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Biomedical subjects

C H Shackleton

Publications and source records attributed to C H Shackleton.

At least 91 records · Page 5Linked to original sources
PubMed1990

5 alpha-reductase activity in polycystic ovary syndrome.

11 patients with polycystic ovary syndrome (hirsutism and oligomenorrhoea), but with no deficiency of 21-hydroxylase or 3 beta-hydroxysteroid dehydrogenase, had abnormal cortisol metabolism. The high ratio of 5 alpha to 5 beta cortisol metabolites in the urine is consistent with enhanced activity of 5 alpha-reductase. Urinary total cortisol metabolites were higher in patients than controls. Increased 5 alpha-reductase activity in liver and skin enhances hepatic cortisol metabolism at the expense of androgen excess and may be the underlying abnormality in polycystic ovary syndrome.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1990

Tandem mass spectrometry in the clinical analysis of variant hemoglobins.

A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.

Hemoglobins, Abnormal↗
PubMed1990

Dehydroepiandrosterone sulfate quantification in serum using high-performance liquid chromatography/mass spectrometry and a deuterated internal standard: a technique suitable for routine use or as a reference method.

A thermospray high-performance liquid chromatography/mass spectrometry method for determination of serum dehydroepiandrosterone sulfate is described. The steroid was measured intact using [7,7-2H2]dehydroepiandrosterone sulfate as internal standard. The analysis was carried out in the negative ion mode by determining the peak height ratio of the molecular anions of the analyte and internal standard. The method was used to determine the steroid in serum from 15 male and female normal adults and the following values were obtained: males, 272 +/- 45 micrograms/dl (range, 197 to 331 micrograms/dl) and females, 215 +/- 67 micrograms/dl (range, 107 to 347 micrograms/dl). In addition, dehydroepiandrosterone sulfate was measured by high-performance liquid chromatography/mass spectrometry and radioimmunoassay (a commercial kit) on 25 individuals of all age groups. There was strong correlation between the values obtained, but the radioimmunoassay values were generally double those obtained by high-performance liquid chromatography/mass spectrometry. Three other steroid sulfates, androsterone sulfate, epiandrosterone sulfate, and androst-5-ene-3 beta, 17 beta-diol sulfate, were also assayed. In males, these had mean values of 112, 44, and 13 micrograms/dl and, in females, they had mean values of 84, 25, and 6 micrograms/dl, respectively. Radioimmunoassay cross-reactivity measurement for these steroids (as reference compounds) showed that they were unlikely to contribute greatly to the discrepancy between radioimmunoassay and high-performance liquid chromatography/mass spectrometry values.

Adult↗
PubMed1990

Synthesis of 6 beta-hydroxyaldosterone by A6 (toad kidney) cells in culture.

Incubation of aldosterone with confluent layers of A6 (toad kidney) cells leads to its hydroxylation at the 6 beta-position. 6 beta-Hydroxyaldosterone is the major metabolite when the incubation is carried out at pH 6.8, whereas the product comprises 6 beta-hydroxy-17-isoaldosterone accompanied by some 6 beta-hydroxyapoaldosterone at pH 7.4. All products were identified by high-field 1H nuclear magnetic resonance spectroscopy. Control experiments indicated that the side-chain isomerization to form the 17-iso and apo derivatives occurs after the cytochrome P 450-dependent synthesis of 6 beta-hydroxyaldosterone.

Aldosterone↗
PubMed1990

Characterization of the steroid-metabolizing capacity of the hepatic cytochrome P450IIC5 expressed in COS-1 cells: 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase type activity.

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroid-hydroxylating enzyme is the ability to convert delta 5-3 beta-hydroxysteroids to the delta 4-3-ketosteroid configuration. The delta 5-3-beta-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the delta 4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17 alpha-hydroxypregnenolone are also 21-hydroxylated, whereas 17 alpha-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19 steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16 alpha position. Contrary to previous reports, no metabolite of estradiol-17 beta was detected, presumably due to the unstable nature of catechol estrogens (2-hydroxyestradiol).

Animals↗
PubMed1990

Correctable subsets of primary aldosteronism. Primary adrenal hyperplasia and renin responsive adenoma.

Among 154 cases of primary aldosteronism seen in the General Clinical Research Center at San Francisco General Hospital, twelve patients did not fulfill established characteristics of an aldosterone producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). Eight patients had nodular adrenocortical hyperplasia; plasma and urinary aldosterone were elevated and responses to stimulatory and suppressive maneuvers demonstrated the same autonomy seen in patients with APA. This subset is designated primary adrenal hyperplasia. Four additional patients also had elevated aldosterone levels that were responsive to these maneuvers, similar to IHA, but had unilateral tumors. This group has been designated as aldosterone-producing renin-responsive adenoma. Eleven patients had unilateral adrenalectomy and one preferred prolonged spironolactone therapy, resulting in a sustained cure or amelioration of hypertension, hypokalemia and normalization of aldosterone production.

18-Hydroxycorticosterone↗
PubMed1989

18-substituted steroids--Part 17. 2 alpha-hydroxylated liver metabolites of aldosterone identified by high-field [1H]NMR spectroscopy.

11 beta,18-Epoxy-2 alpha,3 alpha,18,21-tetrahydroxy-5 alpha,17 alpha- pregnan-20-one (2 alpha-hydroxy-3 alpha,5 alpha-tetrahydro-17-isoaldosterone) and its apo isomer have been identified by high-field NMR studies, supported by thermospray HPLC/MS, to be among the major polar metabolites formed from incubation of aldosterone with rat liver microsomal fraction. Indications that unreduced 2 alpha-hydroxy-aldosterone is also present among the metabolites have still to be confirmed.

Aldosterone↗
PubMed1989

Combined 17-hydroxylase and 17,20-desmolase deficiencies: evidence for synthesis of a defective cytochrome P450c17.

We studied in vivo and in vitro steroidogenesis in six phenotypic female children with 17-hydroxylase deficiency. The diagnosis was suspected as a likely cause of familial low renin hypertension and was confirmed by findings of reduced basal and ACTH-stimulated serum and urinary levels of cortisol and other 17-hydroxysteroids, together with hypergonadotropic hypogonadism in both 46,XY and 46,XX patients, and abnormally increased secretion of 17-desoxysteroids, such as progesterone, 11-deoxycorticosterone, and corticosterone. ACTH stimulation testing demonstrated a lesser degree of 17-hydroxylase deficiency in the obligate heterozygous parents; one father had increased basal serum 17-hydroxyprogesterone values, unresponsive to ACTH, suggesting partial Leydig cell 17,20-desmolase deficiency. In vitro kinetic analysis of testicular microsomal enzymes in the affected 46,XY male pseudohermaphrodites confirmed that both 17-hydroxylase and 17,20-desmolase activities were less than 2% of those in age-matched normal subjects. However, in spite of this virtual absence of both enzymatic activities of cytochrome P450c17, Northern blot analysis demonstrated abundant amounts of RNA in these tests that hybridized to a cDNA specific for this P450 enzyme. Moreover, immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved testicular microsomes showed an apparently normal content of an immunoreactive protein with a mol wt similar to that of authentic P450c17. These results suggest that these patients have a point mutation in the gene for P450c17; the mutant gene is transcribed, but gives rise to a protein defective in normal 17-hydroxylase and 17,20-desmolase activities.

Adrenal Hyperplasia, Congenital↗
PubMed1989

Diagnosis of recessive X-linked ichthyosis: quantitative HPLC/mass spectrometric analysis of plasma for cholesterol sulfate.

A specific, accurate liquid-chromatographic/mass-spectrometric (HPLC/MS) method for measurement of cholesterol sulfate in plasma from normal individuals and patients with recessive X-linked ichthyosis (RXLI) is described. The method is superior to previously described techniques because it measures the analyte intact rather than after hydrolysis. Traces of free cholesterol in the sample analyzed do not add to the measured result. We used either [13C2]cholesterol sulfate or [2H6]cholesterol sulfate as internal standards, which we add to plasma before extraction. Use of such standards makes quantitative extraction unimportant. We use a single solid-phase extraction (SPE) C18 cartridge for plasma extraction. After the cartridge is washed with methanol/ammonium acetate solutions, the fraction containing the steroid sulfates is eluted with methanol, evaporated, and subjected to HPLC/MS analysis, wherein the molecular anions of analyte and internal standards are monitored. The peak ratio gives the cholesterol sulfate concentration directly. Using this method, we have diagnosed 24 patients with RXLI. Their concentration of cholesterol sulfate ranged between 41.7 and 185.3 mumol/L (mean 93.85, SD 31.2 mumol/L). In normal individuals (n = 9) the mean cholesterol sulfate concentration was 2.77 mumol/L (SD 0.62, range 2.05-3.95 mumol/L). The instrumentation required is complex, but the assay is simple: sample preparation takes about 30 min; mass spectrometry, 10 min. About 0.1 mL of plasma is required for cholesterol sulfate measurement in RXLI patients and 0.5-1 mL in normal individuals.

Adult↗
PubMed1988

Dehydroepiandrosterone therapeutics: acetylation of DHA in mouse liver.

This investigation was designed to evaluate the possibility that the therapeutic benefits of dehydroepiandrosterone (DHA)-feeding in mice is mediated by (1) a metabolite of DHA formed in liver or (2) by way of the obligatory hepatic metabolism of DHA fed in large amounts. We found that the pattern of metabolism of DHA is strikingly different when DHA in low (tracer) quantities is incubated with mouse liver compared with that found when DHA in high concentrations is incubated with this tissue. In the former case, the principal metabolites are sulfoconjugates and other polar compounds, e.g. hydroxylated products. In the case of DHA metabolism when the substrate is present in high concentrations (1-100 microM), the principal metabolite formed is delta 5-androstenediol. And in this case, there is the formation of very nonpolar metabolites, which we have identified as the acetates of DHA and delta 5-androstenediol. We find further that the acetates are formed by a transacetylation mechanism in which performed acetylated compounds, e.g. pregnenolone acetate and ethyl acetate, can serve directly as co-substrates; but (at least in short-term incubations), Na+-acetate and acetyl CoA do not serve as co-substrates. We suggest that the therapeutic benefits of DHA-feeding in mice may be, in part, the result of alterations in hepatic intermediary metabolism that is obliged by the metabolism of DHA when this otherwise inert agent is fed in large amounts. Thus, DHA-feeding may serve to cause changes similar to those that are beneficial or therapeutic with dietary manipulations including caloric restriction.

Acetylation↗
PubMed1988

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/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.

Adrenal Glands↗
PubMed1988

Origin of deoxycorticosterone and deoxycorticosterone sulfate in human pregnancy: absence of steroid 21-sulfatase activity in sulfatase-deficient placenta.

The activity of steroid 21-sulfatase, the enzyme that catalyzes the hydrolysis of deoxycorticosterone sulfate (DOC-SO4) is demonstrable in human placenta. Thus, it is possible that this placental enzyme, by way of the hydrolysis of either DOC-SO4 or 21-hydroxypregnenolone mono- or di-sulfate of fetal origin, may be important in the biosynthesis of DOC, which is present in the plasma of pregnant women in high concentration. To investigate this issue further, we evaluated steroid 21-sulfatase activity in microsomal preparations of a sulfatase-deficient placenta. Immediately after delivery, at term, of a living male fetus with sulfatase deficiency, a microsome-enriched fraction of placental tissue was prepared; sulfatase activity was evaluated by use of three substrates, viz. dehydroisoandrosterone sulfate (DS), estrone sulfate (E1-SO4), and DOC-SO4, in various concentrations. Similar incubations were conducted with aliquots of a microsome-enriched fraction prepared from placental tissue of a normal fetus that was delivered, at term, within minutes of the time of delivery of the infant with sulfatase deficiency. In microsomal fractions from the normal placenta, each of the steroid sulfates was hydrolyzed. In the absence of microsomes, and in the presence of microsomal fractions from the sulfatase-deficient placenta, the hydrolysis of DOC-SO4 and DS was not detected. Moreover, in microsomes prepared from the sulfatase-deficient placenta, E1-SO4 was hydrolyzed at a rate that was only 10% of that in incubations with microsomal preparations of the normal placenta. We conclude that with sulfatase deficiency, the placenta is deficient not only in sulfatase activity for steroid-3-sulfates but for steroid 21-sulfates, e.g. DOC-SO4, as well.

17-alpha-Hydroxypregnenolone↗
PubMed1988

Syndrome of apparent mineralocorticoid excess. A defect in the cortisol-cortisone shuttle.

The first adult case of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) deficiency is described. The impaired conversion of cortisol to cortisone (indicated by urinary cortisol and cortisone metabolites and failure to metabolize 11 alpha-[3H]cortisol to [3H]H2O), was associated with hypertension, hypokalemia, and suppression of the renin-angiotensin-aldosterone system. When established on a fixed Na+/K+ intake, dexamethasone, given orally, produced a natriuresis and potassium retention. Plasma renin activity became detectable. When hydrocortisone (10 mg daily s.c. for 4 d) was added, there was marked Na+ retention, a kaliuresis (urinary Na+/K+ falling from 1.2 to 0.15), with suppression of plasma renin activity and an increase in blood pressure. These changes were also seen with the subject on no treatment. Conversion of cortisone to cortisol was not affected. These results suggest that cortisol acts as a potent mineralocorticoid in 11 beta-OHSD deficiency. The major site for the oxidation of cortisol to cortisone is the kidney. In this patient congenital deficiency of 11 beta-OHSD results in high intrarenal cortisol levels which then act on renal type I mineralocorticoid receptors. This condition can be treated with dexamethasone, which suppresses cortisol secretion and binds to the type II glucocorticoid receptor. We suggest that 11 beta-OHSD exerts a critical paracrine role in determining the specificity of the type I receptor. In the normal state cortisol is converted by 11 beta-OHSD to cortisone which thus allows aldosterone to bind preferentially to the type I receptors in the kidney and gut. In this patient deficiency of 11 beta-OHSD results in high intrarenal cortisol concentrations that then bind to the type I receptor.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1987

Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans.

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.

Bacteroides↗
PubMed1987

Mineralocorticoid activity of liquorice: 11-beta-hydroxysteroid dehydrogenase deficiency comes of age.

The sodium retention associated with liquorice ingestion has been thought to be due to a direct mineralocorticoid effect, despite the fact that it does not seem to occur in patients or animals with severe adrenal insufficiency. This study in seven normal subjects given liquorice showed that sodium retention is associated with a significant change in cortisol metabolism indicating inhibition of 11-beta-hydroxysteroid dehydrogenase (11 beta-OHSD). Congenital deficiency of this enzyme produces a syndrome of apparent mineralocorticoid excess. It is suggested that in both conditions there is a defect in the renal conversion of cortisol to cortisone by 11 beta-OHSD which results in high intrarenal cortisol levels, acting on type 1 mineralocorticoid receptors to cause sodium retention.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1987

18-Hydroxy-11-deoxycortisol: a new steroid isolated from incubations of the adrenal with 11-deoxycortisol.

Cortisol has been shown to be metabolized in the zona glomerulosa of the adrenal gland through the same pathway involving the cytochrome P-450, corticosterone methyl oxidase by which corticosterone is transformed to 18-hydroxycorticosterone and aldosterone. When cortisol is the precursor, 18-hydroxycortisol and 18-oxocortisol are formed. 18-Hydroxycortisol can also be made at a similar rate in the bovine zona fasciculata and reticularis as in the zona glomerulosa. We studied the possibility that the formation of 18-hydroxycortisol in the zona fasciculata and reticularis might be through a different pathway involving initial 18-hydroxylation of 11-deoxycortisol before 11 beta-hydroxylation. Rat adrenal capsules or cores were incubated with 10 micrograms of cortisol or 11-deoxycortisol and the formation of 18-hydroxycortisol was measured by radioimmunoassay. Both capsules and cores transformed 11-deoxycortisol to 18-hydroxycortisol, but cortisol was only transformed in the capsular portion. Sixty-two rat adrenals were incubated with 10 mg of 11-deoxycortisol and the putative steroid, 18-hydroxy-11-deoxycortisol, was purified by TLC and HPLC and subjected to gas chromatography mass spectrometry. The mass spectra indicated that the steroid isolated was indeed 18-hydroxy-11-deoxycortisol. The function of this steroid is still unknown.

17-Hydroxycorticosteroids↗
PubMed1987

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.

Chromatography, High Pressure Liquid↗