Cedric H.L. Shackleton

11β-Hydroxysteroid dehydrogenase: fact or fancy?

Abstract Previous attempts to explain the diverse behavior of 11β-hydroxysteroid dehydrogenase (11-HSD) within and between species have not been successful. We now propose that 11-HSD activity is the resultant of the coordinated interaction of two enzyme types, 11-dehydrogenase and 11-reductase. Me have demonstrated their separate existence by physico-chemical and kinetic methods. Based on these findings, two classes of disease in humans that have been recently described can now be characterized as being associated with a deficiency in either 11-dehydrogenase or 11-reductase.

Identification of 17α,20β,21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation

Abstract This study describes the identification of 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-dihydro-11-deoxycortisol, 20β-S) as a major steroid product of the ovary of Atlantic croaker ( Micropogonias undulatus ) incubated in vitro . This is the first report which describes 17α,20β,21-trihydroxy-4-pregnen-3-one as a major product of teleost steroidogenic tissue. The steroid was identified by a variety of methods, including HPLC, TLC, GC-MS, UV absorbance, and reactions with specific enzymes. Full grown oocytes, prior to final oocyte maturation (FOM), accumulated only small amounts of 20β-S. However, a substantial increase in 20β-S production occurred at the time of FOM. These results suggest that 20β-S is a maturational steroid in this species.

Thermospray HPLC/MS: A new mass spectrometric technique for the profiling of steroids

The analysis of various steroid classes by thermospray HPLC-MS using solvent systems containing 0.1 M ammonium acetate has been described. For simple unconjugated 3-oxo-4-ene steroids the positive ion spectra are dominated by a parent ion M + H+ and with increasing numbers of hydroxyl group intense ions formed by sequential losses of water (M + H- n18)+ become important. Steroids with dihydroxyacetone side-chains readily lose these side-chains and the resulting (M + H-60)+ fragment is the base peak in their spectra. The (M + H-60)+ ion is not important for most steroids with glycerol-type side-chains. Although competition between thermal degradation and vaporization was observed at lower concentrations, the effect was minimized after optimizing conditions and the protonated molecular ion was easily detected when as little as 1-10 pmol of material were injected on-column. Steroid glucuronides when analyzed in the negative ion mode give simple spectra with base peak and parent ion (M-H)-. Lack of fragmentation permits facile and sensitive measurement of individual glucoronides by selected-ion-monitoring. Extensive fragmentation is seen in the positive ion mode with sequential losses of H2O from the molecular ions (M + NH4)+ and from the aglycone fragment ion. For simple unconjugated steroids the sensitivity of HPLC-MS in selected-ion-monitoring mode can be excellent. When the protonated molecular ion of testosterone was monitored the signal/noise ratio for 30 pg testosterone was about 10.

Familial male pseudohermaphroditism due to 5-alpha-reductase deficiency in a Turkish Village

Twelve persons with sexual ambiguity were identified in an isolated village in southern Turkey. Eleven were examined and had pseudovaginal perineoscrotal hypospadias; eight were studied. Serum and urine samples from five affected males and urine samples from three affected children were analyzed. Urine samples from another 26 villagers, mostly parents and siblings, were also analyzed. In all but one of the affected adult subjects, serum testosterone levels were either normal or increased, and in all adults, the dihydrotestosterone levels were low (8 to 20 ng/dl) and the testosterone/dihydrotestosterone ratios were elevated (to 36 or more); the levels of 4-androstenedione were normal. Thirty-four urine samples were analyzed for etiocholanolone/androsterone, 11-beta-hydroxyetiocholanolone/11-beta-hydroxyandrosterone, tetrahydrocorticosterone/5-alpha-tetrahydrocorticosterone and tetrahydrocortisol/5-alpha-tetrahydrocortisol ratios. In affected persons, all 5-beta/5-alpha urinary C19 and C21 steroid metabolite ratios measured were elevated. These findings are compatible with the diagnosis of male pseudohermaphroditism due to 5-alpha-reductase deficiency. In parents and some of the siblings of the affected subjects, the 5-beta/5-alpha urinary ratios were between affected and normal levels. The intermediate 5-beta/5-alpha ratios of the parents who were phenotypically normal, together with documented consanguinity, confirm an autosomal recessive mode of inheritance and are useful in identification of the carrier state. The urinary tetrahydrocortisol/5-alpha-tetrahydrocortisol ratios provided the highest index of discrimination between homozygotes (mean +/- SD, adults: 35.80 +/- 20.10; children: 15.48 +/- 7.91), heterozygotes (parents: 4.56 +/- 1.61; siblings and other relatives: 5.97 +/- 3.68), and normal subjects (1.07 +/- 0.36). Thus, this study identified a second community with inherited male pseudohermaphroditism due to 5-alpha-reductase deficiency, confirming the autosomal recessive inheritance of this condition and the generalized abnormality in both C19 and C21 steroid 5-alpha metabolism.

URINARY STEROID METABOLITES IN SUBJECTS WITH MALE PSEUDOHERMAPHRODITISM DUE TO 5α‐REDUCTASE DEFICIENCY

To investigate the enzymatic basis for abnormal steroid metabolism in subjects with male pseudohermaphroditism due to 5α‐reductase deficiency, the ring A reduced urinary 5β and 5α metabolites of testosterone, androstenedione, 11β‐hydroxyandrostenedione, cortisol and corticosterone were measured by gas chromatography. Assays of the four pairs of urinary 5β and 5α steroid metabolites revealed decreased conversion of the parent steroids to 5α‐reduced urinary metabolites, with increased 5β to 5α urinary steroid metabolite ratios. These studies establish that increased urinary 5β/5α ratios are distinctive for this disorder, and represent the most reliable method for confirming the diagnosis of primary inherited 5α‐reductase deficiency. These data also suggest that the conversion on the many Δ4‐3 ketosteroids to 5α‐reduced steroids may be due to a single enzyme with broad specificity, or multiple enzyme reductases with a common regulator.

Mutations in CYP11B1 gene: phenotype-genotype correlations.

11β‐hydroxylase deficiency, an autosomal recessive disorder, is the second most common cause of congenital adrenal hyperplasia. We studied four subjects with classic 11β‐hydroxylase deficiency and severe hypertension: a 46,XX affected subject from a Turkish family with severe ambiguity of the external genitalia and hypertension, and three affected 46,XY subjects from a Dominican kindred with isosexual precocious puberty and severe hypertension. The affected subjects had significantly elevated plasma 11‐desoxycortisol, 11‐desoxycorticosterone, Δ4‐androstenedione, and testosterone. To determine the molecular genetic defects, genomic DNA was isolated from the leukocytes of affected subjects and their family members. The encoding region of the 11β‐hydroxylase gene (CYP11B1) was amplified by PCR with specific primers. Using single‐stranded DNA conformational polymorphism (SSCP) and DNA sequencing, a nonsense mutation in exon 6 of CYP11B1 in the affected 46,XX subject from the Turkish family was identified, where a cytosine was substituted by a thymidine, resulting in the replacement of glutamine (CAG) by a stop codon (TAG) at amino acid position 338 (Q338X). In the three 46,XY Dominican boys, the mutation was also a nonsense mutation in exon 6 of CYP11B1, where a cytosine was substituted by a thymidine, resulting in the replacement of glutamine (CAG) by a stop codon (TAG) at amino acid position 356 (Q356X). Both mutations result in the biosynthesis of a truncated 11β‐hydroxylase protein with loss of enzymatic activity. Heterozygosity was determined in family members of both probands including parents and siblings. These results indicate that mutations of CYP11B1 in these subjects are responsible for their clinical syndromes.

THE COEXISTENCE OF MALE PSEUDOHERMAPHRODITES WITH 17‐KETOSTEROID REDUCTASE DEFICIENCY AND 5α‐REDUCTASE DEFICIENCY WITHIN A TURKISH KINDRED

Two distinct enzyme defects affecting androgen production and resulting in male pseudohermaphroditism were found in a Turkish kindred from a small isolated village in the Taurus mountains of southern Turkey. Pedigree analysis revealed the inter‐relationships of 9 male pseudohermaphrodites. Six affected subjects had adequate steroid hormone analysis. Two adult male pseudohermaphrodites had 17‐ketosteroid reductase deficiency with elevated concentrations of plasma androstenedione relative to testosterone, and elevated concentrations of urinary androsterone (A) and etiocholanolone (E) relative to tetrahydrocortisol (THF), 5α‐tetrahydrocortisol (5α‐THF) and tetrahydrocor‐tisone (THE). Four affected males (three adults, one child) had 5a‐reductase deficiency (elevated ratios of plasma testosterone/dihydrotestosterone and urinary 5β/5αC19 and C21 steroid metabolites). The homozygous state for both enzyme deficiencies was not demonstrable in the same affected subject, suggesting that the enzyme deficiencies are segregating separately within this kindred. Whether the mutant genes are segregating on allelic chromosomes or other autosomes cannot be determined from this study.

Equol: A contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.

Genetic Disorders of Steroid Metabolism Diagnosed by Mass Spectrometry

From a biochemical perspective most genetic disorders of steroid synthesis and metabolism are best studied by measuring individual steroids, or panels in serum, and/or by producing metabolic profiles of urinary steroids. There is increasing interest in further developing these analyses within the developing field of metabolomics. Classical analyses of hormones using immunoassays no longer have the sensitivity and accuracy demanded by physicians and researchers, and tandem mass spectrometry is rapidly taking over this role. However, gas-chromatography/ mass spectrometry (GC/MS) still reins supreme for the metabolic profiling of steroid metabolites, although this may slowly change. This chapter summarises recent developments in HPLC/MS techniques for hormonal steroid analysis as well as providing a comprehensive description of the use of mass spectrometry in the diagnosis and study of almost all single gene disorders of steroid synthesis and metabolism.

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11β-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.