Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is the most common single entity that results in male under-masculinization, but large cohort studies of AIS have rarely been performed. Over the last decade, nationwide cooperation between pediatric endocrinologists in the United Kingdom has allowed the creation of a database of cases of intersex and ambiguous genitalia where detailed clinical information on every notified case has been collected via a questionnaire. Among the 816 entries recorded by January 1999, there were 105 clinically diagnosed cases of complete AIS (CAIS) and 173 cases of partial AIS (PAIS). A masculinization score was devised by scoring the external phenotype, and a score of 12 represented normal masculinization. Androgen receptor (AR) binding was determined by studying binding capacity (Bmax) and receptor affinity (K(d)), and cases were classified as either zero, abnormal, or normal binding. Mutation screening of all eight exons of the AR gene was performed by single-strand conformational polymorphism analysis, followed by direct DNA sequencing. All cases of PAIS presented within the first month of birth. The median age at presentation of children with CAIS was 1 yr (P10,P90: 0.1,10.4). The testes were palpable in the labioscrotal folds or the inguinal region in 77% and 41% of cases of CAIS and PAIS, respectively. There was marked overlap between the masculinization score of those children with PAIS reared as girls [2.5(P10,P90:1, 6)] and those reared as boys [3(P10,P90:2, 7.5)]. Gonadectomy was performed prepubertally in 66% and postpubertally in 29% of the cases of CAIS. The median age of the latter group was older at 14 yr (P10,P90:0.1,18). No cases of malignancy or carcinoma in situ were reported in the 121 cases of AIS where histology results were available. Biochemical endocrine investigations were reported to have been performed in a greater number of cases of PAIS than CAIS (98% vs. 48%). AR binding was abnormal in 44 of 51 (86%) and 40 of 113 (35%) cases of CAIS and PAIS, respectively. Zero binding was encountered in 29 of 43 (67%) and 1 of 55 (2%) cases of CAIS and PAIS, respectively. Mutational analysis of the AR gene, performed in 102 index cases was positive in 57 of 69 (83%) cases of CAIS and 12 of 43 (28%) cases of PAIS. In 24 of these cases, the mutation identified was novel. The mutations in PAIS cases were all missense, whereas in CAIS the mutations were more diverse. AR binding was only normal in 3 of 69 mutation-positive cases. In the PAIS group, mutation-positive cases had a significantly higher Kd and Bmax compared to the mutation negative cases. The clinical diagnosis of AIS can be confirmed in a significant number of cases by a combination of androgen-binding studies and mutational analysis. There is some correlation between the phenotypic features and the abnormalities discovered on mutational analysis of the AR gene, but there is a need to improve this further by developing optimal bioassays of AR function. The phenotypic heterogeneity among clinically diagnosed cases of AIS emphasizes the need for appropriate comprehensive evaluation of male under-masculinization.

The utilization of colon transit scintigraphy in the diagnostic algorithm for patients with chronic constipation.

We performed a retrospective utilization study covering a four-year period to determine how physicians familiar with colon transit scintigraphy (CTS) use it to manage patients with chronic constipation. Following CTS, there was a change is both frequency and likelihood of diagnosis. The diagnosis was changed in 51% of patients-37% of those considered to have slow transit constipation (STC) before CTS, 43% with obstructed defecation, and 64% with functional bowel disease (FBD). CTS increased the diagnostic likelihood in all groups. Of patients with the diagnosis of STC, 16% were considered "almost certain" before CTS while 83% were considered "almost certain" after CTS. For FBD comparable percentages were 13% and 62%. CTS may play a major role in the diagnostic work-up of patients with chronic constipation, both altering diagnosis and increasing diagnostic certainty.

Modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.

The purpose of this study was to measure 3beta-HSD activity in the equine placenta and to assess the effect of fetal and maternal blood plasma progestagens on 3beta-HSD activity was measured in 8 late gestation (collected by caesarian section at 250 to 320 days) and 7 term (collected by caesarian section at 250 to 320 days) and 7 term (collected at birth) equine placentae using a tritium release assay with [3alpha-3H] pregnenolone as substrate. Mean +/- s.d. Km(app) and Vmax for term placentae were in general higher than for late gestation placentae (0.129 +/- 0.217 micromol/l and 23.85 +/- 9.1 nmol/mg/h respectively vs. 0.016 +/- 0.048 micromol/l and 17.36 +/- 20.9 nmol/mg/h) but there was no statistical difference between them. Inhibition studies were performed on 3 term placentae and 3 late gestation ones. Steroid concentrations used for inhibition studies were close to blood plasma concentrations (0.5 to 2 micromol/l). 3beta-hydroxy compounds (5alpha-pregnene-3beta, 20alpha-diol and 3beta-hydroxy-5alpha-pregnan-20-one) showed noncompetitive or mixed inhibition. Mean Ki(app) of 0.7 micromol/l. Inhibition was competitive with 20alpha-hydroxy-5alpha-pregnan-3-one with a mean Ki(app) of 0.1 micromol/l.

Biosynthesis of androgens by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one.

Rat testicular and adrenal gland microsomal preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol) a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatography and crystallized to constant specific radioactivity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesized and incubated with further tissue preparations and shown to be converted to steroid hormones. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.

Gas chromatographic and gas chromatographic/tandem mass spectrometric characterization of precursors on the sesterterpene pathway for steroid biosynthesis.

An increasing amount of evidence is accumulating to support the proposal that steroidogenesis can occur by a sesterterpene pathway as well as the cholesterol pathway. Key intermediates on the sesterterpene pathway are 23,24-dinor-5-cholen-3 beta-ol (guneribol) and some of its metabolites, e.g. 23,24-dinor-4-cholen-3-one (guneribone). It has been reported that these intermediates are biosynthesized and converted to steroid hormones by a range of endocrine tissues in vitro. Monitoring the pentafluorobenzyloxime derivatives by negative ion chemical ionization mass spectrometry in the electron capture mode provided evidence for the presence of guneribone in extracts of bovine testicular and human adrenal tumour tissue. Complementary evidence was obtained from gas chromatographic/tandem mass spectrometric data generated on a triple-quadrupole instrument by monitoring daughter ions (in the multiple ion detection, MID mode) of the molecular anion of derivatized guneribone in both standards and tissue extracts. The present findings that sesterterpene pathway intermediates are present as endogenous compounds in tissue extracts, together with the previously reported radiochemical data, give further support to the sesterterpene pathway hypothesis.

Biosynthesis of adrenal corticosteroids by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one.

Rat adrenal gland preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol), a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatographies and crystallized to constant specific activity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesised and incubated with further tissue preparations and shown to be converted to corticosteroids. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.

Inhibition of cholesterol side-chain cleavage by intermediates of an alternative steroid biosynthetic pathway.

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24-dinor-5-cholen-3 beta-ol (23,24-dinor-5-cholene-3 beta,20-diol and 23,24-dinor-5-cholene-3 beta,21-diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side-chain-hydroxylated derivatives of cholesterol (20 alpha-hydroxycholesterol and 22S-hydroxycholesterol), inhibit cholesterol side-chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24-dinor-5-cholen-3 beta-ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.

Adrenal androgen biosynthesis by a sesterpene pathway.

Rat and human adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by TLC and crystallised to constant specific activity. It was found that rat and human adrenal glands can utilise 23,24-dinor-5-cholen-3 beta-ol to produce androstenedione and dehydroepiandrosterone. Also, it was found that the conversion of 23,24-dinor-5-cholen-3 beta-ol to androgens occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat and human adrenal glands to produce androgens and that the intermediates are converted to androgens in the microsomal fraction.

Evidence of hepatitis virus infection among Australian prisoners of war during World War II.

A sample of Australian male veterans of World War II was surveyed after 40 years. One hundred and seventy veterans had been held by the Japanese as prisoners of war and 172 veterans had served in southeast Asia but had not been taken captive (non-prisoners of war). A medical history was obtained and a physical examination undertaken. Blood was drawn and analysed for standard liver biochemistry and serological markers of hepatitis A and B virus (HAV, HBV) infections. The prevalence of immunoglobulin (Ig)G class antibodies to HAV was 95.2% in non-prisoners of war and 93.3% in prisoners of war. Only three cases of hepatitis B surface antigen (HBsAg) seropositivity were identified (two cases from the prisoner-of-war group). Thirty-six (21.8%) prisoners of war were seropositive for the presence of antibodies to HBsAg (anti-HBs) and 34 (20.0%) prisoners of war for that of antibodies to hepatitis B core antigen (anti-HBc), compared with 16 (9.8%) and eight (4.7%) of the non-prisoners of war, respectively (P = 0.002 and P = 0.0001, respectively). Those veterans who reported jaundice during World War II had a higher prevalence of antibodies to HBV. Among prisoners of war who were forced to work on the Burma-Thailand railway, 24.1% were seropositive for anti-HBc compared with 11.1% of the remaining prisoners of war (P = 0.048). It would appear that hepatitis B was common in prisoners of war but that those who survived 40 years were able to clear the virus and do not appear to have significant liver disease.

Steroid hormone biosynthesis by a sesterterpene pathway in the rat and rabbit testis.

Rat and rabbit testis preparations were incubated with [4-14C]cholesterol and 23,24-dinor-[7 alpha-3H]5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin-layer chromatography and crystallised to constant specific activity. It was found that rat and rabbit testis can utilise 23,24-dinor-5-cholen-3 beta-ol to produce testosterone. The tritium/carbon-14 ratios in the testosterone and androstenedione isolated indicated that these tissues differentiated between the two substrates. This finding is supported by the observation that, on stimulation with HCG, the tritium/carbon-14 ratios in the testosterone isolated were increased compared to the controls. The results of further experiments implied that, while the biosynthesis of testosterone from cholesterol occurred in the rat testis mitochondrial fraction, its biosynthesis from 23,24-dinor-5-cholen-3 beta-ol occurred in the microsomal fraction.

Gastrointestinal morbidity among World War II prisoners of war: 40 years on.

A study of Australian male World War II veterans was conducted to assess clinically the gastrointestinal ill-effects which were present 40 years after the stress of internment as prisoners of war of the Japanese. A random sample of 170 surviving members of the captured Eighth Army Division resident in Sydney in 1983 (ex-POW) was compared with a similar sample of veterans who fought in Southeast Asia during the War, but were not imprisoned (non-POW). Duodenal ulcers and strongyloidiasis were more prevalent in the ex-POW group than in the non-POW group. The increased rate of duodenal ulcer (24.7%, compared with 10.5%; P = 0.0005) was confirmed by a higher proportion of ex-POWs currently taking cimetidine (9.0%, compared with 2.3%; P = 0.008). Strongyloidiasis had been found in 9.7% of all veterans, but in 15% of ex-POWs and in 19% of those who had worked on the Burma-Thailand railway. No other significant differences in gastrointestinal disease were found.

Predictive value of rectal bleeding in screening for rectal and sigmoid polyps.

Overt rectal bleeding is a common symptom of colorectal cancer and polyps but also occurs in apparently healthy people. It is not known how often this represents bleeding from an undiagnosed rectal or sigmoid polyp or cancer. Three hundred and nineteen apparently healthy men aged over 50, selected by random sampling, were interviewed and underwent flexible sigmoidoscopy to at least 30 cm. Polyps of 10 mm or more in diameter were diagnosed in 12, one of whom also had an adenocarcinoma. Rectal bleeding during the previous six months was reported by 48, four of whom were found to have polyps; seven polyps and one cancer were diagnosed among the 271 who reported no rectal bleeding. Rectal bleeding had a specificity of 86%, a sensitivity of 33%, and a positive predictive value of 8% for rectal or sigmoid polyps or cancer. Restricting the analysis to those subjects who regularly inspected their stools did not improve the predictive value. Sigmoidoscopy in apparently healthy subjects with rectal bleeding will not result in the diagnosis of appreciable numbers of rectal and sigmoid polyps or cancers.

The biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one by the horse fetal gonad.

Horse fetal gonadal tissue was incubated with 3 beta-hydroxy-5,7-pregnadien-20-one and 5,7-cholestadien-3 beta-ol and it was shown that both substrates were converted to 3 beta-hydroxy-5,7-androstadien-17-one. These findings support the proposal that in this tissue there is a 5,7-diene pathway producing 3 beta-hydroxy-5,7-androstadien-17-one, the putative precursor of equilin in the placenta.

Steroid biosynthesis by a sesterterpene pathway in the rat adrenal gland in vitro.

Rat adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3 beta-ol. Steroids were isolated, purified by thin-layer and high performance liquid chromatography and crystallised to constant specific activity. It was found that the rat adrenal gland can utilise 23,24-dinor-5-cholen-3 beta-ol to produce corticosterone. Also, in contrast to the conversion of cholesterol to corticosterone which occurs in the mitochondrial fraction, the conversion of 23,24-dinor-5-cholen-3 beta-ol to corticosterone occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat adrenal gland and that the intermediates are converted to steroid hormones in the microsomal fraction.

Serum pancreatic isoamylase estimation by the inhibitor method as a diagnostic test for chronic pancreatitis.

The diagnosis of chronic pancreatitis is often difficult to establish and may involve invasive diagnostic procedures. Serum pancreatic isoamylase has been estimated by the inhibitor method in controls and in 38 patients with known chronic pancreatitis in order to evaluate its use as a diagnostic test. The mean pancreatic isoamylase in those with chronic pancreatitis (68 +/- 77 IU/l) was significantly lower (p less than 0.005) than in controls (113 +/- 60 IU/l). The diagnostic sensitivity of pancreatic isoamylase was 50% (compared to total amylase 9%) and had a specificity of 95%. Stimulation tests with cholecystokinin and secretin failed to increase the sensitivity of the test. A low value of pancreatic isoamylase strongly supports a diagnosis of chronic pancreatitis and may obviate the need for further investigation.

Identification of 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one as endogenous steroids in the fetal horse gonad.

The 5,7-dienes, 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography-mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-diene pathway biosynthesizing 3 beta-hydroxy-5,7-androstadien-17-one, which is the proposed precursor for equilin in the placenta.

Biosynthesis of 3 beta-hydroxy-5,7-pregnadien-20-one by the horse fetal gonad.

The production of equilin and the other ring B-unsaturated estrogens by the pregnant mare is anomalous in that they are biosynthesised by a cholesterol-independent pathway. Fetal horse gonads were incubated with tritiated sodium acetate and radiochemically pure 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were isolated. A fetal gonad--placental system is proposed for equilin production, 3 beta-hydroxy-5,7-pregnadien-20-one being a precursor for 3 beta-hydroxy-5,7-androstadien-17-one in the fetal gonad and the latter being the precursor of equilin in the placenta. The nature of the possible precursor of 3 beta-hydroxy-5,7-pregnadien-20-one is discussed.

Corticosterone-secreting tumours: with and without renal artery stenosis.

Two cases of corticosterone-secreting adrenal adenomata are reported. Both patients were middle-aged females in whom the clinical course spanned several years before the diagnosis was made. Severe hypertension and recurrent hypokalaemia refractory to standard treatments were outstanding features. The importance of measuring the production rates of all mineralocorticoids when a tumour is suspected is emphasized by Case 1 where the production rate of corticosterone alone was elevated. Bilateral renal artery stenosis was responsible for unsuppressed plasma renin activity in Case 2.