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Background: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest., Methods: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration., Results: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations., Conclusions: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Background and Aim: We investigated: (i) the association between severity of cirrhosis and serum levels of free cortisol (SFC) and total cortisol (STC), measured before and 30 min after (T(30)) the low-dose 1-µg short synacthen test (LD-SST); and (ii) the prognostic value of SFC and STC., Methods: Consecutive, hemodynamically stable, cirrhotic patients (34 Child-Pugh class A, 29B, and 32C) underwent the LD-SST. Patients were followed for at least 12 months to assess non-transplant-related mortality., Results: Child-Pugh class C patients had significantly higher basal levels of SFC than Child-Pugh class A or B patients. Prevalence of suspected adrenal dysfunction ranged between 7.4% (T(0) STC < 138 nmol/L) and 49.4% (change in STC < 250 nmol/L) according to the threshold used. In receiver-operator curve analysis, the area-under-the-curve values were 0.67 for T(30) SFC (0.51-0.79), 0.81 for Child-Pugh score (0.70-0.88), and 0.79 for albumin level (0.63-0.88). During the follow-up period, 16 patients with high T(30) SFC (≥ 78.9 nmol/L) (26.2%) and one patient with low T(30) SFC (< 78.9 nmol/L) (3.4%) died (P = 0.027 for high vs low T(30) SFC, log-rank test). Albeit not statistically significant, the risk of death for patients with T(30) SFC ≥ 78.9 nmol/L was fivefold higher than for patients with lower levels after adjusting for cirrhosis severity and level of albumin., Conclusions: One-year, non-transplant-related mortality is high among patients with T(30) levels of SFC ≥ 78.9 nmol/L (26.2%). These findings might result from latent inflammatory stress in hemodynamically stable cirrhotic patients, detected by adrenal testing., (© 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Aims: We have tested the hypothesis that selenium (Se)-containing antioxidative enzymes protect thyroid epithelial cells from oxidative damage associated with enzymatic production of hydrogen peroxide required for thyroid hormone biosynthesis. Thyroid epithelial cells therefore express antioxidative enzymes, including catalase, peroxiredoxins, thioredoxin reductases, and glutathione peroxidases (GPxs). The latter two enzyme families contain highly active peroxide-degrading enzymes that carry selenocysteine (Sec) in their active centers. Since low Se status has been associated with thyroid disorders, selenoproteins are considered essential for thyroid integrity and function. We have conditionally inactivated selenoprotein biosynthesis in thyrocytes by targeting Sec tRNA., Results: Constitutive and inducible Cre/loxP-mediated recombination of tRNA([Ser]Sec) drastically reduced activities of selenoenzymes GPx and type I-deiodinase in thyroid extracts. Immunohistochemical staining revealed increased 4-hydroxynonenal and 3-nitro-tyrosine levels consistent with increased oxidative stress. However, gross thyroid morphology remained intact for at least 6 months after recombination. Circulating thyroid hormone levels remained normal in mutant mice, while thyrotropin (TSH) levels were moderately elevated. Challenging mutant mice with low iodine diet increased TSH, but did not lead to destruction of selenoprotein-deficient thyroids., Innovation: This is the first report probing the assumed physiological roles of selenoproteins in the thyroid using a genetic loss-of-function approach., Conclusion: We conclude that selenoproteins protect thyrocytes from oxidative damage and modulate thyroid hormone biosynthesis, but are not essential for thyrocyte survival.

Context: Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland., Objective: To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice., Design: Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model., Results: We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range., Conclusions: Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes., (Copyright © 2010 Wiley-Liss, Inc.)

Objective: Because over 90% of serum cortisol is bound to albumin and corticosteroid-binding globulin (CBG), changes in these proteins can affect measures of serum total cortisol levels in cirrhotics without altering serum-free and salivary cortisol concentrations., Methods: We assessed basal (T₀) and post-synacthen (T₆₀) serum total cortisol, serum-free and salivary cortisol in 125 consecutive cirrhotics (95 non-septic and 30 septic patients with a Child>8)., Results: Serum total cortisol levels significantly decreased from the Child A-C non-septic group, as did albumin and CBG levels, with a non-significant rise in serum-free cortisol concentrations. Non-septic patients with low albumin (≤25 g/L) or CBG levels (≤35 mg/L) had lower T₀ serum total cortisol levels than patients with near-normal albumin (303.4 vs. 382.6 nmol/L; P=0.0035) or with normal CBG levels (289.9 vs. 441.4 nmol/L; P<0.0001), respectively, despite similar serum-free cortisol or salivary cortisol concentrations. Subnormal T₆₀ serum total cortisol concentrations (<510.4 nmol/L) were measured in 7.2% of all patients (Child C: 14.5% vs. Child A and B: 0%; P=0.0013) but no patients exhibited symptoms suggesting adrenal insufficiency. Patients with or without subnormal T₆₀ total cortisol had similar T₀ salivary cortisol and serum-free cortisol concentrations. A trend was observed towards high serum-free cortisol concentrations and mortality in multivariate analysis., Conclusions: Serum total cortisol levels overestimated the prevalence of adrenal dysfunction in cirrhotics with end-stage liver disease. Since serum-free cortisol cannot be measured routinely, salivary cortisol testing could represent a useful approach but needs to be standardized., (© 2011 John Wiley & Sons A/S.)

Graves' disease autoimmunity is attributable to the presence of serum antibodies (Ab) directed against the TSH receptor (TSHR) measured by a second generation (2G) assay using the human TRAK (hTRAK) with a high sensitivity in the diagnosis of Graves' disease. In this study, we have compared both analytical and clinical performances of hTRAK with those of five new methods using a porcine TSHR: two 2G methods and three assays using the monoclonal M22 directed against the TSHR pocket. We showed a bad reproducibility of these new methods with inter assay CVs higher than 10%. High clinical sensitivity and specificity that appeared similar to those of the hTRAK and next to 100% were observed except for a 2G method that failed to detect five Graves' patients. All these new methods should be avoided since they display a high variability despite their calibration against the same International Standard 90/672. The TRAKh using a human TSHR should be still used for a correct interpretation of results in the follow-up of Graves' disease.

Background: Although numerous reports have discussed the upper limit of the thyrotropin (TSH) reference interval, none have dealt with the lower limit. Recent recommendations regarding subclinical thyroid dysfunction give different advice about its management, depending on whether the TSH concentration is <0.1 mIU/L or 0.1-0.4 mIU/L., Content: We review key studies that have investigated the links between low TSH concentrations, cardiovascular morbidity, and mortality, with a focus on the TSH measurement threshold and assay type., Summary: Despite numerous consensus guidelines and publications of expert opinion, the management of subclinical hyperthyroidism remains largely intuitive and "nonevidence-based." The primary reason for this unsatisfactory situation is the absence of clinical-intervention trials. Important aspects that remain to be addressed are the influence of the method used to measure TSH, the definition of "normality," and the lack of evidence to base the grading of cardiovascular risk on the degree of TSH suppression. A risk-based approach should be adopted to determine the thresholds that would justify interventions. Such considerations assume, of course, that proof will emerge from ongoing clinical trials to support the medical utility of treating subclinical hyperthyroidism.

Objective: Increased reverse tritiodothyronine (T(3)) used to be described as a part of euthyroid sick syndrome (ESS). It was demonstrated to be associated with increased mortality in acutely ill patients. It can also be found with low or normal T(3) in non-severely ill subjects but its significance remains unclear., Patients and Design: The Alsanut study included a representative sample of 440 independently-living subjects aged 65 or over constituted between January 1988 and September 1989. Past and current medical history and nutritional data were collected at inclusion. Baseline thyroid hormone (TSH, FT(4), FT(3) and rT(3)) serum levels were measured. Life status was determined on 1 December 2005., Results: Of the 374 elderly subjects included in the final analysis, 52 had abnormal TSH (43 with hyperthyroidism, nine with hypothyroidism) and 80.7% had died by 1 December 2005. There was no statistical difference in survival between subjects according to thyroid function (P=0.54). Of the 322 elderly subjects with normal TSH, mortality rate was 81.1%. ESS was found in 3.4%, whereas 8.1% of the participants displayed elevated rT(3) with normal FT(3). Time to death was strongly related to rT(3) (P<0.0001) and FT(3) (P<0.0001) in a univariate analysis. After adjusting for other confounding variables, rT(3) was the only thyroid hormone associated with shorter survival (P=0.014)., Conclusions: RT(3) was the only thyroid hormone associated with shorter survival in a representative population of independently-living elderly. In these subjects, isolated elevated rT(3) might be an equivalent of ESS, reflecting declining health.

Background: Chromogranin A (CGA), a stress marker released with catecholamines by the adrenal medulla, has never been associated with acute inflammation in critically ill patients., Aim: To determine evidence for a link between serum concentration of CGA, biomarkers of inflammation, and outcome inpatients admitted with or without the systemic inflammatory response syndrome (SIRS)., Methods: At admission, we measured in 53 patients and 14 healthy controls the serum concentrations of CGA,procalcitonin, and C-reactive protein. We also assessed the Simplified Acute Physiological Score (SAPS) in the patients., Results: Serum CGA concentrations were significantly increased in SIRS patients with a median value of 115 microg/L (68.0-202.8), when compared to healthy controls (PB0.001). In cases where infection was associated with SIRS, patients had the highest increase in CGA with a median value of 138.5 microg/L (65-222.3) (PB0.001). CGA concentrations positively correlated with inflammation markers (procalcitonin, C-reactive protein), but also with SAPS. Receiver operating characteristic (ROC) analysis showed that CGA is equivalent to SAPS as an indicator for 28-day mortality (area under curve (AUC) for both: 0.810)., Conclusions: Patients with CGA concentration superior to 71 microg/L have a significantly shorter survival. A Cox model confirmed that CGA and SAPS were independent predictors of outcome.

Background: We compared the analytical and clinical performance of 3 porcine thyroid receptor antibody (TRAb) methods (1 second- and 2 new third-generation systems) with the conventional TRAb assay based on the human recombinant TSH receptor (hTRAK)., Patients and Methods: We obtained sera from 86 patients with untreated Graves disease (GD) and 71 healthy controls. We measured TRAb concentrations by radioreceptor assay using the hTRAK (Brahms) or the porcine TSH receptor (pRRA) from Beckman-Coulter, by electrochemiluminescence immunoassay (ECLIA) with the Elecsys/Cobas (Roche), and by ELISA using the Medizym TRAb clone (Medipan)., Results: Between-run assay imprecision was < or =10% and < or =7.6% for hTRAK and ECLIA, but reached 14% and 14.9% for ELISA and pRRA, respectively. Maximal specificity and sensitivity close to 100% were obtained for hTRAK, ECLIA, and ELISA. pRRA failed to detect positive TRAbs in 5 GD patients. Although calibrated against the same reference standard 90/672, the assays displayed a high intermethod variability. The results were significantly higher by ECLIA and lower by ELISA and pRRA compared with hTRAK. Patients with ophthalmopathy had higher TRAb results by ELISA and pRRA than those without eye disease., Conclusions: Second- and third-generation TRAb assays had similar diagnostic sensitivities in the diagnostic evaluation of GD. Despite the use of the same reference standard for calibration, high intermethod variability in TRAb assay results was seen in untreated GD patients. Assay harmonization is necessary for correct interpretation in the follow-up of Graves ophthalmopathy.

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T(3)) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T(3) were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2(+) and PSA-NCAM(+) precursors and proliferative cells. Our findings establish a role for T(3) as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Immunoassays are used daily by clinical endocrinologists to refute or confirm a diagnosis, or to follow up the course of a treatment. Immunoassays have become increasingly sensitive, specific and reproducible, so that clinicians have great confidence in their results. However, they do not always yield correct results because interferences still occur. In the first part of this review, immunoassay interferences are described and their mechanisms explained: matrix effects, specificity defaults, interferences from antibodies and binding proteins, or the hook effect. The hormones most frequently concerned by these pitfalls are reviewed, even as strategies used to prevent and minimize interferences. In the second part, the consequences of failure to recognize an interference are reviewed: unjustified clinical decisions (wrong diagnosis, treatment, surgery) and, more exceptionally, publication in the scientific literature of erroneous results. Clinicians may sometimes inform laboratories of an increased risk of interference. In general, clinicians are in the best position to fully validate test results; because only they know all the medical data about their patients. If there is any suspicion of discrepancy between clinical and laboratory data, they must alert the laboratory. This is the only way to better detect and, if possible, eliminate interferences and their regrettable outcomes.

Background: Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients., Methods: We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality., Results: In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r(2) = 0.216; P = 0.02), PCT (r(2) = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r(2) = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r(2) = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 microg/L (163.5-699.5 microg/L) vs 86.0 microg/L (53.8-175.3 microg/L) for CGA, and 6.78 microg/L (2.39-22.92 microg/L) vs 0.54 microg/L (0.16-6.28 microg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r(2) = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004-17.487], SAPS II (HR, 1.046; 95% CI, 1.026-1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731-8.880)., Conclusions: CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.

Objective: The objective of this study was to re-evaluate the adult C(T) reference values determined by five different immunoassays and by introducing criteria for selecting control subjects., Design: A prospective multicenter study., Patients: Three hundred and seventy-five clinically euthyroid subjects., Methods: We used five different C(T) immunoassays. Sera were assayed for the concentration of TSH, gastrin, procalcitonin, urea, calcium, and anti-thyroperoxidase antibodies., Results: Screening for the various potential causes of hypercalcitoninemia led to the exclusion of 23% of the sera. Our reference value analysis dealt with 287 subjects (142 men and 145 women). The proportion of samples in which no C(T) was detected varied from 56% (for assay D) to 88% (for assay C). We observed significant correlations (whose magnitude depended on the assay used) between C(T) levels and age or body mass index (BMI) (primarily in men). The distribution of C(T) levels showed that 4.7, 9.8, 2.5, 6.5, and 8.0% of the values were over 10 pg/ml respectively. These values corresponded essentially to samples from 11 male subjects (median age: 55 years), most of whom were smokers. The highest C(T) values were around twice as high in men than women, and were higher in smokers than non-smokers. Conclusion In clinical practice (and after having excluded the usual causes of raised C(T) levels), the interpretation of C(T) assay results must take into account i) the method used; ii) the patient's gender, age, and weight; and iii) the potential influence of cigarette smoking.

A 25 year old woman consulted for a severe acanthosis nigricans and central distribution of fat. Her masculine type morphology was associated with muscular appearance of the limbs and excess fat deposits in the face and neck. Biological testing confirmed glucose intolerance associated with a severe insulin resistance, hypertriglyceridemia and polycystic ovary syndrome. The detection of a heterozygous missense mutation in LAMIN A/C gene at position 482 confirmed the diagnosis of Familial Partial Lipodystrophy (FPLD2). Due to a deterioration of clinical and metabolic status, 15 and then 30 mg per day of pioglitazone were added to her previous treatment with metformin, bezafibrate and omega-3 fatty acids. Metabolic status improved rapidly after 3 months and continued thereafter. Weight remained stable, body mass composition and waist circumference improved. After 18 months of treatment, glycaemia and triglycerides levels normalized, hepatic enzymes and liver echographic features improved. Insulin sensitivity improved dramatically with a HOMA % S value of 73% with metformin and of 98.2% when pioglitazone was added. Leptin levels increased from 6.6 to 10.2 microg/ml. We report a very rapid and good efficacy of pioglitazone added to metformin without side effects in FPLD2. If confirmed on more patients, early use of pioglitazone in association with metformin could be proposed in FPLD2.

Introduction: The hypospadias procedure are many (more than 300 different procedures) and varied, because of the difficulty of the treatment., Objective: To evaluate our results with two stages and one stage procedure urethroplasty for proximal hypospadias and to recommend two stages procedures for posterior hypospadias with important curvature., Patients and Methods: We retrospectively reviewed the medical record of 35 patients operated on for proximal hypospadias between January 1997 and April 2005. Their age varied between 9 month and 15 years. The hypospadias were proximal penile hypospadias in 31 cases, scrotal hypospadias in 2 cases, and perineal hypospadias in 2 cases. Sixteen patients were treated by one procedure (Onlay, Duckett, or Duplay). The other nineteen were operated on by two stages procedure. In the first stage we corrected the curvature and transferred the prepucial tissue on the ventral aspect of the penis. The second stage was performed six month later by urethroplasty., Results: For the one stage procedure, the fistula's rate was 25% and the complication's rate was 37%. For the two stages procedure the fistula's rate was 15%, and the rate of complication was 26%. The total rate of fistula was 20%, and the total rate of complication was 31%., Conclusion: In this short series the fistula rate was higher in the group of children who had one stage procedure than the group of two stage procedure. We prefer the two stages procedure for the treatment of perineal and scrotal hypospadias with important curvature.

There are numerous etiologies of hyperprolactinemia, a common reason for consultation. Diagnostic measures must be capable of identifying the tumors, the most frequent of which are prolactin adenomas. Hypothalamic-pituitary MRI is the reference morphological examination. In clinical practice, it is usually performed very early, following the discovery of increased plasma concentrations of PRL. This approach is warranted for marked increase in PRL in the absence of drugs with hyperprolactinemic effects (>10 x upper limit of normal) since a diagnosis of PRL adenoma is extremely likely under such circumstances. When hyperprolactinemia is moderate, which is the most common finding in practice, all etiologies are possible in theory and it is important to follow a rational diagnostic plan (history-taking to identify use of any drugs with hyperprolactinemic effects paying attention to renal and hepatic history, investigation for endocrine diseases occasionally associated with hyperprolactinemia such as hypothyroidism or polycystic ovary syndrome (PCOS), confirmation of hyperprolactinemia by a second assay when the initial level is less than five times the upper normal limit, pregnancy testing for women of childbearing age) in order to rule out all non-tumoral causes of hyperprolactinemia before proceeding with imaging. Absence of any consequences of hyperprolactinemia on gonadic function or the existence of a concomitant disease that could account for the clinical signs, demonstration of wide variations in PRL from one assay to another in a single patient could prompt screening for macroprolactinemia before MRI is ordered. Macroprolactinoma could also occur in the case of normal or doubtful MRI or discrepancy in response to medical or surgical treatment. T1- and T2-weighted coronal sections (with or without T1 after gadolinium injection) are generally sufficient for diagnosis of microprolactinoma. Dynamic tests may be useful if MRI is normal or unclear. Gadolinium injection with sagittal and axial sections is essential for examination of large lesions. In this case, when the increase of PRL is moderate (<150 mg/ml), a non-lactotropic lesion may be suspected without misdiagnosing a hook effect. Careful analysis of the images allows differentiation between tumoral lesions and pituitary hyperplasia.

Aim: Glargine, a long-acting insulin analogue, is metabolized in the bloodstream and in subcutaneous tissue. Glargine metabolism and its implications for diabetes therapy remain poorly understood. The aim of our study was to assess in vitro the glargine blood biotransformation and its inter-individual variability., Methods: Formation of M1 glargine metabolite in vitro was studied with Elecsys Insulin immunoassay in pools of sera and sera from patients spiked with glargine. Elecsys Insulin assay is specific of human insulin, does not recognize glargine and its M2 metabolite but does recognize its M1 metabolite., Results: Glargine incubation with serum resulted in M1 metabolite formation which was detected and characterized as an enzymatic process: metabolite kinetics were dependant on temperature, substrate concentration and serum proportion. Carboxypeptidase inhibitors and chelating agents partially inhibited the activity of the enzyme(s). Glargine biotransformation was decreased when blood was collected on EDTA tubes. After 30 min incubation of glargine (100 mU/l) in 69 sera at 37 degrees C, percentage of glargine converted into M1 ranged from 46% to 98% (mean 72%; S.D. 11%)., Conclusion: Glargine blood biotransformation is an enzymatic process probably involving serum carboxypeptidase(s). Metabolite formation is rapid and non negligible. Inter-individual variability of glargine biotransformation is noteworthy and should be confronted to M1 metabolite bioactivity which has not been fully documented yet.

Background: Endogenous TSH and rhTSH stimulate thyroid growth by a direct effect on thyrocytes. Our hypothesis was that rhTSH may also stimulate thyroid angiogenesis., Study Design: A normal human thyroid tissue sample was grafted into the epigastric area of 14 nude mice. Mice were divided in two groups of 7. The first group (treated mice) received rhTSH stimulation (0.014 UI/mouse/day for 3 weeks), while the second group (control mice) had saline. Histological study with special focus on vascular characteristics was performed by image analysis at day 21 for each graft. VEGF immunostaining score, determined by immunohistochemistry, was defined as the percentage of labeled thyrocytes score, plus an intensity score., Results: Thyroid follicles showed signs of increased colloid re-uptake activity in rhTSH group within a larger surface area than controls (p <0.01). Thyrocytes were taller in the rhTSH group (p <0.01). The diameter of capillary vessels was larger and the microvessels expansion more important in the rhTSH group (p <0.02). Relative capillary area, defined as the ratio between capillary area and follicular area, was also higher in the rhTSH group (p <0.02). VEGF immunostaining score was increased in the rhTSH group (p <0.01)., Conclusion: rhTSH stimulates angiogenesis and local VEGF expression in normal human thyroid.

The thyroid gland is rich in selenium (Se) and expresses a variety of selenoproteins that are involved in antioxidative defense and metabolism of thyroid hormones (TH). Se deficiency impairs regular synthesis of selenoproteins and adequate TH metabolism. We recently generated mice that lack the plasma Se carrier, selenoprotein P (SePP). SePP-knockout mice display decreased serum Se levels and manifest growth defects and neurological abnormalities partly reminiscent of thyroid gland dysfunction or profound hypothyroidism. Thus, we probed the TH axis in developing and adult SePP-knockout mice. Surprisingly, expression of Se-dependent 5'-deiodinase type 1 was only slightly altered in liver, kidney, or thyroid at postnatal d 60, and 5'-deiodinase type 2 activity in brain was normal in SePP-knockout mice. Thyroid gland morphology, thyroid glutathione peroxidase activity, thyroid Se concentration, and serum levels of TSH, T4, or T3 were within normal range. Pituitary TSHbeta transcripts and hepatic 5'-deiodinase type 1 mRNA levels were unchanged, indicating regular T3 bioactivity in thyrotropes and hepatocytes. Cerebellar granule cell migration as a sensitive indicator of local T3 action during development was undisturbed. Collectively, these findings demonstrate that low levels of serum Se or SePP in the absence of other challenges do not necessarily interfere with regular functioning of the TH axis. 5'-deiodinase isozymes are preferentially supplied, and Se-dependent enzymes in the thyroid are even less-dependent on serum levels of Se or SePP than in brain. This indicates a top priority of the thyroid gland and its selenoenzymes with respect to the hierarchical Se supply within the organism.

Background: In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls., Methods: Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10 g/L) and incubated for 3 h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined., Results: Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (>or=95%). Anti-insulin antibody interference was significant for RI and RAAs (p

Thyroid hormone (TH) homeostasis depends on peripheral activation and inactivation of iodothyronines by selenoenzymes of the deiodinase (Dio) family. We genetically inactivated hepatic selenoenzyme expression, including Dio1, in order to determine the contribution of hepatic Dio to circulating TH levels. Serum levels of TSH, total T(4), and total T(3) were not different from controls. We measured Dio1 and Dio2 in kidney, skeletal muscle, heart, brown adipose tissue, and brain, but did not find compensatory up-regulation in these tissues. Finally, we determined expression in the liver of the following T(3) target genes: Spot14, alpha-glycerophosphate dehydrogenase (alphaGPD), and malic enzyme (ME). On the transcript level, both Spot14 and alphaGPD were reduced in Dio-deficient liver to about 60-70% of controls. However, mRNA and activity of ME were significantly increased in the same mice. Together, our results indicate that hepatic Dio1 activity is not absolutely required to sustain the euthyroid state in mice.

In connection with a comparative study of nine kits for the measurement of free thyroxin, we determined reference values in a adult control group of 81 women and 73 men. The correlations observed between the kits are associated with very large differences in the results obtained. The reference ranges are more or less broad according to the kits, but narrower than those offered by the manufacturers.

We measured free thyroxine in sera from 29 women in the ninth month of pregnancy. We used the reference method, equilibrium dialysis, and nine immunoassays. The bias between immunoassay and dialysis results was insignificant or modest. With all methods, the results were in the lower part of the non-pregnant female reference interval or below the lower limit of this interval: 10 results with the equilibrium dialysis assay and from 4 to 18 results with the nine immunoassays. Method-specific reference intervals should be determined during pregnancy to be in a position to quantify not only hypothyroidism but also hyperthyroidism and to adjust an appropriate T4 treatment.

DEFINING THE PROBLEM: Sub-clinical hypothyroidism, defined as a moderate and isolated increase in TSH levels, is a common syndrome and is the first phase of a progressive disease. However its treatment remains controversial. Some anamnestic, clinical and biological (anti-thyroperoxidase antibodies) parameters contribute in identifying the patients most likely to progress towards overt hypothyroidism. WHAT CAN BE EXPECTED OF TREATMENT? Several clinical studies have described cardiovascular, neuromuscular and lipid disorders in these patients, but administration of levothyroxine has provided varying results and does not enable the distinction between a pharmacological-like intrinsic effect of the thyroid hormone and the true benefits imputable to the correction of TSH, in the absence of any large interventional study. More studies are required to better identify the patients who will benefit most from hormone replacement.

Objectives: To verify the cut-off values and to determine the clinical sensitivity of antithyroglobulin (TgAb) determinations using our routine RIA and the new electrochemiluminescent Elecsys assay., Design and Methods: We used the DYNOtest anti-Tgn manual RIA from BRAHMS and the new automated Elecsys electrochemiluminescent immunoassay from Roche Diagnostics. We analyzed 452 sera from the following subjects: 193 euthyroid controls, 163 with treated and untreated autoimmune thyroid diseases (AITD) (108 Graves' disease and 55 thyroiditis), 50 with differentiated thyroid carcinoma, 13 with nonautoimmune thyroid disease and 33 with type 1 diabetes mellitus., Results: As expected, using the proposed thresholds (BRAHMS 60 kIU/L, Elecsys 115 kIU/L) approximately 6% of the control subjects were positive for TgAb with both methods. In AITD patients, the sensitivity of TgAb determination was significantly higher with the Elecsys assay (51.5%) than with the BRAHMS assay (39.3%). This difference was not observed in the other patient groups., Conclusion: The Elecsys assay can be preferred not only because it is automated and rapid, but also because of its better clinical performance in AITD patients.

Over the past decade, numerous papers have addressed the various methodological problems encountered with free thyroxine (FT4) assays. We evaluated the clinical performance of nine FT4 assays in five centres, using a panel of 310 sera: 156 from euthyroid controls; 27 from hyperthyroid patients; 34 from untreated hypothyroidism; 22 from patients with renal failure; 30 from women in the last trimester of pregnancy; 23 from patients on thyroid substitutive therapy; and 18 from patients treated with amiodarone. Only three methods showed a Gaussian distribution of FT4 concentrations. Reference ranges were calculated using the 2.5th and 97.5th percentiles. A significant difference was observed between FT4 values in men and women. The areas under the receiver operating characteristic (ROC) curves ranged from 0.996 to 1 for hyperthyroidism and from 0.973 to 1 for hypothyroidism. In sera from patients with renal failure and from pregnant women, method-dependent biases were observed and confirmed with dilution experiments. In conclusion, current FT4 assays show good performance regarding the diagnosis of overt dysthyroidism. Nevertheless, FT4 measurements are still vulnerable to method-dependent artefacts in particular populations such as patients with renal failure and pregnant women.

Hormonal production of the thyroid gland is constituted of thyroxine or T4 (80%) and triiodothyronine or T3 (20%). In the circulation, whole T4 originates from thyroid secretion but most of T3 (80%) is produced extrathyroidally from T4 deiodination. Conversion of T4 to T3 may be influenced by various conditions and circulating T3 is a less reliable reflection of thyroid hormone production than T4. In serum most of T4 and T3 is bound to binding proteins and only 0.02% of T4 and 0.3% of T3 is free. Because of their higher diagnostic performance, free T4 (FT4) and free T3 (FT3) measurements have superseded total (free + bound) hormone determination. Total hormone measurements remain useful for research studies or in case of severe hyperthyroidism. Equilibrium dialysis/RIA is considered as the reference method for free hormone measurements. Routine clinical laboratories use automated direct two-step or one-step immunoassays with a high molecular weight ligand or labelled antibody. Free hormone measurement remains technically demanding, especially in sera from severe non-thyroid ill patients with low serum thyroxine binding capacity. Interference from anti-thyroid hormone antibodies and familial dysalbuminemic hyperthyroxinemia depends on the assay method, but is now less marked and less frequently detected. To be able to correctly interpret the results of an assay, it is necessary to assess its performance in biologically and clinically well-characterised serum samples. FT4, and FT3 measurements, if FT4 is normal and hyperthyroidism suspected, are used to confirm and assess the level of hypo and hyperthyroidism (overt or subclinical). When the thyroidal status is unstable (first months of a thyroid treatment, altered L-T4 dose, subacute thyroiditis) or when the hypothalamic-pituitary function is disturbed (central hypothyroidism), TSH determination is diagnostically misleading and only free hormone measurements are reliable for thyroid function assessment.

Insulin assays play a central role in the investigation of glucose metabolism disorders (investigation of the causes of hypoglycemia, assessment of beta-cell function and determination of pathogenesis of type 1 and type 2 diabetes), and in studies on the pharmacology of insulin itself. Greater convenience and improved reproducibility, sensitivity and specificity have been achieved with human insulin immunometric assays. Besides human insulin, insulin analogues (lispro, aspart and glargine) have been introduced to therapeutic use. The specificity of human insulin assays to these analogues should be assessed. Anti-insulin antibodies are present in a significant proportion of sera to be analyzed for insulin. The sensitivity of insulin assays to interference from these antibodies should also be assessed. Interferences from anti-insulin antibodies and hemolysis, which degrades insulin molecules, remain the main pitfalls of insulin assays. Immunometric assays have also improved the sensitivity and reproducibility of free insulin measurements. Standardization of insulin immunoassays is still called for.

We investigated the interference of insulin antibodies in two insulin immunometric assays (Bio-Rad and Elecsys) by measuring direct and free insulin in plasma from 30 patients without insulin antibodies (group 1), as screened by a sensitive radio-binding assay, and in plasma from 80 patients with insulin antibodies (group 2). In group 1, the direct/free insulin ratio did not differ from 1, showing the equivalence of free and direct insulin results in theses samples. In group 2, this ratio was markedly increased (mean: Bio-Rad 2.63, Elecsys 5.02) and correlated positively with the insulin antibody radio-binding assay result (r=0.92 for the correlation between Bio-Rad and Elecsys assays after log-transformation of the ratios). In samples containing insulin antibodies, direct insulin concentration was frequently lower than total (bound and unbound) insulin measured with the Bio-Rad and Elecsys assays. This study underlines the interference of insulin antibodies in insulin immunometric assays and the importance of assessing an insulin immunometric assay for sensitivity towards the presence of these antibodies.

Background: The Elecsys prolactin (PRL) assay reacts more strongly with macroprolactin than the Centaur PRL assay. We evaluated Elecsys direct and free PRL measurements vs. Centaur direct PRL measurements, in sera with and without macroprolactin., Methods: PRL was measured using Elecsys and Centaur direct assays and the Elecsys assay in the supernatant obtained after PEG precipitation (free PRL) in 34 sera from 34 hyperprolactinemic male subjects (Elecsys direct PRL>434 mIU/l) classified, according to the PRL recovery after PEG precipitation, as: negative, i.e. without predominant macroprolactin (recovery %>50, n=12), positive (recovery %<40, n=18) or indeterminate (n=4)., Results: The positive bias between Elecsys direct and Centaur PRL results was clearly influenced by the presence of macroprolactin and the mean bias between Elecsys free and Centaur prolactin values was near zero in the negative and positive groups. Among 14 patients from the positive group presenting clinical conditions possibly ascribable to hyperprolactinemia, Elecsys free and Centaur PRL levels were normal in seven and increased in five. In the negative and positive groups considered together, Elecsys free PRL agreed well with Centaur prolactin (28/30). The poor concordance observed for the indeterminate samples underlined the heterogeneity of macroprolactin., Conclusion: Elecsys free PRL determination can be used to reduce the marked influence of macroprolactin in this assay.

Background: The cytotoxic T lymphocyte-associated antigen 4 gene (CTLA-4) encode the T cell receptor involved in the control of T cell proliferation and mediates T cell apoptosis. The receptor protein is a specific T lymphocyte surface antigen that is detected on cells only after antigen presentation. Thus, CTLA-4 is directly involved in both immune and autoimmune responses and may be involved in the pathogenesis of multiple T cell-mediated autoimmune disorders. There is polymorphism at position 49 in exon 1 of the CTLA-4 gene, providing an A-G exchange. Moreover, we assessed the CTLA-4 49 (Thr/Ala) polymorphism in diabetic patients and first-degree relatives as compared to control subjects., Research Design and Methods: Three loci (HLA-DQB1, DQA1 and CTLA-4) were analysed in 62 type 1 diabetic patients, 72 first-degree relatives and 84 nondiabetic control subjects by means of PCR-RFLP., Results: A significant enrichment in DQB1 alleles encoding for an amino acid different from Asp in position 57 (NA) and DQA1 alleles encoding for Arg in position 52 was observed in diabetic subjects and first-degree relatives as compared to controls. The genotype and allele frequencies of these polymorphisms in type 1 diabetic patients and first-degree relatives differed significantly from those of controls (p < 0.001 and 0.05 respectively). CTLA-49 Ala alleles frequencies were 75.8% in type 1 diabetic patients and 68.1% in first-degree relatives in comparison to 35.7% in control subjects. The Ala/Ala genotype conferred a relative risk of 18.8 (p < 0.001)., Conclusion: The CTLA-4 49 Ala allele confers an increased risk of type 1 diabetes, independent of age and HLA-DQ genetic markers.

Objective: To assess the antigenicity of the insulin Hoechst 21PH (Hoe21PH) using continuous subcutaneous insulin infusion (CSII) and to compare the antigenicity of this insulin when administered intraperitoneally or subcutaneously. RESEARCH DESIGN AND METHODS; Peritoneal administration of Hoe21PH (Hoechst-Roussel, Somerville, NJ) insulin using implantable devices (continuous peritoneal insulin infusion [CPII]) increases anti-insulin antibody (AIA) levels in type 1 diabetic patients. Intraperitoneal administration, addition of a stabilizer (polyethylene polypropylene glycol), or insulin modifications due to storage in the pump may be involved in this antigenicity. In this nonrandomized study, 24 type 1 diabetic patients were treated with either CSII (n = 11, group 1) or CPII (n = 13, group 2). AIA levels were measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) before starting patients on Hoe21PH and again after 3 and 6 months., Results: Patients were comparable in the two groups. AIA levels (RIA) remained stable (24.3 +/- 8.5% [month 0] to 24.9 +/- 8.5.5% [month 6]) in group 1 and increased (21.8 +/- 6.7% [month 0] to 41.8 +/- 6.9% [month 6]) in group 2 (P = 0.005, Wilcoxon's rank-sum test). Using ELISA, AIA remained stable in the patients in group 1 (n = 9; 3.8 +/- 0.8 units/ml [month 0] and 4.1 +/- 1.0 units/ml [month 6]) and tended to increase in the patients in group 2 (n = 12; 4.1 +/- 0.7 units/ml [month 0] to 17.5 +/- 4.6 units/ml [month 6]) (P = 0.07). Comparison of the evolution of AIA formation between the two groups, using RIA at months 0, 3, and 6 showed a significant difference (analysis of variance, P = 0.009)., Conclusions: No increase in AIA levels was demonstrated when Hoe21PH insulin was administered subcutaneously as assessed by two different assays. CPII is proven to be more antigenic than CSII, and this is not related to a specific antigenicity of Hoe21PH insulin. The intraperitoneal route of administration or insulin modifications due to insulin storage in implantable devices might explain this antigenicity.

Objectives: To assess the presence and magnitude of a serum thyroxine binding capacity (sBC)-dependent bias in five free thyroxine (FT(4)) immunoassays, compared with equilibrium dialysis (ED). The exhibited bias is confronted with clinical results from previous studies to evaluate its impact on FT(4) determination in sera with various sBC., Design and Methods: The sera of three pregnant women and three non thyroidal ill (NTI) patients were serially diluted in an inert buffer to progressively decrease the sBC. FT(4) values were measured in diluted and undiluted samples with the six assays., Results: As a function of increasing dilution performed on pregnancy sera, except for ED and Vitros ECi FT(4,) the other four FT(4) assay results decreased to different degrees, in the following order: two-step GammaCoat RIA< Elecsys< ADVIA:Centaur< Amerlex-MAB RIA. In sera from NTI patients, the decrease was more marked and found at high dilution with the Vitros ECi assay. Data from previous studies showed that FT(4) measured with biased assays were decreased only in samples from very severely NTI patients with low sBC and that FT(4) results in pregnancy sera with high sBC were not significantly biased., Conclusions: The dilution test is a sensitive alarm to assess sBC-dependent bias in FT(4) assays. For all FT4 assays and particularly when a bias is observed, documentation should be sought on the diagnostic performance of the assay and supported by a detailed clinical study including samples with low sBC. Physicians should still be educated about the limitations of FT(4) assays.

Background: The HLA-DQB1 chain, in particular the amino acid in position 57, and genetic variants of the vitamin D-binding protein (DBP) have been reported to be associated with type 1 diabetes. There are two known polymorphisms in exon 11 of the DBP gene resulting in amino acid variants: codons 416 GAT --> GAG (Asp --> Glu) and 420 ACG --> AAG (Thr --> Lys). We compared distribution of DQB1 alleles and amino acid variants of DBP in type 1 diabetic patients (n = 44) in the Alsacian population and in healthy controls (n = 58)., Methods: The second exon of the DQB1 gene and exon 11 of DBP were analyzed by restriction mapping after polymerase chain reaction., Results: A significant enrichment in DQB1 alleles encoding for an amino acid different from Asp in position 57 (NA) was observed in diabetic subjects as compared to controls (94.3 vs. 32.8%; p < 0.001). Combinations other than Ala/Ala carried the highest relative risk (OR = 52; p < 0.001). The analysis of the polymorphism in exon 11 of DBP showed a significant difference in the allele frequency of the HaeIII site, but not of the StyI site between patients and controls. Allele frequencies of HaeIII in diabetic subjects were 36% and 64% for Asp and Glu respectively (p < 0.001; chi(2) = 29.5). The frequency of Asp/Asp and Glu/Glu genotypes was increased in controls and diabetic subjects respectively. DBP alleles in individuals carrying the DQB1 NA combination revealed that 46.6% of diabetics were DBP Asp/Glu, but this was not statistically significant using the Fisher exact test (16/31 vs. 0/3; p = 0.23)., Conclusions: The study of the DQB1 chain confirmed the value of alleles encoding for an amino acid different from Asp in position 57 (NA) in the susceptibility to type 1 diabetes. The allele frequency of the HaeIII site, but not of the StyI site, differed between patients and controls (HaeIII p < 0.001; StyI p > 0.05).

Intraperitoneal insulin infusion of Genapol stabilized insulin via implantable devices significantly improves diabetes control and hypoglycemia frequency in type 1 diabetes while it increases insulin antibody levels. Causes for this particular antigenicity remain unknown. The role of insulin modifications occurring in the reservoir on the antigenicity observed was assessed by comparing the antigenicities of the insulin coming from the vial or from the pump reservoir. Rats were injected intraperitoneally with insulin sampled either from a vial (group 1) or from a pump reservoir during a refill of a clinical trial (group 2). Two control groups, one without insulin, the second one receiving a mixture of silicone and insulin were also studied. Human insulin antibody levels were assessed by RIA 10 days after 4 weekly immunizations. AIA levels were higher in group 1 compared to group 2 (P = 0.003 for the first experiment, P = 0.04 in the second experiment). The increased antigenicity of the insulin sampled from the implanted pump might be due to the insulin modifications occurring during the storage in the device. Insulin aggregates could be involved in this antigenicity since they are known to be antigenic and their concentration was shown to be related to the amplitude of the antigenic response.