Influence of common clinical variables on intraoperative parathyroid hormone monitoring during surgery for primary hyperparathyroidism.

BACKGROUND: Intraoperative monitoring of parathyroid hormone (IOPTH) is a reliable method of predicting the cure of primary hyperparathyroidism (PHPT). The aim of this study is to assess whether common clinical variables (CCV) frequently encountered in patients with PHPT may affect the magnitude of PTH drop or the likelihood of patients meeting the intraoperative cure criterion. DESIGN: Patients who were surgically cured from PHPT caused by single gland disease (SGD) and had full IOPTH protocol (4 measurements) were stratified according to age, gland weight, renal function, vitamin D status and severity of hypercalcemia. The percentage of IOPTH drop and the frequency of patients who had true positive IOPTH test results were compared among groups. RESULTS: 762 patients had surgery for PHPT, of whom 746 were (98%) cured. Of these 746 patients, 511 who had SGD and a full IOPTH protocol were included in this study. The median IOPTH drop was significantly higher among younger patients, those with severe hypercalcaemia at 5, 10, 15 min after gland excision, giant glands (at 5-min only), patients with vitamin D deficiency (at 10, 15 min), and those with normal renal function (at 15 min only). The likelihood of the patients meeting the intraoperative cure criterion was not significantly affected among the groups except in patients with mild hypercalcaemia, who were significantly less likely to have 50% IOPTH drop than those with severe hypercalcaemia at all time points. The frequency of mildly hypercalcaemic patients who met cure criterion was significantly improved by extending measurement to 15 min. CONCLUSIONS: IOPTH monitoring has the ability to mitigate the variability of IOPTH kinetics associated with most clinical variables. Mildly hypercalcemic patients in particular may benefit from waiting for 15-min measurement before any surgical decision is made.

Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

BACKGROUND AND PURPOSE: Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. EXPERIMENTAL APPROACH: Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. KEY RESULTS: Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. CONCLUSIONS AND IMPLICATIONS: Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone.

Spironolactone interference in the immunoassay of androstenedione.

BACKGROUND: In an evaluation of androstenedione results from patient serum samples using the Siemens Immulite 2500 analyser and manual Coat-A-Count (CAC) methods, three outliers were evident with grossly elevated results in the CAC assay. METHODS: The clinic notes of three patients with apparently high serum androstenedione concentrations by the CAC assay were checked for medications. The samples were all from patients with polycystic ovary syndrome taking 100-200 mg/d of a steroidal antiandrogen (spironolactone). Two other patients on 50 mg spironolactone per day had less markedly higher androstendione results with the CAC assay. In a further five patients who were selected since they were on spironolactone and had high androstenedione results by the CAC method, spironolactone was temporarily withdrawn and fresh blood samples obtained for analysis. RESULTS: Spironolactone treatment was associated with higher androstenedione concentrations measured by the CAC assay that reverted to normal on treatment withdrawal. Based on a single test with spironolactone at 1000 ng/mL, the manufacturer reported only 0.109% interference in the CAC assay. CONCLUSIONS: Spironolactone (and/or its metabolites) may interfere in the Siemens CAC assay for androstenedione but not in the Immulite 2500 assay. This experience highlights the need for information from clinicians on drug treatment when laboratory investigations are requested. Drug interferences in immunoassay are common and need evaluation beyond tests performed to certify laboratory reagents.

Plasma aldosterone response to the low-dose adrenocorticotrophin (ACTH 1-24) stimulation test.

BACKGROUND: Endocrine tests for adrenal insufficiency use pharmacological doses of stimulant such as ACTH. More physiological tests have often used high-dose protocols for sampling frequency. AIMS: To evaluate the response of plasma aldosterone concentration to low doses (125, 250 and 500 ng/m(2) body surface area) of synthetic ACTH. DESIGN: A randomised trial in six normal adult males aged 18-27 years. MATERIALS AND METHODS: Aldosterone concentration was measured by radioimmunoassay in serum from blood samples taken at 10 min intervals for 90 min. RESULTS: All three doses produced a significant rise in plasma aldosterone concentration (125 ng/m(2), P = 0.003; 250 ng/m(2), P < 0.001; 500 ng/m(2), P < 0.001) but there was no effect of dose on either the peak or incremental plasma aldosterone concentration. Mean time to peak was similar between the doses and the two higher doses were associated with a longer secretory profile (125 ng/m(2) 56 (26 SD) mins, 250 ng/m(2) 74 (19) mins, 500 ng/m(2) 77 (21) mins; F = 3.39; P = 0.04). Peaks of 100% were detected within 30 min of drug administration and peak response was associated with the prestimulation plasma aldosterone concentration (r = 0.45; P = 0.003). The between- and within-individual coefficients of variation for prestimulation concentrations were 37.0% and 32.8%, and for the peak response were 27.2% and 27.2%, respectively. CONCLUSIONS: The response of plasma aldosterone concentrations to low-dose ACTH administration requires a blood sampling protocol of 0, 10, 20 and 30 min to capture concentrations near the peak response. The high-dose protocol would have missed the response. Over the dose range studied no dose-response was observed so the selection of dose should be based on the dose effective to release steroids in the glucocorticoid pathway if this study is to be used in conjunction with such evaluation.

Identification of neuroactive steroids and their precursors and metabolites in adult male rat brain.

Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3alpha,5alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5alpha-dihydroprogesterone. Furthermore, the presence of 3beta-, 11beta-, 17alpha-, and 20alpha-hydroxylated metabolites of 3alpha,5alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3alpha,5alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.

Potential pitfalls in intraoperative parathyroid hormone measurements during parathyroid surgery.

BACKGROUND: The outcome of parathyroid surgery is often not clear for at least 24 h after the operation. A frozen section does not always distinguish between an adenoma and hyperplasia. Minimally invasive surgical techniques are being refined, so the need for perioperative assurance about the completeness of surgery has increased. The value of intraoperative parathyroid hormone (PTH) measurements in 26 surgical cases undergoing parathyroidectomy has been evaluated. METHODS: Twenty-one patients were diagnosed as having primary hyperparathyroidism, including two patients with multiple endocrine neoplasia type I (MEN I). Five patients had tertiary hyperparathyroidism, including one patient with X-linked hypophosphataemia and four with renal hyperparathyroidism (RHPT). Blood samples were taken at the onset of surgery, at the time of tumour resection and at 5-min intervals following removal of the tumour. PTH was measured using a PTH Turbo assay on the DPC Immulite analyser. RESULTS: Current practice suggests that the PTH concentration should fall to less than 50% of the pre-incision value or to less than 50% of the level at the time of tumour resection (time equals zero). PTH levels were therefore monitored at 5-min intervals following removal of the tumour. In most of the case studies PTH followed the suggested pattern, but not when further exploration was warranted to determine if another adenoma was present. In some cases the PTH levels fell by the appropriate margin to deem the procedure a success but at 10 min post-gland excision the PTH began to rise again. Further exploration was required to confirm the continued source of PTH. CONCLUSION: We recommend that intraoperative PTH measurements continue until at least 15 min post-gland removal in cases of suspected single-gland disease. A decline in PTH concentration to at least 50% of the pre-incision or time of gland resection levels should be observed. If the PTH remains elevated or rises again after an appropriate decrease in levels, then multigland disease or ectopic sources should be considered. Caution is recommended in interpreting intraoperative PTH measurements to ensure complete success of the surgical procedure.

External quality assessment of urinary steroid profile analysis.

BACKGROUND: Clinical samples were distributed on 10 occasions to six UK laboratories that perform urinary steroid profile analysis. Urine samples were from normal adult men and women, normal children and neonates. Samples from patients with Cushing's syndrome, virilization, adrenarche, obesity and congenital adrenal hyperplasia (21 and 17-hydroxylase defects) were also used for evaluation. METHODS: Samples were analysed by capillary column gas chromatography (all laboratories) after hydrolysis of conjugates and derivative formation (five laboratories) or by variation of 17-oxogenic steroid methodology (one laboratory). RESULTS: For each distribution of samples, the performance of the participants was compared for quantitative analysis, and user comments were summarized. Quantitative results showed variation without necessarily biasing the result. Comments varied considerably in length. The interpretations did not always lead to a clear diagnosis or advise about appropriate further tests. CONCLUSIONS: This pilot urine steroid profiling scheme has clearly identified the requirement for external quality assessment. It is now hoped to offer this scheme worldwide in collaboration with the European Research Network for the Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Disorders of Metabolism (ERNDIM).

Effect of red clover-derived isoflavone supplementation on insulin-like growth factor, lipid and antioxidant status in healthy female volunteers: a pilot study.

BACKGROUND: Isoflavones are estrogen-like plant compounds that may protect against cardiovascular disease and endocrine-responsive cancer. Isoflavones may, because of their ability to act as selective estrogen receptor modulators, alter insulin-like growth factor (IGF) status. OBJECTIVE: The aim of this study was to assess the effect of 1-month isoflavone supplementation (86 mg/day red clover-derived isoflavones) on IGF status. DESIGN AND SUBJECTS: Healthy pre- (n=16) and postmenopausal (n=7) women were invited to take part in a randomised, placebo-controlled crossover study with a minimum 2-month washout period. RESULTS: : For premenopausal subjects, the change in IGF-1, IGF-BP1 and IGF-BP3 assessed at different points of the menstrual cycle did not differ between isoflavone and placebo phase. However, the change in IGF-1, when examined pre- and post-supplementation, was nonsignificantly reduced (P=0.06) on the isoflavone supplement compared to placebo. For postmenopausal subjects, the change in IGF-1, IGF-BP1 and IGFBP-3 concentrations over the supplementation period did not differ between isoflavone or placebo phase. Isoflavones increased HDL in postmenopausal women compared to placebo (P=0.02) but did not alter either cholesterol or triacylglycerol concentrations, and had no effect on antioxidant status. CONCLUSIONS: This study shows that 1-month supplementation with red clover isoflavones has a positive effect on HDL cholesterol, but at most a small effect on IGF status in premenopausal and no effect in postmenopausal subjects. Further studies are required to ascertain the role these dietary compounds may have to play in breast cancer prevention.

17-Hydroxylase/17,20 lyase deficiency diagnosed during childhood.

We present a case of familial 17alpha-hydroxylase/17,20 lyase (CYP17) deficiency in which the index case, a 14-year-old XX girl, led to the diagnosis of the condition in a 9-year-old XY sister. No mutations in the CYP 17 gene were found in any of the girls.

Treatment with flutamide decreases cortisol clearance: implications for therapy in congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is characterized by a defect in cortisol and often aldosterone secretion, and adrenal hyperandrogenism. Current treatment is to provide adequate glucocorticoid and mineralocorticoid substitution to prevent adrenal crises and to suppress excess adrenocortical androgen secretion. Anti-androgen therapy with flutamide is an option that allows control of hyperandrogenism without recourse to supraphysiological doses of glucocorticoid. METHODS: We examined the pharmacokinetic parameters of hydrocortisone administered i.v. as a bolus at a dose of 15 mg/m2 in a 17.3 year-old female patient with classic CAH before and four weeks after institution of flutamide treatment by determining serum cortisol concentrations at 10 min intervals for 6 h following the i.v. bolus of hydrocortisone. RESULTS: Treatment with flutamide resulted in a decrease in cortisol clearance from 420 ml/l to 305 ml/l (27% reduction), and a decrease in volume of distribution from 51.61 to 451 (12.9% reduction). The half-life of cortisol increased from 85.3 min to 102.1 min. CONCLUSIONS: Flutamide treatment decreases cortisol clearance, thereby prolonging its half-life. These findings indicate that a reduction in the daily dose of glucocorticoid replacement may need to be considered when flutamide is added to the treatment regimen of patients receiving hydrocortisone.

Urinary steroid profile analysis.

BACKGROUND: A detailed analysis (profile) of the steroid metabolites in urine is useful for diagnosis of adrenal problems. Hospitals from many of UK health regions and around the world use the specialist assay and advisory service at UCLH. According to the total workload, samples are from patients with precocious puberty/premature adrenarche (21%), ambiguous genitalia (17%), Cushing's syndrome (13%), tumors (11%), polycystic ovaries (9%), hypertension (6%), problems of growth and development (5%), salt-loss (3%) and male pseudohermaphroditism (3%). Sixty percent of samples are from children and comprehensive reference data for steroid excretion rates in childhood unique to this laboratory were essential for interpretation of the results. CONCLUSION: The recognition and high excretion rates of certain steroids not easily measured in blood or urine by any other assays was particularly in cases of hypertension and tumors. The assay is cost effective by comparison with the combined costs of several individual hormone measurements but that cost may delay early referral to a specialist centre and that is not in the best interests of the families involved.

Evaluation of neonatal screening for congenital adrenal hyperplasia.

Neonatal screening for congenital hypothyroidism has been effective in early detection and treatment of the condition. The position with respect to neonatal screening for congenital adrenal hyperplasia has been debated for many years. Some countries have performed congenital adrenal hyperplasia screening for many years, others have conducted pilot studies that were then not adopted. This article endeavours to summarize the complex issues behind decisions whether to screen or not and summarizes the findings of neonatal congenital adrenal hyperplasia screening programmes.

Raised urinary glucocorticoid and adrenal androgen precursors in the urine of young hypertensive patients: possible evidence for partial glucocorticoid resistance.

OBJECTIVE: To evaluate urinary glucocorticoid excretion profiles in a cohort of recently diagnosed young hypertensive patients. METHODS: After excluding patients with secondary causes, 60 individuals with premature hypertension were recruited (diagnosed by ambulatory blood pressure monitoring before the age of 36 years). In addition, 30 older hypertensive controls (age of onset > 36 years, "middle aged hypertensive controls"), and 30 normal controls (age matched to the young hypertensive group) were studied. All provided 24 hour urine collections for mass spectrometry for total cortisol metabolites and total androgen metabolites by gas chromatography. RESULTS: Among male patients, those with premature hypertension had higher total urinary excretion of cortisol metabolites (mean (SD), 13 332 (6472) microg/day) than age matched normal controls (7270 (1788) microg/day; p = 0.00001) or middle aged hypertensive controls (8315 (3565) microg/day; p = 0.002). A similar increase was seen among the female patients, although the absolute concentrations were lower. There was no significant difference between middle aged hypertensive patients and normal controls. Urinary total androgen excretion profiles in female patients also showed an unusual increase in the premature hypertension group (2958 (1672) microg/day) compared with the other groups (middle aged hypertensive controls, 1373 (748) microg/day, p = 0.0003; normal controls, 1687 (636) microg/day, p = 0.002). In all subjects, serum sodium and creatinine concentrations were within the normal range; serum potassium concentrations were found to be low before the start of treatment. CONCLUSIONS: Individuals presenting with premature hypertension have an abnormally high excretion of glucocorticoid metabolites in the urine. While the mechanism remains uncertain, these findings are compatible with partial resistance of the glucocorticoid receptors, with a compensatory increase in cortisol and androgen metabolites. The mineralocorticoid effects of the latter (sodium and water retention) may contribute to an abnormally high blood pressure and may have implications for targeted selection of first line treatment in young hypertensive patients.

Mutation of proline 409 to arginine in the meander region of cytochrome p450c17 causes severe 17 alpha-hydroxylase deficiency.

We elucidated the molecular basis of 17 alpha-hydroxylase deficiency in a Chinese patient with male pseudohermaphroditism. The patient is a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. The first mutation, g.6333--6341delGACTCTTTCA, located in exon 8, was reported in a Thai patient living in a rural village in Thailand. We suggest that g.6333--6341delGACTCTTTCA may be a prevalent mutation causing P450c17 deficiency in Southeast Asia. The second mutation is a missense mutation, g.5582C>G, located in exon 7, changing the codon 409 from CCG to CGG, and changing the coded amino acid from proline to arginine, i.e., P409R. This proline residue is conserved in P450c17 of other species and other human P450 proteins. Site-directed mutagenesis, in vitro expression, and functional analysis of the P409R mutant in COS-1 cells show that it has a complete lack of 17 alpha-hydroxylase activity. The proline residue probably causes a turn in the meander region of P450c17, and we hypothesize, by comparison to homologous proteins, that the change in the protein conformation may abolish heme incorporation or may prevent P450c17 from interacting with electron donors.

Plasma cortisol, cortisone and urinary glucocorticoid metabolites in preterm infants.

OBJECTIVE AND METHODS: In the fetal circulation, there is a low cortisol:cortisone (F:E) ratio ( approximately 0.3) suggesting high activity of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2). The circulating F:E ratio rises after birth in term infants, but little is known about infants born prematurely. Our hypothesis was that the low fetal plasma F:E ratio would persist in infants born prematurely, due to persistently high tissue 11betaHSD2 activity. To test this hypothesis, a longitudinal observational study of plasma F, E levels and urinary F and E metabolites was performed in 22 preterm infants of 24-31 weeks gestation. RESULTS: Median plasma F was 234-380 nmol l(-1), median 124-177 nmol l(-1) from 1 to 14 days age. Plasma F fell with increasing postnatal and postconceptional age. The F:E ratio was 3 in the first week of life, and thereafter was 1-2, falling with postnatal age. Urinary glucocorticoid metabolites were low in quantity ( approximately 48-120 microg kg(-1) day(-1)), consisted of E metabolites until term, and did not reflect the plasma F:E ratio. CONCLUSIONS: The fetal plasma F:E ratio did not persist in these preterm infants, due to tenfold higher levels of F. The F:E ratios were similar to those reported in term infants. These data suggest that the low F:E ratio in utero is due to low fetal production of cortisol, and effective placental inactivation of maternal F by 11betaHSD2.

Increased cortisol: cortisone ratio in acute pulmonary tuberculosis.

To evaluate a possible role for altered cortisol metabolism in mediating the immunoparesis associated with progressive tuberculosis (TB), we have studied the hypothalamic-pituitary-adrenal axis, and the activities of the 11beta-hydroxysteroid dehydrogenases (11-HSDs) that interconvert active cortisol and inactive cortisone. In active pulmonary tuberculosis (PTB), the ratio of cortisol/cortisone metabolites in 24-h urine showed a shift towards active cortisol (ratio, 1.19 +/- 0.1, n = 16 versus 0. 89 +/- 0.05 in cured pulmonary tuberculosis (CTB), n = 13, p < 0. 01; and 0.78 +/- 0.04 healthy volunteers (HV), n = 11, p < 0.005). Conversion of cortisone (administered as 25 mg orally) to cortisol in peripheral plasma was higher in PTB (peak 1,157 +/- 55 nM, n = 14 versus 862 +/- 50 nM in CTB, n = 10, p < 0.005, and 882 +/- 73 nM in HV, n = 10; p < 0.005). Cortisol/cortisone ratio was increased in bronchoalveolar lavage fluid in PTB (7.73 +/- 1.48, mean +/- SE, n = 13) compared with HV (4.05 +/- 0.38, n = 11, p < 0.05) but was not different in plasma (PTB, 3.25 +/- 0.68; HV, 4.01 +/- 0.92). Responses of plasma cortisol to dexamethasone, CRH stimulation, and multidose ACTH stimulation were not different. These data suggest that in pulmonary tuberculosis, central control of glucocorticoid production is normal but that peripheral metabolism, in particular in the lung, is deviated in favor of the active metabolite cortisol. This offers a possible mechanism to explain the immunoparesis observed in progressive pulmonary tuberculosis.

Gender differences in the urinary excretion rates of cortisol and androgen metabolites.

Animal studies show significant differences in steroid metabolism between male and female subjects. Similar studies in human subjects are still needed. The aim of this study was to evaluate differences in 24-h urinary excretion of cortisol and androgen metabolites between healthy male and female volunteers to estimate if such differences were significant. Urinary metabolite measurements were performed by gas chromatography. The median urinary excretion of total cortisol metabolites was 6965 microg/day for men and 4595 microg/day for women (P = 0.0005). Urinary excretion of 11beta-hydroxyandrosterone, tetrahydrocortisone, tetrahydrocortisol (5beta), allotetrahydrocortisol (5alpha), alpha-cortolone, beta-cortolone + beta-cortol and alpha-cortol were also significantly different in men compared with women. Total androgen metabolites in men (2660 microg/day) were also higher than in women (1850 microg/day) (P<0.0003). Similarly, urinary excretion of androsterone (5alpha), aetiocholanolone (5beta) and dehydroepiandrosterone were also significantly greater (all P=0.01). This confirms significant differences in the steroid metabolite excretion profiles between men and women. Laboratories should consider adopting gender-related reference ranges for cortisol and androgen metabolite excretion in 24-h urine samples.