A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2014. / The seemingly simple proton abstraction reactions underpin many chemical transformations including isomerization reactions and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid, Δ5- androstene-3,17-dione by the human glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process and the hydrogen bonds postulated to be the stabilizing forces of these transient species. Kinetics studies on Δ5-androstene-17-one, a substrate that is incapable of forming hydrogen bonds reveal that such stabilizing forces are not a requirement to explain the observed rate enhancements. The UV-Vis detection of the intermediate places this specie in the catalytic pathway while fluorescence spectroscopy is used to obtain the binding constant of the intermediate analogue equilenin. Analysis of the kinetics data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mole. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

Thesis (M.S.)--Eastern Illinois University, 2000. / Includes bibliographical references (leaves 47-51).

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Androstenedione, 4-androstenediol, 5-androstenediol, 19-norandrostenediol and 19-norandrostenedione are commonly referred to as "Andro" prohormones. Over the last few years, supplementation using these prohormones has been aggressively marketed to the general public. Supplement manufacturers often claim that Andro use improves serum testosterone concentrations, increases muscular strength and muscle mass, helps to reduce body fatness, enhances mood, and improves sexual performance. However, to date, most studies contradict these claims. In contrast, several studies using oral Andro related prohormones show that Andro use can abnormally elevate estrogen related hormones as well as alterations in hormonal markers (i.e., abnormal elevations in serum estrogen) thought to increase a person's risk for developing prostate or pancreatic cancers. In addition, most studies also indicate that significant declines in high-density lipoproteins occur leading to an increased cardiovascular disease risk. Thus, to date, the current research base suggests that Andro prohormone use does not support manufacturer claims. But it does suggest there should be strong concerns regarding long-term oral Andro prohormone use, especially regarding its effects on blood lipids and estrogen hormone profiles.

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L'un des avantages majeurs des réactions de bioconversion résulte du fait que le milieu réactionnel des biocatalyseurs est l'eau. Ce dit avantage constitue l'une des principales limitations de ces réactions de bioconversion lorsqu'il s'agit de substances lipophiles non solubles dans l'eau connue les stérols. L'efficacité d'un procédé de bioconversion de substances lipophiles solides dépend essentiellement du contact et de 1'interaction entre le biocatalyseur et ce substrat lipophile. Les solutions proposées dans la littérature font appel à des solvants et des produits chimiques de natures souvent toxiques, inflammables et explosives. Ces solutions décrites font perdre à la bioconversion son caractère de biotechnologie blanche. Dans ce travail, nous avons montré qu'en plus de ses avantages connus, l'immobilisation passive de biocatalyseurs au sein d'un support poreux peut favoriser l'interaction cellule-substrat lipophile et augmenter le taux de bioconversion sans utiliser de solvants et de produits chimiques. La réaction modèle étudiée est le clivage de la chaine latérale des stérols par des mycobactéries en vue de l'obtention des androsténones précurseurs naturels des stéroïdes, molécules à forte valeur biologique ajoutée. Le support d'immobilisation le plus performant a été le fruit sec de Luffa cylindrica. Par rapport aux supports organiques classiques tels que les gels de polyacrylamide, les mousses de polyuréthane, la silicone et les plastiques, le fruit sec de Luffa cylindrica présente les avantages suivants : (i) c'est un produit naturel, (ii) biodégradable, (iii) peu onéreux, (iv) non toxique pour les microorganismes, (v) stable du point de vue mécanique et thermique, (vi) et réutilisable. / One of the major advantages of using biocatalysts in organic synthesis is that water constitutes the reaction medium. However, water becomes a serious problem when bioconversion deals with lipophilic compounds, in particular those poorly soluble in water such as sterols. Bioconversion of lipophilic compounds depends on the close contact between the hydrophobic substrate and the biocatalyst. Increasing this contact requires usually the use of huge amounts of chemical which are often toxic, flammable and explosive. In this work, we showed that passive cell immobilization in porous materials can increase the contact between microorganisms and lipophilic substrates without using chemicals. The side chain cleavage of sterols was studied as a model multistep microbial bioconversion of lipophilic compounds. This reaction allows the production of androstenones which are the natural precursors of steroids. Among the studied immobilization carriers, the dried fruit of Luffa cylindrica was the most efficient Compared to other organic support carrier such as alginate beads, polyurethane foams, silicones and plastics, the dried fruit of Luffa cylindrica is advantageous since it is natural, renewable, biodegradable, cheap, mechanically strong, free of toxicity and it doesn't need a chemical pretreatment.

The purpose of this study was to examine the effects of androstenedione supplementation on testosterone levels in older men. Healthy men (n = 11) between the ages of58 and 69 were divided into two groups: 6 taking 300 mg of androstenedione (mean ± SE, 62.33 ± 2.57) supplement and 5 taking the 300 mg cellulose placebo (mean ± SE, 60.2 ± 1.02) for a period of seven days. Subjects in both groups had been participating in the Ball State University Adult Fitness Program (BSUAFP) for at least one year, incorporating both aerobic and resistance training into their workouts. Testing measures involved the subjects performing two exercises (leg extension and leg curl) while having blood drawn prior to, during, and post-exercise for a period of 20 minutes both pre-and post-supplementation. Specific weights for the subjects were determined with a ten-repetition maximum (10-RM) lift on both exercises. It appeared that the subjects in the androstenedione group were stronger with the exercises than the subjects in the placebo group, but with no significance. Testosterone, estradiol, and androstenedione were analyzed via hormone assay pre-and post-supplementation. The analysis of the testosterone revealed a significant difference pre-(mean ± SE, 4.65 ± .51 ng/ml) to post-(mean ± SE, 6.72 ± .58 ng/ml) supplementation for the androstenedione group. Analysis of the androstenedione revealed a significant difference pre-(mean ± SE, 0.88 ± .20) to post-(mean ± SE, 7.46 ± 1.25) supplementation for the androstenedione group. The estradiol assay revealed no significant differences pre-to post-supplementation for either group. The placebo group did not demonstrate any significant differences pre-to post-supplementation for either testosterone or androstenedione. The results of this study concluded that supplementation with 300 mg. of androstenedione for a period of seven days significantly elevated blood testosterone in older men. / School of Physical Education

Recent evidence suggests that declining bone mineral density (BMD) in males is related to declining circulating estrogens. The majority of endogenous plasma estrogens in males result from peripheral aromatization of plasma androgens. Thus, it was hypothesized that dietary supplementation with an aromatizable androgen (androstenedione) may stimulate increases in BMD. BMD (measured by dual energy x-ray absorptiometry) and biochemical markers of bone turnover (1, 25 OH2 Vitamin D, calcitonin, deoxypyrodinoline, and parathyroid hormone) were assessed before and after 12 weeks of dietary androstenedione supplementation (200 mg/d). Twenty-four volunteers were randomized into either an androstenedione supplementation or placebo groups. Study volunteers also performed high intensity resistance training (RT) during the treatment period. Androstenedione supplementation significantly increased plasma estradiol-17β levels by 82%. However, the increase in estradiol-17β did not impact bone turnover. The RT regimen did stimulate significant, local increases in BMD. Spine BMD was significantly increased by 6% for both treatment groups.

Die Reifung der Zona reticularis der Nebennieren-Rinden (NNR) und ihrer Androgen-Sekretion vor der Pubertät unterscheidet sich bei Menschen und höheren Primaten von der NNR-Reifung anderer Species, z.B. der Nager. Die Sekretion der NNR-Androgene leitet die Pubertätsentwicklung ein. Die NNR-Androgene erlangen medizinische Bedeutung dadurch, dass sie bei Frauen zu Hirsutismus und Fertilitätsstörungen führen können. Neben diesen Symptomen stellen sie einen Risikofaktor für die Entwicklung eines Polycystischen-Ovar-Syndroms (PCOS) dar, das ungefähr 7 % der prämenopausalen Frauen betrifft. Lange Zeit war nicht bekannt, wie die Differenzierung der Zona reticularis beim Menschen reguliert wird. Sicher ist ACTH bei weitem das bedeutsamste übergeordnete Hormon für die globale adrenocorticale Differenzierung und Funktion. Weitere Faktoren sind speziell für die Androgensekretion verantwortlich, aber nicht genau definiert. Nun wurde zunächst in zellbiologischen Experimenten belegt, dass die ACTH-Wirkung durch ein Spezies-spezifisches Muster von Wachstumsfaktoren autokrin moduliert wird und so die postnatale Entwicklung der Nebenniere steuern kann. Die vorliegenden Untersuchungen an menschlichen NNR-Zellen von Kindern und Erwachsenen in Primärkultur zeigen erstmals, dass IGF-I und IGF-II differenzierte Funktionen dieser Zellen aufrecht erhalten. IGF-I und, mehr noch, IGF-II steigern die Steroid-Biosynthese und ACTH-Ansprechbarkeit, und sie fördern die Bildung von Androstendion, einem delta5-Androgen der Zona reticularis. Darüberhinaus bewirkt Insulin in physiologischen sowie in micromolaren Konzentrationen den IGFs ähnliche Änderungen der Steroidsynthese. In Querschnittsuntersuchungen an gesunden Kindern vor der Pubertät sowie Kindern mit einfacher Adipositas konnte gezeigt werden, dass die Körperzusammensetzung mit den NNR-Androgenen zusammenhängt. Über die Mediatoren IGF-I, Insulin und Leptin wird offensichtlich der NNR der Zustand von Gewicht und Wachstum des Kindes signalisiert, auch bei pathologischer Körperzusammensetzung, wie dem Prader-Willi-Syndrom. Während die Adipositas die Androgen-Bildung steigern kann, ist sie jedoch selbst nicht der kausale Faktor einer vorzeitigen Nebennierenrindenreifung. Der Prämaturen Pubarche können in 5 - 10 % der untersuchten weiblichen Population ein nicht-klassisches AGS oder NNR-Tumoren zugrunde liegen. Bei den verbleibenden Kindern besteht eine eigentlich harmlose Reifungsbeschleunigung mit normaler Wachstumsprognose. Betrachtet man diese Kinder mit idiopatischer Prämaturer Adrenarche jedoch genauer, so finden sich zwei Untergruppen mit langfristigen Risiken: erstens zeigen Kinder mit einer sogenannte manifesten Heterozygotie für einen 21-Hydroxylase-Defekt Auffälligkeiten des Wachstums, die eine Endgrössenreduktion bewirken könnten, und zweitens wird bei Jugendlichen mit einer Überstimulierbarkeit der NNR diese "Exaggerated Adrenarche" für ein nachfolgendes PCOS verantwortlich gemacht. Schliesslich scheint es vor dem Hintergrund der sich epidemieartig ausbreitenden Zunahme des Übergewichts im Kindesalter angezeigt, den Bezug dieser NNR-Störungen zur Adipositas und der Hyperinsulinämie weiter zu klären. / The prepubertal maturation of the zona reticularis of the adrenal cortex and its androgen secretion in man and higher primates differs from other species, e.g. rodents. The secretion of adrenal androgens induces the pubertal development. The importance of adrenal androgens is derived from them being the cause for hirsutism and fertility disorders in women. In addition they represent a risk factor for the development of the polycystic ovary syndrome (PCOS), that affects about 7% of all pre-menopausal women. The regulation of the differentiation of the zona reticularis was unknown for a long time. However, ACTH is by far the most important hormone to regulate the global adrenocortical differentiation and function. In addition, other yet undefined factors are specifically responsible for the secretion of adrenal androgens. The cell-biological experiments presented here demonstrate that the effects of ACTH can be modulated in an autocrine manner by a species-specific pattern of growth factors so as to allow for the control of the postnatal development of the adrenal gland. The present investigations in human adrenocortical cells of children and adults in primary culture show for the first time that IGF-I and IGF-II maintain the differentiated function of these cells. IGF-I and to an even greater extent IGF-II enhance the biosynthesis of steroids and ACTH-responsiveness, and they promote the production of androstenedione, a delta5-androgen of the zona reticularis. Moreover, insulin, in physiological as well as in micromolar concentrations, induces changes in steroid production similar to the IGFs. In cross-sectional studies of healthy pre-pubertal children and children with simple obesity, it was shown that body composition is associated with adrenal androgens. Mediated by IGF-I, insulin and leptin, body composition apparently signals the child's state of weight and growth to the adrenals, even in patients with abnormal body composition, e.g. the Prader-Willi syndrome. While obesity may enhance androgen production, it is not the direct causal factor to induce premature adrenal maturation. In 5-10% of the female population investigated, premature pubarche is caused by non-classical adrenal hyperplasia or an adrenocortical tumour. In the remaining children, there is merely a harmless acceleration of maturation with normal growth prediction. A closer look at the children with idiopathic premature adrenarche, however, reveals two subgroups with long-term risks: First, children with a so called manifest heterozygosity of a 21-hydroxylase-defect show growth abnormalities, possibly reducing final height. Second, in adolescents with enhanced stimulation of the adrenal cortex, this 'exaggerated adrenarche' is held responsible for the subsequent development of PCOS. Finally, with regard to the rapidly spreading epidemic of overweight in children, it seems essential to study into greater depth the relationship between these adrenal dysfunctions and obesity or hyperinsulinism.