David R Taylor

Enhanced fasting and post-prandial plasma bile acid responses after Roux-en-Y gastric bypass surgery

Abstract Objective. Exogenous bile acid (BA) administration is associated with beneficial metabolic effects very similar to those seen after Roux-en-Y gastric bypass (RYGB) surgery. Re-routing of bile into a biliopancreatic limb with simultaneous exclusion of food occurs after RYGB, with subsequent increased fasting plasma BAs. The study assessed fasting and post-prandial plasma BA response before and 15 months after RYGB. Material and methods. The prospective study recruited 63 obese individuals (43 females), aged 43 (36–56) [median (IQR)] years. Blood samples were collected before and every 30 min for 120 min after a standard 400 kcal meal. Fasting and post-prandial plasma BAs, glucagons like peptide-1 (GLP-1), –tyrosine (PYY), fasting C-reactive protein (CRP), glucose and insulin were measured and homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. Results. Following RYGB, body mass index, CRP, fasting glucose and HOMA-IR decreased; 43.7 (39.3–49.2) kg/m2 to 29.2 (25.1–35.0) kg/m2, 7.9 (4.1–11.9) mg/L to 0.4 (0.2–1.0) mg/L, 5.5 (5.0–6.0) mmol/L to 4.6 (4.3–4.9) mmol/L and 5.9 (3.5–9.2) to 1.7 (1.1–2.2), respectively, all P < 0.001. Fasting total BAs, GLP-1 and PYY increased after RYGB; 1.69 (0.70–2.56) µmol/L to 2.43 (1.23–3.82) µmol/L (P = 0.02), 6.8 (1.5–15.3) pmol/L to 17.1 (12.6–23.9) pmol/L (P < 0.001) and 4.0 (1.0–7.1) pmol/L to 15.2 (10.0–28.3) pmol/L (P < 0.001), respectively. The area under the curve for post-prandial total BAs, total glycine-conjugated BAs, GLP-1 and PYY were greater after RYGB; 486 (312–732) µmol/L/min versus 1012 (684–1921) µmol/L/min, 315 (221–466) µmol/L/min versus 686 (424–877) µmol/L/min, 3679 (3162–4537) pmol/L/min versus 5347 (4727–5781) pmol/L/min and 1887 (1423–2092) pmol/L/min versus 3296 (2534–3834) pmol/L/min, respectively, all P < 0.0001. Conclusion. Weight loss following RYGB is associated with an increase in post-prandial plasma BA response due to larger amounts of glycine-conjugated BAs. This suggests up regulation of BA production and conjugation after RYGB.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Higher circulating bile acid concentrations in obese patients with type 2 diabetes.

Background Bile acids (BAs) play an important role in releasing incretin hormones via the enteroendocrine L-cell surface TGR5 receptors. The aim of this study was to investigate the difference in BA concentration at baseline and in response to a meal stimulus between type 2 diabetes mellitus (T2DM) and a matched normoglycaemic group. Materials and methods A cross-sectional study of 12 patients with known T2DM and 12 matched normoglycaemic controls compared BA fractions after an overnight fast and following a standard meal. Results The T2DM group had higher baseline glucose (P < 0.001), but baseline total BA, total glycine conjugated BAs (GCBA) and total taurine conjugated BA (TCBA) were similar between both groups. The T2DM group compared to the normoglycaemic group had a higher post-prandial peak change in total BAs 4.28 (3.51–5.38) µmol/L vs. 0.88 (0.60–1.57) µmol/L (P < 0.001) and peak total GCBA 2.77 (1.07–4.19) µmol/L vs. 0.94 (0.34–1.15) µmol/L (P < 0.0001), but similar peak total TCBA 0.36 (0.02–0.76) µmol/L vs. 0.08 (0.04–0.22) µmol/L (P=0.91). Conclusion The post-prandial bile acid response is elevated in obese patients with T2DM compared to matched normoglycaemic individuals.

LC-MS candidate reference methods for the harmonisation of parathyroid hormone (PTH) measurement: a review of recent developments and future considerations

Abstract The analysis of intact parathyroid hormone (PTH) (PTH1-84) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. The analysis is complicated by the presence of PTH fragments, which may accumulate in renal failure and cross-react in immunoassays, including the most recent third-generation immunoassays. Large variability exists between different commercially available assays. This article reviews the current literature on PTH testing, with emphasis on the use of mass spectrometry-based methods, and considers the important sources of variation which still need to be addressed prior to the development of much needed candidate reference methods for PTH analysis. Recently, mass spectrometric methods have been developed for the quantitation of PTH1-84 using surrogate tryptic peptides, but even these methods are subject to significant interferences due to the presence of newly observed modified PTH species, such as oxidised and phosphorylated PTH variants, which can accumulate in patient samples. Further work, including: 1) the use of high-resolution mass spectrometry; and 2) the analysis of PTH without prior protease digestion, is required before these approaches can be considered as reference methods against which other methods should be harmonised.

Urine bile acids relate to glucose control in patients with type 2 diabetes mellitus and a body mass index below 30 kg/m2.

Bile acids are important endocrine signalling molecules, modulating glucose homeostasis through activation of cell surface and nuclear receptors. Bile acid metabolism is altered in type 2 diabetes mellitus; however, whether this is of pathogenic consequence is not fully established. In this study urinary bile acid excretion in individuals with type 2 diabetes and matched healthy volunteers was assessed. Urinary bile acid excretion in type 2 diabetes patients was considered in the context of prevailing glycaemia and the patient body mass index. Urine bile acids were measured by liquid chromatography-tandem mass spectrometry, allowing individual quantification of 15 bile acid species. Urinary bile acid excretion in patients with type 2 diabetes who were normal weight (BMI 18.5–24.9 kg/m2) and overweight (BMI 25–29.9 kg/m2) were elevated compared to healthy normal weight volunteers, both p<0.0001. In obese (BMI≥30 kg/m2) type 2 diabetes patients, urinary bile acid excretion was significantly lower than in the normal and overweight type 2 diabetes groups (both p<0.01). Total bile acid excretion positively correlated with HbA1c in normal (rs = 0.85, p = <0.001) and overweight (rs = 0.61, p = 0.02) but not obese type 2 diabetes patients (rs = −0.08, p = 0.73). The glycaemia-associated increases in urine bile acid excretion in normal weight and overweight type 2 diabetes seen in this study may represent compensatory increases in bile acid signalling to maintain glucose homeostasis. As such alterations appear blunted by obesity; further investigation of weight-dependent effects of bile acid signalling on type 2 diabetes pathogenesis is warranted.

A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma

BACKGROUND Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capacity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC-MS/MS is increasingly replacing immunoassay, in particular for steroids most subject to cross-reaction. METHODS We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phaeochromocytoma/paraganglioma, cortisol-producing adenoma, and lesions demonstrating no hormonal excess) was then investigated. RESULTS In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was markedly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non-ACC adrenal lesions. CONCLUSIONS Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.

Predicting refeeding hypophosphataemia: insulin growth factor 1 (IGF-1) as a diagnostic biochemical marker for clinical practice:

Background Refeeding syndrome (RS) is a potentially fatal condition that can occur following the re-introduction of nutrition after a period of starvation. Hypophosphataemia following the reintroduction of nutrition is often the only reliable biochemical marker of RS. Refeeding index (RI) generated from baseline insulin-like growth factor-1 (IGF-1) and leptin has been proposed as a useful biochemical marker for the identification of patients at risk of developing refeeding hypophosphataemia (RH). Methods A prospective study included 52 patients referred for parenteral nutrition (PN). The sensitivity and specificity of IGF-1 measured using a sensitive assay was compared to the RI in predicting the development of RH (a ≥30% drop in PO4 during the first 36-h of PN administration). Leptin and IGF-1 were analysed on baseline samples using a quantitative enzyme-linked immunoassay. Daily blood samples were collected from all patients for routine biochemistry for the full duration of PN administration. Results High sensitivity IGF-1 measurement alone was comparable with the RI, using receiver–operating characteristic (ROC) curve analysis, with areas under the curve being 0.79 and 0.80, respectively, and superior to leptin alone (0.72) for predicting ≥30% drop in PO4. The cut-off value for IGF-1 that gave best sensitivity (91% [95% CI 75–98%]) and specificity (65% [95% CI 41–85%]) was 63.7 µg/L, with a likelihood ratio of 2.59. Conclusion Baseline IGF-1 is an objective, sensitive and specific biochemical marker in identifying patients who are at high risk of developing RH prior to PN administration and therefore may have a role in clinical practice.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells

Summary Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

Mineralocorticoid hypertension and hypokalaemia induced by posaconazole

Summary We describe severe hypokalaemia and hypertension due to a mineralocorticoid effect in a patient with myelodysplastic syndrome taking posaconazole as antifungal prophylaxis. Two distinct mechanisms due to posaconazole are identified: inhibition of 11β hydroxylase leading to the accumulation of the mineralocorticoid hormone 11-deoxycorticosterone (DOC) and secondly, inhibition of 11β hydroxysteroid dehydrogenase type 2 (11βHSD2), as demonstrated by an elevated serum cortisol-to-cortisone ratio. The effects were ameliorated by spironolactone. We also suggest that posaconazole may cause cortisol insufficiency. Patients taking posaconazole should therefore be monitored for hypokalaemia, hypertension and symptoms of hypocortisolaemia, at the onset of treatment and on a monthly basis. Treatment with mineralocorticoid antagonists (spironolactone or eplerenone), supplementation of glucocorticoids (e.g. hydrocortisone) or dose reduction or cessation of posaconazole should all be considered as management strategies. Learning points: Combined hypertension and hypokalaemia are suggestive of mineralocorticoid excess; further investigation is appropriate. If serum aldosterone is suppressed, then further investigation to assess for an alternative mineralocorticoid is appropriate, potentially using urine steroid profiling and/or serum steroid panelling. Posaconazole can cause both hypokalaemia and hypertension, and we propose that this is due to two mechanisms – both 11β hydroxylase inhibition and 11β HSD2 inhibition. Posaconazole treatment may lead to cortisol insufficiency, which may require treatment; however, in this clinical case, the effect was mild. First-line treatment of this presentation would likely be use of a mineralocorticoid antagonist. Patients taking posaconazole should be monitored for hypertension and hypokalaemia on initiation and monthly thereafter.

Measurement of serum 7α-hydroxy-4-cholesten-3-one as a marker of bile acid malabsorption in dogs with chronic diarrhoea: a pilot study

Bile acid malabsorption is a common cause of chronic diarrhoea in people, however it has never previously been investigated in dogs, despite clinical suspicion of its existence. The goal of this study was to assess the feasibility of measuring serum 7α-hydroxy-4-cholesten-3-one (C4) in dogs, as a potential marker of bile acid malabsorption, and to see whether this is related to clinical disease severity or the presence of hypocobalaminaemia. Serum C4 concentration was measured in 20 clinically healthy control dogs and 17 dogs with chronic diarrhoea. Three of the 17 affected dogs (17.6 per cent) had a C4 concentration significantly above the range of clinically healthy dogs; these dogs were all poorly responsive to conventional therapy. These results suggest that bile acid malabsorption may be a clinically relevant disorder in dogs with chronic diarrhoea and serum C4 may be a useful tool to investigate this further.