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C H Shackleton

Publications and source records attributed to C H Shackleton.

At least 37 records · Page 2Linked to original sources
PubMed1997

Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: potential use in gas chromatography isotope ratio mass spectrometry.

The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and beta-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5 alpha (and beta)-androstane-3 alpha,17 alpha-diol (epitestosterone as precursor); 5 alpha (and beta)-androstane-3 alpha,17 beta-diol (testosterone as precursor); 5-androstene-3 beta,17 beta-diol (dehydroepiandrosterone precursor); and 5 alpha-androstane-3 alpha,17 beta- (and 17 alpha-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of delta 13C/1000 value compared to normal values. Typically, in the male individual studied, delta 13C/1000 values for all components were -26 to -27 before drug administration and -29 to -30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5 alpha-dihydrotestosterone. A major metabolite was 5 alpha-androstane-3 alpha,17 alpha-diol, which had presumably been formed by 17 beta/17 alpha isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.

Androstane-3,17-diol↗
PubMed1997

Apparent mineralocorticoid excess in a Brazilian kindred: hypertension in the heterozygote state.

BACKGROUND: Apparent mineralocorticoid excess (AME) is a cause of low-renin, low-aldosterone hypertension in which cortisol acts as a mineralocorticoid. The condition reflects an inability to inactivate cortisol to cortisone due to defective activity of the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2). Homozygous mutations in 11beta-HSD2 gene in patients with AME have been described. A 7-year-old Brazilian girl had previously been found to have AME. Her father recently presented with mineralocorticoid hypertension at age 38 years. OBJECTIVE: To describe the clinical details, to perform steroid analyses and to assess the molecular basis for the hypertension in this kindred. METHODS: The 11beta-HSD2 gene was amplified from genomic DNA by the polymerase chain reaction and sequenced by direct chain-termination sequencing on an automatic DNA sequencer. The sequencing results were validated by restriction-site polymorphism. The mutant 11beta-HSD2 protein was expressed in Chinese hamster ovary polyoma cells and enzymatic activity was assessed by metabolizing cortisol in vitro. RESULTS: Sequence analysis of genomic DNA revealed a novel C1061T point mutation in exon V of the human 11beta-HSD2 gene, resulting in an amino acid substitution of alanine by valine at codon 328 of the enzyme protein (A328V). Expression studies confirmed that the mutant protein was devoid of 11beta-HSD2 activity. A HhaI restriction-site polymorphism confirmed that the proband was homozygous for the mutation whereas both parents were heterozygotes. The father of the proband had hypertension, a normal serum potassium level, suppressed plasma renin activity and plasma aldosterone level and a moderately elevated urinary cortisol: cortisone metabolite ratio. CONCLUSIONS: AME in this kindred is caused by a novel mutation in the 11beta-HSD2 gene. Detection of hypokalaemia, at least in this kindred, is an insensitive screening test for mineralocorticoid-based hypertension. In contrast to results from previously investigated kindreds, we have demonstrated that this kindred has an abnormal phenotype in the heterozygote state. Further studies are now required in order to evaluate the role of 11beta-HSD2 activity in the pathophysiology of 'essential' hypertension.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Measurement of hepatic Ra UDP-glucose in vivo in rats: relation to glycogen deposition and labeling patterns.

We previously described an isotopic method for quantifying the rate of appearance of hepatic UDP-glucose (Ra UDP-Glc) and the direct entry of glucose into hepatic UDP-Glc in humans. Here, the method is tested in depth in rats. The basic principles are that dilution of labeled galactose in hepatic UDP-Glc, sampled noninvasively by the xenobiotic glucuronate (GlcUA) method, reveals Ra UDP-Glc. First, labeling patterns in secreted acetaminophen-GlcUA were compared with hepatic glycogen and plasma glucose by use of mass isotopomer distribution analysis from [2-(13)C]glycerol. Labeling was consistent with common precursor pools of glucose 6-phosphate and triose-phosphate for all end products studied in fasted and in intravenous glucose- and fructose-infused states. Next, [1-(3)H]galactose was administered. After a 24-h fast, Ra UDP-Glc was 25.0 +/- 1.7 mumol.kg body wt-1.min-1 and rose to 57.7 and 72.7 mumol.kg-1.min-1 at intravenous glucose infusion rates of 111 and 167-194 mumol.kg-1.min-1, respectively. Liver glycogen deposition correlated closely with Ra UDP-Glc (R2 = 0.76), although the turnover value was approximately 50% higher than the net deposition rate. In conclusion, the turnover of an intrahepatic metabolite, UDP-Glc, can be measured noninvasively, and Ra UDP-Glc correlates with liver glycogen deposition in rats.

Animals↗
PubMed1997

Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1.

The secosteroid hormone, 1,25-dihydroxyvitamin D [1,25(OH)2D], plays a crucial role in normal bone growth, calcium metabolism, and tissue differentiation. The key step in the biosynthesis of 1,25(OH)2D is its 1 alpha-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney. Because its expression in the kidney is very low, we cloned and sequenced cDNA for 25-OHD-1 alpha-hydroxylase (P450c1 alpha) from human keratinocytes, in which 1 alpha-hydroxylase activity and mRNA expression can be induced to be much greater. P450c1 alpha mRNA was expressed at much lower levels in human kidney, brain, and testis. Mammalian cells transfected with the cloned P450c1 alpha cDNA exhibit robust 1 alpha-hydroxylase activity. The identity of the 1,25(OH)2D3 product synthesized in transfected cells was confirmed by HPLC and gas chromatography-mass spectrometry. The gene encoding P450c1 alpha was localized to chromosome 12, where the 1 alpha-hydroxylase deficiency syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been localized. Primary cultures of human adult and neonatal keratinocytes exhibit abundant 1 alpha-hydroxylase activity, whereas those from a patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1 alpha cDNA from the patient with VDDR-1 contained deletion/frameshift mutations either at codon 211 or at codon 231, indicating that the patient was a compound heterozygote for two null mutations. These findings establish the molecular genetic basis of VDDR-1, establish a novel means for its study in keratinocytes, and provide the sequence of the key enzyme in the biological activation of vitamin D.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1996

Effects of dietary n-3 fatty acid supplementation in men with weight loss associated with the acquired immune deficiency syndrome: Relation to indices of cytokine production.

Cytokines may be involved in weight loss and disturbances of metabolism associated with human immunodeficiency virus (HIV) infection. Dietary n-3 fatty acids reduce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMC) in normal humans and prevent IL-1 and TNF anorexia in animals. Accordingly, we studied the nutritional and metabolic effects of a 10-week trial of dietary fish oil (MaxEPA 18 g/day) in men with weight loss due to acquired immune deficiency syndrome (AIDS). Twenty men were enrolled, and 16 completed the 10-week supplementation period. Prior weight loss was 13.7 +/- 1.8 kg(17.4 +/- 1.6% body weight, means +/- SE). Food intake, body composition, blood chemistries, serum cytokine concentrations, in vitro production of IL-1 and TNF by PBMC, and clinical course were followed. A subset of subjects (n=12) underwent stable isotope infusions to measure de novo hepatic lipogenesis (DNL), an in vivo metabolic index that is influenced by cytokine presence and has previously been found to be elevated in AIDS. An unsupplemented group of men with AIDS wasting (10.4 +/- 2.4 kg weight loss, 13.1 +/- 2.2% body weight) was monitored for 10 weeks as controls. Baseline food intake (2,395 +/- 177 kcal/day and 95.1 +/- 7.2 g protein/day), body weight, percent fat, and fat-free mass were unchanged over the 10-week supplementation period. Serum triglycerides were reduced in hypertriglyceridemic subjects, confirming compliance with fish oil supplementation and suggesting that their hypertriglyceridemia was at least in part due to overproduction. Serum TNF and IL-1 were undetectable before or after fish oil supplementation. Serum interferon alpha (IFN) was measurable but did not change. In vitro production of IL-1 and TNF by PBMC was markedly reduced both at baseline and after fish oil supplementation in this population, even in the presence of new AIDS complications, compared with normal controls. The metabolic measurement DNL fell and weight was gained (2.1 +/- 1.3 kg) in subjects who did not develop new AIDS-related complications, but further increases in DNL and further weight loss were observed in subjects who developed a new AIDS complication (p<0.05 for interaction between new complication and change in DNL). No changes in body weight, food intake, serum triglycerides, serum cytokines, or DNL were observed in the unsupplemented group. We conclude that fish oil is a weak anticytokine agent that is unable to overcome the metabolic and nutritional consequences of acute AIDS-related complications but may exert a clinical anticytokine effect in stable AIDS patients. Cytokine production by PBMC is not a useful or reliable marker of in vivo cytokine activity in AIDS patients with weight loss. In contrast, an integrative functional index that is sensitive to cytokine presence in tissues (hepatic DNL) correlated with clinical response. These findings are relevant to the design of future studies of more potent anticytokine agents, such as thalidomide.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Apparent mineralocorticoid excess: type I and type II.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension due to an inborn error of cortisol metabolism and is characterized by hypokalemia and low renin levels despite subnormal or normal levels of aldosterone and other known mineralocorticoids. The syndrome is attributable to congenital deficiency of the enzyme 11 beta-hydroxydehydrogenase (11 beta-HSD), which converts cortisol (F) to biologically inactive cortisone. This results in a prolonged half-life of F, which acts at the kidney level as a potent mineralocorticoid (MC). In fact, both F and aldosterone have similar affinities in vitro for type I MC receptor (MR), and 11 beta-HSD activity protects the MR in vivo from the higher circulating levels of F. The biochemical marker of this disorder is an increased ratio of tetrahydrocortisol (THF) + allo-THF/tetrahydrocortisone (THE) in the urine, which has been found in more than 20 patients described to date, together with evidence of a more general defect in steroid ring A reduction. Only a few cases (the so-called type II form) described in Italy differ from the classic form having a normal THF/THE ratio, but in both forms the ratio of free urinary F/E has recently been found to be similarly high. Dexamethasone is the treatment of choice but is often inadequate in long term control of high blood pressure. Acquired forms of AME are those consequent on abuse of licorice or carbenoxolone, which both inhibit 11 beta-HSD; the latter also inhibits the reverse 11-oxoreductase reaction leading to somewhat different abnormalities of urinary cortisol/cortisone. So far, two isoenzymes of 11 beta-HSD have been purified and cloned; 11 beta-HSD type 1 is NADP-dependent, abundant in liver, lung, and testis, and catalyzes both 11 beta-dehydrogenation and 11 beta-oxoreduction; no mutation in its gene was detected in patients with AME. A second NAD-dependent isoenzyme is present in kidney and placenta and catalyzes dehydrogenation only. Very recently (1995) two groups have independently demonstrated the presence of mutations in its gene, located in chromosome 16q22. New and co-workers found a point mutation in exon 6 of two affected siblings of an Iranian family, while White and co-workers in parallel studies showed point mutations or small deletions in both alleles in nine unrelated patients; importantly, expression studies showed minimal or absent activity for almost all the mutant sequences. No definite mutations have been so far identified in patients with AME type II. AME is thus the third single gene cause of human hypertension to be described, after glucocorticoid remediable aldosteronism in 1992 and Liddle's syndrome in 1994.

Child, Preschool↗
PubMed1996

Carboxymethylation of the human estrogen receptor ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows that cysteine 447 does not react with iodoacetic acid.

Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.

Alkylation↗
PubMed1996

Intact noncovalent dimer of estrogen receptor ligand-binding domain can be detected by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESMS) of the estrogen receptor ligand binding domain (ER LBD) in its estradiol-binding form was performed. A dimeric ER LBD was observed, with a greatly reduced capacity for protonation (major charge state for dimer +16 vs. +23 for a monomer). Peak broadening (probably due to heterogeneity resulting from salt and water adduct formation) adversely affected our ability to distinguish between multiple discreet dimeric species and thus prevented us from establishing an accurate average mass for the dimerized domain. A mixture of species with molecular masses between 57,240 Da and 57,900 Da was observed, which would compare to 57,274 Da, 57,546 Da, and 57,818 Da for the calculated masses of the dimer without estradiol, or with one or two bound ligand molecules, respectively. Hence, nonliganded ER LBD dimer appeared to constitute the major species. The presence of low levels of a singly liganded ER LBD dimer cannot be ruled out, but the data argue against the possibility of the ER LBD dimer carrying two molecules of estradiol. Allowing for current limitations in the technology, our data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range (4000 Da for singly charged ions) has potential utility for studying ligand-binding proteins. In particular, in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.

Binding Sites↗
PubMed1996

Quantitation of cortisol and related 3-oxo-4-ene steroids in urine using gas chromatography/mass spectrometry with stable isotope-labeled internal standards.

A method for the profiling of several important 3-oxo-4-ene urinary steroids is reported. The methodology is combined gas chromatography/mass spectrometry (GC/MS) utilizing stable isotope-labeled internal standards. The following standards were obtained or easily synthesized: [9, 11, 12, 12-2H4]cortisol, [1,2-2H2] and [9, 12, 12-2H2]cortisone, [1,2-2H2]6 beta-hydroxycortisol, and [1,2-2H2]18-hydroxycortisol. We found the following excretions of free steroids for normal adult males and females: cortisol (males mean +/- SD, 35 +/- 13; females mean +/- SD, 23 +/- 13), cortisone (males mean +/- SD, 58 +/- 23; females mean +/- SD, 50 +/- 22), 6 beta-hydroxycortisol (males mean +/- SD, 164 +/- 59; females mean +/- SD, 108 +/- 55), and 18-hydroxycortisol (males mean +/- SD, 148 +/- 55; females mean +/- SD, 71 +/- 30). For 18-hydroxycortisol in particular, the excretions were much higher for males than for females. We found that the larger part of urinary cortisol and cortisone is not free but is released from conjugation by enzymes present in snail digestive juice. Using a pooled urine sample from an equal number of male and female subjects, we found that for cortisol 29% was excreted free, 28% as glucuronide and 43% as other conjugates (probably sulfates). For cortisone 41% was free, 45% beta-glucuronide and 14% as other conjugates. Relatively little (3-8%) of the hydroxylated cortisols were excreted conjugated.

Adolescent↗
PubMed1996

Urinary free cortisone and the assessment of 11 beta-hydroxysteroid dehydrogenase activity in man.

OBJECTIVE: Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyse the interconversion of cortisol to hormonally inactive cortisone; defects in the 11 beta-HSD2 isoform result in hypertension. The kidney, expressing high levels of 11 beta-HSD2, is the principal source of cortisone in man. We have validated the measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pitultary-adrenal axis in an attempt to more accurately measure the activity of 11 beta-HSD2 in vivo. SUBJECTS: Forty-one normal adults, 12 normal children < 12 years of age, 15 patients with Cushing's syndrome, 12 with hypopitultarism on replacement hydrocortisone, 12 with the syndrome of apparent mineralocorticoid excess (AME) and 7 volunteers consuming liquorice. MEASUREMENTS: A complete 24-hour urine collection was analysed by gas chromatography/mass spectrometry for "A-ring' reduced cortisol and cortisone metabolites, i.e. tetrahydrocortisols (THF and allo-THF) and tetrahydrocortisone (THE). In addition, urinary free cortisol (UFF) and urinary free cortisone were quantified using deuterium-labelled internal standards. RESULTS: In normal adults and children, UFE excretion exceeded that of UFF (UFF 30.4 +/- 2.4 micrograms/24h (mean +/- SE), UFE 54.6 +/- 4.1 micrograms/24h, adults) (for conversion to nmol/24h multiply E by 2.78 and F by 2.76 respectively). Thus the normal UFF/UFE ratio was 0.54 +/- 0.05 in contrast to the (THF + allo-THF)/THE ratio of 1.21 +/- 0.06. UFE excretion was normal in hypopituitary patients on replacement hydrocortisone. Although UFE was elevated in all forms of Cushing's syndrome, the UFF/UFE ratio was grossly elevated in patients with the ectopic ACTH syndrome (14.0 +/- 6.7, n = 6). UFE was below the lower limit of the assay (< 1 microgram/24h) in most patients with the so-called type 1 variant of AME and significantly reduced in 4 patients described as having the type 2 variant of AME (10.5 +/- 3.5 micrograms/h, P < 0.05) and in 7 volunteers consuming liquorice (26.8 +/- 10.0 micrograms/24h, P < 0.01). In ectopic ACTH syndrome, AME, and liquorice ingestion the UFF/UFE ratio was more deranged than the (THF + allo-THF)/THE ratio. CONCLUSION: In normals the discrepant THF + allo-THF/ THE and UFF/UFE ratio suggests that much more of the UFE is derived from the kidney. Reduction in UFE excretion is seen following liquorice ingestion and in both variants of AME, though it is more profound in AME1. The high UFF/UFE ratio in the mineralocorticoid excess state seen in the ectopic ACTH syndrome is compatible with substrate-saturation of renal 11 beta-HSD2. The measurement of UFE and the UFF/UFE ratio is a significant advance in the analysis of human 11 beta-HSD activity in vivo; in particular, the UFF/UFE ratio appears to be a more sensitive index than the (THF + allo-THF)/THE ratio of renal 11 beta-HSD2 activity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Increased vasoconstrictor sensitivity to glucocorticoids in essential hypertension.

Glucocorticoids raise blood pressure but were thought not to play a pathophysiological role in essential hypertension when it was demonstrated that cortisol secretion rates and circulating concentrations are normal in this disease. However, recent observations suggest that increased tissue sensitivity to cortisol, mediated by either abnormal glucocorticoid receptors or impaired inactivation of cortisol by 11 beta-dehydrogenase, may allow cortisol to raise blood pressure despite normal circulating concentrations. We studied 11 patients with essential hypertension and 11 matched normotensive control subjects. Dermal vasoconstriction after topical application of both cortisol (16 +/- 4 versus 32 +/- 5 U, control subjects versus hypertensive patients; P < .02) and beclomethasone dipropionate (75 +/- 10 versus 100 +/- 7 U; P < .05) was increased in the hypertensive patients. Hypothalamic-pituitary glucocorticoid receptor sensitivity was normal, as judged by basal cortisol secretion rates and suppression of plasma cortisol during sequential overnight dexamethasone suppression tests. 11 beta-Dehydrogenase activity was impaired in essential hypertension, as judged by prolonged half-lives of [11 alpha-3H]cortisol (44 +/- 4 versus 58 +/- 4 minutes, control subjects versus hypertensive patients; P < .02). However, this did not correlate with the dermal vasoconstrictor response. We conclude that vasoconstrictor sensitivity to glucocorticoids is increased in essential hypertension and that this may initiate and/or sustain the increased peripheral vascular resistance that characterizes this disease. The mechanism of increased sensitivity remains uncertain, but it will be important to establish whether it relates to genetic abnormalities of the glucocorticoid receptor that have been observed in animal models and young individuals who are predisposed to essential hypertension.

Administration, Oral↗
PubMed1996

Metabolism of 5 alpha-dihydroprogesterone in women and men: 3 beta- and 3 alpha-,6 alpha-dihydroxy-5 alpha-pregnan-20-ones are major urinary metabolites.

The metabolism of 5 alpha-dihydroprogesterone (5 alpha-DHP) in women and men was evaluated by defining the pattern and identity of selected metabolites excreted in urine after the iv infusion of radiolabeled 5 alpha-DHP. Virtually all of the radioactivity in urine (approximately 37% of the administered dose) was excreted within 72 h. Quantitatively, the 2 major urinary metabolites of 5 alpha-DHP in each of 13 studies conducted in 7 women and 2 men were 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one and 5 alpha-pregnane-3 alpha,20 alpha-diol, which could be extracted after beta-glucuronidase, but not solvolysis, treatment of the urine. Radiolabeled 3 alpha,6 alpha dihydroxy-5 alpha-pregnan-20-one (glucuronoside), in lesser amounts, also was identified in the urine of each subject. The 3 alpha/beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-ones arise through specific extrahepatic pathways of progesterone/5 alpha-DHP metabolism. These metabolites are not the products of the enzyme reaction catalyzed by the cytochrome P450 steroid 6 alpha-hydroxylase of human liver (and other tissues), which affects the 6 alpha-hydroxylation of C19- and C21-delta 4-3-ketosteroids (e.g., progesterone, testosterone, and cortisol), but does not act upon 5 alpha-reduced steroids. Moreover, the steroid 5 alpha-reductases do not act upon 6 alpha-hydroxy-delta 4-3-ketosteroids. In addition, the 6 alpha-hydroxylation of 5 alpha-reduced-3 alpha/beta-hydroxysteroids is not demonstrable in adult liver tissue. Rather, the formation of 6 alpha-hydroxylated-5 alpha-pregnane-3 alpha/beta-ol-20-ones is indicative of an extrahepatic pathway of progesterone metabolism, viz. progesterone-->5 alpha-DHP-->5 alpha-pregnan-3 beta/alpha-ol-20-one(s)-->3 beta/alpha,6 alpha-dihydroxy-5 alpha-pregnan-20-one(s), in which 5 alpha-pregnan-3 alpha/beta-ol-20-ones are metabolized by an enzyme(s) that catalyzes the 6 alpha-hydroxylation of saturated substrates. There are important differences among mammalian species in the enzymes that catalyze the C-6-hydroxylation of 5 alpha-reduced C19- and C(21)-3 beta/alpha-hydroxysteroids, but in all species studied, these enzymatic reactions are the final steps in the extrahepatic inactivation of 5 alpha-reduced bioactive metabolites of progesterone (or testosterone).

5-alpha-Dihydroprogesterone↗
PubMed1996

Apparent cortisone reductase deficiency: a unique form of hypercortisolism.

We describe two female siblings who had production of cortisol (F; as determined from excretion of urinary metabolites) high enough to give rise to Cushing's disease, but who had no clinical indications of the condition. The teenage patients were hirsute as a result of adrenal hyperandrogenism. A notable feature of the condition was the elevated excretion of corticosteroid metabolites with 11-carbonyl groups and very low excretion of 11 beta-hydroxylated steroids. We termed this disorder apparent cortisone (E) reductase disorder. The steroid metabolite phenotype appeared to be the opposite of that seen in the apparent mineralocorticoid excess syndrome, in which the excretion of 11-keto compounds is attenuated. As an example, the tetrahydrocortisol plus 5 alpha-tetrahydrocortisol/tetrahydrocortisone ratio was about 0.04 compared to normal values of about 1.0 and apparent mineralocorticoid excess syndrome values of 5.0-50.0. Paradoxically, among the F metabolites that had not undergone A-ring reduction, 11 beta-hydroxylated steroids dominated over 11-carbonyl compounds. The F/E ratio was about 1.8 compared to an average normal value of 0.54. Neither the father nor the mother of the patient had abnormal F metabolite/E metabolite ratios, although the father did excrete highly elevated free E and F, possibly an unrelated condition. A conclusion was not reached regarding the basis of the disorder. We considered that the most likely causes were 1) defective hepatic 11 beta-hydroxysteroid dehydrogenase-1, 2) failure to develop the adult form of F metabolism, or 3) excessive activity of A ring reduction enzymes acting on E.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

5 alpha-reductase-2 gene mutations in the Dominican Republic.

Male pseudohermaphroditism due to 5 alpha-reductase deficiency was clinically and biochemically described in a large Dominican kindred of 23 families with 38 affected subjects in 1974. Recently, the 5 alpha-reductase-2 gene defect in the large Dominican kindred was found to be due to a single base substitution of thymidine (TGG) for cytosine (CGG) on exon 5 of the 5 alpha-reductase-2 gene, causing a tryptophan replacement of arginine at amino acid 246 (R246W) of the enzyme. In the present report, affected subjects from four additional Dominican families were studied to determine whether they carried the same 5 alpha-reductase-2 gene defect as the large kindred, suggesting a common ancestry for the gene defect within this small country. Using single strand conformational polymorphism and DNA sequencing, two other mutations of the 5 alpha-reductase-2 gene were found in affected subjects from two of the four families. A point mutation on exon 2 of the 5 alpha-reductase-2 gene, in which substitution of adenine (GAC) for guanine (GGC) caused an aspartic acid replacement of glycine at amino acid 115 (G115D), was demonstrated in one of these families, and a substitution of adenine (AGT) for guanine (GGT) on exon 3 causing a serine replacement for glycine at amino acid 183 (G183S) was detected in the other family. Affected subjects from the two remaining families demonstrated the same exon 5 mutation of the 5 alpha-reductase-2 gene as previously detected in the large Dominican kindred. The phenotypic and biochemical characteristics of the male pseudohermaphrodites were similar regardless of the genetic defect, except that one affected subject (C-VI-2) with the same exon 5 mutation as the large Dominican kindred had much more facial and body hair. Thus, the identification of multiple mutations in the 5 alpha-reductase-2 gene in male pseudohermaphrodites from the Dominican Republic demonstrates a lack of common ancestry, as had been previously postulated.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

[Hyperaldosteronism sensitive to dexamethasone with adrenal adenoma. Clinical, biological and genetic study].

OBJECTIVES: Dexamethasone-sensitive hyperaldosteronism is associated with early onset hypertension and primary hyperaldosteronism. Diagnosis is difficult but can be improved by genetic testing for the mutant gene. METHODS: We collected the clinical, biological and genetic elements observed in a family with dexamethasone-sensible hyperaldosteronism. Complete data were obtained in 5 adult subjects with the disease. Degree of hypertension varied, more so in the second generations as did hypokaliaemia and hyperaldosteronism. In affected patients, there was a 10 to 50 fold increase in urinary 18-OH components and 18 oxocortisol. RESULTS: Single dose (1.5 mg) dexamethasone led to a greater than 80% drop in aldosterone levels in the blood and urine, confirming the abnormal effect of ACTH on mineralocorticoid secretion. At the dose of 1 mg/d for 10 weeks, dexamethasone lowered mean 24-H ambulatory arterial pressure (11.8/9.6 mmHg) and corrected for the hypokaliaemia (+0.54 mmol/l) and the hyperaldosteronism (mean decrease -36% and -75% in blood and urine respectively). An adrenal tumour was identified in hyperplasic glands in two subjects and a micronodular formation was identified in two others. The specific molecular diagnosis of the disease was done with Southern blotting. Among the 18 families in 3 generations, 8 carried a 11 beta OHase-aldosterone synthetase chimeric gene. This mutation cosegregates with hormonal abnormalities and confirms the autosomal dominant inheritance of the disease. CONCLUSION: The simplicity and rapidity of genetic testing allows early diagnosis of this disease among families with early onset hypertension and associated hyperaldosteronism with or without adrenal hyperplasia and/or a tumoral formation.

Adolescent↗
PubMed1995

Long-lived testosterone esters in the rat.

Over the past decade it has become increasingly clear that steroid hormones are enzymatically esterified with fatty acids. These steroidal esters are the natural analogs of synthetic esters that are used therapeutically. One such family of pharmacological steroids is the synthetic alkyl esters of testosterone, androgens with great hormonal potency. We have investigated whether testosterone esters exist naturally by using the rat as a model. Most tissues of male rats, including blood, have very little if any ester (quantified by immunoassay as a nonpolar saponifiable metabolite), but fat and testes have sizable quantities, approximately 3 ng of testosterone equivalents per g of tissue. Testosterone in fat averages 9 ng/g. The fat from female rats and long-term (> 2 weeks) castrated males has no detectable testosterone ester. The presence of testosterone esters was confirmed by GC/MS, which clearly showed the presence of testosterone in the hydrolyzed ester fraction of fat from intact males but not long-term castrates. Upon castration, testosterone levels in the fat completely disappear within 6 hr. To the contrary, it is not until 48 hr after castration that a measurable fall in the testosterone ester fraction was observed; even after 10 days a small amount of ester is still present in the fat. These experiments demonstrate the existence of a previously unknown androgen with a potentially important physiological impact; testosterone esters, natural analogs of potent therapeutic agents, occur in the fat where they can serve as a reservoir of preformed androgen to stimulate neighboring target tissues.

Adipose Tissue↗
PubMed1995

Rate of glucose entry into hepatic uridine diphosphoglucose by the direct pathway in fasted and fed states in normal humans.

We used the glucuronate (GlcUA) probe technique to measure the rate of glucose entry into hepatic uridine diphosphoglucose (UDP-glc) by the direct pathway, to quantify the rate of appearance (Ra) of hepatic UDP-glc, and to calculate hepatic glucose cycling in vivo in normal humans. The direct pathway contribution to UDP-glc as determined by the ratio of [1-d1]-GlcUA to plasma [LD1]-glucose enrichments was minor (15% to 20%) in normal men after an overnight fast. After 9 hours of refeeding with intravenous (IV) glucose or an oral liquid formula meal each at a rate of 7 mg carbohydrate/kg/min, the direct pathway increased to 66.3% +/- 6.7% and 61.6% +/- 6.0% (mean +/- SE), respectively. Plasma glucose concentrations remained below 7.8 mmol/L and could not account for most of the variability in direct pathway contribution. The dilution of labeled [L-D1]-galactose in excreted acetaminophen-GlcUA was used to measure Ra UDP-glc, on the assumption that labeled galactose passes through the liver during its assimilation. Ra UDP-glc was 1.1 +/- 0.1 mg/kg/min after an overnight fast and increased to 2.0 +/- 0.1 with i.v. glucose and 2.6 +/- 0.2 with the oral liquid mixed meal. By combining the fractional glucose contribution with the Ra of hepatic UDP-glc, the rate of direct glucose entry into hepatic UDP-glc was 0.2 mg/kg/min (fasted) and increased to 1.3 to 1.6 (fed). This represented approximately 18% to 21% of systemic glucose disposal or 19% to 23% of the administered carbohydrate load during i.v. or oral refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗