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C H Shackleton

Publications and source records attributed to C H Shackleton.

At least 127 records · Page 7Linked to original sources
PubMed1983

Analysis of intact steroid conjugates by secondary ion mass spectrometry (including FABMS) and by gas chromatography.

The analysis of intact steroid conjugates by two different methods is described. One method employed secondary ion mass spectrometry (SIMS) using a Cs+ beam for ionisation, although comparable data were obtained by fast atom bombardment (FAB) using a Xe0 beam. In both of these mass spectrometric techniques the samples were analysed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulphate and glucuronide analysis. The negative ion spectrum of each steroid is dominated by a pseudomolecular ion at m/z [M - H]- (M of free acid) and a lack of marked fragmentation. Mixtures of steroids can be resolved in a single spectrum, providing the individual steroids differ in mass. The second method was gas chromatography. The carboxylic acid moieties of the steroid glucuronides were derivatised with diazomethane and the remaining functional groups in the steroids were thermally protected by methyloxime formation (for carbonyls) and trimethylsilylation (for all steroidal and glucuronic acid hydroxyls). Satisfactory analysis of steroid glucuronides was achieved through the use of glass or fused silica columns stable at high temperature (330 degrees C). Conveniently, trimethylsilylation resulted in exchange of the sulphate in 3 beta-hydroxy-5-ene steroid sulphates for a trimethylsilyl group so these could effectively be analysed as "free" steroids.

Chromatography, Gas↗
PubMed1982

Direct analysis of steroid conjugates: the use of secondary ion mass spectrometry.

Data are presented on the mass spectrometry of intact steroid conjugates. The principal technique used was secondary ion mass spectrometry (SIMS) using a Cs+ ion beam for ionization, although comparable data were obtained by fast atom bombardment (FAB) using a Xeo beam. In both techniques the samples were analyzed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulfate and glucuronide analysis. The negative ion spectra are dominated by a pseudomolecular ion at m/z [M-H]- (M of free acid) and the lack of marked fragmentation permits mixtures of steroids to be resolved in a single spectrum, providing they differ in mass. Preliminary data on the separate analysis of individual components from urine and plasma of patients with assorted disorders of steroid synthesis and metabolism are presented. This technique shows great promise for the clinical analysis of steroid conjugates without the need for enzymic hydrolysis or chromatographic separation of individual steroids.

Glucuronates↗
PubMed1982

Identification of 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone as products of deoxycorticosterone metabolism by rat adrenals.

The formation of 19-hydroxydeoxycorticosterone (19,21-dihydroxy-4-pregnen-3,20-dione), 19-oxo-deoxycorticosterone (21-hydroxy-4-pregnen-3,19,20-trione), and 19-oic-deoxycorticosterone (19-oic-21-hydroxy-4-pregnen-3,20-dione) from precursor deoxycorticosterone by adrenal glands obtained from intact rats and from rats undergoing adrenal regeneration was demonstrated. These metabolites were isopolar with corresponding authentic steroid standards on thin layer chromatography, gas chromatography, and high pressure liquid chromatography. They were further characterized by either mass spectrometry or gas chromatography-mass spectrometry. Therefore, rat adrenals have the enzymes required to convert deoxycorticosterone to 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone; however, rat adrenals do not convert deoxycorticosterone or any of the oxygenated metabolites to 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnen-3,20-dione). It is possible, however, that 19-nor-deoxycorticosterone is formed at peripheral sites from the oxygenated deoxycorticosterone precursors.

Adrenal Glands↗
PubMed1982

Analysis of steroids in urine for differentiation of pseudohypoaldosteronism and aldosterone biosynthetic defect.

The salt-losing syndromes in the neonatal period and early infancy due to adrenal disease can be differentiated by the pattern of excretion of steroids in urine. The presence or absence of metabolites of cortisol, aldosterone, and corticosterone as well as certain precursors can be established in a single analysis of steroids in urine by using gas chromatography with open tubular capillary columns. The profiles of steroid excretion in the urine of 8 infants with renal tubular insensitivity to aldosterone were compared with those in 5 infants with isolated aldosterone biosynthetic defects. The excretion in urine of 18 hydroxytetrahydro-compound A was elevated in all 13 children, but relative to the excretion of tetrahydroaldosterone, a high ratio was found for the biosynthetic defect and clearly distinguished the 2 conditions. Age-related changes in steroid metabolism are described. The diagnosis in each case was supported by clinical investigation together with determinations of PRA and aldosterone concentrations.

Aldosterone↗
PubMed1982

Evidence against significant 19-nor-deoxycorticosterone production in patients with 17 alpha-hydroxylase deficiency.

Tetrahydro-19-nor-deoxycorticosterone (3 alpha, 21-dihydroxy-19-nor-5 beta-pregnan-20-one, a presumed metabolite of 19-nor-DOC was sought in the conjugated steroid fractions of urine from patients with 17 alpha-hydroxylase deficiency syndrome. The reference material was prepared by microbial reduction (Clostridium paraputrificum) of 19-nor-DOC. Urinary steroid fractions of appropriate polarity were examined by high resolution gas chromatography and gas chromatography/mass spectrometry (GC/MS) but no tetrahydro-19-nor-DOC was found. The high selectivity of the GC/MS technique ensured that the excretion of this compound could not exceed about 1% of the excretion of tetrahydroDOC. Assuming that tetrahydro-19-nor-DOC is a major metabolite of 19-nor-DOC, it can be stated that 19-nor-DOC production is not a significant feature of 17 alpha-hydroxylase deficiency. This assumption may not be valid if 19-nor-DOC is formed from DOC in the kidney and is excreted unmetabolized soon after synthesis.

Adrenal Hyperplasia, Congenital↗
PubMed1981

Derivatization of estrogen conjugates for analysis by capillary gas chromatography.

By using 20 meter wall-coated open tubular glass capillary columns of high stability, analysis of methyl ester, methyloxime trimethylsilyl ether derivatives of estrogen glucuronides had been achieved. Relative retention times of five glucuronide conjugates on OV-1 stationary phase are reported. Estrogen sulfates conjugated at the 3-position were shown to be quantitatively hydrolyzed and derivatized in a single trimethylsilylation step, and this method of direct derivatization was compared to two solvolysis methods. These analytical methods could be further developed to allow rapid and quantitative analysis of estrogens in biological fluids, and may prove particularly useful for analysis of labile compounds.

Chromatography, Gas↗
PubMed1981

Familial congenital Cushing's syndrome due to bilateral nodular adrenal hyperplasia.

Two siblings with congenital Cushing's syndrome due to bilateral nodular adrenal hyperplasia are described. The older, a boy, presented with severe hypertension and died soon after subtotal adrenalectomy. His sister, who had clitoral enlargement and showed persistent hyponatraemia, had a two-stage total adrenalectomy and is still alive. Investigations in the second case showed grossly elevated urinary cortisol metabolites, 17-oxosteroids and 3 beta-hydroxy-5-ene-steroids. These were not suppressed by dexamethasone, and plasma ACTH was undetectable, indicating that the disorder was not due to excessive ACTH secretion. Cell culture studies on the resected adrenals failed to demonstrate an abnormal pattern of steroid synthesis in vitro, and normal trophic responses were obtained with 1-24 ACTH and monobutyryl cyclic AMP. No stimulation of steroid synthesis was obtained with a range of polypeptide hormones, and the cause of the adrenal hyperplasia remains unknown.

Adrenal Glands↗
PubMed1980

Use of Sep-pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis.

A method is described for the rapid and quantitative extraction of free and conjugated steroids from urine using Sep-pak C18 cartridges. The method was evaluated by determining the efficiency of recovery of (1) radiolabeled steroid glucuronides, (2) radiolabeled steroids freed by enzymatic hydrolysis, (3) steroid sulphates, (4) selected reference neutral free steroids of varied structure and polarity, and (5) oestrogens. In all cases the cartridges gave results equal to or better than those obtained by solvent or Amberlite XAD-2 extraction methods. Each urine extraction could be completed in 2-3 minutes and no further purification of extracts was required prior to derivatisation and gas chromatographic analysis.

Chromatography, Gas↗
PubMed1980

Gas chromatographic and mass spectrometric analysis of urinary acidic metabolites of cortisol.

A method is described suitable for the analysis of the urinary acidic metabolites of cortisol which are amongst the major metabolites of this hormone (5-25% of secretion). Following hydrolysis of the urinary glucuronide conjugates and extraction of the freed steroids, methyl ester-trimethylsilyl ethers were prepared for gas chromatographic analysis. This analysis was carried out on open tubular columns coated with Carbowax stationary phase. The polar phase column permitted the complete resolution of the four acidic metabolites: alpha-cortolonic, beta-cortolonic, alpha-cortolic and beta-cortolic acids.

Carboxylic Acids↗
PubMed1980

The causes of low oestrogen excretion in pregnancy: assessment of the fetal contribution by steroid measurements post partum.

Oestrogen levels in urine from 21 normotensive and 13 hypertensive pregnant women were moderately correlated (r = 0.48) with levels of 3 beta-hydroxy-5-ene steroids (oestrogen precursors) in urine from their infants. In five infants from otherwise normal pregnancies in which oestrogen excretion was very low, levels of 3 beta-hydroxy-5-ene steroids were significantly lower than normal while there was no difference between hypertensives and normals. Levels of urinary cortisol metabolites in the infants were moderately correlated with 3 beta-hydroxy-5-ene steroids (r = 0.55) and were especially low in 2 out of 5 infants in the series suffering from distress during delivery. We conclude that subnormal fetal steroidogenesis rather than reduced placental metabolism is the most common cause of low oestrogen excretion of unknown aetiology. A factor in the increased perinatal risk in this group may be an associated insufficient cortisol synthesis by the fetus.

Birth Weight↗
PubMed1980

Pseudohypoaldosteronism.

10 infants are described with pseudohypoaldosteronism, 5 in detail and a further 5 briefly. They all presented with hyperkalaemia, urinary salt-wasting disease, and ostensibly normal renal and adrenocortical function. Diagnosis was established by demonstrating the greatly increased values of plasma renin activity and plasma aldosterone concentration, plus the increased excretion of aldosterone and its metabolites on gas chromatographic and mass spectrometric analyses of urine. The children were treated with sodium chloride supplements, up to 60 mmol/day, but by the time most of the infants were about a year old these could be stopped. Exogenous mineralocorticoids were without effect in those to whom they were administered. The precise aetiology of the condition remains conjectural; lack of renal tubular response to aldosterone seems probable. Pseudohypoaldosteronism may be more common than has been thought and new techniques for investigating salt-wasting disorders may show its true incidence.

Aldosterone↗
PubMed1980

Hypertension in a four-year-old child: gas chromatographic and mass spectrometric evidence for deficient hepatic metabolism of steroids.

A 4-yr-old boy with hypertension and hypokalaemic alkalosis had low plasma aldosterone levels and renin activity. The hypertension and hypokalemia responded to spironolactone and triamterene therapy. A partial response to dexamethasone was observed. Analysis of urinary steroid metabolites by gas chromatography-mass spectrometry showed that the excretion of metabolites of deoxycorticosterone and aldosterone was subnormal, and there was no evidence for sizeable excretion of unusual steroids with potential mineralocorticoid activity. The cortisol excretion rate, however, was subnormal, and the relative excretions of individual metabolites of this hormone were not typical. In particular, the excretion of tetrahydrocortisone was markedly reduced, and the excretions of allotetrahydrocortisol and free cortisol and metabolites were elevated. These findings suggest that modified or deficient metabolism of adrenal steroids could give rise to elevated blood pressure. It is not known whether the inappropriate production of unusual cortisol metabolites were responsbile for the high blood pressure or whether the altered metabolism is indicative of similar abnormality in the metabolism of other adrenal steroids, resulting in hyperproduction or extended half-life of minor but highly active mineralocorticoids of unknown structures.

Aldosterone↗