Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome.

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

Measurement of selected androgens using liquid chromatography-tandem mass spectrometry in reproductive-age women with Type 1 diabetes.

STUDY QUESTION: What information does androgen profiling using liquid chromatography tandem mass spectrometry (LC-MS/MS) provide in reproductive-age women with Type 1 diabetes (T1D)? SUMMARY ANSWER: In T1D women, androstenedione proved most useful of the measured androgens in differentiating subgroups based on clinical phenotypes of hyperandrogenism (HA) and polycystic ovary syndrome (PCOS). WHAT IS KNOWN ALREADY: The prevalence of HA and PCOS are increased in women with T1D. These observations are based on measurement of serum androgens using immunoassays, to-date no studies using LC-MS/MS have been reported in reproductive-age women with T1D. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study with recruitment of three groups of reproductive-age women: women with T1D (n = 87), non-diabetic women with (N = 97) and without PCOS (N = 101). PARTICIPANTS/MATERIALS, SETTING, METHODS: Using LC-MS/MS, we aimed to characterize androgen profiles and PCOS status in women with T1D, and interpret findings in relation to cohorts of non-diabetic women with and without PCOS. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to non-diabetic women, dehydroepiandrosterone/dehydroepiandrosterone sulphate (DHEA/DHEAS) ratio was lower (P < 0.05) in women with T1D. Testosterone levels were greater in T1D women with clinical HA and anovulation compared to those without clinical HA and with regular cycles, while androstenedione levels were greater in T1D women with HA and anovulation compared to those with HA and regular cycles and also those without HA and with regular cycles (P < 0.05 for all). Compared to T1D women without PCOS, the 18% of T1D women who had PCOS were younger with lower BMI, an older age of menarche, and were more likely to have a positive family history of PCOS (P < 0.05 for all). Androgen levels did not differ between women with T1D and PCOS compared to BMI-matched non-diabetic women with PCOS, but androstenedione levels were greater in T1D women with PCOS compared to obese women with PCOS (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Relatively small subgroups of patients were studied, reducing the power to detect small differences. Free testosterone levels were not measured using equilibrium dialysis, and were not calculated - commonly used formulae have not been validated in T1D. WIDER IMPLICATIONS OF THE FINDINGS: Androstenedione is a sensitive biochemical marker of clinical hyperandrogenism and PCOS in T1D. T1D women with PCOS are leaner than those without PCOS but are more likely to have a family history of PCOS. Women with T1D and PCOS have a similar biochemical phenotype to lean non-diabetic women with PCOS but differ from obese women with PCOS. The mechanisms underlying PCOS in T1D and its clinical significance require further investigation. STUDY FUNDING/COMPETING INTEREST(S): The study was part-funded by the Meath Foundation. The authors have no competing interests.

Dexamethasone-related adrenal insufficiency in patients with brain and skull base tumours.

PURPOSE: This study aimed to evaluate the prevalence of glucocorticoid-induced adrenal insufficiency in a cohort of patients with brain and skull base tumours and to identify factors which may predict its occurrence. METHODS: Patients with brain or skull base tumours attending for a short synacthen test (SST) (adrenocorticotropin hormone (ACTH) stimulation test) at a single institution over a 3-year period were retrospectively identified. Baseline demographics and dexamethasone exposure were examined. Only patients with dexamethasone exposure were included in the final analysis looking at the primary end point of SST failure. Fisher's exact test, Student's t test, Mann-Whitney test and the Kendall's tau-b test were used to evaluate the influence of age, gender, diagnosis and mean pituitary radiation dose on the primary endpoint. Receiver operating characteristic (ROC) curves were generated to explore the impact of duration and total exposure to dexamethasone on likelihood of SST failure. RESULTS: Thirty-one of 51 patients with previous dexamethasone exposure failed their first SST (61%). No significant relationship was demonstrated between age, gender, diagnosis or mean pituitary radiation dose and SST failure. Duration of and total exposure to dexamethasone were significantly associated with SST failure (p = 0.001 and p = 0.007, respectively). ROC curves generated values of 78 days and 171 mg days to give a sensitivity of 94 and 97%, respectively, to detect SST failure. CONCLUSIONS: Duration of dexamethasone use and total exposure predict for adrenal insufficiency in patients with brain and skull base tumours. Values derived from this study may be useful to identify patients at higher risk of adrenal suppression who require empirical hydrocortisone pending formal testing of the hypothalamic-pituitary-adrenal axis.

Serum fibroblastic growth factor 23 in acute Sarcoidosis and normal kidney function.

BACKGROUND: Serum fibroblastic growth factor (FGF) 23 has recently been established as a major physiological regulator of phosphate homeostasis and may have a causal role in adverse cardiovascular and bone outcomes. However its role in states of disordered phosphate homeostasis and normal kidney function is as yet under characterised. AIMS: To investigate whether this biomarker of vascular calcification and adverse bone outcomes is detectable in patients with sarcoidosis. DESIGN: We conducted a cross sectional study on a convenience sample of patients presenting with acute sarcoidosis to a respiratory tertiary referral unit. METHODS: We set out to systematically examine the characteristics and determinants of serum FGF-23 in patients presenting with acute sarcoidosis. RESULTS: We studied 39 patients, 26 were male. Mean (SD) age was 33 (9.6) years. 15.4% of patients had a serum level of FGF-23 ≥ 9.9 pg/mL. The remaining 84.6% of patients had a serum FGF-23 < 9.9 pg/mL. Those with a detectable serum FGF-23 had a significantly higher serum calcium (P = 0.007), and lower serum iPTH (P<0.001). Serum phosphate and 25-hydroxyvitamin D were not statistically significantly different between groups (P=0.25 and P=0.83). The proportion of patients with stage II disease on CXR was higher in those with a detectable FGF-23 (P<0.001). CONCLUSIONS: Serum FGF-23 was below the level of detection in the majority of this cohort of patients presenting with acute sarcoidosis. A detectable serum FGF-23 was associated with a higher serum calcium and lower serum iPTH.

Radiological remission and recovery of thirst appreciation after infliximab therapy in adipsic diabetes insipidus secondary to neurosarcoidosis.

BACKGROUND: Neurosarcoidosis is a rare and aggressive variant of systemic sarcoidosis which may result in hypothalamic-pituitary dysfunction. We report a case of hypothalamic hypopituitarism secondary to neurosarcoidosis complicated by adipsic diabetes insipidus (ADI). Initiation of anti-tumour necrosis factor-α (TNF-α) therapy resulted in both radiological disease remission and recovery of osmoregulated thirst appreciation after 3 months. CASE SUMMARY: A 22-year-old man was referred to the endocrinology service with profound weight gain, polyuria and lethargy. Biochemical testing confirmed anterior hypopituitarism while posterior pituitary failure was confirmed by hypotonic polyuria responding to desmopressin. Magnetic resonance imaging (MRI) demonstrated extensive hypothalamic infiltration; neurosarcoidosis was confirmed histologically after excisional cervical lymph node biopsy. Osmoregulated thirst appreciation was normal early in the disease course despite severe hypotonic polyuria. However, subsequent subjective loss of thirst appreciation and development of severe hypernatraemia in the setting of normal cognitive function indicated onset of ADI. MANAGEMENT: Clinical management involved daily weighing, regular plasma sodium measurement, fixed daily fluid intake and oral desmopressin. We initiated immunosuppressive therapy with pulsed intravenous anti-TNF-α therapy (infliximab) after multidisciplinary team consultation. OUTCOME: Infliximab therapy resulted in successful radiological disease remission and complete recovery of osmoregulated thirst appreciation. This was confirmed by subjective return of thirst response and maintenance of plasma sodium in the normal range in the absence of close biochemical monitoring.

The impact of maternal obesity on gestational outcomes.

To investigate the effects of raised maternal BMI on pregnancy outcome in glucose tolerant women, using the IADPSG criteria. Prospective observational study of fetal and maternal outcome in a cohort of pregnant women recruited to a universal screening programme for gestational diabetes under the ATLANTIC-DIP partnership. Maternal outcomes included glucose, delivery mode, pregnancy induced hypertension (PIH), preeclampsia (PET), antepartum hemorrhage (APH) and postpartum hemorrhage (PPH). Fetal outcomes included birthweight, congenital malformation, fetal death, neonatal jaundice, hypoglycemia and respiratory distress. Increasing maternal BMI was associated with adverse pregnancy outcomes: higher cesarean section rates, pre-eclamptic toxemia, pregnancy induced hypertension, increased birth weight and congenital malformation. There was also an association between normal range glucose and emergency cesarean section, hypertension of pregnancy and birthweight. In spite of tightening criteria for hyperglycemia during pregnancy, raised BMI is associated with adverse pregnancy outcome.