Fit-for-Purpose Radio Receptor Assay for the Determination of Growth Hormone Secretagogues in Urine.

The everlasting pharmacological development is continuously producing new substances with potential doping abuse. Among these, secretagogues are very prone to misuse by athletes for their properties to release growth hormone (GH) and some limitations in the actual analytical methods to detect them. In this paper, an in-depth study on the key variables of the radio receptor method previously developed by our group is performed and a fit-for-purpose protocol is established. Thus, this sensitive and robust screening method is proposed as an intelligent and preventive antidoping method to detect new growth hormone secretagogues (GHSs) in exceptional suspicious urine samples obtained from athletes and will support the current detection methods based on liquid chromatography-mass spectrometry (LC-MS).

Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports-doping control.

Detection of misuse of peptides and proteins as growth promoters is a major issue for sport and food regulatory agencies. The limitations of current analytical detection strategies for this class of compounds, in combination with their efficacy in growth-promoting effects, make peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative analysis of peptides and proteins is well-established, particularly due to tremendous efforts in the proteomics community. Similarly, due to advancements in targeted proteomic strategies and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of peptides and proteins is becoming more widely accepted. These continuous advances in MS instrumentation and MS-based methodologies offer enormous opportunities for detection and confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to improve the qualitative and quantitative analysis of peptide and proteins with growth-promoting properties. This review aims to address the opportunities of MS for peptide and protein analysis in veterinary control and sports-doping control with a particular focus on detection of illicit growth promotion. An overview of potential peptide and protein targets, including their amino acid sequence characteristics and current MS-based detection strategies is, therefore, provided. Furthermore, improvements of current and new detection strategies with state-of-the-art MS instrumentation are discussed for qualitative and quantitative approaches.

Metabolomics in food analysis: application to the control of forbidden substances.

Metabolomics is a science of interest in food analysis to describe and predict properties of food products and processes. It includes the development of analytical methods with the ultimate goal being the identification of so-called 'quality markers', (i.e. sets of metabolites that correlate with, for example, quality, safety, taste, or fragrance of foodstuffs). In turn, these metabolites are influenced by factors as genetic differences of the raw food ingredients (such as animal breed or crop species differences), growth conditions (such as climate, irrigation strategy, or feeding) or production conditions (such as temperature, acidity, or pressure). In cases where the routine-based measurement of a food property faces some limitations such as the lack of knowledge regarding the target compounds to monitor, monitoring based on a limited set of crucial biomarkers is a good alternative, which is of great interest for food safety purposes regarding growth promoting practices. Such an approach may be more efficient than using a classic approach based on a limited set of known metabolites of anabolic compounds. In this context, screening strategies allowing detection of the physiological response resulting from anabolic compound administration are promising approaches to detect their misuse. The global metabolomics workflow implemented for such studies is presented and illustrated through various examples of biological matrices profiling (tissue, blood, urine) and for different classes of anabolic compounds (steroids, ß-agonists and somatotropin).

Multifunctional core-shell nanoparticles: discovery of previously invisible biomarkers.

Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core-shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(N-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (K(D) < 10(-11) M) for specific low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable.

The role of urinary soluble endoglin in the diagnosis of pre-eclampsia: comparison with soluble fms-like tyrosine kinase 1 to placental growth factor ratio.

OBJECTIVE: Endoglin, an anti-angiogenic glycoprotein expressed on endothelial cells, has been proposed recently as a biomarker of pre-eclampsia (PE). Given that PE is characterised by an imbalance of angiogenic factors, we sought to determine the clinical utility of urinary soluble endoglin, relative to the soluble fms-like tyrosine kinase 1 to placental growth factor (PlGF) ratio, in the diagnosis of PE during gestation. DESIGN: Prospective observational cohort. SETTING: Tertiary referral university hospital. POPULATION: Two hundred and thirty-four pregnant women were enrolled prospectively in the following groups: healthy controls, n = 63; gestational age (GA), median (interquartile range), 33 weeks (27-39 weeks); chronic hypertension, n = 27; GA, 33 weeks (30-36 weeks); mild PE, n = 38; GA, 37 weeks (34-40 weeks); severe PE, n = 106; GA, 32 weeks (29-37 weeks). METHODS: Free urinary levels of soluble endoglin, soluble fms-like tyrosine kinase 1 and PlGF were measured by sensitive and specific immunoassay. Levels for all urinary analytes were normalised to creatinine. MAIN OUTCOME MEASURES: Urinary soluble endoglin, and the soluble fms-like tyrosine kinase 1 to PlGF ratio. RESULTS: In healthy controls, urinary soluble endoglin levels were increased significantly at term relative to those earlier in gestation. Severe PE was characterised by an increased urinary level of soluble endoglin, soluble fms-like tyrosine kinase 1, protein to creatinine ratio and soluble fms-like tyrosine kinase 1 to PlGF ratio compared with all other groups. There was a direct correlation between urinary soluble endoglin and proteinuria that remained after GA correction (R = 0.382, P < 0.001). Urinary soluble endoglin could not differentiate mild PE from severe preterm PE. Overall, soluble endoglin had the ability to discriminate PE from chronic hypertension and healthy controls only in women who were evaluated at <37 weeks of GA. The sensitivity, specificity and accuracy of urinary soluble endoglin alone in the diagnosis of PE or in the identification of women with PE requiring a mandated delivery before 37 weeks of gestation were 70%, 86% and 76%, respectively. These values were inferior to those of the soluble fms-like tyrosine kinase 1 to PlGF ratio (P < 0.001). The addition of urinary soluble endoglin did not improve the diagnostic accuracy of the soluble fms-like tyrosine kinase 1 to PlGF ratio alone. CONCLUSIONS: We have provided evidence that soluble endoglin is present and elevated in the urine of women who develop preterm PE. Urinary soluble endoglin has only limited ability to determine the severity of PE and to distinguish between PE and chronic hypertension both preterm and at term. Compared with urinary soluble endoglin, the soluble fms-like tyrosine kinase 1 to PlGF ratio remains a better marker of disease presence, severity and outcome.

Development of a metabonomic approach based on LC-ESI-HRMS measurements for profiling of metabolic changes induced by recombinant equine growth hormone in horse urine.

Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter, it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH-treated horses by liquid chromatography-electrospray-high-resolution mass spectrometry (LC-ESI-HRMS) is described and assessed in this paper in order to develop a new screening tool for growth hormone abuse in horseracing. The strategy involves a limited sample preparation of the urine samples and the use of appropriate software for data processing and analysis. As preliminary work, reproducibility of both sample preparation and mass spectrometry (MS) measurements was evaluated in order to demonstrate the reliability of the method. Application of the developed protocol on two horses demonstrated the suitability of the developed strategy and preliminary results showed significant modifications of the metabolome after treatment with reGH.

Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.

BACKGROUND: Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor. METHODS: A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection. RESULTS: The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%. CONCLUSIONS: Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.

[Possible reasons for the presence of nandrolone metabolites in urine]. / Mozliwe przyczyny obecnosci metabolitów nandrolonu w moczu.

The report presents the case of a sportswoman who accused her coach of having administered to her doping substances. During the judicial proceedings, biological samples of urine and hair were collected from this sportswoman. In the urine sample, a nandrolone metabolite was detected, but the result of hair analysis was negative. The paper presents possible reasons for the presence of 19-norandrosterone in urine, as well as the difficulties associated with interpretation of 19-norandrosterone detection during the doping control.

Effects of growth hormone and insulin-like growth factor 1 on progesterone production in human luteinized granulosa cells.

OBJECTIVE: To investigate the relationship between growth hormone (GH) and luteal function. DESIGN: In vivo comparative and in vitro culture studies. SETTING: University hospital. PATIENT(S): Eighteen women who were interested in becoming pregnant and visited our clinic voluntarily participated in the study for urinary GH measurement. Ten women undergoing in vitro fertilization and embryo transfer (IVF-ET) were enrolled in in vitro culture studies. INTERVENTION(S): Ten women received IVF-ET. MAIN OUTCOME MEASURE(S): The GH concentrations in the urine collected for 24 hours, and serum progesterone and estradiol concentrations were measured during the midluteal phase. Effects of GH and insulin-like growth factor 1 (IGF-1) on progesterone and estradiol production were examined in the presence or absence of human chorionic gonadotropin (hCG) in human luteinized granulosa cells. RESULT(S): Urinary GH concentrations were statistically significantly correlated with serum progesterone and estradiol concentrations. Progesterone production by luteinized granulosa cells was increased by IGF-1 (1, 10, 100 ng/mL) but not by GH (100 ng/mL) in a dose-dependent manner. Progesterone production stimulated by IGF-1 (100 ng/mL) was further increased in the presence of hCG (1 IU/mL). Estradiol production was increased by IGF-1 and GH. CONCLUSION(S): Growth hormone may influence luteal function directly or indirectly via IGF-1.

[Correlation between growth hormone/insulin-like growth factor-1 resistance and insulin resistance in non catch-up growth rats born small for gestational age].

OBJECTIVE: To investigate the post-receptor signaling mechanism responsible for insulin resistance-induced growth hormone (GH) resistance in non-catch-up (NCU) growth rats born small for gestational age (SGA). METHODS: Twenty pregnant female SD rats were fed with restricted food (40% of normal intake, 9 g/d) throughout the pregnancy so as to develop NCU-SGA rats. The rats with their length and body weight < or = -2SD were out into the NCU-SGA group, and those with their length and body weight > -2SD were out into the catch-up (CU) growth group. Rats born to normally-fed pregnant rats were set as normal control (control Group, C Group, n = 17). The body weight and length were measured every 2 weeks. At the age of 4 weeks, 24 h urine was collected to measure the urine GH (U-GH). Then blood samples were collected to measure the serum insulin-like growth factor-1 (IGF-1), fasting insulin (FINS), and glucose levels, and the liver was taken out to detect the expression of STAT5 signal. Twelve 3-week NCU-SGA rats were divided into 2 equal groups: P13K blocking group, undergoing intraperitoneal injection of LY294002, blocker of P13K twice every 3 days, and solvent control group, undergoing intraperitoneal injection of DMSO. At the age of 4 weeks, blood samples were collected and then the liver was taken out to detect the IGF-1 mRNA and STAT5 signal. RESULTS: (1) The body weight and length at birth of the NCU-SGA group were (4.4 +/- 0.5) g and (4.5 +/- 0.2) cm, both significantly lower than those of Group C [(6.8 +/- 0.6) g and (5.3 +/- 0.2) cm respectively], and the body weight and length at 4 weeks of age of the NCU-SGA group were (63 +/- 12) g and (13.2 +/- 1.0) cm respectively, both significantly lower than those of the C group [(88 +/- 12) g and (15.3 +/- 0.5) cm respectively, all P < 0.01]. The serum IGF-1 level, IGF-1 mRNA expression, and total and phosphate STAT5 level in liver of the NCU-SGA group were (248 +/- 58) ng/ml, (6.1 +/- 0.3) copies, and (61 +/- 22)% respectively, all significantly lower than those of the C group [(383 +/- 62) ng/ml, (6.6 +/- 0.4) copies, and (91 +/- 29)%, all P < 0.01]. There was no statistic difference in 24 h U-GH between the NCU-SGA and C groups (P > 0.05). The FINS and glucose level of the NCU-SGA group were (24.7 +/- 9.6) mU/ml and (5.4 +/- 0.3) mmol/L respectively, both significantly higher than those of the C group [(9.8 +/- 2.8) mU/ml and (4.5 +/- 1.7) mmol/L respectively, both P < 0.05]. The level of 24 h U-GH was positively correlated with FINS (r = 0.680, P = 0.000). No correlation was found between IGF-1 and fasting insulin level. (2) After the PI3K pathway was chronically blocked, the NCU-SGA rats lost weight and developed a more severe insulin resistance, decreased serum IGF-1 level and the IGF-1 mRNA expression level of the PI3K inhibitor group were (218 +/- 60) ng/ml and (6.1 +/- 0.3) copies respectively, both significantly lower than those of the solvent control group [(286 +/- 45) ng/ml and (6.3 +/- 0.3) copies, both P < 0.05]. No statistically significant difference in total and phosphate STAT5 levels in liver between the P13K blocker and solvent groups. CONCLUSION: GH resistance is closely associated with insulin resistance in the NCU-SGA rats. GH resistance-induced failure of catch-up growth is related to the impairment of JAK2-STAT5 pathway. Insulin resistance exacerbates growth axis resistance and growth retardation in NCU-SGA rats via a non-STAT5 dependent pathway.

Growth hormone deficiency (GHD) in adult thalassaemic patients.

BACKGROUND AND OBJECTIVE: Short stature and growth hormone deficiency (GHD) are frequent occurrences in thalassaemic children, while data on the prevalence of GHD in adult patients are lacking. Therefore, we elected to study the growth hormone and insulin-like growth factor-I (GH-IGF-I) axis in a large group of adult thalassaemic subjects. DESIGN: Cross-sectional study on the prevalence of GHD in 94 adult thalassaemic patients (69 with thalassaemia major and 25 with thalassaemia intermedia, 39 men and 55 women, aged 31.5 +/- 6.8 years, on sex steroid replacement when necessary). METHODS: All patients underwent GHRH (1 microg/kg as an i.v. bolus) plus arginine (0.5 g/kg as a 30 min i.v. infusion) testing. Severe GHD was defined by GH peaks lower than 9 microg/l, whereas partial GHD was defined by GH peaks ranging from 9-16.5 microg/l. Blood samples for IGF-I, ferritin and pseudocholinesterase measurements were collected. Urinary free cortisol (UFC) levels were also assayed. RESULTS: Severe GHD was demonstrated in 21 of the 94 patients (22.3%), while 18 additional patients (19.1%) displayed partial GHD. GH peaks were positively correlated with IGF-I standard deviation score (SDS) (r = 0.22, P < 0.05), although 1 of the 21 patients with severe GHD showed normal IGF-I SDS values, and 44 of the 55 patients with normal GH reserve displayed low IGF-I SDS. A strong positive correlation (r = 0.48, P < 0.0001) between IGF-I SDS and pseudocholinesterase was identified. No correlations were found between ferritin and UFC levels on the one hand and GH peaks and IGF-I SDS on the other. CONCLUSION: Findings from this study demonstrate that GHD, either partial or severe, is not a rare occurrence in adult thalassaemic patients. GHD is associated with a higher prevalence of low serum IGF-I levels, recorded also in patients with normal GH secretion. The lack of correlation between ferritin and both GH peaks and IGF-I SDS suggests that mechanisms additional to iron overload, whose relevance cannot however be definitely ruled out, play a role in the pathophysiology of somatotrophin-somatomedin deficiency in this clinical condition. The positive correlation between IGF-I SDS on the one hand and GH peaks and pseudocholinesterase values on the other hand indicates that reduced liver protidosynthetic activity, in addition to somatotrophin secretory status, is a major determinant of the impaired IGF-I production in thalassaemia. Therefore biosynthetic GH replacement therapy in GH-deficient thalassaemic adults is worth considering.

Effects of arginine treatment on nutrition, growth and urea cycle function in seven Japanese boys with late-onset ornithine transcarbamylase deficiency.

BACKGROUND: The aim of this study was to investigate the effects of arginine on nutrition, growth and urea cycle function in boys with late-onset ornithine transcarbamylase deficiency (OTCD). Seven Japanese boys with late-onset OTCD enrolled in this study resumed arginine treatment after the cessation of this therapy for a few years. Clinical presentations such as vomiting and unconsciousness, plasma amino acids and urinary orotate excretion were followed chronologically to evaluate urea cycle function and protein synthesis with and without this therapy. In addition to height and body weight, blood levels of proteins, lipids, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein -3 (IGFBP-3) were monitored. RESULTS: The frequency of hyperammonemic attacks and urinary orotate excretion decreased significantly following the resumption of arginine treatment. Despite showing no marked change in body weight, height increased gradually. Extremely low plasma arginine increased to normal levels, while plasma glutamine and alanine levels decreased considerably. Except for a slight increase in high-density lipoprotein cholesterol level, blood levels of markers for nutrition did not change. In contrast, low serum IGF-I and IGFBP-3 levels increased to age-matched control levels, and normal urinary GH secretion became greater than the level observed in the controls. CONCLUSION: Arginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.

Growth impairment and adrenal suppression on low-dose inhaled beclomethasone.

Growth impairment and adrenal suppression secondary to inhaled corticosteroids (ICS) is a well-recognised phenomenon. We report a 13-year-old boy, treated long-term for asthma, who presented with short stature while on low-dose inhaled corticosteroid (beclomethasone 200 microg/d). Investigations revealed evidence of severe adrenal suppression. Weaning off the steroid treatment resulted in recovery of adrenal activity and rapid growth. While low-dose ICS are normally considered to have few side effects, this case illustrates the extreme variability of individual sensitivity and the need for careful surveillance of all children treated with long-term steroids.

Investigating the role of the growth hormone-insulin-like growth factor (GH-IGF) axis as a determinant of male bone mineral density (BMD).

INTRODUCTION: The GH-IGF axis has profound effects on bone metabolism and may be important in the etiology of idiopathic osteoporosis. Serum IGF-I is often low in men with osteoporosis, which may be attributable to GH hypo-secretion or hepatic GH insensitivity. We studied the GH-IGF axis in depth to look for evidence to support these hypotheses. MATERIALS AND METHODS: 28 healthy 60- to 70-year-old men with low, intermediate, or normal BMD were studied. GH secretion was measured by overnight urine collection. GH reserve was assessed by exercise and glucagon stimulation tests. Hepatic IGF-I production was investigated using a GH-IGF-I generation test. Data were analyzed using Pearson's correlation coefficient, linear regression, and analysis of variance. RESULTS: Serum IGF-I was reduced in subjects with low BMD (P = 0.009). There was no difference in GH secretion or reserve between the groups. Overall, GH reserve and IGF-I were positively related but this was attenuated in the low BMD group. However, no statistically significant difference in IGF-I generation capacity between BMD groups was found. CONCLUSIONS: Men with reduced BMD have low IGF-I but normal GH secretion and reserve. Our data suggested, but could not confirm, hepatic resistance to GH as a mechanism for this association.

Growth hormone abuse: methods of detection.

In the past two decades, growth hormone (GH) has been considered as a performance-enhancing drug in the sport world, certainly favoured by the awareness that there is not yet an approved method for detecting its abuse. Because resting or random measurements of plasma GH concentrations per se are meaningless, new methods have been devised to evaluate plasma levels of GH-sensitive substances that are more stable, and hence detectable, than the hormone itself. This review discusses some of the most recently proposed approaches, including a diagnostic algorithm, based on the timed application of different tests, which, collectively, would have a high diagnostic capability.

Laboratory measurement of growth hormone.

Growth hormone (GH) measurements are complicated by the heterogeneous nature of GH, as well as by the presence of the GH binding protein in plasma. Several isoforms of GH exist, and specific assays for each are currently either unavailable, impractical, or not clinically indicated. Bioassays include the in vivo assays based on rat weight gain, tibial line widening, or IGF-I generation. In vitro bioassays, based on the proliferation of cell lines expressing the prolactin receptor or GH receptor, are sensitive but prone to nonspecific interference by factors present in serum. Immunoassays (RIA, IRMA, ELISA, and immunofunctional assay design) are widely used in the clinical laboratory because of speed, sensitivity, and convenience. Discrepancies among results rendered by different immunoassays have become more apparent as monoclonal assays have superseded polyclonal assays, presumably because different antibodies recognize different epitopes among the heterogeneous mixture of GH isoforms in serum. Some assays, especially those with short, nonequilibrium incubation times are vulnerable to interference by the GH binding protein present in serum. Recommendations are given for strategies designed to minimize disparity of results obtained by different GH immunoassays applied to serum. Urinary GH measurements, while technically feasible, are of limited clinical utility because of biological variation in urinary GH excretion.

Renal tubule-specific expression and urinary secretion of human growth hormone: a kidney-based transgenic bioreactor growth.

Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.

Hormonal changes during 17 days of head-down bed-rest.

We investigated in six men the impact of 17 days of head-down bed rest (HDBR) on the daily rhythms of the hormones involved in hydroelectrolytic regulation. This HDBR study was designed to mimic a real space flight. Urine samples were collected at each voiding before, during and after HDBR. Urinary excretion of Growth Hormone (GH), Cortisol, 6 Sulfatoxymelatonin, Normetadrenaline (NMN) and Metadrenaline (NM) was determined. A decrease in urinary cortisol excretion during the night of HDBR was noted. For GH, a rhythm was found before and during HDBR. The rhythm of melatonin, evaluated with the urine excretion of 6 Sulfatoxymelatonin (aMT6S), the main hepatic metabolite, persisted throughout the experiment without any modification to the level of phase. A decrease during the night was noted for normetadrenaline urinary derivates, but only during the HDBR.

Insulin sensitivity and lipolysis in adolescent girls with poorly controlled type 1 diabetes: effect of anticholinergic treatment.

OBJECTIVES: Increased GH secretion could be one factor behind the impaired glycaemic control often seen in adolescent girls with type 1 diabetes. Because GH induces insulin resistance, treatment with anticholinergic agents, such as pirenzepine (PZP), has been used to reduce GH secretion. However, in a previous study of adolescent girls with type 1 diabetes, we observed an improvement in glycaemic control during 12 weeks of PZP therapy despite unchanged excretion of GH in urine. Considering the complex mechanisms behind urinary GH excretion, the effects of PZP on pituitary GH secretion or secretory pattern cannot be excluded. Thus, to assess the effect of anticholinergic treatment on metabolic control in adolescent girls with diabetes, we have investigated GH secretion, insulin sensitivity and lipolysis before and during treatment with PZP. PATIENTS: Eleven adolescent girls with type 1 diabetes and poor metabolic control were investigated before and after treatment with PZP, 100 mg orally, twice a day for 3 weeks. DESIGN: Serum samples for analysis of haemoglobin A1c and IGF-I were obtained in addition to serum profiles of GH, insulin and IGFBP-1 before and after 3 weeks of PZP treatment. Effects on insulin sensitivity and lipolysis were also assessed. MEASUREMENTS: IGFBP-1 was measured every hour, whereas serum GH and insulin were measured every 20 min for 24 h. Insulin sensitivity was analysed with the hyperinsulinaemic euglycaemic clamp technique. The rate of lipolysis was assessed under basal conditions following a constant rate infusion of [1,1,2,3,3-2H5]-glycerol. In five girls, lipolysis was also estimated during the hyperinsulinaemic euglycaemic clamp. RESULTS: There was a significant reduction in haemoglobin A1c levels (9.9 +/- 0.2%vs. 9.1 +/- 0.2; P < 0.0001) during 3 weeks of PZP treatment. In additional, the glucose requirement during the euglycaemic hyperinsulinaemic clamp increased by more than 30% (72.5 +/- 4.9 vs. 96.8 +/- 8.5 mg/m2/min; P = 0.003). However, we could not demonstrate any significant changes in GH secretion (area under the curve, basal levels or peak amplitude) or in the GH secretory pattern (peak height, peak length or interpeak interval). Concordantly, the IGF-I levels were statistically unchanged, as were IGFBP-1 concentrations. The rate of lipolysis did not change under basal conditions (3.40 +/- 0.53 vs. 3.04 +/- 0.54 micro mol/kg/min, n = 11, P = 0.54) or during the hyperinsulinaemic euglycaemic clamp (1.58 +/- 0.21 vs. 2.08 +/- 0.26 micro mol/kg/min; n = 5, P = 0.32). CONCLUSIONS: Our observations of an increased glucose requirement during the clamp as well as a decrease in haemoglobin A1c demonstrate improved insulin sensitivity in the adolescent girls with diabetes following pirenzepine therapy. The mechanism behind the improvement is not clear, as neither secretion nor the secretory pattern of GH changed significantly. The persistently high levels of GH might explain the unaltered rate of lipolysis despite the improved insulin sensitivity. The observed improvement in glycaemic control in adolescent girls with type 1 diabetes following pirenzepine therapy is promising, although more studies on this topic are needed.

Clinical and biochemical determinants of bone metabolism and bone mass in adolescent female patients with anorexia nervosa.

Among pathologies prevalent in western societies, anorexia nervosa has increased over the last decade. Its effects on bone mass need to be defined, and prognostic factors, either clinical or biochemical, could aid clinicians in individual patient management. To determine which clinical and/or biochemical parameters could be related to bone mass status in adolescent female anorexia nervosa patients, 73 female patients were classified according to different stages of their illness and studied in terms of clinical and biochemical parameters and bone densitometric mineral content at lumbar spine. Patients (age 17.2 +/- 1.7 y, mean +/- SD) with Tanner pubertal stage 5, regular menstruation for more than 3 mo before the onset of secondary amenorrhea, and diagnosed with anorexia nervosa were consecutively studied and classified in three clinical situations: I) active phase (34 patients): undernourished and amenorrheic; II) weight recovered but still amenorrheic (20 patients); III) fully recovered (19 patients). Clinical data were recorded at the time of bone density measurement, concomitant with blood sample extraction for study of IGF-I, IGF-binding protein 3 (IGFBP-3), IGFBP-1, estradiol, sex hormone-binding globulin, dehydroepiandrosterone sulfate, prealbumin, amino-terminal propeptide of procollagen III, osteocalcin, bone alkaline phosphatase, carboxy-terminal propeptide of procollagen I, amino-terminal propeptide of procollagen I, carboxy-terminal telopeptide of collagen I, 25-OH-vitamin D, 1,25(OH)(2)-vitamin D, and parathormone. In addition, a 24-h urine collection was made for cortisol, GH, deoxypyridinoline, amino-terminal telopeptide of collagen I, and calcium and creatinine content analysis. IGF-I, estradiol, and biochemical bone formation markers were higher and IGFBP-1, sex hormone-binding globulin, and biochemical bone resorption markers were lower in the weight-recovered stages (stages II and III) compared with the active phase (stage I). Bone formation markers correlated positively with body mass index SD score and IGF-I, whereas bone resorption markers correlated negatively with body mass index SD score and estradiol. Although no statistically significant differences regarding lumbar spine bone mineral density SD score values were recorded among the three stages of the illness, the proportion of osteopenic patients was clearly lower among stage III patients. The actual bone mineral density was inversely related to the duration of amenorrhea and directly related to duration of postmenarcheal menses before amenorrhea. In addition, a subset of osteopenic patients (five of 19) in the fully clinically recovered group with accelerated bone turnover was identified. Normal circulating estrogen level exposure time predicts actual bone mineral density at lumbar spine in young adolescent anorexia nervosa patients. In addition to psychiatric and nutritional interventions, estrogen-deprivation periods must be shortened to less than 20 mo. Patients remaining osteopenic at full clinical recovery require additional follow-up studies.