LC-MSMS assays of urinary cortisol, a comparison between four in-house assays.

BACKGROUND: Twenty-four hour urinary free cortisol (UFC) determination can be used for screening and follow-up of Cushing syndrome (CS). As immunoassay methods lack specificity for UFC measurement, the use of high-performance liquid chromatography coupled to mass spectrometer (LC-MSMS) is recommended. The aim of our study was to compare UFC results using four LC-MSMS methods performed in four independent laboratories in order to evaluate interlaboratory agreement. METHODS: Frozen aliquots of 24-h urine samples (78 healthy volunteers and 20 patients with CS) were sent to four different laboratories for analysis. Following liquid-liquid or solid-liquid extraction, UFC were determined using four different LC-MSMS assay. RESULTS: UFC intra- and interassays variation coefficients were lower than 10% for each centre. External quality control results were not significantly different. UFC normal ranges (established from healthy volunteers) were 17-126, 15-134, 12-118 and 27-157 nmol/day, respectively. Classification of UFC from healthy volunteers and patients with CS using a 95th percentile threshold was similar. However, for extreme UFC values (<50 or >270 nmol/day), negative or positive bias was noted. CONCLUSIONS: Even for highly specific methods such as LC-MSMS, variations of results can be found depending on analytical process. Validation of LC-MSMS methods including determination of the reference range is essential.

Lower Circulating Sertoli and Leydig Cell Hormone Levels During Puberty in Obese Boys: A Cross-sectional Study.

CONTEXT: Alterations in semen characteristics and circulating Sertoli and Leydig cell hormones have been described in obese male adults. Whether hormonal alterations occur before adulthood has not been fully evaluated. OBJECTIVE: We describe circulating Sertoli and Leydig cell hormone levels in overweight-obese (ow/ob) boys through childhood and adolescence in a cross-sectional study. METHODS: Monocentric study in the Pediatric Endocrinology Unit of Angers University Hospital. Three hundred and fifty-one obese and overweight boys aged 5-19 years underwent physical examination, dual-energy X-ray absorptiometry for body composition, oral glucose tolerance test on insulin and glucose, and measurements of follicle-stimulating hormone, luteinizing hormone, anti-Müllerian hormone (AMH), inhibin B, testosterone, and estradiol. Hormonal levels were compared with normative data obtained from 652 healthy nonoverweight nonobese boys of similar age or Tanner stage. RESULTS: Median inhibin B and testosterone levels during puberty were significantly lower in ow/ob than in healthy boys (1) from age >12 years and thereafter for inhibin B, and (2) from age >14 years and thereafter for testosterone. At Tanner stages 4 and 5, 26%, 31%, and 18% of inhibin B, testosterone, and AMH values were below the 5th percentile in ow/ob subjects (P < .01). In multiple regression analyses, estradiol and total bone mineral density Z-score were negative predictors of inhibin B, fat mass percentage was a negative predictor of testosterone, and insulin was a negative predictor of AMH. CONCLUSION: Lower Sertoli and Leydig cell hormone levels during puberty were observed in the ow/ob boys.

Analytical validation of eight methods of thyroglobulin measurement in fine-needle aspiration washouts.

BACKGROUND: Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. METHODS: Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. RESULTS: In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between -24.5 and 10.2% around 1 µg/L. Total analytical error was ≤40% near the functional sensitivity values. CONCLUSION: No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

Familial Dysalbuminemic Hyperthyroxinemia: An Underdiagnosed Entity.

Resistance to thyroid hormone (RTH) is a syndrome characterized by impaired sensitivity of tissues to thyroid hormone (TH). The alteration of TH-binding proteins, such as in Familial Dysalbuminemic Hyperthyroxinemia (FDH), can mimic the abnormal serum thyroid tests typical of RTH. We aimed to characterize a population referred to our center with suspected RTH and estimate the proportion of patients with FDH. For 303 different families, we collected clinical and hormonal data and sequenced the thyroid hormone receptor ß gene (THRB) and exon 7 of the albumin gene (ALB). We found 56 THRB variants (i.e., 38% of the 303 index cases, called RTHß group). Among the samples screened for FDH variants, 18% had the variant R218H in ALB (FDH group); in addition, 71% of the cases had neither variant (non-FDH/RTHß group). Patients with FDH had significantly lower free T3 (fT3) and free T4 (fT4) levels and more often an isolated elevation of fT4 than RTHß patients. Clinically, patients with FDH had fewer symptoms than patients with RTHß. Our study suggests that FDH should be systematically considered when examining patients suspected of having RTH. In most cases, they present no clinical symptoms, and their biochemical alterations show an elevation of fT4 levels, while fT3 levels are 1.11 times below the upper limit of the assay.

Apparent resistance to thyroid hormones: From biological interference to genetics.

Resistance to thyroid hormones syndrome is defined as increased thyroxine (T4) and triiodothyronine (T3) concentrations associated with normal or sometimes increased thyrotropin (TSH) concentration. This is usually due to a pathogenic variant with loss of function of the gene coding for thyroid hormone receptor ß (THRB). This discrepancy in thyroid hormones (TH) and TSH concentrations is also frequently observed in the presence of analytical interference, notably alteration of TH transport proteins in serum. During 2017, 58 samples were sent to our laboratory in the Angers University Hospital Rare Thyroid and Hormone Receptor Disease Reference Center in order to identify an etiology for discrepant TSH and TH results. We sequenced the genes involved in TH regulation, action and transport (THRB,THRA, SECISBP2, SLC16A, ALB, TTR, SERPINA7). Free T4 and free T3 assay were performed with a second immunoassay (Siemens ADVIA Centaur). A genetic cause of discrepancy in TH and TSH concentrations, with mutation in THRB, was found in 26% of cases (15/58). Biological interference due to TH serum transport protein variant was found in 24% (14/58) of cases. No pathogenic variants were found in the other genes studied. Biological interference was also suspected in 26% of cases without genetic variant, in which the biological discrepancy was not confirmed by a second analytical technique (15/58). Finally, no etiology for the biological discrepancy could be found in 24% of cases (14/58). Clinically, patients in whom biological discrepancy was due to analytic interference were more often asymptomatic, and patients with no identified etiology tended to be older. To limit diagnostic errors associated with the finding of discrepant TSH and TH, we recommend initially conducting a second thyroid function test (TSH, free T4 and free T3) with a different assay, and then screening for a genetic variant in gene coding for thyroid hormone receptor ß (THRB) and the TH serum transport proteins (ALB, TTR, SERPINA7).

The insulin-like growth factor-I response to growth hormone is increased in prepubertal children with obesity and tall stature.

CONTEXT: Children with obesity [body mass index (BMI) > +2 sd score (SDS)] and children with constitutional tall stature [CTS; height > +2 SDS)] have normal-high serum IGF-I levels, associated with a low and broad range of GH secretion, respectively. This suggests increased sensitivity to GH, whereas children with idiopathic short stature (ISS; height < -2 SDS) are believed to have decreased GH sensitivity. OBJECTIVE, DESIGN, AND MAIN OUTCOME MEASURE: To compare the responsiveness to GH in 62 prepubertal children (43 females, 19 males) with obesity, CTS, or ISS and 26 controls (15 females, 11 males; height and BMI -2 to +2 SDS), we used an IGF-I generation test and studied the IGF-I concentration 24 h after a single injection of GH (2 mg/m2). PATIENTS: Twenty patients with obesity, 20 with CTS, 22 with ISS, and 26 controls were studied. The mean age was 8.3 +/- 2.9 yr, with no difference in age or gender between groups. RESULTS: Compared with controls, the mean IGF-I increment was 80% higher in obese children and 36% higher in tall children (P < 0.05 obese or tall vs. control children; P = 0.05 obese vs. tall children). Conversely, the IGF-I increment was similar in short compared with control children, despite a mean baseline IGF-I 62% lower in short children (P < 0.05 vs. controls). In all groups, the IGF-I increment was correlated with the BMI SDS or the fat mass percentage (r = 0.51-0.58, P < 0.05). CONCLUSION: Obese children tend to have greater GH responsiveness than tall children, and both have greater GH responsiveness than controls. GH responsiveness was similar in controls and short children, despite a lower baseline IGF-I in short children. Whether the differences in the IGF-I response to GH between these children reflect differences in the respective anabolic (growth promotion) and metabolic (i.e. insulin action modulation) roles of circulating IGF-I is unknown.

Pituitary stalk interruption syndrome: a rare and severe cause of pituitary deficiency Laboratory diagnosis of a newborn case.

We report the case of a newborn with neonatal hypotonia associated to a micropenis and a bilateral cryptorchidia. The discovery of severe hypoglycemia at 0.22 mmol/L led to further biological investigations that revealed sharply decreased levels of several hypophyseal hormones. Altered corticotropic, somatotropic, thyreotropic, and gonadotropic axes finally suggested congenital hypophyseal insufficiency. This diagnostic was confirmed by a brain MRI (magnectic resonance imaging), which revealed a total interruption of the pituitary stalk. Immediate substitutive hormonal treatment allowed a clinical improvement of the condition and limited the risk of further episodes of hypoglycemia. The pituitary stalk interruption syndrome (PSIS), a very rare congenital disorder, has an estimated incidence of about 1:200.000. This developmental anomaly of the hypophysis calls for urgent diagnosis since prognosis depends on the rapid implementation of substitutive hormonal therapy. The hormonal deficit in the newborn affected by PSIS is often of a multiple nature with a constant somatotropic deficit, thus requiring the exploration of the different antehypophyseal axes. Despite the fact that PSIS is a rare disorder, it should always be kept in the differential diagnosis of newborn presenting with hypoglycemia. Since the interpretation of hormonal assays is particularly delicate at birth, close clinico-biological cooperation is essential for rapid diagnosis of PSIS and appropriate adaptation of the short- and long-term therapeutic management of the newborn.

Birth Weight Is Associated With the IGF-1 Response to GH in Children: Programming of the Anabolic Action of GH?

CONTEXT: Intrauterine programming of the somatotropic axis has been hypothesized in cases of intrauterine growth retardation. OBJECTIVE: The objective of the study was to study the effects of birth weight and body composition on GH sensitivity. DESIGN: This was a cross-sectional study with a single GH administration to assess GH sensitivity. SETTING: The study was conducted at the Department of Pediatric Endocrinology of an academic medical center. PATIENTS: One hundred normal short children aged from 4 to 17 years old (44 girls, 56 boys) separated into four groups: early childhood (aged 4-8 y, n = 14), late childhood (aged 9-12 y, pubertal stage 1, n = 30), early puberty (aged 10-15 y, stage 2, n = 32), and midpuberty (aged 12-17 y, stages 3 and 4, n = 24). INTERVENTION AND MAIN OUTCOME MEASURE: Serum IGF-1 at baseline and 24 hours after a single administration of GH (2 mg/m(2)) were measured. RESULTS: δIGF-1 significantly increased across the groups (P < .0001) with no gender difference, whereas the percentage of change in IGF-1 was similar (47% ± 32%). Independent predictors of δIGF-1 were birth weight SD score, fat percentage, fasting insulin (all positive predictors), and free fatty acids (negative predictor), with age, puberty, and baseline IGF-1 as adjusting variables (multiple R = 0.73, P < .0001). Independent predictors of the percentage of change in IGF-1 were birth weight SD score, fat percentage, and baseline IGF-1 (multiple R = 0.43, P < .001). CONCLUSION: This study suggests that in cases of low birth weight, intrauterine programming of GH sensitivity may be an adaptation to an expected poor postnatal nutritional environment, serving to restrict the anabolic action of GH. Conversely, postnatal excess energy stores may promote the anabolic action of GH.

[Mass spectrometry for steroid assays]. / Dosages des stéroïdes par spectrométrie de masse.

Steroid hormone measurement, first developed with radioimmunoassay, is now becoming easier with the use of automated platforms of immunoassay. However, some hormones remain uneasily detectable because of their low blood concentration, their structural homology or the presence of interferences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be considered as an alternative to immunoassays. This approach allows the simultaneous determination of several parameters thanks to its selectivity led by the detector mass spectrometer and the separate dimension of chromatography liquid. In addition, recourse to UHPLC (ultra high performance liquid chromatography) allows improving selectivity and sensitivity while limiting the samples volumes. The "ready-to-use" kits are now available and added to the "homemade" techniques developed by laboratories, thus giving opportunity for measurement of a wide steroid panel with only one sample. Finally, mass spectrometry methods, including a prior extraction step, allow the use of varied biological fluids (blood, urine, saliva…). Also, several clinical indications could gain from mass spectrometry, especially when hormone levels are low, when several steroids have to be identified, when the sample volume is low. However, this technology represents an important financial investment and in-depth staff training. In addition, some steroids are not easily quantifiable by mass spectrometry. It is likely by immunoassay and mass spectrometry, well-matched technologies, that we could answer the best to clinical questions about steroids.

Maternal and cord blood ghrelin in the pregnancies of smoking mothers: possible markers of nutrient availability for the fetus.

AIMS: To investigate the role of ghrelin in maternal and fetal metabolism, we determined its value in maternal smoking, a specific cause of reduced placenta function and fetal growth. METHODS: In 85 normal term pregnancies, 42 in smoking and 43 in non-smoking mothers, we measured ghrelin in the maternal blood at the onset of labor and in the cord blood of their 85 singletons immediately after birth. We determined the relationships between ghrelin and placental GH (PGH), pituitary GH (pitGH), and IGF-I. RESULTS: The newborns of smoking mothers weighed 0.24 kg less (p < 0.05) than those of non-smoking mothers. Cord blood ghrelin was 71% higher and PGH and cord blood IGF-I were 34% and 32% lower, respectively, in the pregnancies of smoking compared with non-smoking mothers (p < 0.05). Cord blood ghrelin was unrelated to pitGH and cord blood IGF-I. Maternal ghrelin was unchanged in smoking mothers, increased with maternal fasting duration (r = 0.26, p < 0.05), showed no correlation with PGH and negative correlation with cord blood IGF-I (r = -0.42, p < 0.01). CONCLUSION: The decrease in placental function and fetal growth in smoking mothers is associated with an increase in cord blood ghrelin, and no change in maternal ghrelin. Maternal ghrelin concentration increases with fasting, and is negatively correlated with cord blood IGF-I: it may signal a reduction in the level of nutrients available for placental transfer. No correlation supports a role for ghrelin in PGH or pitGH secretion.