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Background. Urinary collagen peptides, the breakdown products of endogenous collagen, have been used as biomarkers for various diseases. These non-invasive biomarkers are easily measured via mass spectrometry, aiding in diagnostics and therapy effectiveness. Objectives. The objective of this study was to investigate the effects of consuming collagen-containing meat on collagen peptide composition in human blood and urine. Methods. Ten collagen peptides in 24 h urine were quantified. Results. Prolyl-hydroxyproline (Pro-Hyp) was the most abundant peptide. Except for hydroxyprolyl-glycine (Hyp-Gly), levels of other minor collagen peptides showed high correlation coefficients with Pro-Hyp (r = 0.42 vs. r > 0.8). Notably, 24 h urinary Hyp-Gly showed a correlation coefficient of r = 0.72 with meat consumption, significantly higher than the coefficient for Pro-Hyp (r = 0.37). Additionally, the levels of Pro-Hyp and Hyp-Gly in the blood of seven young women participants increased similarly after consuming fish meat, while before ingestion, only negligible amounts of Hyp-Gly were present. To examine which peptides are generated by the degradation of endogenous collagen, mouse skin was cultured. The amount of Pro-Hyp released from the skin was approximately 1000-fold higher than that of Hyp-Gly. Following consumption of collagen-containing meat, both Pro-Hyp and Hyp-Gly are released in blood and excreted into urine, although Pro-Hyp is primarily generated from endogenous collagen even under physiological conditions. Conclusions. Therefore, in 24 h urine samples, the non-negligible fraction of Pro-Hyp is contributed by endogenous collagen, making 24 h urine Hyp-Gly level a potential biomarker for evaluating meat consumption on the day.

A highly sensitive detection system for α-amylase was developed via host-guest complexation between γ-cyclodextrin and dansyl-modified diphenylalanine (FF-Dns). The host-guest inclusion of FF-Dns into the cavity of γ-CD in a HEPES buffer solution (10 mM, pH 7.4) significantly enhanced the fluorescence intensity, and the emission wavelength gradually shifted from 558 to 535 nm. The hydrolysis of γ-CD by the addition of α-amylase released FF-Dns, leading to the recovery of the fluorescence emission characteristics. Therefore, the FF-Dns/γ-CD host-guest complexation system can serve as a platform for the sensitive detection of α-amylase with good selectivity against potential interference. The limit of detection (LOD) of the system was 0.004 U/mL, with a linear working range of 0-6 U/mL. The detection assay was successfully applied in 0.1 % serum, achieving an LOD of 0.017 U/mL and a linear working range of 0-10 U/mL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A dipeptide-based bifunctional material immobilized with Ti 4+ (denoted as APE-MBA-VPA-Ti 4+ ) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol μL -1 for glycopeptides, 0.005 fmol μL -1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, β-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Purposes: It is unclear whether the parent Saxagliptin (SAX) in vivo is the same as that in vitro, which is twice that of 5-hydroxy Saxagliptin (5-OH SAX). This study is to construct a Pharmacokinetic-Pharmacodynamic (PK-PD) link model to evaluate the genuine relationship between the concentration of parent SAX in vivo and the effect., Methods: First, we established a reliable Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS) method and DPP-4 inhibition ratio determination method. Then, the T2DM rats were randomly divided into four groups, intravenous injection of 5-OH SAX (0.5 mg/kg) and saline group, intragastric administration of SAX (10 mg/kg) and Sodium carboxymethyl cellulose (CMC-Na) group. Plasma samples were collected at different time points for subsequent testing. Finally, we used the measured concentrations and inhibition ratios to construct a PK-PD link model for 5-OH SAX and parent SAX., Results: A two-compartment with additive model showed the pharmacokinetic process of SAX and 5-OH SAX, the concentration-effect relationship was represented by a sigmoidal E max model and sigmoidal E max with E 0 model for SAX and 5-OH SAX, respectively. Fitting parameters showed SAX was rapidly absorbed after administration (T max =0.11 h, t 1/2, ka =0.07 h), widely distributed in the body (V ≈ 20 L/kg), plasma exposure reached 3282.06 ng*h/mL, and the elimination half-life was 6.13 h. The maximum plasma dipeptidyl peptidase IV (DPP-4) inhibition ratio of parent SAX was 71.47%. According to the final fitting parameter EC 50 , EC 50, 5-OH SAX =0.46EC 50, SAX(parent) , it was believed that the inhibitory effect of 5-OH SAX was about half of the parent SAX, which is consistent with the literature., Conclusions: The PK-PD link model of the parent SAX established in this study can predict its pharmacokinetic process in T2DM rats and the strength of the inhibitory effect of DPP-4 based on non-clinical data., (© 2024. The Author(s).)

Background: Acute ischemic stroke (AIS) is one of the most common cerebrovascular diseases which accompanied by a disruption of aminothiols homeostasis. To explore the relationship of aminothiols with neurologic impairment severity, we investigated four aminothiols, homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CG) and glutathione (GSH) in plasma and its influence on ischemic stroke severity in AIS patients., Methods: A total of 150 clinical samples from AIS patients were selected for our study. The concentrations of free reduced Hcy (Hcy), own oxidized Hcy (HHcy), free reduced Cys (Cys), own oxidized Cys (cysteine, Cyss), free reduced CG (CG) and free reduced GSH (GSH) were measured by our previously developed hollow fiber centrifugal ultrafiltration (HFCF-UF) method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The concentration ratio of Hcy to HHcy (Hcy/HHcy), Cys to Cyss (Cys/Cyss) were also calculated. The neurologic impairment severity of AIS was evaluated using National Institutes of Health Stroke Scale (NIHSS). The Spearman correlation coefficient and logistic regression analysis was used to estimate and perform the correlation between Hcy, HHcy, Cys, Cyss, CG, GSH, Hcy/HHcy, Cys/Cyss and total Hcy with NIHSS score., Results: The reduced Hcy and Hcy/HHcy was both negatively correlated with NIHSS score in AIS patients with P = 0.008, r=-0.215 and P = 0.002, r=-0.249, respectively. There was no significant correlation of Cys, CG, GSH, HHcy, Cyss, Cys/Cyss and total Hcy with NIHSS score in AIS patients with P value > 0.05., Conclusions: The reduced Hcy and Hcy/HHcy, not total Hcy concentration should be used to evaluate neurologic impairment severity of AIS patient., (© 2024. The Author(s).)

The COVID-19 pandemic has incurred tremendous costs worldwide and is still threatening public health in the "new normal." The association between neutralizing antibody levels and metabolic alterations in convalescent patients with COVID-19 is still poorly understood. In the present work, we conducted absolutely quantitative profiling to compare the plasma cytokines and metabolome of ordinary convalescent patients with antibodies (CA), convalescents with rapidly faded antibodies (CO), and healthy subjects. As a result, we identified that cytokines such as M-CSF and IL-12p40 and plasma metabolites such as glycylproline (gly-pro) and long-chain acylcarnitines could be associated with antibody fading in COVID-19 convalescent patients. Following feature selection, we built machine-learning-based classification models using 17 features (six cytokines and 11 metabolites). Overall accuracies of more than 90% were attained in at least six machine-learning models. Of note, the dipeptide gly-pro, a product of enzymatic peptide cleavage catalyzed by dipeptidyl peptidase 4 (DPP4), strongly accumulated in CO individuals compared with the CA group. Furthermore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination experiments in healthy mice demonstrated that supplementation of gly-pro down-regulates SARS-CoV-2-specific receptor-binding domain antibody levels and suppresses immune responses, whereas the DPP4 inhibitor sitagliptin can counteract the inhibitory effects of gly-pro upon SARS-CoV-2 vaccination. Our findings not only reveal the important role of gly-pro in the immune responses to SARS-CoV-2 infection but also indicate a possible mechanism underlying the beneficial outcomes of treatment with DPP4 inhibitors in convalescent COVID-19 patients, shedding light on therapeutic and vaccination strategies against COVID-19.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: Aminothiols (glutathione (GSH), cysteinylglycine (CG)) may play an important role in the pathogenesis of coronavirus disease 2019 (COVID-19), but the possible association of these indicators with the severity of COVID-19 has not yet been investigated., Methods: The total content ( t ) and reduced forms ( r ) of aminothiols were determined in patients with COVID-19 ( n = 59) on admission. Lung injury was characterized by computed tomography (CT) findings in accordance with the CT0-4 classification., Results: Low tGSH level was associated with the risk of severe COVID-19 (tGSH ≤ 1.5  μ M, mild vs. moderate/severe: risk ratio (RR) = 3.09, p = 0.007) and degree of lung damage (tGSH ≤ 1.8  μ M, CT < 2 vs. CT ≥ 2: RR = 2.14, p = 0.0094). The rGSH level showed a negative association with D-dimer levels ( ρ = -0.599, p = 0.014). Low rCG level was also associated with the risk of lung damage (rCG ≤ 1.3  μ M, CT < 2 vs. CT ≥ 2: RR = 2.28, p = 0.001). Levels of rCG ( ρ = -0.339, p = 0.012) and especially tCG ( ρ = -0.551, p = 0.004) were negatively associated with platelet count. In addition, a significant relationship was found between the advanced oxidation protein product level and tGSH in patients with moderate or severe but not in patients with mild COVID-19., Conclusion: Thus, tGSH and rCG can be seen as potential markers for the risk of severe COVID-19. GSH appears to be an important factor to oxidative damage prevention as infection progresses. This suggests the potential clinical efficacy of correcting glutathione metabolism as an adjunct therapy for COVID-19., Competing Interests: The authors have declared no conflict of interest., (Copyright © 2021 Evgeny Vladimirovich Kryukov et al.)

Objective: To appraise the utility as biomarkers of blood antibodies and immune complexes to neurofilaments and dipeptide repeat proteins, the products of translation of the most common genetic mutation in amyotrophic lateral sclerosis (ALS)., Methods: Antibodies and immune complexes against neurofilament light, medium, heavy chains as well as poly-(GP)-(GR) dipeptide repeats were measured in blood samples from the ALS Biomarkers (n = 107) and the phenotype-genotype biomarker (n = 129) studies and in 140 healthy controls. Target analyte levels were studied longitudinally in 37 ALS cases. Participants were stratified according to the rate of disease progression estimated before and after baseline and C9orf72 genetic status. Survival and longitudinal analyses were undertaken with reference to matched neurofilament protein expression., Results: Compared to healthy controls, total neurofilament proteins and antibodies, neurofilament light immune complexes (p < 0.0001), and neurofilament heavy antibodies (p = 0.0061) were significantly elevated in ALS, patients with faster progressing disease (p < 0.0001) and in ALS cases with a C9orf72 mutation (p < 0.0003). Blood neurofilament light protein discriminated better ALS from healthy controls (AUC: 0.92; p < 0.0001) and faster from slower progressing ALS (AUC: 0.86; p < 0.0001) compared to heavy-chain antibodies and light-chain immune complexes (AUC: 0.79; p < 0.0001 and AUC: 0.74; p < 0.0001). Lower neurofilament heavy antibodies were associated with longer survival (Log-rank Chi-square: 7.39; p = 0.0065). Increasing levels of antibodies and immune complexes between time points were observed in faster progressing ALS., Conclusions: We report a distinctive humoral response characterized by raising antibodies against neurofilaments and dipeptide repeats in faster progressing and C9orf72 genetic mutation carriers ALS patients. We confirm the significance of plasma neurofilament proteins in the clinical stratification of ALS., (© 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Abstract: Major depressive disorder (MDD) is a common disease with both affective and cognitive disorders. Alterations in metabolic systems of MDD patients have been reported, but the underlying mechanisms still remains unclear. We sought to identify abnormal metabolites in MDD by metabolomics and to explore the association between differential metabolites and neurocognitive dysfunction.Plasma samples from 53 MDD patients and 83 sex-, gender-, BMI-matched healthy controls (HCs) were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was then used to detect metabolites in those samples. Two different algorithms were applied to identify differential metabolites in 2 groups. Of the 136 participants, 35 MDD patients and 48 HCs had completed spatial working memory test. Spearman rank correlation coefficient was applied to explore the relationship between differential metabolites and working memory in these 2 groups.The top 5 metabolites which were found in sparse partial least squares-discriminant analysis (sPLS-DA) model and random forest (RF) model were the same, and significant difference was found in 3 metabolites between MDD and HCs, namely, gamma-glutamyl leucine, leucine-enkephalin, and valeric acid. In addition, MDD patients had higher scores in spatial working memory (SWM) between errors and total errors than HCs. Valeric acid was positively correlated with working memory in MDD group.Gamma-glutamyl leucine, leucine-enkephalin, and valeric acid were preliminarily proven to be decreased in MDD patients. In addition, MDD patients performed worse in working memory than HCs. Dysfunction in working memory of MDD individuals was associated with valeric acid., Competing Interests: The authors have no conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Sugar consumption is associated with a whole range of negative health effects and should be reduced and the natural sweetener xylitol might be helpful in achieving this goal. The present study was conducted as a randomized, placebo-controlled, double-blind, cross-over trial. Twelve healthy, lean volunteers received intragastric solutions with 7, 17 or 35 g xylitol or tap water on four separate days. We examined effects on: gut hormones, glucose, insulin, glucagon, uric acid, lipid profile, as well as gastric emptying rates, appetite-related sensations and gastrointestinal symptoms. We found: (i) a dose-dependent stimulation of cholecystokinin (CCK), active glucagon-like peptide-1 (aGLP-1), peptide tyrosine tyrosine (PYY)-release, and decelerated gastric emptying rates, (ii) a dose-dependent increase in blood glucose and insulin, (iii) no effect on motilin, glucagon, or glucose-dependent insulinotropic peptide (GIP)-release, (iv) no effect on blood lipids, but a rise in uric acid, and (v) increased bowel sounds as only side effects. In conclusion, low doses of xylitol stimulate the secretion of gut hormones and induce a deceleration in gastric emptying rates. There is no effect on blood lipids and only little effect on plasma glucose and insulin. This combination of properties (low-glycemic sweetener which stimulates satiation hormone release) makes xylitol an attractive candidate for sugar replacement.

BACKGROUND The relative efficacy of carotid endarterectomy (CEA)/thromboendarterectomy (TEA) and carotid artery stenting (CAS) already has been compared in randomized controlled trials and a meta-analysis, but only limited data exist describing the status of cerebral metabolism before and after these interventions. The aim of the present study was to compare metabolic changes before and after treatment of carotid stenosis and assess their potential clinical implications.   MATERIAL AND METHODS Patients with asymptomatic unilateral critical internal CAS were imaged with proton 3T magnetic resonance spectroscopy (H-MRS) because the technique is more sensitive than regular magnetic resonance imaging for detection of the early signs of ischemic events. Abnormal metabolite ratios detected with H-MRS may precede actual morphological changes associated with hypoperfusion as well as reperfusion changes. Ipsilateral and contralateral middle cerebral artery vascular territories were both evaluated before and after vascular intervention. H-MRS was performed within 24 h before and after surgery. Correlations in the metabolic data from H-MRS for N-acetylaspartic acid (NAA)+N-acetylaspartylglutamate, creatinine (Cr)+phosphocreatinine, and phosphocholine+glycerophosphocholine (Cho) were sought. RESULTS H-MRS voxels from 11 subjects were analyzed. Values for dCho/CrI, dCho/CrC and Cho/Naal (P<0.001) were significantly higher ipsilaterally than contralaterally. Ratios for dNaa/ChoC and Cho/NaaC were significantly higher on the non-operated side (P<0.001). CONCLUSIONS H-MRS may be helpful for assessment of patients with CAS, particularly because unlike other modalities, it reveals postoperative changes in metabolic brain status. Initial results indicate the important role of perioperative neuroprotective treatment.

Objective: Gout is the most common inflammatory arthritis and the worldwide incidence is increasing. By revealing the metabolic alterations in serum and urine of gout patients, the first aim of our study was to discover novel molecular biomarkers allowing for early diagnosis. We also aimed to investigate the underlying pathogenic pathways., Methods: Serum and urine samples from gout patients (n = 30) and age-matched healthy controls (n = 30) were analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to screen the differential metabolites and construct a diagnostic model. Next, the model was verified and optimized in the second validation cohort (n = 100). The pathways were illustrated to understand the underlying pathogenesis of gout., Results: In general, serum metabolomics demonstrated a clearer distinction than urine metabolomics. In the discovery cohort, 40 differential serum metabolites were identified that could distinguish gout patients from healthy controls. Among them, eight serum metabolites were verified in the validation cohort. Through regression analysis, the final model consisted of three serum metabolites-pyroglutamic acid, 2-methylbutyryl carnitine and Phe-Phe-that presented optimal diagnostic power. The three proposed metabolites produced an area under the curve of 0.956 (95% CI 0.911, 1.000). Additionally, the proposed metabolic pathways were primarily involved in purine metabolism, branched-chain amino acids (BCAAs) metabolism, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, bile secretion and arachidonic acid metabolism., Conclusion: The metabolomics signatures could serve as an efficient tool for early diagnosis and provide novel insights into the pathogenesis of gout., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects., Results: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ -glutamyltranspeptidase ( γ -GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH., Conclusion: s. The prominent in vivo pharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ -GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage., Competing Interests: The authors declare they have not conflicts of interest., (Copyright © 2020 Daniela Giustarini et al.)

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APP Sw/Ind ) J20 and wild-type mice with VX-765 delays both APP Sw/Ind - and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aβ) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.

This study investigated metabolic, endocrine, appetite and mood responses to a maximal eating occasion in fourteen men (mean: age 28 (sd 5) years, body mass 77·2 (sd 6·6) kg and BMI 24·2 (sd 2·2) kg/m2) who completed two trials in a randomised crossover design. On each occasion, participants ate a homogenous mixed-macronutrient meal (pizza). On one occasion, they ate until 'comfortably full' (ad libitum) and on the other, until they 'could not eat another bite' (maximal). Mean energy intake was double in the maximal (13 024 (95 % CI 10 964, 15 084) kJ; 3113 (95 % CI 2620, 3605) kcal) compared with the ad libitum trial (6627 (95 % CI 5708, 7547) kJ; 1584 (95 % CI 1364, 1804) kcal). Serum insulin incremental AUC (iAUC) increased approximately 1·5-fold in the maximal compared with ad libitum trial (mean: ad libitum 43·8 (95 % CI 28·3, 59·3) nmol/l × 240 min and maximal 67·7 (95 % CI 47·0, 88·5) nmol/l × 240 min, P < 0·01), but glucose iAUC did not differ between trials (ad libitum 94·3 (95 % CI 30·3, 158·2) mmol/l × 240 min and maximal 126·5 (95 % CI 76·9, 176·0) mmol/l × 240 min, P = 0·19). TAG iAUC was approximately 1·5-fold greater in the maximal v. ad libitum trial (ad libitum 98·6 (95 % CI 69·9, 127·2) mmol/l × 240 min and maximal 146·4 (95 % CI 88·6, 204·1) mmol/l × 240 min, P < 0·01). Total glucagon-like peptide-1, glucose-dependent insulinotropic peptide and peptide tyrosine-tyrosine iAUC were greater in the maximal compared with ad libitum trial (P < 0·05). Total ghrelin concentrations decreased to a similar extent, but AUC was slightly lower in the maximal v. ad libitum trial (P = 0·02). There were marked differences on appetite and mood between trials, most notably maximal eating caused a prolonged increase in lethargy. Healthy men have the capacity to eat twice the energy content required to achieve comfortable fullness at a single meal. Postprandial glycaemia is well regulated following initial overeating, with elevated postprandial insulinaemia probably contributing.

Background: The effect of nonnutritive sweeteners on appetite is controversial. Some studies have found changes in certain appetite control hormones with sucralose intake that may be through interaction with sweet taste receptors located in the intestine., Objective: The aim of this study was to evaluate whether sucralose consumption could produce changes in fasting plasma concentrations of appetite-regulating hormones, including glucagon-like peptide 1, ghrelin, peptide tyrosine tyrosine, and leptin, and secondarily in insulin resistance., Design: A 2-week parallel randomized clinical trial with an additional visit conducted 1 week after dosing termination., Participants/setting: Sixty healthy, normal-weight individuals, without habitual consumption of nonnutritive sweeteners were recruited from July 2015 to March 2017 in Mexico City., Intervention: Daily sucralose consumption at 15% of the acceptable daily intake by using commercial sachets added to food. The control group followed the same protocol without an intervention., Main Outcomes Measured: Fasting concentrations of appetite regulating hormones before and after the intervention. Fasting glucose and insulin concentrations were measured to assess insulin resistance as a secondary outcome., Statistical Analysis Performed: Basal and final concentrations were compared using Wilcoxon matched-pairs test and Mann-Whitney U test for analysis between groups. Repeated measures analysis of variance was used to evaluate changes in the homeostasis model assessment of insulin resistance., Results: Sucralose was not associated with changes in any of the hormones measured. One week postintervention, an incremental change (P=0.04) in the homeostasis model assessment of insulin resistance was found in the intervention group., Conclusions: Sucralose intake is not associated with changes in fasting concentrations of glucagon-like peptide 1, ghrelin, peptide tyrosine tyrosine, or leptin. An increase in the homeostasis model assessment of insulin resistance observed only at 1 week postdosing is of unknown clinical significance, if any., (Copyright © 2020 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Hyperhomocysteinemia is recognized as risk factor for cardiovascular and age-associated diseases. Folic acid supplementation efficiently lowers plasma homocysteine (Hcy) levels, but high intake may negatively affect health because of unnatural levels of unmetabolized folic acid in the systemic circulation. Oxoproline (Oxo) provides by glutamic acid production an increase of intracellular folic acid trapping. Aim of this study was to compare the efficacy of three supplementation protocols: (1) traditional therapy (5-methyl-tetrahydrofolate: 15 mg/day); (2) 5 mL/day of Oxo with 300 μg folic acid (oxifolic); (3) 5 mL/day of Oxo alone (magnesio+) in a 90 days randomized trial on thirty-two moderate hyperhomocysteinemic (18.6 ± 2.4 μmol.L -1 ) patients (age 48 ± 14 yrs). Thiols: cysteine (Cys), cysteinylglycine (Cys-Gly) and glutathione levels were assessed too. Every supplementation induced significant ( p range <0.05-0.0001) reductions of Hcy level and Cys concentration after the three protocols adopted. Otherwise glutathione concentration significantly increased after oxifolic ( p < 0.01) and traditional ( p < 0.05) supplementation. The integration of Oxo resulted an interesting alternative to traditional therapy because absence or minimal number of folates in the integrator eliminates any chance of excess that can constitute a long-term risk., Competing Interests: The authors declare no conflicts of interest.

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Scope: The role of PEPT1 in the uptake of intact peptides as compared to hydrolysis prior to uptake of their constituents is unknown. Here, dipeptides, tripeptides, and amino acids are quantified to study the fate of selected peptides in different intestinal models., Methods and Results: An LC-MS/MS-based method is applied for the simultaneous assessment of rates of hydrolysis and transport of a peptide panel in Caco-2 transwell cell culture, in vitro and in vivo in mice expressing or lacking PEPT1, and in hydrolysis studies in vitro using human intestinal samples. It is shown that susceptibility to hydrolysis of peptides at the brush border membrane or within epithelial cells is practically identical in all tested models and strictly structure-dependent. Peptides with high luminal disappearance show substantial hydrolysis and low basolateral appearance, while peptides with low disappearance show strong PEPT1 dependency and high basolateral appearance in intact form in Caco-2 transwell culture., Conclusion: Hydrolysis and transport of intact peptides are highly variable and structure-dependent. For peptides possessing less polar N-terminal residues, hydrolysis usually dominates over transport via PEPT1. For other peptides with high intrinsic hydrolysis resistance, including anserine, carnosine, ɣ-glutamyl-dipeptides, and aminocephalosporins, PEPT1 is the main determinant for appearance in peripheral blood., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pharmacokinetics and relative bioavailability of antidepressant GSB-106 (hexamethylendiamide bis-(N-monosuccinyl-L-seryl-L-lysine) were determined in rabbits after single oral administration of the pharmaceutical substance and tablet mass. GSB-106 concentrations in the blood plasma were determined by HPLC-mass spectrometry. Relative bioavailability of GSB-106 tablet form was 160.79±24.33%.

Purpose: Fixed-combination drug products (FCDPs) for patients with type 2 diabetes mellitus (T2DM) may show efficacy comparable to their individual components (ICs) while improving adherence to treatment. This study evaluated the bioequivalence and safety of 2 dapagliflozin/saxagliptin/metformin extended-release (XR) FCDPs relative to their ICs: saxagliptin and dapagliflozin/metformin XR., Methods: This randomized, open-label, single-dose, single-center crossover study was conducted in 84 healthy subjects aged 18-55 years. The primary objective was to evaluate the fed-state bioequivalence of a dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP and a dapagliflozin 10-mg/saxagliptin 5-mg/metformin 1000-mg XR FCDP relative to the ICs. Secondary objectives included the evaluation of the effect of food on the pharmacokinetic (PK) parameters of saxagliptin, dapagliflozin, and metformin in both FCDPs and characterization of the PK parameters of the active metabolite of saxagliptin, 5-hydroxy saxagliptin, in healthy subjects. PK parameters (AUC 0-∞ , AUC 0-t , and C max ) were used to assess the bioequivalence of the 2 FCDPs with their ICs. The C max and AUC 0-t of the study drugs were compared between female and male subjects to assess sex differences in exposure. Safety and tolerability of both FCDPs and ICs were also assessed with adverse events, vital signs (systolic and diastolic blood pressures and pulse rate), 12-lead ECG, physical examinations, and laboratory assessments., Findings: Both dapagliflozin/saxagliptin/metformin XR FCDPs were bioequivalent to their ICs. For the dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP, the 90% CI for the geometric mean ratio of dapagliflozin C max was slightly above the 80%-125% bioequivalence limit, which is unlikely to be clinically relevant. Food delayed the absorption of the study drugs in both FCDPs, which is unlikely to have a clinically relevant impact on efficacy. In both cohorts, exposure was higher in female subjects compared with male subjects, potentially due to the lower body weight of the female subjects. The safety profile and tolerability of the FCDPs were similar to those of their ICs, and no deaths or serious adverse events were reported., Implications: These data support the use of the dapagliflozin/saxagliptin/metformin XR FCDP in patients with T2DM. ClinicalTrials.gov identifier: NCT03169959., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The citrulline generation test (CGT) has been proposed as a tool to determine gut function. However, the increase in plasma citrulline concentration that follows a bolus dose of alanyl-glutamine may also result from a reduction in citrulline clearance due to competition with glutamine for transport., Materials and Methods: A swine model was developed, and stable isotope tracers were used to determine the mechanism behind the increase in plasma citrulline that follows a bolus dose of alanyl-glutamine. Plasma concentrations and enrichments were determined, and a non-steady-state model was used to calculate rates of appearance, disappearance, and conversion., Results: The pig model recapitulated the increase in plasma citrulline observed in humans after a dose of alanyl-glutamine. The dipeptide was rapidly hydrolyzed to its constitutive amino acids. Both citrulline plasma concentration and citrulline rate of appearance increased by ≈45% after the bolus dose of alanyl-glutamine. The conversion of citrulline to arginine and the rate of appearance of arginine also increased. Glutamine contributed up to 25% ± 2% of the rate of appearance of citrulline. No changes in the rate of disappearance of citrulline were observed., Conclusion: Our results indicate that a single bolus dose of alanyl-glutamine increases plasma citrulline concentration by increasing citrulline production without any effect on citrulline disposal. Our findings strongly indicate that the CGT assesses the metabolic response of the gut and that CGT can become a useful tool to evaluate gut mass and function., (© 2018 American Society for Parenteral and Enteral Nutrition.)

New Findings: What is the central question of this study? What are the interactions between sleep and appetite following early evening high-intensity interval exercise (HIIE)? What is the main finding and its importance? HIIE can be performed in the early evening without subsequent sleep disruptions and may favourably alter appetite-related hormone concentrations. Nonetheless, perceived appetite and energy intake do not change with acute HIIE regardless of time of day., Abstract: Despite exercise benefits for sleep and appetite, due to increased time restraints, many adults remain inactive. Methods to improve exercise compliance include preferential time-of-day or engaging in short-duration, high-intensity interval exercise (HIIE). Hence, this study aimed to compare effects of HIIE time-of-day on sleep and appetite. Eleven inactive men undertook sleep monitoring to determine baseline (BASE) sleep stages and exclude sleep disorders. On separate days, participants completed 30 min HIIE (60 s work at 100% V ̇ O 2 peak , 240 s rest at 50% V ̇ O 2 peak ) in (1) the morning (MORN; 06.00-07.00 h), (2) the afternoon (AFT; 14.00-16.00 h) and (3) the early evening (EVEN: 19.00-20.00 h). Measures included appetite-related hormones (acylated ghrelin, leptin, peptide tyrosine tyrosine) and glucose pre-exercise, 30 min post-exercise and the next morning; overnight polysomnography (PSG; sleep stages); and actigraphy, self-reported sleep and food diaries for 48 h post-exercise. There were no between-trial differences for total sleep time (P = 0.46). Greater stage N3 sleep was recorded for MORN (23 ± 7%) compared to BASE (18 ± 7%; P = 0.02); however, no between-trial differences existed (P > 0.05). Rapid eye movement (REM) sleep was lower and non-REM sleep was higher for EVEN compared to BASE (P ≤ 0.05). At 30 min post-exercise, ghrelin was higher for AFT compared to MORN and EVEN (P = 0.01), while glucose was higher for MORN compared to AFT and EVEN (P ≤ 0.02). No between-trial differences were observed for perceived appetite (P ≥ 0.21) or energy intake (P = 0.57). Early evening HIIE can be performed without subsequent sleep disruptions and reduces acylated ghrelin. However, perceived appetite and energy intake appear to be unaffected by HIIE time of day., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Collagen hydrolysate is a well-known nutritional supplement for the improvement of healthy skin. Here, collagen peptide NS (CPNS) from fish scale was prepared, and its physicochemical properties were investigated. Gly-Pro was revealed as a representative low molecular weight peptide of CPNS, by performing prep-HPLC and LC-MS/MS. CPNS treatment attenuated matrix metalloproteinase-1 production and increased the synthesis of type 1 procollagen in HDF cells. After orally administering CPNS to rats, the plasma concentrations of Gly-Pro and Pro-Hyp increased dramatically. To examine the protective effects of CPNS against ultraviolet B (UVB)-induced photoaging in vivo, the dorsal skins of hairless mice were exposed to UVB and supplemented with CPNS for 12 weeks. The CPNS consumption significantly attenuated UVB-induced wrinkle formation, transepidermal water loss, and epidermis thickness, and increased skin hydration. Collectively, these results suggest that bioactive peptides of CPNS, Gly-Pro and Pro-Hyp, exert beneficial effects on skin health.

Saxagliptin is an orally administered, highly potent, and selective dipeptidyl peptidase-4 inhibitor for the management of type 2 diabetes mellitus. This study was conducted to determine the effect of magnesium and aluminum hydroxides plus simethicone, famotidine, and omeprazole on the pharmacokinetics of saxagliptin and its active metabolite, 5-hydroxy saxagliptin. This was an open-label, randomized, 5-treatment, 5-period, 3-way crossover study in 15 healthy subjects. Mean C max of saxagliptin was 26% lower, but AUC was almost unchanged when saxagliptin was coadministered with Maalox Max. Mean C max was 14% higher, but AUC was almost unchanged when saxagliptin was coadministered with famotidine. Changes in pharmacokinetics of 5-hydroxy saxagliptin generally paralleled the changes in saxagliptin. These pharmacokinetic changes were unlikely to be clinically meaningful. Coadministration of omeprazole did not affect saxagliptin C max or AUC. Saxagliptin in combination with these medicines resulted in no unexpected safety or tolerability findings in these healthy subjects. No dose adjustment of saxagliptin or separation in the time of saxagliptin dosing is necessary with medicines that raise gastric pH when coadministered with saxagliptin., (© 2018, The American College of Clinical Pharmacology.)

Prior epidemiologic findings for plasma folate and B-vitamins and breast cancer risk are inconsistent and have not assessed the influence of folic acid fortification. Therefore, we examined the associations of plasma folate, B 12 , pyridoxal 5'-phosphate (PLP), homocysteine, cysteine and cysteinylglycine with breast cancer risk, before and after fortification. We conducted a nested case-control study within the prospective Nurses' Health Study. In 1989-1990 (pre-fortification), 32,826 women donated a blood sample and 18,743 donated an additional blood sample in 2000-2001 (post-fortification). Between the first blood collection and 2006, 1874 incident breast cancer cases with at least one blood sample and 367 with two were 1:1 matched to controls. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) adjusting for breast cancer risk factors. Overall, higher plasma folate, B 12 , PLP, homocysteine, cysteine and cysteinylglycine levels were not associated with breast cancer risk. Associations did not vary by in situ/invasive, hormone receptor status, or tumor molecular subtype. Additionally, associations were null before and after fortification. For example, the RR (95% CI) for the highest versus lowest tertile of 1990 (pre-fortification) plasma folate with 1990-2000 follow-up was 0.93 (0.75-1.16) and for the 2000 plasma folate (post-fortification) with 2000-2006 follow-up the RR (95% CI) was 1.17 (0.79-1.74). Plasma folate, B 12 , PLP, homocysteine, cysteine and cysteinylglycine were not significantly associated with breast cancer overall, before and after fortification, or with specific tumor molecular subtypes. However, long term associations (>8 years) after the implementation of fortification could not be examined., (© 2018 UICC.)

Scope: Peptide transporter 1 (PEPT1) function is well understood, yet little is known about its contribution toward the absorption of dietary amino acids in the form of di- and tripeptides. In the present human study, changes in plasma concentrations of a representative oligopeptide panel are investigated after meat intake., Methods and Results: Based on a method for quantitative analysis of a panel of selected di- and tripeptides in biological samples, the kinetics of plasma changes of peptides derived from a widely accessible dietary protein source are described. The findings demonstrate postprandial changes of a whole spectrum of dipeptides of different size, charge, and polarity in peripheral blood in a dose-dependent manner after consumption of chicken breast in healthy human volunteers. Although the substrate specificity of PEPT1 is well known, the spectrum of peptides appearing in blood cannot be matched to the affinity to PEPT1. Stability against hydrolysis by exo- and endopeptidases appears to be another factor influencing their presence in blood. In addition, the study shows that dipeptides, including gamma-glutamyl-peptides, as well as tripeptides are common components present in human plasma., Conclusion: Besides amino acids, human peripheral blood contains numerous di- and tripeptides. The dietary source determines their abundance and composition., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An ultra-high performance hydrophilic interaction liquid chromatography - triple quadrupole tandem mass spectrometry method was developed for the determination of biologically important thiols, namely cysteine, homocysteine, cysteinyl-glycine, glutathione, in rat plasma. The sample preparation procedure as well as the analytical method were comprehensively optimized and subsequently validated. An optimum sample preparation protocol was based on the simple and fast derivatization of the thiols with new derivatization reagent, N-phenylmaleimide, enabling highly selective and sensitive quantification in plasma matrices. The method, characterized by favourable performance parameters and meeting the FDA criteria for biomedical analysis, was successfully applied for monitoring the concentration levels of the selected thiols in the samples from transgenic rat model for tauopathy. The study revealed significant changes in homocysteine and glutathione levels related to tauopathy while other thiols did not indicate such relationship. Indeed, these findings could play an important role in further understanding of tauopathy process in the brain. Moreover, the proposed highly effective, reliable and robust analytical protocol can be easily adapted for other thiol compounds, spreading its application range in this biomedical field., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A method is described allowing forensic analysis of plasma samples to prove human poisoning with the organophosphorus pesticides omethoate (OM) and dimethoate (DIM). Upon incubation of human serum albumin (HSA) with both pesticides tyrosine residues were phosphorylated. In addition, a novel disulfide-adduct between the identical thiol-containing leaving group of OM and DIM (2-mercapto-N-methylacetamide, MNMA) and the only free cysteine residue in HSA (Cys 34 ) was formed. Following pronase-catalyzed proteolysis either O,O-dimethyl phosphotyrosine (Tyr-dmp) or O,O-dimethyl thiophosphotyrosine (Tyr-dmsp) as well as the cysteine-proline dipeptide disulfide-adduct (MNMA-CysPro) were produced. All biomarkers were simultaneously detected using modern microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). Corresponding limits of identification (LOI) for tyrosine-adducts (LOI OM : 30 μM, LOI DIM : 120 μM) and disulfide-adducts (LOI OM : 1.2 μM, LOI DIM : 30 μM) demonstrated that MNMA-CysPro allowed a considerably more sensitive detection. Finally, this novel method was applied to a plasma sample of an 87-year-old man, who had unintentionally ingested the pesticide Roxion ® containing DIM as active ingredient. Unambiguous proof of poisoning demonstrated suitability of the novel biomarkers for sensitive verification analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Anxiety-related disorders, including fearfulness are common and leading welfare problems among the worldwide dog population. The etiology of anxieties is complex and affected by genetic and environmental factors. Thus, there is a need for more comprehensive approaches, such as metabolomics, to understand the causes of anxiety and to identify anxiety-related biomarkers for more efficient diagnostic and treatment options. To study metabolic alterations related to canine fearfulness, a non-targeted plasma metabolite profiling was performed in a cohort of 20 fearful and 21 non-fearful dogs. The results showed that nine metabolic features were significantly associated with fearfulness. The most prominent change included increased plasma glutamine and γ-glutamyl glutamine (γ-Glu Gln) in fearful dogs across breeds. Alterations in glutamine metabolism have previously been associated with several psychiatric disorders, indicating the relevance of this finding also in dogs. In addition, we describe a novel breed-specific association between renal biomarker symmetric dimethylarginine (SDMA) and canine fearfulness. These observed metabolic alterations may result from high levels of prolonged psychological stress in fearful dogs.

Levels of short linear hydroxyproline (Hyp)-containing peptides, such as prolyl-hydroxyproline (Pro-Hyp), increase in human blood after the ingestion of collagen hydrolysate, which has been associated with beneficial effects for human skin and joints. The present study demonstrates the presence of a novel food-derived collagen peptide, cyclic Pro-Hyp, in human blood after the ingestion of collagen hydrolysate. The cyclic Pro-Hyp levels in plasma samples were estimated by liquid chromatography mass spectrometry (LC-MS). Cyclic Pro-Hyp levels significantly increased in the plasma after ingestion of collagen hydrolysate, reaching a maximum level after 2 h and then decreasing. The maximum level of cyclic Pro-Hyp in plasma ranged from 0.1413 to 0.3443 nmol/mL, representing approximately 5% of linear Pro-Hyp in plasma after ingestion of collagen hydrolysate. Addition of cyclic Pro-Hyp in medium at 7 nmol/mL significantly enhanced the growth rate of mouse skin fibroblasts on collagen gel more extensively compared to linear Pro-Hyp.

The dipeptide amide H-Phe-Phe-NH 2 (1) that previously was identified as a ligand for the substance P 1-7 (SP 1-7 ) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP 1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP 1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH 2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED 50 ) of 4 was superior as compared to gabapentin and morphine that are used in clinic., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Oxidative stress is evident from an early stage in chronic kidney disease (CKD). Therefore, we investigated redox biomarkers in polycystic kidney disease (ADPKD) and IgA nephropathy (IGAN)., Methods: This is a case-control study with three groups: ADPKD (n = 54), IGAN (n = 58) and healthy controls (n = 86). The major plasma aminothiols with their redox species were examined: homocysteine (Hcy), cysteinglycine (CG), cysteine (Cys) and glutathione (GSH). The redox ratio was the ratio of reduced free and oxidized aminothiols in plasma. We investigated malonedialdehyde (MDA) and advanced oxidation protein products (AOPP), and ten single nucleotide polymorphisms of antioxidant enzymes., Results: Patients had elevated oxidized free Hcy and Cys with associated low redox ratios - most pronounced in IGAN. Patients with IGAN had elevated AOPP and possibly MDA. Oxidized free Hcy and Cys with redox ratios were correlated to AOPP, MDA and proteinuria. Furthermore, there was an independent relationship to parathyroid hormone (PTH). IGAN had an elevated frequency of Val16Ala SNP rs4880, which influence the function of mitochondrial superoxide dismutase 2 (p = 0.03)., Conclusions: Patients with ADPKD and IGAN have evidence of oxidative stress from stage 1 to 4 - most pronounced in IGAN. In patients, aminothiol redox biomarkers were correlated to AOPP, proteinuria and PTH, which are known prognostic markers in CKD. It may be possible that oxidative stress influences PTH dysregulation in CKD. The association between IGAN and the redox related variant allele rs4880(C) might indicate a new susceptibility locus for IGAN, but this needs verification., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

A simple, sensitive and specific hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric (HILIC-MS) method was developed and validated to determine the plasma concentrations of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously in clinical studies. Plasma samples were first acidified and then protein precipitated with acetonitrile. Chromatographic separation was achieved on a HILIC Chrom Matrix HP amide column (5 μm, 3.0 × 100 mm I.D.). The mobile phase consisted of acetonitrile and 5 mM ammonium formate buffer containing 0.1% formic acid. Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r ≥ 0.999) over the established concentration range of 1.0-1000 ng/mL for metformin and 0.1-100 ng/mL for saxagliptin and its active metabolite 5-hydroxy saxagliptin. The extraction recovery for all of the analytes was >92% and the matrix effect ranged from 91.0 to 110.0%. After validation, the method was successfully applied to a bioequivalence study with a single-pill combination (SPC) consisting of 5 mg saxagliptin and 500 mg metformin in 10 healthy Chinese subjects., (Copyright © 2018. Published by Elsevier B.V.)

The main target of this work is to examine blood clearance and external exposure for 177 Lu-DOTATATE compared with new emerging 177 Lu-PSMA therapy. Blood clearance and radiation exposure of 31 patients treated with 5.5 ± 1.1 GBq 177 Lu-DOTATATE were compared to those of 23 patients treated with 7.4 GBq 177 Lu-PSMA. Dose rates were measured at several distances and time points up to 120 h after treatment. Blood samples were collected conjunctively after infusion. Caregiver's cumulative dose was measured by means of an OSL (optically stimulated luminescence) dosimeter for 4-5 days and medical staff's dose was also estimated using electronic personal dosimeters. Finger dose was determined via ring TLD (Thermoluminescence Dosimeter) for radiopharmacists and nurses. Dose rates due to 177 Lu-DOTATATE at a distance of 1 m, 4 h and 6 h after infusion, were 3.0 ± 2.8 and 2 ± 1.9 µSv/(h GBq), respectively, while those due to 177 Lu-PSMA were 3.1 ± 0.8 and 2.2 ± 0.9 µSv/(h GBq). Total effective dose of 17 caregivers was 100-200 µSv for 177 Lu-DOTATATE therapy. Mean effective doses to nurses and radiopharmacists were 5 and 4 µSv per patient, respectively, while those for physicists and physicians were 2 µSv per patient. For 177 Lu-DOTATATE, effective half-life in blood and early elimination phase were 0.31 ± 0.13 and 4.5 ± 1 h, while they were found as 0.4 ± 0.1 and 5 ± 1 h, respectively, for 177 Lu-PSMA. The first micturition time following 177 Lu-DOTATATE infusion was noted after 36 ± 14 min, while the second and third voiding times were after 74 ± 9 and 128 ± 41 min, respectively. It is concluded that blood clearance and radiation exposure for 177 Lu-DOTATATE are very similar to those for 177 Lu-PSMA, and both treatment modalities are reasonably reliable for outpatient treatment, since the mean dose rate [2.1 µSv/(h GBq)] decreased below the dose rate that allows release of the patient from the hospital (20 µSv/h) after 6 h at 1 m distance.

A rapid and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of a novel anti-HBV compound Y101 and its metabolites M8 and M9 in human plasma. The plasma samples were deproteinated with acetonitrile after addition of Peramivir (internal standard, IS) and separated on a 40 °C ACQUITY UPLC BEH C 18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase consisted of water (containing 5 mM ammonium acetate and 0.1% formic acid) and acetonitrile (74:26, v/v) at a flow rate of 0.3 mL/min. The detection was performed on a Triple Quad 5500 tandem mass spectrometer coupled with electrospray ionization (ESI) source in positive mode. Quantification was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 490.1 → 339.0 for Y101, m/z 357.2 → 105.2 for metabolite M8, m/z 373.1 → 105.1 for metabolite M9 and m/z 329.1 → 270.2 for IS, respectively. The method was validated over the calibration curve range of 1.000-1000 ng/mL for Y101, 2.000-2000 ng/mL for metabolite M8 and 0.3000-300.0 ng/mL for metabolite M9, using linear regression and 1/x 2 weighting. No matrix effect and carryover effect was observed. The intra- and inter-batch precision and accuracy of Y101, metabolite M8 and M9 were all within the acceptable criteria. This method allows a rapid and simple determination of Y101 and its metabolites M8 and M9 in human plasma. It was successfully applied in a pharmacokinetic study in human for the first time., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Migraine is a highly disabling primary headache associated with a high socioeconomic burden and a generally high prevalence. The clinical management of migraine remains a challenge. This study was undertaken to identify potential serum biomarkers of migraine. Using Liquid Chromatography coupled to Mass Spectrometry (LC-MS), the metabolomic profile of migraine was compared with healthy individuals. Principal component analysis (PCA) and Orthogonal partial least squares-discriminant analysis (orthoPLS-DA) showed the metabolomic profile of migraine is distinguishable from controls. Volcano plot analysis identified 10 serum metabolites significantly decreased during migraine. One of these was serotonin, and the other 9 were amino acids. Pathway analysis and enrichment analysis showed tryptophan metabolism (serotonin metabolism), arginine and proline metabolism, and aminoacyl-tRNA biosynthesis are the three most prominently altered pathways in migraine. ROC curve analysis indicated Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine are potential sensitive and specific biomarkers for migraine. Our results show Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine may be as specific or more specific for migraine than serotonin which is the traditional biomarker of migraine. We propose that therapeutic manipulation of these metabolites or metabolic pathways may be helpful in the prevention and treatment of migraine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent evidence suggests that increased brain serotonin synthesis impairs performance in high-intensity intermittent exercise and specific amino acids may modulate this condition, delaying fatigue. This study investigated the effects of glutamine and alanine supplementation on central fatigue markers in rats submitted to resistance training (RT). Wistar rats were distributed in: sedentary (SED), trained (CON), trained and supplemented with alanine (ALA), glutamine and alanine in their free form (G + A), or as dipeptide (DIP). Trained groups underwent a ladder-climbing exercise for eight weeks, with progressive loads. In the last 21 days, supplementations were offered in water with a 4% concentration. Albeit without statistically significance difference, RT decreased liver glycogen, and enhanced the concentrations of plasma glucose, free fatty acids (FFA), hypothalamic serotonin, and ammonia in muscle and the liver. Amino acids affected fatigue parameters depending on the supplementation form. G + A prevented the muscle ammonia increase by RT, whereas ALA and DIP augmented ammonia and glycogen concentrations in muscle. DIP also increased liver ammonia. ALA and G + A reduced plasma FFA, whereas DIP increased this parameter, free tryptophan/total tryptophan ratio, hypothalamic serotonin, and the serotonin/dopamine ratio. The supplementations did not affect physical performance. In conclusion, glutamine and alanine may improve or impair central fatigue markers depending on their supplementation form., Competing Interests: The authors declare they have no conflict of interest.

Background: It remains unknown whether sustained daily feeding-fasting patterns modify the acute response to specific feedings on a given day., Objective: We conducted a randomized controlled trial to establish if daily breakfast consumption or fasting until noon modifies the acute metabolic and appetitive responses to a fixed breakfast and ad libitum lunch., Methods: With the use of a parallel group design, we randomly assigned 31 healthy, lean men and women (22-56 y) to 6 wk of either consuming ≥700 kcal of self-selected items before 1100 or fasting (0 kcal) until 1200 daily. Following 48 h of diet and physical activity standardization, we examined metabolic and appetite responses to a standardized breakfast and ad libitum lunch before and after the intervention. Data were analyzed using 3- and 2-way ANCOVA., Results: Systemic concentrations of energy balance regulatory hormones total and acylated ghrelin, leptin, and peptide tyrosine-tyrosine) responded similarly to breakfast and lunch before and after 6 wk of either morning fasting or regular breakfast, with the exception of a tendency for increased glucagon-like peptide-1 concentrations from baseline to follow-up in the Breakfast Group compared with a decrease over that period in the Fasting Group [P = 0.06, partial eta squared value (ƞ2) = 0.16]. Subjective appetite sensations also did not differ over the course of the day, and ad libitum energy intake at lunch was not systematically affected by either intervention, decreasing by 27 kcal (95% CI: -203, 149 kcal) with fasting and by 77 kcal (95% CI: -210, 56 kcal) with breakfast. Similarly, glycemic, insulinemic, lipemic, and thermogenic responses to breakfast and lunch were very stable at baseline and follow-up and, thus, did not differ between treatment groups., Conclusions: Our results indicate that a sustained period of either extended morning fasting or eating a daily breakfast has minimal effect upon acute metabolic and appetite responses in lean adults. This trial was registered at www.isrctn.org as ISRCTN31521726., (© 2018 American Society for Nutrition.)

Bariatric surgery is currently the most effective strategy for treating morbid obesity. Weight regain following significant weight loss, however, remains a problem, with the outcome proportional to the period of follow-up. This review revisits a well-established physiological neurohormonally-mediated feedback loop, the so called ileal brake mechanism, with a special emphasis on the gut hormone peptide tyrosine tyrosine. The manuscript not only highlights the potential role of the ileal brake mechanism in weight loss and weight maintenance thereafter following obesity surgery, it also provides a compelling argument for using this appetite suppressing feedback loop to enable sustained long-term weight loss in patients undergoing surgery for morbid obesity., (© 2017 Royal Australasian College of Surgeons.)

Background/objectives: Fatty liver disease (FLD) is an important intermediate trait along the cardiometabolic disease spectrum and strongly associates with type 2 diabetes. Knowledge of biological pathways implicated in FLD is limited. An untargeted metabolomic approach might unravel novel pathways related to FLD., Subjects/methods: In a population-based sample (n=555) from Northern Germany, liver fat content was quantified as liver signal intensity using magnetic resonance imaging. Serum metabolites were determined using a non-targeted approach. Partial least squares regression was applied to derive a metabolomic score, explaining variation in serum metabolites and liver signal intensity. Associations of the metabolomic score with liver signal intensity and FLD were investigated in multivariable-adjusted robust linear and logistic regression models, respectively. Metabolites with a variable importance in the projection >1 were entered in in silico overrepresentation and pathway analyses., Results: In univariate analysis, the metabolomics score explained 23.9% variation in liver signal intensity. A 1-unit increment in the metabolomic score was positively associated with FLD (n=219; odds ratio: 1.36; 95% confidence interval: 1.27-1.45) adjusting for age, sex, education, smoking and physical activity. A simplified score based on the 15 metabolites with highest variable importance in the projection statistic showed similar associations. Overrepresentation and pathway analyses highlighted branched-chain amino acids and derived gamma-glutamyl dipeptides as significant correlates of FLD., Conclusions: A serum metabolomic profile was associated with FLD and liver fat content. We identified a simplified metabolomics score, which should be evaluated in prospective studies.

Introduction: Homocysteine increase and glutathione derivative cysteinyl-glycine fall are indirect biomarkers for oxidative stress, for instance due to dopamine D 1 receptor stimulation., Objectives: To investigate the influence of the D 1 receptor agonists levodopa and rotigotine compared with placebo on homocysteine and cysteinyl-glycine in plasma of patients with Parkinson's disease., Methods: Patients received 100 mg levodopa, 4 mg rotigotine or placebo. Cysteinyl-glycine and homocysteine were measured every 30 min over three hours., Results: Homocysteine rose during levodopa- and placebo administration. Rotigotine had no effect. Cysteine-glycine only increased after placebo- but not after levodopa- or rotigotine., Discussion: Homocysteine elevation results from hepatic and gastrointestinal methylation processes. Transdermal rotigotine circumvents these methylation locations. Turnover of segregated alkyl residuals from rotigotine serves as methyl group donors, which counteract homocysteine increment. The placebo-related cysteinyl-glycine increase results from reduced free radical exposure. Low levodopa dosing and antioxidants in the rotigotine patch matrix prevented cysteinyl-glycine fall.

Collagen hydrolysate is a well-known dietary supplement for the treatment of skin aging; however, its mode of action remains unknown. Previous studies have shown that the oral ingestion of collagen hydrolysate leads to elevated levels of collagen-derived peptides in the blood, but whether these peptides reach the skin remains unclear. Here, we analyzed the plasma concentration of collagen-derived peptides after ingestion of high tripeptide containing collagen hydrolysate in humans. We identified 17 types of collagen-derived peptides transiently, with a particular enrichment in Gly-Pro-Hyp. This was also observed using an in vivo mouse model in the plasma and skin, albeit with a higher enrichment of Pro-Hyp in the skin. Interestingly, this Pro-Hyp enrichment in the skin was derived from Gly-Pro-Hyp hydrolysis, as the administration of pure Gly-Pro-Hyp peptide led to similar results. Therefore, we propose that functional peptides can be transferred to the skin by dietary supplements of collagen.

A specific and rapid liquid chromatography-tandem mass spectrometry method is proposed for the simultaneous determination of metformin (MET), saxagliptin (SAXA) and its active metabolite, 5-hydroxy saxagliptin (5-OH SAXA) in human plasma. Sample preparation was accomplished from 50 μL plasma sample by solid-phase extraction using sodium dodecyl sulfate as an ion-pair reagent. Reversed-phase chromatographic resolution of analytes was possible within 3.5 min on ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile and10.0 mm ammonium formate buffer, pH 5.0 (80:20, v/v) as the mobile phase. Triple quadrupole mass spectrometric detection was performed using electrospray ionization in the positive ionization mode. The calibration curves showed good linearity (r 2  ≥ 0.9992) over the established concentration range with limit of quantification of 1.50, 0.10 and 0.20 ng/mL for MET, SAXA and 5-OH SAXA respectively. The extraction recoveries obtained from spiked plasma samples were highly consistent for MET (75.12-77.84%), SAXA (85.90-87.84%) and 5-OH SAXA (80.32-82.69%) across quality controls. The validated method was successfully applied to a bioequivalence study with a fixed-dose formulation consisting of 5 mg SAXA and 500 mg MET in 18 healthy subjects. The reproducibility of the assay was demonstrated by reanalysis of 87 incurred samples., (Copyright © 2016 John Wiley & Sons, Ltd.)

Polydextrose (PDX) reduces subsequent energy intake (EI) when administered at midmorning in single-blind trials of primarily normal-weight men. However, it is unclear if this effect also occurs when PDX is given at breakfast time. Furthermore, for ecological validity, it is desirable to study a female population, including those at risk for obesity. We studied the effects of PDX, served as part of a breakfast or midmorning preload, on subsequent EI and other appetite-related parameters in healthy normal-weight and overweight females. Per earlier studies, the primary outcome was defined as the difference in subsequent EI when PDX was consumed at midmorning versus placebo. Thirty-two volunteers were enrolled in this acute, double-blind, placebo-controlled, randomized, and crossover trial to examine the effects of 12.5 g of PDX, administered as part of a breakfast or midmorning preload, on subsequent EI, subjective feelings of appetite, well-being, and mood. Gastric emptying rates and the blood concentrations of glucose, insulin, cholecystokinin, ghrelin, glucagon-like peptide 1 (GLP-1), and peptide tyrosine-tyrosine were measured in the group that received PDX as part of their breakfast. There were no differences in EI between volunteers who were fed PDX and placebo. PDX intake with breakfast tended to elevate blood glucose (P = 0.06) during the postabsorptive phase, significantly lowered insulin by 15.7% (P = 0.04), and increased GLP-1 by 39.9% (P = 0.02); no other effects on blood parameters or gastric emptying rates were observed. PDX intake at midmorning reduced hunger by 31.4% during the satiation period (P = 0.02); all other subjective feelings of appetite were unaffected. Volunteers had a uniform mood profile during the study. PDX was well tolerated, causing one mild adverse event throughout the trial., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH 2 was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimize the N terminus. Using two successive Sonogashira cross-coupling reactions on a solid-supported peptide, we were able to introduce various alkynes at the N terminus to afford compounds with sub-micromolar affinities for hGPR54. However, functional assays indicated the benzoylated dipeptide Bz-Arg-Trp-NH 2 as the most promising compound in terms of agonistic properties. Interestingly, this compound appeared much more stable than the endogenous neuropeptide kisspeptin, both in serum and in liver microsomes of rats. This compound was also found to be able to induce a significant in vivo increase in testosterone levels in male rats., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic arsenic toxicity is a serious environmental health problem across the world. Bangladesh and India (particularly the state of West Bengal) are the worst affected countries with such problem. The present study reports plasma-aminothiols (p-aminothiols) like L-cysteine (L-Cys), cysteinyl glycine (Cys-gly), total homocysteine (t-Hcy) and glutathione (GSH) status, and the inverse relationship of t-Hcy with B-vitamins (B1, B6, B9 and B12) in arsenic exposed population of West Bengal, India. Reverse phase HPLC was used to measure p-aminothiols and serum B-vitamins in different arsenic exposed population. Arsenic in drinking water and urine were measured by flow injection analysis system - Atomic Absorption Spectrometry (FIAS-AAS) and Transversely heated graphite atomizer (THGA-AAS) techniques, respectively. Water arsenic exposure was >50 µg/L in 50% population, of which majority (33.58%) belong to the range of >50-500 µg/L and more than 8% were even >1000 µg/L. Urine arsenic (µg/g creatinine) levels increased with arsenic exposure. The variability among p-aminothiols was also observed with higher exposure to arsenic in drinking water. A significant difference between exposed and control population was noticed for plasma L-Cys. The difference of B-vitamins between the population exposed to <50 and >50 µg/L arsenic in drinking water was also found to be significant. B9 and B12 deficiency with increased consumption of arsenic in water corroborates the anemic conditions commonly observed among arsenic exposed population. The aminothiol status indicated oxidative stress in exposed population. This study demonstrated progressive increase in plasma t-Hcy as well as inverse relationships of serum B-vitamins with increased water arsenic concentration.