Arpad G. Fazekas

Studies on the biosynthesis of 18-oxygenated steroids from exogenous corticosterone by domestic duck (Anas platyrhynchos) adrenal gland mitochondria

Abstract The transformation of exogenous, isotopically labelled corticosterone to 18-hydroxycorti-costerone and aldosterone by domestic duck ( Anas platyrhynchos ) adrenal gland mitochondria was studied. The mitochondrial 18-oxygenating system was NADPH dependent. K + , Na + , Ca 2+ and Mg 2+ were necessary for maximal enzymatic activity. The enzyme present in 1 mg of mitochondrial protein became saturated with 13.2 βM of substrate (13.8 μg). Under saturation conditions the maximal production of 18-hydroxycorticosterone was found as 1.43 nmole/min/ mg protein and that of aldosterone 0.33 nmole/min/mg protein. The Michaelis constant for both 18-hydroxycorticosterone and aldosterone was 6.6 × 10 −6 M. Q 10 (average between 20°C and 40°C) was 2.16 for the reaction corticosterone → 18-hydroxycorticosterone and 1.34 for the reaction corticosterone → aldosterone. The mitochondrial enzyme system did not 18-oxygenate exogenous 11-deoxycorticosterone, 11-dehydrocorticosterone, 20β-dihydro-corticosterone. Exogenous 18-hydroxycorticosterone and aldosterone were not metabolized. The kinetics of the transformation of corticosterone to 18-oxygenated metabolites suggested a parallel pseudo-first order reaction rather than a series pseudo-first order reaction. 18-Oxygenation of corticosterone was strongly inhibited by d,1-18-hydroxycorticosterone, p-chloromercuribenzoate, carbon monoxide, metopirone (competitive inhibition; K i = 3.0 × 10 −6 M for 18-hydroxycorticosterone and 9.0 × 10 −6 M for aldosterone) and by aminopterin (noncompetitive inhibition; K i = for both metabolites 2.0 × 10 −5 M). Protein synthesis inhibitors did not have any effect. Cytochrome-P450 was shown to be present in mitochondria by spectrophotometric measurements. Addition of corticosterone. 11-deoxycorticosterone and metopirone produced type II difference spectra. The mitochondrial P450 became saturated with either corticosterone or metopirone at a concentration of 21.8 nmoles/mg protein. From these studies it was concluded that the duck adrenal mitochondrial 18-oxygenating system differed from the mammalian adrenal system previously described, especially in regard of substrate specificity. 18-Hydroxycorticosterone, either endogenous or exogenous, could not serve as substrate for aldosterone production with duck adrenal mitochondria. However, under ordinary circumstances. 18-hydroxycorticosterone synthesis was always associated with aldosterone synthesis in rather fixed proportions. Circumstantial evidence suggested the role of cytochrome-P450 in 18-oxygenation. As to the mechanism of the corticosterone → 18-hydroxycorticosterone → aldosterone reaction, this sequence could not be proven. The possibility should not be discarded that atmospheric oxygen is introduced into a hitherto unknown intermediary substance, which by enzymatic action and/or chemical rearrangement gives rise simultaneously to 18-hydroxy-corticosterone and aldosterone.

Metabolism of androgens by isolated human beard hair follicles

Abstract Human beard hair follicles were incubated in vitro with testosterone-7- 3 H, androstenedione-1,2- 3 H and dehydroepiandrosterone-4- 14 C.The principal metabolites of testosterone were androstenedione, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. The conversion of androstenedione into testosterone and 5α-dihydrotestosterone was very limited. Dehydroepiandrosterone was metabolized to 5-androstene-3β,17β-diol, 7α-hydroxy-dehydroepiandrosterone and androstenedione, with rates of formation far exceeding those reported for human skin. The significance of these metabolic transformations is discussed with special reference to the mechanism of action of androgens.

Corticosteroid-binding macromolecules in the salt-activated nasal gland of the domestic duck (Anas platyrhynchos).

The interaction of tritiated corticosterone and tritiated 11-dehydrocorticosterone with salt-activated nasal glands of the domestic duck was studied. Nasal gland cytosol (105,000g supernatant) bound corticosterone and 11-dehydrocorticosterone at 0° with apparent Kd values of 10−9 and 10−11 M, respectively. The cytosols transformed [3H]corticosterone to [3H]11-dehydrocorticosterone (average transformation: 95% in 2 hr). Competition studies have shown that radioinert corticosterone is a more efficient competitor for cytosol binding sites than radioinert 11-dehydrocorticosterone. Cytosols labeled with [3H]corticosterone showed two major peaks following sucrose density gradient centrifugation: a heavy peak at 9–11S and a lighter peak at 3–4S. In sucrose gradients containing 0.4 M KCl, part of the heavier peak became transformed to the 3–4S form. Following incubation of [3H]corticosterone-labeled cytosols with crude nuclei, the cytosols became depleted of the label and the tritium activity, in the form of [3H]11-dehydrocorticosterone, accumulated in the nuclear Tris-soluble fraction and in the chromatin-bound (Tris-insoluble, 0.4 M KCl-soluble) fraction. Following the incubation of nasal gland slices with either [3H]corticosterone or [3H]11-dehydrocorticosterone, the cytosol, nuclear Tris-soluble fraction, and chromatin-bound fraction became labeled with [3H]11-dehydrocorticosterone only. Both steroids seemed to be taken up by the tissue slices at identical rates. Administration in vivo of [3H]corticosterone to a saltwater-maintained bird showed the accumulation of [3H]11-dehydrocorticosterone and, to a smaller extent, of [3H]corticosterone in the nasal gland intracellular fractions. It is suggested that the duck nasal gland is a corticosteroid target organ and the cytoplasmic protein-bound corticosterone is transported to the nucleus mostly as 11-dehydrocorticosterone in a manner similar to the intracellular transport of aldosterone in the rat and duck kidney.

Chromatographic and Radioisotopic Methods for the Analysis of Riboflavin and the Flavin Coenzymes

This chapter will describe chromatographic and radioisotopic methods for the analysis of riboflavin (RF) and the flavin coenzymes in animal tissues. Chromatographic separations of the three major tissue flavins—flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and RF—have been in wide use since the early 1950s and are summarized in excellent review articles(21) and textbooks.(16,14) The first part of this chapter, therefore, deals only with the most important and practical chromatographic methods, including those that have been successfully used in the author’s laboratory.

Unusual pathways of aldosterone biosynthesis in the rabbit adrenal

Abstract The in vitro biosynthesis of aldosterone and other 18-oxygenated corticosteroids from labeled steroid precursors was investigated by rabbit adrenal slices, homogenates and mitochondrial preparations. Exogenous 18-hydroxy-corticosterone and 18-hydroxy-11-dehydrocorticosterone were both converted to aldosterone to a limited extent only. Both corticosterone and 11-dehydrocorticosterone were efficiently 18-hydroxylated by adrenal slices and mitochondria. The two 18-hydroxylated derivatives were interconvertible. Investigation of isotope ratios of steroids synthesized in substrate pair experiments, utilizing 11-dehydrocorticosterone-4-14C and corticosterone-1,2-3H have shown, that aldosterone was formed mainly from corticosterone via 18-hydroxy-corticosterone. However, the contribution of 11-dehydrocorticosterone to aldosterone was higher than to 18-hydroxy-corticosterone. This could have been mediated through 11-dehydroaldosterone which originated from 11-dehydrocorticosterone and was shown to be efficiently converted to aldosterone in a separate incubation.

The influence of corticosteroids on flavin nucleotide biosynthesis in rat liver and kidney

Abstract The biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) from exogenous [2- 14 C]-riboflavin (RF) was investigated in the liver and kidney of intact, adrenalectomized (AX), AX-aldosterone treated, AX-corticosterone acetate treated and AX-cortisol acetate treated rats. Radioactive flavins were determined by a new method using reverse isotope dilution and ion exchange chromatography. It was found that 3 days after adrenalectomy, hepatic FAD formation was reduced by 39% as compared to the intact group. Treatment of AX animals with aldosterone (10μg/day for 3 days) partially reversed the effect of adrenalectomy (to 82% of the control value). Treatment of AX animals with corticosterone acetate or cortisol acetate decreased hepatic FAD synthesis below the adrenalectomized level. Adrenalectomy and adrenalectomy followed by aldosterone treatment did not affect hepatic FMN formation, while corticosterone or cortisol treatment of AX animals decreased the formation of this flavin nucleotide. No significant changes were observed in renal FMN and FAD biosynthesis by any of the above procedures. These results indicate an extrarenal effect of aldosterone connected to flavoprotein dependent oxidative processes.

Pteridins and steroid hydroxylation—I: I—The effect of folic acid and aminopterin on the steroid 11β- and 21-hydroxylation by domestic duck (Anas platyrhynchos) adrenocort1cal tissue preparations

Abstract The effects of folic acid and aminopterin were studied on the steroid 11β and 21-hydroxylases of domestic duck adrenals. Aminopterin partially inhibited the transformation of labelled, exogenous progesterone to 11β-, 18- and 21-hydroxylated metabolites by a duck adrenal gland whole homogenate, while the presence of folic acid resulted in a slight stimulation of the sequential hydroxylation of progesterone. When added to duck adrenal mitochondrial or microsomal preparations aminopterin effected a concentration dependent inhibition of the 11β- or 21-hydroxylase activity while the presence of folic acid resulted in a concentration dependent stimulation. Aminopterin inhibition of mitochondrial 11β-hydroxylase was competitive, while that of the microsomal 21-hydroxylation was non-competitive. Neither folic acid or aminopterin induced difference spectra of mitochondrial or microsomal cytochrome P-450.