Urinary detection of rapid-acting insulin analogs in healthy humans.

Human insulin and its synthetic analogs are considered as life-saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non-scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid-acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano-flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25-48%; matrix effect: -3-(-8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.

Detection of recombinant insulins in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry after immunoaffinity purification based on monolithic microcolumns.

This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.

Recent advances in the determination of insulins from biological fluids.

The qualitative and quantitative determination of insulin and its related substances (e. g., C-peptide) is of great importance in many different areas of analytical chemistry. In particular, due to the steadily increasing prevalence of metabolic disorders such as diabetes mellitus, an adequate control of the circulating amount of insulin is desirable. In addition, also in forensics and doping control analysis, the determination of insulin in blood, urine or other biological matrices plays a major role. However, in order to establish general reference values for insulin and C-peptide for diabetology, the comparability of measured concentrations is indispensable. This has not yet been fully implemented, although enormous progress has been made in recent years, and the search for a "gold standard" method is still ongoing. In addition to established ligand-binding assays, an increasing number of mass-spectrometric methods have been developed and employed as the to-date available systems (for example, high-resolution/high accuracy mass spectrometers) provide the sensitivity required to determine analyte concentrations in the sub-ng/mL (sub-100pmol/L) level. Meanwhile, also high-throughput measurements have been realized to meet the requirement of testing a high number of samples in a short period of time. Further developments aim at enabling the online measurement of insulin in the blood with the help of an insulin sensor and, in the following, in addition to a brief review, today's state of the art testing developments are summarized.

Polyphenol-rich curry made with mixed spices and vegetables benefits glucose homeostasis in Chinese males (Polyspice Study): a dose-response randomized controlled crossover trial.

PURPOSE: To investigate acute effects of two doses of a polyphenol-rich curry made with seven different spices and four base vegetables, eaten with white rice, on 24 h glucose response, postprandial insulinemia, triglyceridemia and 24 h urinary total polyphenol excretion (TPE). METHODS: Randomized, controlled, dose-response crossover trial in healthy, Chinese men [n = 20, mean ± standard deviation (SD) age 23.7 ± 2.30 years, BMI 23.0 ± 2.31 kg/m2] who consumed test meals matched for calories, macronutrients and total vegetables content, consisting either Dose 0 Control (D0C) or Dose 1 Curry (D1C) or Dose 2 Curry (D2C) meal. 24 h glucose concentration was measured using continuous glucose monitoring (CGM), together with postprandial plasma insulin and triglyceride for up to 7 h. Total polyphenol content (TPC) of test meals and urinary TPE were measured using the Folin-Ciocalteu assay. RESULTS: TPC for D0C, D1C and D2C were 130 ± 18, 556 ± 19.7 and 1113 ± 211.6 mg gallic acid equivalent (GAE) per portion served, respectively (p < 0.0001). Compared with D0C meal, we found significant linear dose-response reductions in the 3-h postprandial incremental AUC (iAUC) for CGM glucose of 19% and 32% during D1C and D2C meals respectively (p < 0.05) and non-significant linear dose response reductions in iAUC of insulin (p = 0.089). Notably, we found significant dose-dependent increases in postprandial triglyceride with increasing curry doses (p < 0.01). Significant increases in TPE with increasing curry doses were also observed (p < 0.01). CONCLUSION: Polyphenol-rich curry intake can improve postprandial glucose homeostasis. The longer term effects remain to be established.

Impaired metabolic profile is a predictor of capillary rarefaction in a population of hypertensive and normotensive individuals.

Capillary rarefaction is typically encountered in essential hypertension, yet identification of factors interfering with this phenomenon remains substantially underinvestigated. We examined whether components of metabolic profile (dyslipidemia, insulin resistance), inflammatory (high-sensitivity C-reactive protein, high-sensitivity C-reactive protein), and angiogenic (vascular endothelial growth factor) factors are implicated in this phenomenon in a population of newly diagnosed, never-treated hypertensive patients and normotensive controls. Nailfold capillary density was estimated with nailfold capillaroscopy using specifically designed software. A total of 159 individuals, 93 hypertensives, and 66 normotensives were included. Nailfold capillary density was lower among hypertensives compared to normotensives (146.4 ± 31.0 vs. 155.4 ± 26.9, respectively; P = .047). In the total population, capillary density significantly correlated with high-density lipoprotein (HDL) (r = 0.232; P = .003), HDL/low-density lipoprotein ratio (r = 0.175; P = .025), age (r = 0.236; P = .003), but neither with vascular endothelial growth factor or high-sensitivity C-reactive protein. An inverse association was found with body mass index (r = -0.174; P = .029), insulin levels (r = -0.200; P = .018), and homeostasis model assessment-insulin resistance (r = -0.223; P = .009). In the separate analysis for the hypertensive population, sex (P = .014) and homeostasis model assessment-insulin resistance (P = .011) were identified as significant predictors of capillary rarefaction after adjustment for other factors. On the contrary, only HDL levels (P = .036) predicted capillary density in the multiple regression model for the normotensive population. Different aspects of impaired metabolic profile, that is, insulin resistance and low HDL levels, but not angiogenic or inflammatory markers, appear to be independently associated with capillary rarefaction in hypertensive and normotensive individuals.

Elevated glucose levels in early puerperium, and association with high cortisol levels during parturition.

Background Gestational diabetes is one of the commonest metabolic problems associated with pregnancy and an accurate diagnosis is critical for the care. Research has shown that pregnant women have high levels of cortisol during the last stage of parturition. As cortisol is a diabetogenic hormone causing increased glucose levels, we wanted to study the association between cortisol and glucose levels during parturition. Materials and methods Glucose and cortisol were analyzed during parturition in 50 females divided according to slow (n = 11) and normal labors (n = 39). Blood samples were analyzed three times during the parturition and four times in the first day after delivery. Glucose levels were also measured once in each trimester. Results In the normal group, the glucose concentration increased from 6.2 (IQR 5.6-8.0) mmol/L in the latency phase to 11.6 (10.0-13.3) mmol/L at aftercare (p < 0.05). After parturition the glucose concentrations decreased gradually. There were significant Spearman rank correlations between glucose and cortisol values. Conclusions The changes associated with birth cause significant elevations of cortisol and glucose around parturition.

Early pregnancy maternal urinary metabolomic profile and later insulin resistance and fetal adiposity.

OBJECTIVE: Our objective was to determine if early pregnancy urinary metabolomic profiles could predict fetal adiposity and macrosomia. METHODS: This is a prospective study of 50 healthy women in their second pregnancy. Fasting urine samples taken during early pregnancy were analyzed using NMR spectroscopy. Maternal glucose and insulin were measured in early pregnancy and at 28 weeks and the HOMA index for insulin resistance calculated. At 34 weeks ultrasound assessed fetal anthropometry including fetal anterior abdominal wall width (AAW). At delivery birth weight was recorded. Probabilistic principal component with covariates analysis (PPCCA), a novel extension of principal component analysis, which facilitates joint modeling of metabolomic data and additional covariate information, was employed to analyze the data. RESULTS: This analysis revealed that maternal HOMA and AAW significantly covaried with the (1)H NMR derived metabolomic profile of the urine. As such, in this cohort of healthy, non-diabetic women, early pregnancy urinary metabolomic profile differed significantly according to both maternal insulin resistance and fetal fat deposition in utero. CONCLUSION: These findings hold potential for early pregnancy identification of those at risk of fetal macrosomia.

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.

Geração e caracterização de linhagens de células-tronco mesenquimais de camundongo geneticamente modificadas para expressão / Generation and characterization of mousemesenchymal stem cells lines genetically modified for the ectopic expression of hG-CSFand hIGF-1ectópica de hIGF-1 ou hG-CSF

As células-tronco mesenquimais (CTM) constituem uma ferramenta promissora para o campo de terapia celular. Além de seu potencial de diferenciação em diferentes tipos celulares, as CTM apresentam a habilidade de secretar moléculas bioativas e, assim, exercer múltiplos efeitos biológicos, tais como indução da regeneração de tecidos lesionados, redução de fibrose e modulação do sistema imune. A superexpressão dos fatores de crescimento G-CSF e IGF-1, conhecidos por seus efeitos sobre os processos de imunomodulação, sobrevivência celular e reparo tecidual, pode ampliar as ações terapêuticas das CTM. O objetivo deste trabalho consiste em gerar e caracterizar linhagens de CTM de camundongo superexpressando hGCSF ou hIGF-1. Um sistema lentiviral de segunda geração foi utilizado para modificação de CTM para expressão ectópica dos genes de interesse. As sequências codificantes de hG-CSF e hIGF-1 foram amplificadas por PCR e subclonadas em um vetor lentiviral de transferência, contendo um promotor constitutivo. As partículas lentivirais foram produzidas a partir da cotransfecção de células da linhagem HEK293FT...

Mesenchymal stem cells (MSCs) are a promising tool for the cell therapy field. In addition to their potential for differentiation into different cell types, MSCs have the ability to secrete bioactive molecules and thus exert multiple biological effects such as induction of the injured tissue regeneration, fibrosis reduction and modulation of the immune system. The overexpression of the growth factors G-CSF and IGF-1, known for their effects on immune modulation processes, cell survival and tissue repair, can result in a magnification of MSCs' therapeutic actions. The objective of this work is to generate and characterize mouse MSCs lines overexpressing hG-CSF or hIGF-1. A second generation lentiviral system was used to modify MSCs derived from mice for the ectopic expression of the genes of interest. The coding sequences of hG-CSF and hIGF-1 were amplified by PCR and subcloned into a lentiviral transfer vector containing a constitutive promoter. The lentiviral particles were produced from the co-transfection of HEK293FT...

Association of infertile patients having polycystic ovarian syndrome with recurrent miscarriage.

Polycystic ovarian syndrome (PCOS) has a pivotal role in the development of various complications during pregnancy. Polycystic ovarian syndrome women having elevated LH and hyper insulineuia may be at increased risk of miscarriage. The study was done to find out the recurrent pregnancy loss among the PCOS patient. This was a cross sectional case control study in total 100 infertile patients between age 20-40 years attending BSMMU out patient Department from July 2011 to June 2012, among them 50 infertile patients with PCOS regarding as a case and 50 infertile patients without PCOS selected as a control. Regarding case (infertile patients with PCOS) shows 20(40%) recurrent miscarriage and among control (infertile patients without PCOS) shows recurrent miscarriage 6(12%). And also among case group shows insulin resistance 8(16%) and control group insulin resistance 1(2%). Six (75%) abortion occur among PCOS with insulin resistance and 5(62.5%) abortion occur among PCOS with raised testosterone level. It is observed that recurrent miscarriage is higher in PCOS group. And also concluded that insulin resistance and raised testosterone level is responsible for this condition. So, further large scale study would be needed to reduce the chance of recurrent pregnancy loss by treatment with insulin sensitizer in case of obese PCOS with insulin resistance patient.

Sensitive detection of human insulin using a designed combined pore approach.

A unique combined pore approach to the sensitive detection of human insulin is developed. Through a systematic study to understand the impact of pore size and surface chemistry of nanoporous materials on their enrichment and purification performance, the advantages of selected porous materials are integrated to enhance detection sensitivity in a unified two-step process. In the first purification step, a rationally designed large pore material (ca. 100 nm in diameter) is chosen to repel the interferences from nontarget molecules. In the second enrichment step, a hydrophobically modified mesoporous material with a pore size of 5 nm is selected to enrich insulin molecules. A low detection limit of 0.05 ng mL(-1) in artificial urine is achieved by this advanced approach, similar to most antibody-based analysis protocols. This designer approach is efficient and low cost, and thus has great potential in the sensitive detection of biomolecules in complex biological systems.

Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part II: Polymer permeation-ion exchange separation adsorbents with polyethylene glycol and strong anion exchange groups.

In chromatographic separations, the most general problem in small biomolecule isolation and purification is that such biomolecules are usually found in extremely low concentrations together with high concentrations of large molecular weight proteins. In the first part of this work, adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide Immobilized Metal Affinity Chromatography (IMAC) matrix was synthesized and used to develop chromatographic adsorbents that preferentially adsorb and separate low molecular weight biomolecules while rejecting large molecular weight proteins. In this second part, we expand the concept of controlled access polymer permeation adsorption (CAPPA) media by grafting polyethylene glycol (PEG) on a high capacity polysaccharide ion exchange (IEX) chromatographic resin where PEG acts as a semi-permeable barrier that preferentially allows the permeation of small molecules while rejecting large ones. The IEX resin bearing quaternary ammonium groups binds permeated biomolecules according to their ion exchange affinity while excluding large biomolecules by the PEG barrier and thus cannot compete for the binding sites. This new AdSEC media was used to study the retention of peptides and proteins covering a wide range of molecular weights from 1 to 150 kDa. The effect of protein molecular weight towards retention by ion exchange was performed using pure protein solutions. Recovery of insulin from insulin-spiked human serum and insulin-spiked human urine was evaluated under polymer controlled permeation conditions. The CAPPA media consisted of agarose beads modified with amino-PEG-methoxy and with trimethyl ammonium groups, having chloride capacities between 20 and 40 µeq/mL and were effective in rejecting high molecular weight proteins while allowing the preferential adsorption of small proteins and peptides.

Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry.

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.

Effect of physical fitness and endurance exercise on indirect biomarkers of growth hormone and insulin misuse: Immunoassay-based measurement in urine samples.

Indirect biomarkers of recombinant human growth hormone (rhGH), insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBP-2 and IGFBP-3) and insulin (C-peptide) were measured together with urinary parameters of renal damage (beta(2)-microglobulin and proteinuria) by immunoassays, in house validated for the purpose, in 61 subjects (36 elite athletes, 18 recreational athletes and 7 sedentary individuals) with different levels of physical fitness and endurance exercise. Validation parameters were good for the evaluated assays, excluding a high inter-assay imprecision and inaccuracy of 24 and 26% obtained for GH assay. The range of concentrations found in urine samples under investigation was generally covered by the calibration curves of the studied immunoassays. However, for the samples below or above the calibration curve, opportune dilution or concentration were performed. Particularly, C-peptide samples had to be diluted 1:5 and beta(2)-microglobulin ones assayed using a triple sample volume, to fall within the calibration range. Urinary C-peptide was the only biomarker statistically higher in samples of elite athletes when compared to recreational athletes and sedentary individuals. Among elite athletes, tae-kwon-do athletes showed the highest IGF-II basal values while weightlifting athletes showed the lower IGF-I and IGFBP-3 basal values. The trend observed in weightlifters' basal samples was confirmed in their training samples: IGF-I, IGF-II, IGFBP-3 and beta(2)-microglobulin were lower in with respect to those from synchronised swimming. Over the training season, within athlete variability was observed for IGFBP-3 for weightlifting athletes. In the studied subjects, no direct associations were found between biomarkers of GH or insulin misuse and urinary parameters of renal damage, eventually due to high-workload endurance training. The variations observed in different biomarkers should be taken in consideration in the hypothesis of setting reference concentration ranges for doping detection.

Insulin.

Due to its versatile nature and its corresponding anabolic and anticatabolic properties, insulin has been prohibited in sports since 1999. Numerous studies concerning its impact on glycogen formation, protein biosynthesis, and inhibition of protein breakdown have illustrated its importance for healthy humans and diabetics as well as elite athletes. Various reports described the misuse of insulin to improve performance and muscle strength, and synthetic analogs were the subject of several studies describing the beneficial effects of biotechnologically modified insulins. Rapid- or long-acting insulins were developed to enhance the injection-to-onset profile as well as the controllability of administered insulin, where the slightest alterations in primary amino acid sequences allowed the inhibition of noncovalent aggregation of insulin monomers (rapid-acting analogs) or promoted microprecipitation of insulin variants upon subcutaneous application (long-acting analogs). Information on the metabolic fate and renal elimination of insulins has been rather limited, and detection assays for doping control purposes were primarily established using the intact compounds as target analytes in plasma and urine specimens. However, recent studies revealed the presence of urinary metabolites that have been implemented in confirmation methods of sports drug testing procedures. So far, no screening tool is available providing fast and reliable information on possible insulin misuse; only sophisticated procedures including immunoaffinity purification followed by liquid chromatography and tandem mass spectrometry have enabled the unambiguous detection of synthetic insulins in doping control blood or urine samples.

A novel ultra-sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement in urine from healthy subjects and patients with diabetes mellitus.

OBJECTIVE: For the early identification of patients at risk of developing diabetes mellitus, and to prevent the onset of diabetes by performing dietary counseling and exercise guidance, we have developed an ultra-sensitive immune complex transfer enzyme immunoassay (ICT-EIA) to measure soluble human insulin receptor ectodomain (sIRalpha) in urine which is collected non-invasively. DESIGN AND METHODS: We developed ICT-EIA for sIRalpha and measured urinary sIRalpha from 106 healthy volunteers, 35 obese volunteers and 42 patients with diabetes. RESULTS: The detection limit of ICT-EIA (0.04 pg/mL), using a urine sample of as little as 100 microL, was a few hundred-fold higher than that of conventional ELISA. Using ICT-EIA, the urinary sIRalpha level in patients with diabetes (9.7+/-20.1 pg/mg creatinine) was significantly higher than those in healthy volunteers (1.4+/-0.9; P<0.001). CONCLUSION: ICT-EIA for sIRalpha may be useful as a good marker for evaluating diabetes risk.

Sensitive and fast identification of urinary human, synthetic and animal insulin by means of nano-UPLC coupled with high-resolution/high-accuracy mass spectrometry.

The double-chain polypeptide insulin and its synthetic (Insulin Glulisine, Insulin Aspart, Insulin Glargine, or Insulin Lispro) or animal analogues (porcine insulin or bovine insulin) are potential performance-enhancing agents in elite sports or potentially effective toxins in forensic science.The present study demonstrates an analytical method to purify the insulins simultaneously from urine specimens with an approach based on immunoaffinity isolation, using coated magnetic beads (anti-mouse) and a primary anti-insulin antibody (IgG, monoclonal). The extracts were purified sufficiently for separation by means of nano-flow liquid chromatography coupled with nano-scale high-resolution, high-accuracy ESI-MS/MS. Elucidation of collision-induced dissociations with product ion experiments using the fivefold protonated precursor ion of each target analyte enabled all synthetic and animal insulins to be differentiated from their human counterpart, which was particularly important for Lispro, possessing the same molecular mass as human insulin.The method was fully validated for specificity, limit of detection (LOD, 0.5 fmol/mL), precision (<20%), recovery (approximately 30%) and linearity (2-40 fmol/mL) for all target analytes.

Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing.

Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown. In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains.

Mass spectrometric determination of insulins and their degradation products in sports drug testing.

Insulins' anabolic and anti-catabolic properties have supposedly led to its misuse in sport. Hence, doping control assays were developed to allow the unequivocal identification of synthetic insulin analogs and metabolic products derived from human insulin and its artificial counterparts in urine and plasma specimens. Analyses were based on immunoaffinity purification and subsequent characterization of target analytes by top-down sequencing-based approaches, which were conducted with hybrid tandem mass spectrometers that consisted of either quadrupole-linear ion trap or linear ion trap-orbitrap analyzers. Diagnostic product ions and analytical strategies are presented and discussed in light of the need to unambiguously identify misused drugs in urine and plasma specimens for doping control.

The uridine diphosphate glucuronosyltransferase 2B15 D85Y and 2B17 deletion polymorphisms predict the glucuronidation pattern of androgens and fat mass in men.

CONTEXT: Previous in vitro studies have demonstrated that the UDP glucuronosyltransferase (UGT)2B15 and UGT2B17 glucuronidate androgens and their metabolites. OBJECTIVE: Our objective was to determine in vivo whether the UGT2B15 D85Y and the UGT2B17 deletion polymorphisms predict androgen glucuronidation and body composition. PARTICIPANTS: Two population-based cohorts including young adult (n = 1068; age = 18.9 yr) and elderly (n = 1001; age = 75.3 yr) men were included in the study. MAIN OUTCOME MEASURES: Serum and urine levels of testosterone (T) and dihydrotestosterone (DHT) were measured by gas chromatography-mass spectrometry, and serum levels of the major glucuronidated androgen metabolites androstane-3alpha,17beta-diol(androstanediol)-3-glucuronide, androstanediol-17-glucuronide, and androsterone-glucuronide were measured by liquid chromatography-tandem mass spectrometry. Body composition was measured by dual-energy x-ray absorptiometry. RESULTS: Both the UGT2B15 D85Y and the UGT2B17 deletion polymorphisms were associated with serum levels of androstanediol-17-glucuronide (P < 0.001) but not with levels of androstanediol-3-glucuronide or androsterone-glucuronide in both cohorts. Glucuronidation of T and DHT was associated with the UGT2B17 deletion but not with the UGT2B15 D85Y polymorphism, suggested by strong associations between the deletion polymorphism and urine levels of these two hormones. Both polymorphisms were associated with several different measures of fat mass (P < 0.01). The UGT2B17 deletion polymorphism was associated with insulin sensitivity (P < 0.05) as indicated by the homeostasis model assessment index. CONCLUSIONS: The UGT2B15 D85Y and the UGT2B17 deletion polymorphisms are both predictors of the glucuronidation pattern of androgens/androgen metabolites. Our findings indicate that UGT2B17 is involved in 17-glucuronidation of mainly T but also of DHT and androstanediol and that UGT2B15 is involved in the 17-glucuronidation of androstanediol. Furthermore, these two polymorphisms are predictors of fat mass in men.