Global FT4 immunoassay standardization: an expert opinion review.

Abstract Objectives Results can vary between different free thyroxine (FT4) assays; global standardization would improve comparability of results between laboratories, allowing development of common clinical decision limits in evidence-based guidelines. Content We summarize the path to standardization of FT4 assays, and challenges associated with FT4 testing in special populations, including the need for collaborative efforts toward establishing population-specific reference intervals. The International Federation of Clinical Chemistry and Laboratory Medicine Committee for Standardization of Thyroid Function Tests has undertaken FT4 immunoassay method comparison and recalibration studies and developed a reference measurement procedure that is currently being validated. Further studies are needed to establish common reference intervals/clinical decision limits. Standardization of FT4 assays will change test results substantially; therefore, a major education program will be required to ensure stakeholders are aware of the benefits of FT4 standardization, planned transition procedure, and potential clinical impact of the changes. Assay recalibration by manufacturers and approval process simplification by regulatory authorities will help minimize the clinical impact of standardization. Summary Significant progress has been made toward standardization of FT4 testing, but technical and logistical challenges remain. Outlook Collaborative efforts by manufacturers, laboratories, and clinicians are required to achieve successful global standardization of the FT4 assays

Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding.Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases

Characterization of the metabolic profile associated with serum 25-hydroxyvitamin D : a cross-sectional analysis in population-based data

Background: Numerous observational studies have observed associations between vitamin D deficiency and cardiometabolic diseases, but these findings might be confounded by obesity. A characterization of the metabolic profile associated with serum 25-hydroxyvitamin D [25(OH)D] levels, in general and stratified by abdominal obesity, may help to untangle the relationship between vitamin D, obesity and cardiometabolic health. Methods: Serum metabolomics measurements were obtained from a nuclear magnetic resonance spectroscopy (NMR)- and a mass spectrometry (MS)-based platform. The discovery was conducted in 1726 participants of the population-based KORA-F4 study, in which the associations of the concentrations of 415 metabolites with 25(OH)D levels were assessed in linear models. The results were replicated in 6759 participants (NMR) and 609 (MS) participants, respectively, of the population-based FINRISK 1997 study. Results: Mean [standard deviation (SD)] 25(OH)D levels were 15.2 (7.5) ng/ml in KORA F4 and 13.8 (5.9) ng/ml in FINRISK 1997; 37 metabolites were associated with 25(OH) D in KORA F4 at P <0.05/415. Of these, 30 associations were replicated in FINRISK 1997 at P <0.05/37. Among these were constituents of (very) large very-low-density lipoprotein and small low-density lipoprotein subclasses and related measures like serum triglycerides as well as fatty acids and measures reflecting the degree of fatty acid saturation. The observed associations were independent of waist circumference and generally similar in abdominally obese and non-obese participants. Conclusions: Independently of abdominal obesity, higher 25(OH)D levels were associated with a metabolite profile characterized by lower concentrations of atherogenic lipids and a higher degree of fatty acid polyunsaturation. These results indicate that the relationship between vitamin D deficiency and cardiometabolic diseases is unlikely to merely reflect obesity-related pathomechanisms.Peer reviewe

Ectopic Cushing' syndrome caused by a neuroendocrine carcinoma of the mesentery

BACKGROUND: ACTH overproduction within the pituitary gland or ectopically leads to hypercortisolism. Here, we report the first case of Cushing' syndrome caused by an ectopic ACTH-secreting neuroendocrine carcinoma of the mesentery. Moreover, diagnostic procedures and pitfalls associated with ectopic ACTH-secreting tumors are demonstrated and discussed. CASE PRESENTATION: A 41 year-old man presented with clinical features and biochemical tests suggestive of ectopic Cushing's syndrome. First, subtotal thyroidectomy was performed without remission of hypercortisolism, because an octreotide scan showed increased activity in the left thyroid gland and an ultrasound revealed nodules in both thyroid lobes one of which was autonomous. In addition, the patient had a 3 mm hypoenhancing lesion of the neurohypophysis and a 1 cm large adrenal tumor. Surgical removal of the pituitary lesion within the posterior lobe did not improve hypercortisolism and we continued to treat the patient with metyrapone to block cortisol production. At 18-months follow-up from initial presentation, we detected an ACTH-producing neuroendocrine carcinoma of the mesentery by using a combination of octreotide scan, computed tomography scan, and positron emission tomography. Intraoperatively, use of a gamma probe after administration of radiolabeled (111)In-pentetreotide helped identify the mesenteric neuroendocrine tumor. After removal of this carcinoma, the patient improved clinically. Laboratory testing confirmed remission of hypercortisolism. An octreotide scan 7 months after surgery showed normal results. CONCLUSION: This case underscores the diagnostic challenge in identifying an ectopic ACTH-producing tumor and the pluripotency of cells, in this case of mesenteric cells that can start producing and secreting ACTH. It thereby helps elucidate the pathogenesis of neuroendocrine tumors. This case also suggests that patients with ectopic Cushing's syndrome and an octreotide scan positive in atypical locations may benefit from explorative radioguided surgery using (111)In-pentetreotide and a gamma probe

Leptin inhibits sOb-R release from Ob-Rfl or Ob-R219.3 transfected cells into the supernatant.

<p>Ob-Rfl or Ob-R219.3 transfected cells were incubated with leptin (5–100 ng/ml) for 24 h. Following this incubation sOb-R in the supernatant was determined. sOb-R levels of leptin-treated cells are shown relative to those of non-treated cells. Data are presented as means ± SD of n≥3 experiments.</p

Enhanced release of sOb-R after palmitate incubation is mediated by ADAM10 and ADAM17 and inhibited by oleate.

<p>(A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (0.5–1 mM), oleate (0.5 mM–1 mM or methanol (2%) for 24 and or 48 h. sOb-R levels of palmitate- or oleate-treated cells are displayed relative to those of cells treated with methanol. (B) sOb-R levels in the supernatant of HEK cells transfected with a non-targeting, ADAM10 or ADAM17 specific siRNA (50 nM) and subsequently with Ob-R219.3, after incubation with palmitate (1 mM) or methanol (2%) for 48 h. sOb-R levels of ADAM10 and ADAM17 siRNA transfected cells are shown relative to sOb-R levels of cells transfected with a non-targeting siRNA and treated with methanol. (C) Representative western blot of cleaved PARP after palmitate incubation. Ob-R transfected cells were incubated with palmitate (0.5–1 mM) or Methanol (2%) for 24 and 48 h. Following this incubation cleaved PARP in cell lysates was determined by western blot analysis. (D) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate 1 mM and Z-VAD 100 µM or methanol (2%) for 48 h. sOb-R levels of palmitate- and or Z-VAD-stimulated cells are displayed relative to those of cells treated with methanol. (E) Representative western blot of cleaved caspase-3 and cleaved PARP. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and Z-VAD (100 µM) or Methanol (2%) for 48 h. Following this incubation cleaved caspase-3 and cleaved PARP in cell lysates was determined by western blot analysis. (F and G) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (1 mM), oleate (0.1–0.5 mM), palmitate (1 mM) and oleate (0.1–0.5 mM) or methanol (2%) for 48 h. sOb-R levels of palmitate-, oleate– or palmitate- and oleate-treated cells are displayed relative to those of cells treated with methanol. (H) Representative western blot of cleaved PARP after co-incubation with palmitate and oleate. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and oleate (0.1–0.5 mM) or Methanol (2 %) for 48 h. Following this incubation cleaved PARP in cell lysates was determined by western Blot analysis. Data are presented as means ± SD of n≥3 experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034787#pone-0034787-g005" target="_blank">Fig. 5</a> D and E, n = 2).</p

Co-expression of Ob-R219.3 to Ob-Rfl and increased sOb-R levels modulate leptin signaling of Ob-Rfl.

<p>(A) Ob-R transfected cells were serum-starved for 16 h and subsequently incubated with leptin (100 ng/ml) for 30 min. P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with equimolar amounts of Ob-Rfl + GFP (equimolar to Ob-R219.3) or Ob-Rfl + Ob-R219.3 were determined by ELISA and western blot. P-STAT3/total STAT3 ratios are displayed relative to P-STAT3/total STAT3 ratio of non-treated cells transfected with Ob-Rfl + GFP. (B) P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with Ob-Rfl. Ob-Rfl-transfected cells were stimulated with media containing leptin (10 ng/ml) and increasing concentrations of sOb-R (0–1000 ng/ml). P-STAT3 and total STAT3 levels are displayed relative to those of leptin-treated control without sOb-R. Data are presented as means ± SD of n≥2 experiments.</p