Stephen Ball

SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.

Pituitary Apoplexy: A Review of Clinical Presentation, Management and Outcome in 45 Cases

Objective: To review clinical presentation, management and outcomes following different therapies in patients with pituitary apoplexy. Methods: Retrospective analysis of case-records of patients with classical pituitary apoplexy treated in our hospitals between 1983–2004. Results: Forty-five patients (28 men; mean age 49 years, range 16–72 years) were identified. Only 8 (18%) were known to have pituitary adenomas at presentation. Thirty-four (81%) patients had hypopituitarism at presentation. CT and MRI identified pituitary apoplexy in 28% and 91% cases, respectively. Twenty-seven (60%) patients underwent surgical decompression, whilst 18 (40%) were managed conservatively. Median time from presentation to surgery was 6 days (range 1–121 days). Patients with visual field defects were more likely than those without these signs to be managed surgically (p = 0.01). Complete or near-complete resolution occurred in 93% (13/14), 94% (15/16) and 93% (13/14) of the surgically treated patients with reduced visual acuity, visual field deficit and ocular palsy, respectively. All patients with reduced visual acuity (4/4), visual field deficit (4/4) and ocular palsy (8/8) in the conservative group had complete or near-complete recovery. Only 5 (19%) patients in the surgical group and 2 (11%) in the conservative group had normal pituitary function at follow up. One (4%) patient in the surgical group and 4 (22%) in the conservative group had a recurrence of pituitary adenoma. Conclusions: This large series suggests that the patients with classical pituitary apoplexy, who are without neuro-ophthalmic signs or exhibit mild and non-progressive signs, can be managed conservatively in the acute stage.

Expression of steroidogenic factor 1 and Wilms' tumour 1 during early human gonadal development and sex determination

The transcription factors SF-1 and WT1 play pivotal roles in mammalian gonadal development and sexual differentiation. In human embryos, both SF-1 and WT1 are expressed when the indifferent gonadal ridge first forms at 32 days post-ovulation. As the sex cords develop - providing morphological evidence of testis differentiation - SF-1 localises predominantly to developing Sertoli cells in the sex cords, whereas WT1 retains a broader pattern of expression. Later, SF-1 localises predominantly to steroidogenic Leydig cells, and WT1 localises to the sex cords. In the ovary, SF-1 and WT1 transcripts persist in the gonadal ridge from the earliest developmental stages throughout the critical period of sex determination. These studies, which delineate for the first time the sequential expression profiles of SF-1 and WT1 during human gonadal development, provide a framework for understanding human sex reversal phenotypes associated with their mutations.

Novel SOX9 expression during human pancreas development correlates to abnormalities in Campomelic dysplasia.

Haploinsufficiency of SOX9, which encodes a homeodomain transcription factor, results in Campomelic dysplasia. Classical features of this disorder (e.g. skeletal dysplasia and 46,XY sex reversal) are in concordance with SOX9 expression profiles during human embryonic development. We report the robust expression of SOX9 throughout the pancreas during human embryogenesis, at levels of detection equivalent to the developing skeleton and testis. In the early foetal period, SOX9 expression declines and, in particular, is not apparent within the pancreatic islets. In keeping with this profile, examination of three cases with Campomelic dysplasia revealed abnormal pancreatic morphology. Epithelial cells were less densely packed within the mesenchymal stroma and islets less clearly formed with variable expression of hormone and beta cell markers. Taken together, these data indicate a novel potential role for SOX9 in pancreas development during human embryogenesis and early foetal life.

Improving the Vitamin D Status of Vitamin D Deficient Adults Is Associated With Improved Mitochondrial Oxidative Function in Skeletal Muscle

OBJECTIVE Suboptimal mitochondrial function has been implicated in several disorders in which fatigue is a prominent feature. Vitamin D deficiency is a well-recognized cause of fatigue and myopathy. The aim of this study was to examine the effects of cholecalciferol therapy on skeletal mitochondrial oxidative function in symptomatic, vitamin D-deficient individuals. DESIGN This longitudinal study assessed mitochondrial oxidative phosphorylation in the gastrosoleus compartment using phosphorus-31 magnetic resonance spectroscopy measurements of phosphocreatine recovery kinetics in 12 symptomatic, severely vitamin D-deficient subjects before and after treatment with cholecalciferol. All subjects had serum assays before and after cholecalciferol therapy to document serum 25-hydroxyvitamin D (25OHD) and bone profiles. Fifteen healthy controls also underwent (31)P-magnetic resonance spectroscopy and serum 25OHD assessment. RESULTS The phosphocreatine recovery half-time (τ1/2PCr) was significantly reduced after cholecalciferol therapy in the subjects indicating an improvement in maximal oxidative phosphorylation (34.44 ± 8.18 sec to 27.84 ± 9.54 sec, P < .001). This was associated with an improvement in mean serum 25OHD levels (8.8 ± 4.2 nmol/L to 113.8 ± 51.5 nmol/L, P < .001). There was no difference in phosphate metabolites at rest. A linear regression model showed that decreasing serum 25OHD levels was associated with increasing τ1/2PCr (r = -0.41, P = .009). All patients reported an improvement in fatigue after cholecalciferol therapy. CONCLUSIONS Cholecalciferol therapy augments muscle mitochondrial maximal oxidative phosphorylation after exercise in symptomatic, vitamin D-deficient individuals. This finding suggests that changes in mitochondrial oxidative phosphorylation in skeletal muscle could at least be partly responsible for the fatigue experienced by these patients. For the first time, we demonstrate a link between vitamin D and the mitochondria in human skeletal muscle.

Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma

BACKGROUND Plasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma. METHODS Off-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization. RESULTS Detection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay. CONCLUSIONS Plasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma.

Vasopressin and disorders of water balance: the physiology and pathophysiology of vasopressin

Disorders of water balance are a common feature of clinical practice. An understanding of the physiology and pathophysiology of the key endocrine regulator of water balance vasopressin (VP) is key to diagnosis and management of these disorders. Diabetes insipidus is the result of a lack of VP or (less commonly) resistance to the renal effects of the hormone. Diagnostic testing can clarify aetiology and direct appropriate management. VP production can be associated with hyponatraemia. A comprehensive assessment of cardiovascular status and pharmacological influences are needed in these circumstances to differentiate between primary (inappropriate) and secondary (appropriate) physiological VP production. As with diabetes insipidus, diagnostic testing can help define the aetiology of hyponatraemia and direct appropriate management. Patients with disorders of water balance benefit from a joint clinical and laboratory medicine approach to diagnosis and management.

Biochemical detection of phaeochromocytoma: Why are we continuing to ignore the evidence?

Abstract Phaeochromocytomas are rare tumours that require consideration among large numbers of patients with hypertension. If not diagnosed, the excessive secretion of catecholamines by these tumours can cause considerable morbidity and mortality. With a wide clinical variability in presentation, diagnosis can be difficult and invariably requires the biochemical confirmation of excessive catecholamine production by the tumour. At the First International Symposium on Phaeochromocytoma in October 2005, a panel of experts recommended that initial biochemical testing for phaeochromocytoma should include measurements of plasma and urinary metadrenalines. The accumulated evidence clearly indicates that measurement of fractionated metadrenalines in urine or plasma provides superior diagnostic sensitivity over plasma or urine measurements of catecholamines and metabolites. The low prevalence of phaeochromocytoma and paraganglioma (PGL) emphasizes the need to use biochemical tests of the highest sensitivity. To achieve this, it is recommended that the initial biochemical testing for phaeochromocytoma and secreting PGL should always include the measurements of metadrenalines in plasma or urine or both.

Cardiac biomarkers at high altitude.

BACKGROUND Classically, biomarkers such as the natriuretic peptides (NPs) BNP/NT-proBNP are associated with the diagnosis of heart failure and hs-cTnT with acute coronary syndromes. NPs are also elevated in pulmonary hypertension. High pulmonary artery systolic pressure (PASP) is a key feature of high altitude pulmonary edema (HAPE), which may be difficult to diagnose in the field. We have previously demonstrated that NPs are associated with high PASP and the presence of acute mountain sickness (AMS) in a small cohort at HA. We aimed to investigate the utility of several common cardiac biomarkers in diagnosing high PASP and AMS. METHODS 48 participants were assessed post-trekking and at rest at three altitudes: 3833 m, 4450 m, and 5129 m. NPs, hs-cTnT and hsCRP, were quantified using immunoassays, PASP was measured by echocardiography, and AMS scores were recorded. RESULTS Significant changes occurred with ascent in NPs, hs-cTnT, hsCRP (all p<0.001) and PASP (p=0.006). A high PASP (≥40 mm Hg) was associated with higher NPs, NT-proBNP: 137±195 vs. 71.8±68 (p=0.001); BNP 15.3±18.1 vs. 8.7±6.6 (p=0.001). NPs were significantly higher in those with AMS or severe AMS vs. those without (severe AMS: NT-proBNP: 161.2±264 vs. 76.4±82.5 (p=0.008)). The NPs correlated with hsCRP. cTnT increased with exercise at HA and was also higher in those with a high PASP (13.8±21 vs. 7.8±6.5, p=0.018). CONCLUSION The NPs and hs-cTnT are associated with high PASP at HA and the NPs with AMS.

The investigation and management of severe hyperandrogenism pre- and postmenopause: Non-tumor disease is strongly associated with metabolic syndrome and typically responds to insulin-sensitization with metformin

Background. An androgen-secreting tumor needs to be excluded in any woman with severe hyperandrogenism. We sought to characterize patients with biochemical hyperandrogenism in respect of tumor versus non-tumor etiologies, explore possible links between non-tumor hyperandrogenism and metabolic syndrome, and ascertain whether metformin therapy can elicit diagnostic reductions in serum testosterone (T). Patients and methods. Seven-year retrospective study of all women referred to a university hospital endocrinology service with baseline T >4.0 nmol/l. Dataset comprised age, menopausal status, body mass index (BMI), presence/absence of hypertension, diabetes, acanthosis or dyslipidemia, along with changes in BMI and serum T following intervention with metformin, oophorectomy or dexamethasone. Non-tumor hyperandrogenism was defined by normalization of serum T or >40% reduction from baseline. Results. Four out of 18 cases had adrenal carcinoma that was clinically obvious at initial presentation (one virilized, three Cushingoid). The remaining 14 were characterized by metabolic syndrome (BMI: 39.9 ± 8.1 kg/m2), serum T of 6.14 ± 1.6 nmol/l, and nadir serum T following intervention of 2.2 ± 1.04 nmol/l. Diagnostic reductions in serum T occurred in 11/12 patients treated with metformin. Conclusions. Non-tumor hyperandrogenism with markedly elevated serum T and associated metabolic syndrome is a defined clinical entity in postmenopause as well as in premenopausal women with polycystic ovary syndrome. This has hitherto been only sparsely documented in the published literature. A fall in serum T level in response to insulin-sensitizing therapy with metformin and lifestyle change may be a reassuring indicator that such women are highly unlikely to harbor an androgen-secreting tumor.