptom record before and after hysterectomy. Psychological and physical symptom patterns were analysed by Fourier analysis and the response to hysterectomy by maximum likelihood ANOVA. Hysterectomy was associated with a 66% reduction in mean premenstrual tension (PMT) severity for both psychological and physical symptoms (p less than 0.005). The milder symptoms appeared unrelated to post-operative changes in health or ovarian function. Seven women experienced mood-related PMT in every pre-hysterectomy cycle; the persistence of unfailing mood-related PMT in one of these women after hysterectomy suggests that her symptoms were hormonally controlled and the loss of regular PMT in the other six women suggests that a uterine factor, or psychological factors associated with the menstrual cycle were implicated. We conclude that a uterus is not essential for the expression of PMT, but that its removal often results in the amelioration of symptoms.

[Use of an ion exchanger as a rapid means of extraction, applied to the determination of urinary estrogens and pregnanediol in pregnancy, using colorimetry or gas chromatography]. / Utilisation d'un échangeur d'ions comme moyen rapide d'extraction appliqué au dosage des estrogènes et du prégnandiol urinaires de grossesse par colorimétrie ou par chromatographie en phase gazeuse

The method described permits, after classical hydrolysis, extraction without a solvent of estrogens during pregnancy. It also permits estimation of pregnanediol which is eluted in a different fraction. Twenty estimations may be carried out in half a day on test samples of from 2 to 10 ml of urine. The estimation of total estrogen by colorimetry takes into consideration the efficiency of extraction by the use of a standard range treated under the same conditions. For gas chromatography, two internal standards are added to the urine after hydrolysis: these are 16,17-epiestriol and 5 beta pregnane-3 alpha, 17 alpha, 20 beta triol (PGTS) have respectively the same behaviour as estriol on the one hand and pregnanediol and pregnanetriol, on the other hand. They take into consideration resin extraction and chromatographic behaviour and may be used for quantitative estimation. The graphs of standardisation and recovery have been studied for the main estrogens together with pregnanediol. The various eluates were controlled by gas chromatography coupled to mass spectrometry.