Search

Aiming at advancing protocols for safer, environmentally sensible peptide synthesis we report our findings with regard to the occurrence of hydrogen cyanide (HCN, prussic acid) in amide bond-forming reactions mediated by diisopropylcarbodiimide (DIC) and ethyl (hydroxyimino)cyanoacetate (Oxyma). We determined that HCN is always formed in amide bond-forming reactions on solid support in N,N-dimethylformamide (DMF) when DIC/Oxyma is employed. In an attempt to minimize the formation of prussic acid by means of preventing the linear DIC/Oxyma adduct 2 from cyclizing to oxadiazole 3 and in turnreleasing HCN, we evaluated a series of greener solvents such as N-butylpyrrolidinone (NBP), NBP/ethyl acetate (EtOAc, 1:1), methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate (PolarClean, PC), and PC/EtOAc (1:1). We found that the ratio between 2 and 3 greatly depends on the solvent used, and consequently, we further examined DMF, NBP, NBP/EtOAc (1:1), and NBP/EtOAc (1:4) as solvents for DIC/Oxyma-mediated amidations on solid support and in solution. We found that using carboxylic acid/Oxyma/DIC in a 1:1:1 ratio the rate of HCN formation decreases in the following order DMF > NBP > NBP/EtOAc (1:1) > NBP/EtOAc (1:4), while the reaction rate increases in the order of DMF ≈ NBP < NBP/EtOAc (1:1) < NBP/EtOAc (1:4). Of thesolvents examined, the NBP/EtOAc (1:4) mixture gave the lowest rate of HCN formation and the highest rate of amide bond formation both in solution and on solid support. As altering the solvent for DIC/Oxyma-mediated amidations resulted in suppressing HCN rather than its full elimination we evaluated the concept of in situ scavenging of the HCN formed. We performed DIC/Oxyma-mediated amidation of Fmoc-Gly-OH + (S)-(−)-1-phenylethylamine in deuterated DMF with 0, 5, and 10 equiv of dimethyl trisulfide (DMTS) as HCN scavenger. The formation of HCN and rate of amidation was monitored by 1H NMR, revealing that DMTS scavenges HCN without inhibiting the rate of amidation. DIC/Oxyma-mediated amidations of Fmoc-Ser(t-Bu)−OHwith (S)-(−)-1-phenylethylamine in DMF and NBP/EtOAc (1:4) with and without 10 equiv of DMTS were performed and found to be comparable.

Aiming at greener synthesis of complex peptides we report that conventional one-dimensional (1D) green solid-phase peptide synthesis (SPPS) is elevated to a two-dimensional (2D) concept in which side chains in peptide resins are functionalized by suitable green tactics. Specifically, we disclose on-resin deblocking using trifluoroacetic acid (TFA)/triisopropylsilane (TIS) in EtOAc/MeCN used in a synthesis of a melanocortin receptor agonist comprising (i) 1D green SPPS (ii) 2D green SPPS by an on-resin TFA/TIS in EtOAc/MeCN deprotection of Lys(Mtt) and Asp(O-2-PhiPr) followed by a lactamization using PyBOP/DIEA in NBP/EtOAc (iii) TFA cleavage followed by green precipitation using 4-methyltetrahydropyran (MTHP)/n-heptane. A further application for our green deprotection protocol was found in peptide fragment cleavages off CTC resins.

Aiming at advancing protocols for safer, environmentally sensible peptide synthesis we report our findings with regards to the occurrence of hydrogen cyanide (HCN, prussic acid) in amide bond forming reactions mediated by diisopropylcarbodiimide (DIC) and ethyl (hydroxyimino)cyanoacetate (Oxyma). We have determined that HCN is always formed in amide bond forming reactions on solid support in N,N-dimethylformamide (DMF) when employing DIC/Oxyma. In an attempt to minimize the formation of prussic acid by means of preventing the linear DIC/Oxyma adduct 2 from cyclizing to oxadiazole 3 and in turn releasing HCN, we evaluated a series of greener solvents such as N-butylpyrrolidinone (NBP), NBP/ethyl acetate (EtOAc, 1:1), methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate (PolarClean, PC), and PC/EtOAc (1:1). We found that the ratio between 2 and 3 greatly depends on the solvent used and consequently, we further examined DMF, NBP, NBP/EtOAc (1:1) and NBP/EtOAc (1:4) as solvents for DIC/Oxyma mediated amidations on solid support and in solution. We found that using carboxylic acid/Oxyma/DIC in a 1:1:1 ratio the rate of HCN formation decreases in the following order DMF>NBP>NBP/EtOAc (1:1)>NBP/EtOAc (1:4) while the reaction rate increases in order of DMF~NBPin situ scavenging of the HCN formed. We carried out DIC/Oxyma mediated amidation of Fmoc-Gly-OH + (S)-(-)-1-phenylethylamine in DMF-d7 with 0, 5 and 10 equiv of dimethyl trisulfide (DMTS) as HCN scavenger. The formation of HCN and rate of amidation was monitored by 1H NMR, revealing that DMTS scavenges HCN without inhibiting the rate of amidation. DIC/Oxyma mediated amidations of Fmoc‑Ser(t‑Bu)‑OH with (S)-(‑)-1-phenylethylamine in DMF and NBP/EtOAc (1:4) with and without 10 equiv of DMTS were carried out and found to be comparable.

Introduction The female sexual response is complex and influenced by several biological, psychological, and social factors. Testosterone is believed to modulate a woman's sexual response and desire, because low levels are considered a risk factor for impaired sexual function, but previous studies have been inconclusive. Aim To investigate how androgen levels and psychosocial factors are associated with female sexual dysfunction (FSD), including hypoactive sexual desire disorder (HSDD). Methods The cross-sectional study included 428 premenopausal women 19 to 58 years old who completed a questionnaire on psychosocial factors and had blood sampled at days 6 to 10 in their menstrual cycle. Logistic regression models were built to test the association among hormone levels, psychosocial factors, and sexual end points. Main Outcome Measures Five different sexual end points were measured using the Female Sexual Function Index and the Female Sexual Distress Scale: impaired sexual function, sexual distress, FSD, low sexual desire, and HSDD. Serum levels of total and free testosterone, androstenedione, dehydroepiandrosterone sulfate, and androsterone glucuronide were analyzed using mass spectrometry. Results After adjusting for psychosocial factors, women with low sexual desire had significantly lower mean levels of free testosterone and androstenedione compared with women without low sexual desire. None of the androgens were associated with FSD in general or with HSDD in particular. Relationship duration longer than 2 years and mild depressive symptoms increased the risk of having all the sexual end points, including FSD in general and HSDD in particular in multivariate analyses. Conclusion In this large cross-sectional study, low sexual desire was significantly associated with levels of free testosterone and androstenedione, but FSD in general and HSDD in particular were not associated with androgen levels. Length of relationship and depression were associated with FSD including HSDD.

Objective:As previous research has suggested that exposure to vitamin D insufficiency in utero may have relevance for the risk of multiple sclerosis (MS), we aimed to examine the direct association between level of neonatal vitamin D and risk of MS.Methods:We carried out a matched case-control study. Dried blood spots samples (DBSS) belonging to 521 patients with MS were identified in the Danish Newborn Screening Biobank. For every patient with MS, 1–2 controls with the same sex and birth date were retrieved from the Biobank (n = 972). Level of 25-hydroxyvitamin D (25[OH]D) in the DBSS was measured using liquid chromatography tandem mass spectroscopy. The association between different levels of 25(OH)D and risk of MS was evaluated by odds ratios (OR) calculated in conditional logistic regression models.Results:We observed that lower levels of 25(OH)D in neonates were associated with an increased risk of MS. In the analysis by quintiles, MS risk was highest among individuals in the bottom quintile (Conclusion:Low concentrations of neonatal vitamin D are associated with an increased risk of MS. In light of the high prevalence of vitamin D insufficiency among pregnant women, our observation may have importance for public health.

Aims/hypothesis: The aim of this work was to assess whether neonatal levels of 25-hydroxyvitamin D (25(OH)D) are associated with risk of developing type 1 diabetes before the age of 18 years. Methods: Two large-scale studies with different designs—a case-cohort and a case–control—were conducted using Danish national register data and biobank material. Weighted Cox regression and conditional logistic regression were used to calculate HRs and ORs, respectively. The concentration of 25(OH)D was assessed from neonatal dried blood spots using highly sensitive liquid chromatography–tandem mass spectrometry. Quintiles of 25(OH)D3were used in the main analyses. Results: The case-cohort study included 912 type 1 diabetes cases and 2866 individuals without type 1 diabetes born in Denmark between 1981 and 2002 and followed up until the end of 2012. The case–control study included 527 matched case–control pairs born between 1981 and 1999 and followed up until May 2004. Both studies found no association between 25(OH)D3levels and later risk of developing type 1 diabetes. The neonatal total 25(OH)D levels in the studies were low: 46% (case-cohort study) and 51% (case–control study) of individuals had 25(OH)D levels

BACKGROUND AND AIMS: Low levels of vitamin D are suspected to be a risk factor for cardiovascular disease and atherosclerosis. The aim of this study was to assess the prevalence of subclinical atherosclerosis among Inuit in Greenland, and to evaluate the association with vitamin D status. We hypothesized that low vitamin D status could be associated with higher carotid intima-media thickness (IMT) as a marker of atherosclerosis.METHODS: 756 adults from the Inuit Health in Transition (IHIT) study carried out in Greenland in the period 2005-2010 were included. A blood sample donated in 1987 was available for a sub-sample of 102 individuals. Serum 25(OH)D3 from the IHIT study and the 1987 survey was used as a measure of vitamin D status. IMT measurements were conducted by ultrasound scanning. The prevalence of atherosclerosis was estimated, and the association between serum 25(OH)D3 and IMT measurements was examined by linear regression.RESULTS: The overall prevalence of subclinical atherosclerosis was 20.1% (n = 152). The linear regression analyses indicated a weak positive association between serum 25(OH)D3 level and IMT measurements from the IHIT study, though not statistically significant after adjustment for potential confounders (β = 0.35% per 10 nmoL/L 25(OH)D3, p = 0.06). Linear regression analyses of the association between serum 25(OH)D3 level in the 1987 survey and IMT measurements also indicated a positive, though not statistically significant, association after adjustment (β = 0.07% per 10 nmoL/L 25(OH)D3, p = 0.86).CONCLUSIONS: Our findings did not support the hypothesis of an association between low vitamin D levels and risk of atherosclerosis.

Introduction For women, the correlation between circulating androgens and sexual desire is inconclusive. Substitution with androgens at physiological levels improves sexual function in women who experience decreased sexual desire and androgen deficiency from surgical menopause, pituitary disease, and age-related decline in androgen production in the ovaries. Measuring bioactive testosterone is difficult and new methods have been proposed, including measuring the primary androgen metabolite androsterone glucuronide (ADT-G). Aim The aim of this study was to investigate a possible correlation between serum levels of androgens and sexual desire in women and whether the level of ADT-G is better correlated than the level of circulating androgens with sexual desire. Methods This was a cross-sectional study including 560 healthy women aged 19–65 years divided into three age groups. Correlations were considered to be statistically significant at P < 0.05. Main Outcome Measure Sexual desire was determined as the total score of the sexual desire domain of the Female Sexual Function Index. Total testosterone (TT), calculated free testosterone (FT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), and ADT-G were analyzed using mass spectrometry. Results Sexual desire correlated overall with FT and androstenedione in the total cohort of women. In a subgroup of women aged 25–44 years with no use of systemic hormonal contraception, sexual desire correlated with TT, FT, androstenedione, and DHEAS. In women aged 45–65 years, androstenedione correlated with sexual desire. No correlations between ADT-G and sexual desire were identified. Conclusions In the present study, FT and androstenedione were statistically significantly correlated with sexual desire in the total cohort of women. ADT-G did not correlate more strongly than circulating androgens with sexual desire and is therefore not superior to measuring circulating androgens by mass spectrometry.

Studies on vitamin D status during pregnancy and risk of type 1 diabetes mellitus (T1D) lack consistency and are limited by small sample sizes or single measures of 25-hydroxyvitamin D (25(OH)D). We investigated whether average maternal 25(OH)D plasma concentrations during pregnancy are associated with risk of childhood T1D. In a case-cohort design, we identified 459 children with T1D and a random sample (n = 1,561) from the Danish National Birth Cohort (n = 97,127) and Norwegian Mother and Child Cohort Study (n = 113,053). Participants were born between 1996 and 2009. The primary exposure was the estimated average 25(OH)D concentration, based on serial samples from the first trimester until delivery and on umbilical cord plasma. We estimated hazard ratios using weighted Cox regression adjusting for multiple confounders. The adjusted hazard ratio for T1D per 10-nmol/L increase in the estimated average 25(OH)D concentration was 1.00 (95% confidence interval: 0.90, 1.10). Results were consistent in both cohorts, in multiple sensitivity analyses, and when we analyzed mid-pregnancy or cord blood separately. In conclusion, our large study demonstrated that normal variation in maternal or neonatal 25(OH)D is unlikely to have a clinically important effect on risk of childhood T1D. This is a pre-copyedited, author-produced version of an article accepted for publication following peer review.

A remediable cause of poor treatment response in drug-susceptible tuberculosis (TB) patients may be low plasma levels of one or more of the first-line anti-TB drugs. The aim of this work was to develop an accurate and precise LC-MS/MS method for simultaneous quantification of all four first-line anti-TB drugs in plasma suitable for therapeutic drug monitoring (TDM). To adjust for degradation and losses during sample preparation, isotopically labeled compounds were used as internal standards. Plasma samples spiked with internal standards were extracted using protein precipitation with methanol and acetonitrile. Simultaneous separation of all four drugs was accomplished with a Chromolith Reversed-Phase column and mobile phases consisting of water, methanol, ammonium acetate and formic acid with subsequent mass spectrometric quantification. The linear range of the calibration curve for isoniazid was 0.5-10 mg/L, for rifampicin 0.75-30 mg/L, for ethambutol 0.25-10 mg/L and for pyrazinamide 4-80 mg/L. The lower limit of quantification was 0.5 mg/L, 0.75 mg/L, 0.25 mg/L and 4.0 mg/L, respectively. Precision estimated by the coefficient of variation was

Objective Basic and epidemiologic studies on inflammatory bowel disease (IBD) have suggested an association between vitamin D and IBD risk. Though, the literature on IBD – especially pediatric-onset IBD – and vitamin D is still in its cradle. We therefore wanted to examine if levels of 25(OH)D at birth were associated with increased risk of developing pediatric-onset IBD. Material and methods A case-cohort study composed of cases diagnosed with Crohn’s disease, ulcerative colitis or indeterminate/unclassified colitis and healthy controls. Cases and controls were matched on date of birth and were born in the period 1981–2004. Cases were diagnosed before the age of 18 years. The concentration of 25(OH)D was assessed from neonatal dried blood spots using a highly sensitive liquid chromatography tandem mass spectrometry. Odds ratios (OR) were calculated using conditional logistic regression and two-way ANOVA were used to test for season and birth year 25(OH)D variations. A total of 384 matched pairs were included in the statistical analyses. Results No significant association were found between levels of 25(OH)D and IBD risk in the adjusted model (OR [95% CI] (per 25 nmol/L increase), 1.12 [0.88; 1.42], p = 0.35). 25(OH)D levels were found to fluctuate significantly with season (p

Little is known on how vitamin D status is affected by adherence to UVB-limiting sun exposure guidelines. Our aim was to investigate the relationship between adherence to the Danish sun exposure guidelines and vitamin D status. In total, 3194 Danes (2625 adults, 569 children) were recruited among the general population, and more than 92% had blood samples taken both autumn and spring. Using linear regression, we associated serum vitamin D concentrations to questionnaire responses on: seeking shade, wearing a sunhat, wearing protective clothing or using sunscreen. The odds ratio (OR) of either low (

OBJECTIVE: Epidemiological studies have provided evidence of an association between vitamin D insufficiency and type 2 diabetes. Vitamin D levels have decreased among Inuit in Greenland, and type 2 diabetes is increasing. We hypothesized that the decline in vitamin D could have contributed to the increase in type 2 diabetes, and therefore investigated associations between serum 25(OH)D3 as a measure of vitamin D status and glucose homeostasis and glucose intolerance in an adult Inuit population.METHODS: 2877 Inuit (≥18 years) randomly selected for participation in the Inuit Health in Transition study were included. Fasting- and 2hour plasma glucose and insulin, C-peptide and HbA1c were measured, and associations with serum 25(OH)D3 were analysed using linear and logistic regression. A subsample of 330 individuals who also donated a blood sample in 1987, were furthermore included.RESULTS: After adjustment, increasing serum 25(OH)D3 (per 10 nmol/L) was associated with higher fasting plasma glucose (0.02 mmol/L, p = 0.004), 2hour plasma glucose (0.05 nmol/L, p = 0.002) and HbA1c (0.39%, pCONCLUSIONS: Our results did not support an association between low vitamin D levels and risk of type 2 diabetes. Instead, we found weak positive associations between vitamin D levels and fasting- and 2hour plasma glucose levels, HbA1c and impaired fasting glycaemia, and a negative association with beta-cell function, underlining the need for determination of the causal relationship.

Epidemiological studies have provided evidence of an association between vitamin D insufficiency and depression and other mood disorders, and a role for vitamin D in various brain functions has been suggested. We hypothesized that low vitamin D status during pregnancy might increase the risk of postpartum depression (PPD). The objective of the study was thus to determine whether low vitamin D status during pregnancy was associated with postpartum depression. In a case-control study nested in the Danish National Birth Cohort, we measured late pregnancy serum concentrations of 25[OH]D3 in 605 women with PPD and 875 controls. Odds ratios [OR) for PPD were calculated for six levels of 25[OH]D3. Overall, we found no association between vitamin D concentrations and risk of PPD (p = 0.08). Compared with women with vitamin D concentrations between 50 and 79 nmol/L, the adjusted odds ratios for PPD were 1.35 (95% CI: 0.64; 2.85), 0.83 (CI: 0.50; 1.39) and 1.13 (CI: 0.84; 1.51) among women with vitamin D concentrations Epidemiological studies have provided evidence of an association between vitamin D insufficiency and depression and other mood disorders, and a role for vitamin D in various brain functions has been suggested. We hypothesized that low vitamin D status during pregnancy might increase the risk of postpartum depression (PPD). The objective of the study was thus to determine whether low vitamin D status during pregnancy was associated with postpartum depression. In a case-control study nested in the Danish National Birth Cohort, we measured late pregnancy serum concentrations of 25[OH]D3 in 605 women with PPD and 875 controls. Odds ratios [OR) for PPD were calculated for six levels of 25[OH]D3. Overall, we found no association between vitamin D concentrations and risk of PPD (p = 0.08). Compared with women with vitamin D concentrations between 50 and 79 nmol/L, the adjusted odds ratios for PPD were 1.35 (95% CI: 0.64; 2.85), 0.83 (CI: 0.50; 1.39) and 1.13 (CI: 0.84; 1.51) among women with vitamin D concentrations

Objectives: Basic and epidemiological studies on rheumatic autoimmune diseases have suggested an association between vitamin D levels around time of birth and disease risk. The literature on vitamin D and juvenile idiopathic arthritis (JIA) is scarce. We hypothesized that low levels of 25-hydroxyvitamin D [25(OH)D] around time of birth would be associated with increased risk of oligo- or polyarticular JIA. Method: We conducted a case–cohort study of validated cases diagnosed with oligo- and polyarticular JIA (1993–2012) and controls matched on date of birth. Cases and controls were born in the period 1983–2010. Cases were diagnosed using international criteria. The concentration of 25(OH)D was assessed from neonatal dried blood spot (DBS) samples using high-sensitivity liquid chromatography tandem mass spectrometry (LC-MS/MS). Odds ratios (ORs) were calculated using conditional logistic regression and a two-way analysis of variance (ANOVA) was used to test for season and birth year 25(OH)D variations. A total of 300 matched pairs were included in the statistical analyses. Results: No significant association was found between levels of 25(OH)D and JIA risk in the adjusted model [OR (per 25 nmol/L increase) 1.2, 95% confidence interval (CI) 0.9–1.6, p = 0.2]. 25(OH)D levels were found to fluctuate significantly with season (p