Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification.

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.

High serum levels of free cortisol indicate severity of cirrhosis in hemodynamically stable patients.

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.

Thyroid function is maintained despite increased oxidative stress in mice lacking selenoprotein biosynthesis in thyroid epithelial cells.

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.

Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy.

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.

Assessment of adrenal function in cirrhotic patients using concentration of serum-free and salivary cortisol.

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.

Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells.

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.

Subclinical hyperthyroidism: considerations in defining the lower limit of the thyrotropin reference interval.

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.

Serum concentration of chromogranin A at admission: an early biomarker of severity in critically ill patients.

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.

Intermethod variability in TSH-receptor antibody measurement: implication for the diagnosis of Graves disease and for the follow-up of Graves ophthalmopathy.

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.

Recovery from chronic demyelination by thyroid hormone therapy: myelinogenesis induction and assessment by diffusion tensor magnetic resonance imaging.

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.

Prognostic value of chromogranin A at admission in critically ill patients: a cohort study in a medical intensive care unit.

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.

Reference range of serum calcitonin levels in humans: influence of calcitonin assays, sex, age, and cigarette smoking.

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.

Use of insulin immunoassays in clinical studies involving rapid-acting insulin analogues: Bi-insulin IRMA preliminary assessment.

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

Recombinant human thyrotropin stimulates thyroid angiogenesis in vivo.

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.