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Background: Several recent studies reported the potential adverse effects of titanium exposure on glucose homeostasis among the non-pregnant population, but the association of titanium exposure with gestational diabetes mellitus (GDM) is scarce., Methods: The present study of 1,449 pregnant women was conducted within the Jiangsu Birth Cohort (JBC) study in China. Urine samples were collected in the early pregnancy, and urinary titanium concentration and non-targeted metabolomics were measured. Poisson regression estimated the association of titanium exposure in the early pregnancy with subsequent risk of GDM. Multiple linear regression screened for titanium-related urine metabolites. Mediation analyses assessed the mediating effects of candidate metabolites and pathways., Results: As parameterized in tertiles, titanium showed positive dose-response relationship with GDM risk (P for trend = 0.008), with women at the highest tertile of titanium exposure having 30% increased risk of GDM [relative risk (RR) = 1.30 (95% CI: 1.06, 1.61)] when compared to those exposure at the first tertile level. Meanwhile, we identified the titanium-related metabolites involved in four amino acid metabolic pathways. Notably, the perturbation of the aminoacyl-tRNA biosynthesis and alanine, aspartate and glutamate metabolism mediated 27.1% and 31.0%, respectively, of the relative effect of titanium exposure on GDM. Specifically, three titanium-related metabolites, choline, creatine and L-alanine, demonstrated predominant mediation effects on the association between titanium exposure and GDM risk., Conclusions: In this prospective study, we uniquely identified a correlation between early pregnancy titanium exposure and increased GDM risk. We unveiled novel insights into how perturbations in amino acid metabolism may mediate the link between titanium exposure and GDM. Notably, choline, creatine, and L-alanine emerged as key mediators influencing this association. Our findings imply that elevated titanium exposure in early pregnancy can lead to amino acid dysmetabolism, thereby elevating GDM risk., (© 2024. The Author(s).)

Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract. Changes in amino acid metabolites have been implicated in tumorigenesis and disease progression. Biomarkers on the basis of chiral amino acids, especially D-amino acids, have not been established for early diagnosis of CRC. Quantification of chiral amino acids, especially very low concentrations of endogenous D-amino acids, is technically challenging. We report here the quantification of L- and D-amino acids in urine samples collected from 115 CRC patients and 155 healthy volunteers, using an improved method. The method of chiral labeling, liquid chromatography, and tandem mass spectrometry enabled separation and detection of 28 amino acids (14 L-amino acids, 13 D-amino acids and Gly). Orthogonal partial least squares discriminant analysis identified 14 targeted variables among these chiral amino acids that distinguished the CRC from the healthy controls. Binary logistic regression analysis revealed that D-α-aminobutyric acid (D-AABA), L-alanine (L-Ala), D-alanine (D-Ala), D-glutamine (D-Gln) and D-serine (D-Ser) could be potential biomarkers for CRC. A receiver operating characteristic curve analysis of combined multi-variables contributed to an area under the curve (AUC) of 0.995 with 98.3 % sensitivity and 96.8 % specificity. A model constructed with D-AABA, D-Ala, D-Gln, and D-Ser achieved an AUC of 0.988, indicating important contributions of D-amino acids to the association with CRC. Further analysis also demonstrated that the metabolic aberration was associated with age and the development of CRC, D-methionine (D-Met) was decreased in CRC patients with age over 50, and D/L-Gln in patients at stage IV was higher than patients at stage I. This study provides the signature of D-amino acids in urine samples and offers a promising strategy for developing non-invasive diagnosis of CRC., 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.)

The contrasting relationships of plant and animal protein intake with blood pressure (BP) may be partially attributed to the differential non-protein (e.g., saturated fat and fibre) and amino acid (AA) compositions. This study determined whether animal and plant protein intake were related to differential metabolomic profiles associated with BP. This study included 1008 adults from the African-PREDICT study (aged 20-30 years). Protein intake was determined using 24-h dietary recalls. Twenty-four-hour ambulatory BP was measured. Amino acids and acylcarnitines were analysed in spot urine samples using liquid chromatography-tandem mass spectrometry-based metabolomics. Participants with a low plant, high animal protein intake had higher SBP (by 3 mmHg, p = 0.011) than those with high plant, low animal protein intake (low-risk group). We found that the relationships of plant and animal protein intake with 24-h SBP were partially mediated by BMI and saturated fat intake, which were independently associated with SBP. Protein intake was therefore not related to SBP in multiple regression analysis after adjusting for confounders. In the low-risk group, methionine (Std. β = -0.217; p = 0.034), glutamic acid (Std. β = -0.220; p = 0.031), glycine (Std. β = -0.234; p = 0.025), and proline (Std. β = -0.266; p = 0.010) were inversely related to SBP, and beta-alanine (Std. β = -0.277; p = 0.020) to DBP. Ultimately a diet high in animal and low in plant protein intake may contribute to higher BP by means of increased BMI and saturated fat intake. Conversely, higher levels of urinary AAs observed in adults consuming a plant rich diet may contribute to lower BP., (© 2024. The Author(s).)

The exposure to modifiable risk factors at young ages have been linked to premature fatal and non-fatal cardiovascular and kidney outcomes. The use of urinary metabolomics has shown strong predictability of kidney function and cardiovascular disease (CVD). We therefore determined the associations between estimated glomerular filtration rate (eGFR) and urinary metabolites in young adults with and without CVD risk factors. Apparently healthy Black and White sexes were included (aged 20-30 years) and categorised by the presence or absence of risk factors, i.e., obesity, physical inactivity, smoking, excessive alcohol intake, masked hypertension, hyperglycemia, dyslipidemia and low socio-economic status, forming the CVD risk group (N = 1036), CVD risk clusters (i.e. presenting with 1 CVD risk factor (N = 344), 2 CVD risk factors (N = 360) and 3 + CVD risk factors (N = 332)) and the control group (N = 166). eGFR was calculated with CKD-EPI equations. A targeted metabolomics approach using liquid chromatography-tandem mass spectrometry was used to measure amino acids and acylcarnitines. Lower cystatin C-based eGFR were indicated in the CVD risk group, 2 and 3 + CVD risk clusters compared to the control group (all P ≤ 0.033). In the CVD risk group, eGFR associated positively with histidine, lysine, asparagine, glycine, serine, glutamine, dimethylglycine, threonine, alanine, creatine, cystine, methionine, tyrosine, pyroglutamic acid, leucine/isoleucine, aspartic acid, tryptophan, glutamic acid, free carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, octanoylcarnitine and decanoylcarnitine (all P ≤ 0.044), with similar results found in the CVD risk clusters, particularly the 2 CVD risk cluster. eGFR was positively associated with metabolites linked to aromatic amino acid and branched-chain amino acid metabolism, energy metabolism and oxidative stress. These findings may indicate altered reabsorption of these metabolites or altered metabolic regulation to preserve renal health in the setting of CVD risk factors at this young age without established CVD., (© 2024. The Author(s).)

Introduction: The human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches., Objective: This study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC-MS) metabolomics., Methods: Urine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC-MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups., Results: The study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients., Conclusion: The metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations., (© 2024. The Author(s).)

Chronic kidney disease (CKD) is highly prevalent in domestic cats. This study aimed to compare urinary D-amino acid levels between control and CKD-afflicted cats as a novel noninvasive method for assessing CKD. Cats were divided into control and CKD stage II groups in accordance with the International Renal Interest Society guidelines. The urinary DL-amino acid levels of the cats were analyzed using chiral tandem liquid chromatography-tandem mass spectrometry, and their medical records were investigated. The CKD group had considerably lower urinary D-amino acid concentrations and enantiomeric ratios than the control group. The total urinary D-amino acid contents significantly correlated with blood parameters (creatinine and urea nitrogen). These findings may contribute towards the detection of CKD stage II in domestic cats.

Rationale: The mechanisms underlying major depressive disorder (MDD) in children and adolescents are unclear. Metabolomics has been utilized to capture metabolic signatures of various psychiatric disorders; however, urinary metabolic profile of MDD in children and adolescents has not been studied., Objectives: We analyzed urinary metabolites in children and adolescents with MDD to identify potential biomarkers and metabolic signatures., Methods: Here, liquid chromatography-mass spectrometry was used to profile metabolites in urine samples from 192 subjects, comprising 80 individuals with antidepressant-naïve MDD (AN-MDD), 37 with antidepressant-treated MDD (AT-MDD) and 75 healthy controls (HC). We performed orthogonal partial least squares discriminant analysis to identify differential metabolites and employed logistic regression and receiver operating characteristic analysis to establish a diagnostic panel., Results: In total, 143 and 71 differential metabolites were identified in AN-MDD and AT-MDD, respectively. These were primarily linked to lipid metabolism, molecular transport, and small molecule biochemistry. AN-MDD additionally exhibited dysregulated amino acid metabolism. Compared to HC, a diagnostic panel of seven metabolites displayed area under the receiver operating characteristic curves of 0.792 for AN-MDD, 0.828 for AT-MDD, and 0.799 for all MDD. Furthermore, the urinary metabolic profiles of children and adolescents with MDD significantly differed from those of adult MDD., Conclusions: Our research suggests dysregulated amino acid metabolism and lipid metabolism in the urine of children and adolescents with MDD, similar to results in plasma metabolomics studies. This contributes to the comprehension of mechanisms underlying children and adolescents with MDD., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Neoadjuvant chemotherapy (NAC) is the preferred treatment option in locally advanced breast cancer (BC). The administration of NAC is associated with a wide range of adverse effects. This pilot observational prospective study examined the effect of NAC using anthracycline + cyclophosphamide (AC) followed by paclitaxel (PTx) on a portfolio of 22 plasma and urinary amino acids, plasma proteins (albumin, prealbumin, transferrin), and products of nitrogen metabolism (urea, creatinine, uric acid) in plasma and urine. Plasma and 24-h urine samples were obtained from ten patients with early breast cancer (N1-3 N0-2 M0), at the following time points: before the start of NAC and during the AC/PTx treatment period (a total of 8 measurements at three-weekly intervals). Amino acids were analyzed using ion exchange chromatography. There were no significant differences in the measured parameters in plasma and urine between pre-NAC and during AC- and PTx-treatment. No trend was detected. A significant difference in the portfolio of plasma and urinary amino acids was found only in the pre-treatment period compared to the control group. Levels of eight plasma amino acids (8/22) were significantly reduced and those of nine urine amino acids were increased (9/22). Nitrogenous catabolites in plasma and urine were not indicative of increased protein catabolism during the anthracycline and taxane treatment periods. A slightly positive nitrogen balance was accompanied by an average weight gain of 3.3 kg (range 0-6 kg). The AC/PTx treatment regimen did not cause significant changes in the monitored laboratory parameters.

Glucagon plays a central role in amino acid (AA) homeostasis. The dog is an established model of glucagon biology, and recently, metabolomic changes in people associated with glucagon infusions have been reported. Glucagon also has effects on the kidney; however, changes in urinary AA concentrations associated with glucagon remain under investigation. Therefore, we aimed to fill these gaps in the canine model by determining the effects of glucagon on the canine plasma metabolome and measuring urine AA concentrations. Employing two constant rate glucagon infusions (CRI) - low-dose (CRI-LO: 3 ng/kg/min) and high-dose (CRI-HI: 50 ng/kg/min) on five research beagles, we monitored interstitial glucose and conducted untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) on plasma samples and urine AA concentrations collected pre- and post-infusion. The CRI-HI induced a transient glucose peak (90-120 min), returning near baseline by infusion end, while only the CRI-LO resulted in 372 significantly altered plasma metabolites, primarily reductions (333). Similarly, CRI-HI affected 414 metabolites, with 369 reductions, evidenced by distinct clustering post-infusion via data reduction (PCA and sPLS-DA). CRI-HI notably decreased circulating AA levels, impacting various AA-related and energy-generating metabolic pathways. Urine analysis revealed increased 3-methyl-l-histidine and glutamine, and decreased alanine concentrations post-infusion. These findings demonstrate glucagon's glucose-independent modulation of the canine plasma metabolome and highlight the dog's relevance as a translational model for glucagon biology. Understanding these effects contributes to managing dysregulated glucagon conditions and informs treatments impacting glucagon homeostasis.

Background: Mucopolysaccharidosis (MPS) and glycoproteinosis are 2 groups of heterogenous lysosomal storage disorders (LSDs) caused by defective degradation of glycosaminoglycans (GAGs) and glycoproteins, respectively. Oligosaccharides and glycoamino acids have been recognized as biomarkers for MPS and glycoproteinosis. Given that both groups of LSDs have overlapping clinical features, a multiplexed assay capable of unambiguous subtyping is desired for accurate diagnosis, and potentially for severity stratification and treatment monitoring., Methods: Urinary oligosaccharides were derivatized with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) together with the underivatized glycoamino acids. Novel biomarkers were identified with a semi-targeted approach with precursor mass scanning, the fragmentation pattern (if applicable), and the biochemical basis of the condition., Results: A UPLC-MS/MS analysis with improved chromatographic separation was developed. Novel biomarkers for MPS-IIIA, IIIB, IIIC, and VII were identified and validated. A total of 28 oligosaccharides, 2 glycoamino acids, and 2 ratios were selected as key diagnostic biomarkers. Validation studies including linearity, lower limit of quantitation (LLOQ), and precision were carried out with the assay performance meeting the required criteria. Age-specific reference ranges were collected. In the 76 untreated patients, unambiguous diagnosis was achieved with 100% sensitivity and specificity. Additionally, the levels of disease-specific biomarkers were substantially reduced in the treated patients., Conclusions: A multiplexed UPLC-MS/MS assay for urinary oligosaccharides and glycoamino acids measurement was developed and validated. The assay is suitable for the accurate diagnosis and subtyping of MPS and glycoproteinosis, and potentially for severity stratification and monitoring response to treatment., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Immunoglobulin nephropathy (IgAN) stands as the most prevalent primary glomerular nephropathy globally, typically diagnosed through an invasive renal biopsy. Emerging research suggests the significant involvement of chiral amino acids in kidney disease progression. This study introduces a nonderivative LC-tandem mass spectrometry approach, offering efficient separation outcomes within 15 min for identifying chiral amino acids in human urine samples. Subsequently, using this method, the analysis of l- and d-amino acids in the urine of both patients with IgAN and healthy individuals was conducted. Fourteen d-amino acids and 20 l-amino acids were identified in the urine samples obtained from 17 patients with IgAN and 21 healthy individuals. The results indicated notable variances in the concentrations of both l- and d-amino acids between the IgAN and healthy control groups. In contrast to the healthy group, the IgAN group exhibited higher mean urine concentrations of most l-amino acids and lower concentrations of d-amino acids. Furthermore, correlations between amino acids and clinical markers were investigated. These results propose a novel method for monitoring trace amino acids in urine samples and introduce a new concept for potential markers of IgAN., (© 2024 John Wiley & Sons Ltd.)

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Abs max ) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Abs max in healthy males (R 2  = 0.439, P = 0.000), females (R 2  = 0.245, P = 0.000), female stroke patients (R 2  = 0.187, P = 0.000), but not in male stroke patients (R 2  = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Abs max correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Abs max , while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Abs max , while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Abs max were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke., (© 2024. The Author(s).)

Objective To develop a method for the quantification of amino acids and organic acids in trace urine by high performance liquid chromatography-tandem mass spectrometry.Methods Random urine samples(10 μl each)were precipitated by acetonitrile and underwent derivatization with 3 mol/L HCl in n-butanol.The analytes were separated by ACE Excel 2 AQ column(50×2.1 mm,2 μm).Electrospray ionization in positive ion mode was carried out and the analytes were detected in multiple reaction monitoring mode.According to existing guidelines,the method was systematically evaluated in terms of sensitivity,specificity,accuracy,precision,recovery,matrix effect,and stability.Then,the established method was employed to detect 19 target compounds in urine samples from 70 healthy children,27 children with suspected vitamin B12 deficiency,and 3 children with cblC type methylmalonic acidemia.Results The lower limit of quantification of the method for the 19 compounds ranged from 0.01 μmol/L to 1.00 μmol/L,and the calibration curves were linear, R 2 >0.990.The method showed good accuracy with relative error less than ±15% and the intra-day and intra-day precision less than 15%.The run time was 8 min.No obvious matrix effect was detected except for arginine,and the recovery ranged from 80.20% to 114.97%.The samples were stable after 8 h at room temperature and 3 freeze-thaw cycles.The measured values of the compounds in the urine of healthy children were within the children's reference intervals published by Labcorp.The levels of methylmalonic acid( P =0.030)and homocysteine( P <0.001)in the urine samples of children with suspected vitamin B12 deficiency were higher than those in healthy children.The levels of methylmalonic acid,methylcitric acid,and homocysteine in the urine samples of children with cblC type methylmalonic acidemia were 5.14-76.52 times higher than the median levels of healthy children. Conclusions The method established in this study has small sample demand and short run time,which can accurately quantify the levels of amino acids and metabolites in the urine of children.Moreover,it can provide data support for related studies about the metabolic characteristics of urine amino acids and their metabolites in children with vitamin B12 deficiency.

The scope of non-hydrogenative parahydrogen hyperpolarization (nhPHIP) techniques has been expanding over the last years, with the continuous addition of important classes of substrates. For example, pyruvate can now be hyperpolarized using the Signal Amplification By Reversible Exchange (SABRE) technique, offering a fast, efficient and low-cost PHIP alternative to Dynamic Nuclear Polarization for metabolic imaging studies. Still, important biomolecules such as amino acids have so far resisted PHIP, unless properly functionalized. Here, we report on an approach to nhPHIP for unmodified α-amino acids that allows their detection and quantification in complex mixtures at sub-micromolar concentrations. This method was tested on human urine, in which natural α-amino acids could be measured after dilution with methanol without any additional sample treatment., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Metabolomics has become an important tool for clinical research, especially for analyzing inherited metabolic disorders (IMDs). The purpose of this study was to explore the performance of metabolomics in diagnosing IMDs using an untargeted metabolomic approach. A total of 40 urine samples were collected: 20 samples from healthy children and 20 from pediatric patients, of whom 13 had confirmed IMDs and seven had suspected IMDs. Samples were analyzed by Orbitrap mass spectrometry in positive and negative mode alternately, coupled with ultra-high liquid chromatography. Raw data were processed using Compound Discovery 2.0 ™ and then exported for partial least squares discriminant analysis (PLS-DA) by SIMCA-P 14.1. After comparing with m/zCloud and chemSpider libraries, compounds with similarity above 80% were selected and normalized for subsequent relative quantification analysis. The uncommon compounds discovered were analyzed based on the Kyoto Encyclopedia of Genes and Genomes to explore their possible metabolic pathways. All IMDs patients were successfully distinguished from controls in the PLS-DA. Untargeted metabolomics revealed a broader metabolic spectrum in patients than what is observed using routine chromatographic methods for detecting IMDs. Higher levels of certain compounds were found in all 13 confirmed IMD patients and 5 of 7 suspected IMD patients. Several potential novel markers emerged after relative quantification. Untargeted metabolomics may be able to diagnose IMDs from urine and may deepen insights into the disease by revealing changes in various compounds such as amino acids, acylcarnitines, organic acids, and nucleosides. Such analyses may identify biomarkers to improve the study and treatment of IMDs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sepsis, defined as a dysregulated host response to infection, causes the interruption of homeostasis resulting in metabolic changes. An examination of patient metabolites, such as amino acids, during the early stage of sepsis may facilitate diagnosing and assessing the severity of the sepsis. The aim of this study was to compare patterns of urine and serum amino acids relative to sepsis, septic shock and survival. Urine and serum samples were obtained from healthy volunteers (n = 15) once or patients (n = 15) within 24 h of a diagnosis of sepsis or septic shock. Concentrations of 25 amino acids were measured in urine and serum samples with liquid chromatography-electrospray mass spectrometry. On admission in the whole cohort, AAA, ABA, mHis, APA, Gly-Pro and tPro concentrations were significantly lower in the serum than in the urine and Arg, Gly, His, hPro, Leu, Ile, Lys, Orn, Phe, Sarc, Thr, Tyr, Asn and Gln were significantly higher in the serum than in the urine. The urine Gly-Pro concentration was significantly higher in septic shock than in sepsis. The serum Cit concentration was significantly lower in septic shock than in sepsis. The urine ABA, mHis and Gly-Pro, and serum Arg, hPro and Orn concentrations were over two-fold higher in the septic group compared to the control group. Urine and serum amino acids measured in septic patients on admission to the ICU may shed light on a patient's metabolic condition during sepsis or septic shock., (© 2021. The Author(s).)

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). An optimized derivatization protocol has been successfully applied using multiple isotope labelled analytical internal standards of selected deuterated and 13 C selected compounds, covering a range of different groups of metabolites for non-automated GC metabolomics (off-line). Moreover, the study was also realized in a pooled urine sample, following metabolic profiling. A study of thermal degradation of metabolites due to GC inlet and oven programs (fast, slow) was performed, where the results indicated that both GC oven programs (fast and slow) negatively affected the thermal stability of the metabolites, while the fast-ramp GC program also suppressed MS signals. However, the use of multiple internal standards can overcome this drawback. The application of extended temperature ramp GC program presented identical behaviour on metabolite stability and better chromatographic separation combined with much lower signal suppression, compared to a short temperature ramp program. No effects were observed for organic acids, fatty acids, sugars and sugar alcohols, while significant differences were observed for amino acids. GC metabolomics is a strong tool that can facilitate analysis, but special attention is required for sampling handling and heating, before and during the GC analysis. The use and application of multiple multi-group internal standards is highly recommended., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Obesity is associated with changes in amino acid metabolism, and studies show that ingestion of fish proteins influence amino acid composition in plasma and urine, in addition to affecting risk factors for metabolic syndrome. Since the majority of fish proteins consumed by humans are as fish fillet, it is of interest to investigate if cod fillet intake affects amino acid composition and metabolic disorders. We hypothesized that a modified AIN-93G diet containing cod fillet would affect amino acid compositions in plasma and urine in obese rats, and also affect risk factors for metabolic syndrome when compared to rats fed a regular AIN-93G diet with casein as the protein source. Obese Zucker fa/fa rats, a rat model of metabolic syndrome, received diets containing 25% protein from lyophilized baked cod fillet and 75% protein from casein (Baked cod diet), or a Control diet with casein for four weeks. The Baked cod diet affected the amino acid composition in plasma, with e.g., lower glycine, histidine, homoarginine, homocysteine, methionine, proline and tyrosine concentrations, but did not affect amino acid concentrations in urine. The concentrations of markers for kidney and liver dysfunction were lower in the Baked cod group, however blood pressure development, fasting and postprandial glucose, and hepatic triacylglycerol concentrations were similar to the Control group. To conclude, substituting 25% of dietary protein with baked cod fillet affected concentrations of some amino acids in plasma and delayed development of kidney and liver dysfunction, but did not affect blood pressure, glucose concentration or fatty liver., (Copyright © 2021. Published by Elsevier Inc.)

The World Health Organization has shown that coronary heart disease (CHD) is a more common cause of death than cancer. In traditional Chinese medicine (TCM), CHD is classified as a form of thoracic obstruction that can be divided in different subtypes including Qi stagnation with blood stasis (QS) and Qi deficiency with blood stasis (QD). Different treatment strategies are used based on this subtyping. Owing to the lack of scientific markers in the diagnosis of these subtypes, subjective judgments made by clinicians have limited the objective manner for utility of TCM in the treatment of CHD. Untargeted (UHPLC-QTOF-MS) and targeted (UHPLC-MS/MS) metabolomics approaches were employed to search significantly different metabolites related to the QS or QD subtypes of CHD with angina pectoris in this study. A total of 42 metabolites were obtained in the untargeted metabolomics analysis and 34 amino acids were detected in the targeted metabolomics analysis. In total, 16 metabolites were found significantly different among different groups. The results showed distinct metabolic profiles of urine samples not only between CHD patients and healthy controls, but also between the two subtypes of CHD. Pathway analysis of the significantly varied metabolites revealed that there were subtype-related differences in the activity of pathways. Therefore, urinary metabolomics can reveal the pathological changes of CHD in different subtypes, make the diagnosis of CHD in different subtypes in an objective manner and comprehensive and contribute to personalized treatment by providing scientific evidence., (Copyright © 2021. Published by Elsevier B.V.)

Aminoacids and their derivatives are key biologically important metabolites and reliable, rapid and accurate, quantification for these analytes in urine remains an important analytical challenge. Here a fast and reliable HILIC-tandem MS method is presented for application in clinical or nutritional studies. The developed method was validated according to existing guidelines adapted for endogenous analytes. The validation strategy provided evidence of linearity, LOD and LOQ, accuracy, precision, matrix effect and recovery. The surrogate matrix approach was applied for calibration proving satisfactory accuracy and precision based on standard criteria over the working concentration ranges. Intra and inter day accuracy was found to range between 0.8 and 20% for the LQC (low QC) and between 0.05 and 15 % for MQC (medium QC) and HQC (high QC). Inter and intraday precision were found to be between 3 and 20 % for the LQC and between 1 and 15% for the MQC and HQC. The stability of the analytes, in both surrogate and pooled urine QC samples, was found to be within 15% over a short period at 4 °C or after a up to 3 freeze-thaw cycles. The uncertainty of the method was also assessed to provide increased confidence for the acquired measurements. The method was successfully applied to a subset of human urine samples involved in a study of amino acids dietary uptake. This method may provide a valuable tool for many applications or studies where amino acid metabolic signatures in the excreted urine are under investigation., 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 © 2021. Published by Elsevier B.V.)

Taking into account the growing demand for new analytical procedures that are appropriate for analysis of complex biological samples with increased sensitivity, accuracy and precision, a novel analytical method was described for the determination of underivatized amino acids in human plasma and urine samples. The presented analytical procedure involved the direct analysis of urine samples and the analysis of plasma samples followed by a simple protein precipitation protocol. Samples were analyzed using a simple and fast chromatographic method developed for the determination of 17 different amino acids by liquid chromatography - tandem mass spectrometry. The limit of detection and quantification values for amino acids were ranged between 0.03-2.26 µmol kg -1 and 0.09-7.54 µmol kg -1 . Matrix effects of plasma and urine on the quantification of analytes were determined by spiking experiments. The accuracy of method was evaluated by matrix matching and quadruple isotope dilution strategies. Excellent accuracy and precision were obtained with the use isotope labeled amino acids demonstrating the high reliability and reproducibility of the proposed method. The percent recovery values were found to be between 98.70 - 101.68% with%RSD below than 1.62% for human plasma and 99.14 - 101.78% with%RSD below than 2.44% for urine samples., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Background and Objectives: Genetic variants in NAT8 , a liver- and kidney-specific acetyltransferase encoding gene, have been associated with eGFR and CKD in European populations. Higher circulating levels of two NAT8 -associated metabolites, N- δ -acetylornithine and N-acetyl-1-methylhistidine, have been linked to lower eGFR and higher risk of incident CKD in the Black population. We aimed to expand upon prior studies to investigate associations between rs13538, a missense variant in NAT8 , N-acetylated amino acids, and kidney failure in multiple, well-characterized cohorts., Design, Setting, Participants, & Measurements: We conducted analyses among participants with genetic and/or serum metabolomic data in the African American Study of Kidney Disease and Hypertension (AASK; n =962), the Atherosclerosis Risk in Communities (ARIC) study ( n =1050), and Bio Me , an electronic health record-linked biorepository ( n =680). Separately, we evaluated associations between rs13538, urinary N-acetylated amino acids, and kidney failure in participants in the German CKD (GCKD) study ( n =1624)., Results: Of 31 N-acetylated amino acids evaluated, the circulating and urinary levels of 14 were associated with rs13538 ( P <0.05/31). Higher circulating levels of five of these N-acetylated amino acids, namely, N- δ -acetylornithine, N-acetyl-1-methylhistidine, N-acetyl-3-methylhistidine, N-acetylhistidine, and N2,N5-diacetylornithine, were associated with kidney failure, after adjustment for confounders and combining results in meta-analysis (combined hazard ratios per two-fold higher amino acid levels: 1.48, 1.44, 1.21, 1.65, and 1.41, respectively; 95% confidence intervals: 1.21 to 1.81, 1.22 to 1.70, 1.08 to 1.37, 1.29 to 2.10, and 1.17 to 1.71, respectively; all P values <0.05/14). None of the urinary levels of these N-acetylated amino acids were associated with kidney failure in the GCKD study., Conclusions: We demonstrate significant associations between an NAT8 gene variant and 14 N-acetylated amino acids, five of which had circulation levels that were associated with kidney failure., (Copyright © 2021 by the American Society of Nephrology.)

Background: Efficacy of protein absorption and subsequent amino acid utilization may be reduced in the elderly. Higher protein intakes have been suggested to counteract this., Objectives: We aimed to elucidate how habituated amounts of protein intake affect the fasted state of, and the stimulatory effect of a protein-rich meal on, protein absorption, whole-body protein turnover, and splanchnic amino acid metabolism., Methods: Twelve men (65-70 y) were included in a double-blinded crossover intervention study, consisting of a 20-d habituation period to a protein intake at the RDA or a high amount [1.1 g · kg lean body mass (LBM)-1 · d-1 or >2.1 g · kg LBM-1 · d-1, respectively], each followed by an experimental trial with a primed, constant infusion of D8-phenylalanine and D2-tyrosine. Arterial and hepatic venous blood samples were obtained after an overnight fast and repeatedly 4 h after a standardized meal including intrinsically labeled whey protein concentrate and calcium-caseinate proteins. Blood was analyzed for amino acid concentrations and phenylalanine and tyrosine tracer enrichments from which whole-body and splanchnic amino acid and protein kinetics were calculated., Results: High (compared with the recommended amount of) protein intake resulted in a higher fasting whole-body protein turnover with a resultant mean ± SEM 0.03 ± 0.01 μmol · kg LBM-1 · min-1 lower net balance (P < 0.05), which was not rescued by the intake of a protein-dense meal. The mean ± SEM plasma protein fractional synthesis rate was 0.13 ± 0.06%/h lower (P < 0.05) after habituation to high protein. Furthermore, higher fasting and postprandial amino acid removal were observed after habituation to high protein, yielding higher urea excretion and increased phenylalanine oxidation rates (P < 0.01)., Conclusions: Three weeks of habituation to high protein intake (>2.1 g protein · kg LBM-1 · d-1) led to a significantly higher net protein loss in the fasted state. This was not compensated for in the 4-h postprandial period after intake of a meal high in protein.This trial was registered at clinicaltrials.gov as NCT02587156., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Propionic acidemia (PA) is caused by inherited deficiency of mitochondrial propionyl-CoA carboxylase (PCC) and results in significant neurodevelopmental and cardiac morbidity. However, relationships among therapeutic intervention, biochemical markers, and disease progression are poorly understood. Sixteen individuals homozygous for PCCB c.1606A > G (p.Asn536Asp) variant PA participated in a two-week suspension of therapy. Standard metabolic markers (plasma amino acids, blood spot methylcitrate, plasma/urine acylcarnitines, urine organic acids) were obtained before and after stopping treatment. These same markers were obtained in sixteen unaffected siblings. Echocardiography and electrocardiography were obtained from all subjects. We characterized the baseline biochemical phenotype of untreated PCCB c.1606A > G homozygotes and impact of treatment on PCC deficiency biomarkers. Therapeutic regimens varied widely. Suspension of therapy did not significantly alter branched chain amino acid levels, their alpha-ketoacid derivatives, or urine ketones. Carnitine supplementation significantly increased urine propionylcarnitine and its ratio to total carnitine. Methylcitrate blood spot and urine levels did not correlate with other biochemical measures or cardiac outcomes. Treatment of PCCB c.1606A > G homozygotes with protein restriction, prescription formula, and/or various dietary supplements has a limited effect on core biomarkers of PCC deficiency. These patients require further longitudinal study with standardized approaches to better understand the relationship between biomarkers and disease burden., (Copyright © 2020. Published by Elsevier Inc.)

Quantitative analysis of amino acids in blood and urine is primarily indicated for the diagnosis of amino acid disorders. The high-performance liquid chromatography (HPLC) technique is frequently used for this detection. The frequency of sample collection on filter paper has been increasing exponentially, and there are many advantages attributed to processing biological samples in this way. The aim of this study was to validate a quantitative analysis of amino acids by HPLC in blood and urine collected on filter paper and to establish reference values in the neonatal period. Dried blood and dried urine samples of respectively 58 and 45 healthy newborns (2-9 days) were collected. Pre-treatment and extraction of samples were done according to the literature. Separation and analysis of amino acids were carried out by HPLC with fluorescence detection. The developed method demonstrated excellent separation, linearity, limits of detection and quantification, repeatability and recovery. The reference values for 17 amino acids were defined in dried blood and urine samples of newborns. This work presents a simple, fast and effective method for the simultaneous analysis of 17 amino acids in blood and urine collected on filter paper in a single run. The reference values were established and validated., (© 2020 John Wiley & Sons, Ltd.)

Background Urine amino acid analysis is used for the assessment of various diseases. The aim of this study was to estimate the valid biological variation (BV) components (within- and between-subjects) required for the safe clinical application of free urine amino acids. Methods First morning void urine samples were taken from 12 healthy subjects (five females, seven males) once a week for 10 consecutive weeks, and amino acid analysis was performed using an Agilent 6470 triple quadrupole tandem mass spectrometer instrument. The obtained data were subjected to normality, outlier and variance homogeneity analyses prior to coefficient of variation (CV) analysis. Within- and between-subject BV values (CVI and CVG) of 39 amino acids were determined for all subjects. In addition, the index of individuality (II), reference change value (RCV), imprecision, bias and total error were estimated using BV data obtained from our study. Results The CVI values ranged from 8.9 (histidine) to 36.8% (trans-4-hydroxyprolin), while the CVG values ranged from 25.0 (1-methyl-L-histidine) to 63.3% (phenylalanine). The II value of most amino acids was less than 0.6 and ranged between 0.21 and 0.88. The imprecision, bias and total error ranged between 4.45 and 16.6, between 7.69 and 16.6, and between 18.4 and 43.2, respectively. Conclusions This study, designed according to a rigorous protocol, has the feature of being the first to give information about BV data of urine amino acids. We believe that the reference intervals have a limitation in the evaluation of consecutive results from an individual, so the use of RCV would be more appropriate.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

We studied changes in the bone tissue in patients with diffuse large B-cell lymphoma at the onset of the disease (N=41; before chemotherapy) and 5-16 years after the end of treatment (N=47). Osteodensitometry, biochemical markers of osteoporosis in the blood and urine, and gene expression in multipotent mesenchymal stromal cells were analyzed. In multipotent mesenchymal stromal cells of all patients, the expression of genes associated with bone and cartilage differentiation (FGF2, FGFR1, FGFR2, BGLAP, SPP1, TGFB1, and SOX9) was changed. In primary patients, the ratio of deoxypyridinoline/creatinine in the urine and blood level of β-cross-laps were increased, while plasma concentration of vitamin D was reduced, which indicates activation of bone resorption. No differences between the groups were revealed by osteodensitometry. No direct relationship between changes in gene expression in multipotent mesenchymal stromal cells and osteoporosis markers was found. The presence of a tumor in the body affects the bone marrow stroma, but achievement of remission and compensatory mechanisms provide age-appropriate condition of the bone tissue.

As a severe metabolic disease, type 2 diabetes mellitus (T2DM) has aroused increasing public attentions. Resistant starch 3 (RS3), as a starch resistant to enzymatic hydrolysis owing to its special structure, has a good effect on improving insulin resistance and reducing blood sugar in T2DM patients. However, the possible mechanisms were barely interpreted yet. In our research, we aimed to evaluate the effects and the possible mechanisms of RS3 on the treatment of T2DM. ICR mice treated with high-fat diet (HFD) for eight weeks, and then injected with streptozotocin (STZ) (100 mg/kg) to establish the T2DM. We choose the mice with the fast blood glucose (FBG) more than 11 mmol/L as T2DM. After treated for 11 weeks the relevant data was analyzed. According to the results, the FBG was dramatically reduced (p < 0.05), which also downregulated triglyceride (p < 0.01) and total cholesterol (p < 0.01). Additionally, the insulin resistance indexes were significantly reduced (p < 0.01), the homeostasis model assessment-β and insulin-sensitive index were significantly improved (p < 0.01) in RS3 group. Meanwhile, the metabolic profiles of urine were analyzed and 29 potential biomarkers were screened out, including amino acids and lipids. In conclusion, we speculated that the tricarboxylic acid cycle, amino acid metabolism and lipid metabolism played roles in the therapeutic mechanisms of RS3 on T2DM., Competing Interests: Declaration of Competing Interest All authors report no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Celiac disease (CeD) is an autoimmune enteropathy caused by gluten intake in genetically predisposed individuals. We investigated the metabolism of CeD by metabolic profiling of intestinal mucosa, blood plasma and urine using NMR spectroscopy and multivariate analysis. The metabolic profile of the small intestinal mucosa was compared between patients with CeD (n = 64) and disease controls (DCs, n = 30). The blood plasma and urinary metabolomes of CeD patients were compared with healthy controls (HCs, n = 39). Twelve metabolites (proline (Pro), arginine (Arg), glycine (Gly), histidine (His), glutamate (Glu), aspartate, tryptophan (Trp), fumarate, formate, succinate (Succ), glycerophosphocholine (GPC) and allantoin (Alln)) of intestinal mucosa differentiated CeD from controls. The metabolome of blood plasma with 18 metabolites (Pro, Arg, Gly, alanine, Glu, glutamine, glucose (Glc), lactate (Lac), acetate (Ace), acetoacetate (AcAc), β-hydroxybutyrate (β-OHB), pyruvate (Pyr), Succ, citrate (Cit), choline (Cho), creatine (Cr), phosphocreatine (PCr) and creatinine) and 9 metabolites of urine (Pro, Trp, β-OHB, Pyr, Succ, N-methylnicotinamide (NMN), aminohippurate (AHA), indoxyl sulfate (IS) and Alln) distinguished CeD from HCs. Our data demonstrated changes in nine metabolic pathways. The altered metabolites were associated with increased oxidative stress (Alln), impaired healing and repair mechanisms (Pro, Arg), compromised anti-inflammatory and cytoprotective processes (Gly, His, NMN), altered energy metabolism (Glc, Lac, β-OHB, Ace, AcAc, Pyr, Succ, Cit, Cho, Cr and PCr), impaired membrane metabolism (GPC and Cho) and intestinal dysbiosis (AHA and IS). An orthogonal partial least square discriminant analysis model provided clear differentiation between patients with CeD and controls in all three specimens. A classification model built by combining the distinguishing metabolites of blood plasma and urine samples gave an AUC of 0.99 with 97.7% sensitivity, 93.3% specificity and a predictive accuracy of 95.1%, which was higher than for the models built separately using small intestinal mucosa, blood plasma and urine. In conclusion, a panel of metabolic biomarkers in intestinal biopsies, plasma and urine samples has potential to differentiate CeD from controls and may complement traditional tests to improve the diagnosis of CeD., (© 2020 John Wiley & Sons, Ltd.)

High fructose intake has been shown to increase circulating alanine transaminase in humans, which could reflect damage to the liver by fructose but could also be linked to higher level of transamination of amino acids in liver. Therefore, we hypothesized that a diet with high content of fructose would affect the amino acid composition in rat plasma and urine differently from a diet with high sucrose content. Because high intake of sucrose and fructose is often accompanied with high intake of saturated fat in the Western-style diet, we wanted to compare the effects of high fructose/sucrose in diets with normal or high content of coconut oil on individual free amino acids plasma and urine. Male Wistar rats were fed diets with normal (10 wt%) or high (40 wt%) content of sucrose or fructose, with normal or high fat content (7 or 22 wt%) and 20 wt% protein (casein). Rats fed high-fructose high-fat diet had higher plasma concentrations of aspartic acid, cystine, glutamic acid, ornithine, and phenylalanine and higher urine concentrations of arginine and citrulline when compared to rats fed high-sucrose high-fat diet. Substituting normal content of sucrose with fructose in the diets had little impact on amino acids in plasma and urine. Serum concentrations of alanine transaminase, aspartate transaminase, and creatinine, and urine cystatin C and T cell immunoglobulin mucin-1 concentrations were comparable between the groups and within normal ranges. To conclude, substituting high-dose sucrose with high-dose fructose in high-fat diets affected amino acid compositions in plasma and urine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aim: Specific amino acids have been linked to regulation of insulin secretion from pancreatic β-cells; on the contrary, increased concentration of certain amino acids is associated with insulin resistance (IR) and development of type 2 diabetes mellitus (T2DM). Nowadays, urine as a biological sample has attracted more attention for diagnosis of disease for its special superiority; insufficient research in the study of urinary amino acid (UAA) pattern in patients with T2DM has led to the present study with the aim to determine the levels of UAAs, their excretory patterns and the association of UAA to plasma glucose and IR in patients with T2DM., Methods: Quantification of total urinary amino acids was done spectrophotometrically and the patterns of amino acid excretion was elucidated by thin layer chromatography technique. Fasting blood samples were used for plasma glucose and insulin estimation by fully automated analyzer., Result: The levels of UAA in patients with T2DM in comparison to healthy controls were higher (p < 0.0001). The frequency of urinary phenylalanine, arginine, tryptophan, tyrosine and cysteine were significantly higher in patients with T2DM than controls. There was also a strong positive correlation of UAA levels with blood glucose levels and HOMA-IR in patients with T2DM., Conclusion: Our study has shown subtle abnormalities in UAA patterns in patients with T2DM. The measurement of UAA levels and excretory pattern can be used as an index of hyperglycemia and IR which could serve as an inexpensive and non-invasive marker for T2DM. More studies are required to confirm this association., Competing Interests: Declaration of competing interest None of the authors have conflicts of interest associated with the work., (Copyright © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Background: Urolithiasis is the process of stone formation in the urinary tract. Its etiology is only partly known, and efficient therapeutic approaches are currently lacking. Metabolomics is increasingly used in biomarkers discovery for its ability to identify mediators of relevant (patho)physiological processes. Amino acids may be involved in kidney stone formation. The aim of the present study was to investigate the presence of an amino acid signature in stone former urine through a targeted metabolomic approach., Methods: A panel of 35 amino acids and derivatives was assessed in urines from 15 stone former patients and 12 healthy subjects by UPLC-MS. Partial Least Squares Discriminant Analysis (PLS-DA) was used to define amino acid profiles of cases and controls. Results and Discussion. Our approach led to the definition of a specific amino acid fingerprint in people with kidney stones. A urinary amino acid profile of stone formers was characterized by lower levels of α -aminobutyric acid, asparagine, ethanolamine, isoleucine, methionine, phenylalanine, serine, tryptophan, and valine. Metabolomic analysis may lend insights into the pathophysiology of urolithiasis and allow tracking this prevalent condition over time., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Aniello Primiano et al.)

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (R s = 0.9-3.2 for catecholamines and R s = 1.7-2.8 for amino acids) and efficiencies (330-520 ×10 3 t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 μM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: Methylated amino compounds and basic amino acids are important analyte classes with high relevance in nutrition, physical activity and physiology. Reliable and easy quantification methods covering a variety of metabolites in body fluids are a prerequisite for efficient investigations in the field of food and nutrition., Methods: Targeted ultra-performance liquid chromatography/tandem mass spectrometric (UHPLC/MS) analysis was performed using HILIC separation and timed ESI-MRM detection, combined with a short sample preparation. Calibration in urine and blood plasma was achieved by matrix-matched standards, isotope-labelled internal standards and standard addition. The method was fully validated and the performance was evaluated using a subset from the Karlsruhe Metabolomics and Nutrition (KarMeN) study., Results: Within this method, a total of 30 compounds could be quantified simultaneously in a short run of 9 min in both body fluids. This covers a variety of free amino compounds which are present in very different concentrations. The method is easy, precise and robust, and has a broad working range. As a proof of principle, literature-based associations of certain metabolites with dietary intake of respective foods were clearly confirmed in the KarMeN subset., Conclusions: Overall, the method turned out to be well suited for application in nutrition studies, as shown for the example of food intake biomarkers in KarMeN. Application to a variety of questions such as food-related effects or physical activity will support future studies in the context of nutrition and health., (© 2019 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Background: Metabolome profiling is used to identify biomarkers for acute ischemic stroke (AIS). Previous studies compared metabolite profiles in AIS and healthy controls, which did not account for factors that affect metabolome (genetics, medications). This pilot project evaluates the change in metabolite concentrations between the acute and chronic stage of stroke in the same cohort in order to minimize other factors impact., Methods: We performed global metabolome profile on serum of 20 and urine of 12 stroke patients in acute (72 hours) and chronic (3-5.2 months) stage and compared relative peak values using Wilcoxon and orthogonal partial least squares discriminant analysis methods. Chronic stage metabolite concentrations were considered baseline. We performed analysis to identify significantly overrepresented pathways using MetaboAnalyst., Results: Three serum metabolites asparagine (P = .045), tyrosine (P = .015), and xylose (P = .003) had significantly higher concentrations in acute stage. Seven out of top 10 serum metabolites ranked by Wilcoxon test P value were related to amino acid (AA) metabolism. Two urine metabolites glycine (P = .03) and acetylcarnitine (P = .05) had significantly different concentrations in the acute stage. Five of the top 10 urine metabolites related to AA metabolism. We identified 6 significant pathways after false discovery rate correction that were upregulated in the acute stage: (1) Aminoacyl-tRNA synthesis, (2) nitrogen, (3) alanine, aspartate, and glutamate, (4) branched-chain AA, (5) arginine and proline, and (6) phenylalanine metabolism., Conclusion: Longitudinal study design confirms that AA metabolism heavily involved in the pathophysiology of acute brain ischemia. Prospective longitudinal studies with a higher number of participants are needed to establish useful stroke biomarkers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: The design of training programs for football players is not straightforward due to intra- and inter-individual variability that leads to different physiological responses under similar training loads., Objective: To study the association between the external load, defined by variables obtained using electronic performance tracking systems (EPTS), and the urinary metabolome as a surrogate of the metabolic adaptation to training., Methods: Urine metabolic and EPTS data from 80 professional football players collected in an observational longitudinal study were analyzed by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry and assessed by partial least squares (PLS) regression., Results: PLS models identified steroid hormone metabolites, hypoxanthine metabolites, acetylated amino acids, intermediates in phenylalanine metabolism, tyrosine, tryptophan metabolites, and riboflavin among the most relevant variables associated with external load. Metabolic network analysis identified enriched pathways including steroid hormone biosynthesis and metabolism of tyrosine and tryptophan. The ratio of players showing a deviation from the PLS model of adaptation to exercise was higher among those who suffered a muscular lesion compared to those who did not., Conclusions: There was a significant association between the external load and the urinary metabolic profile, with alteration of biochemical pathways associated with long-term adaptation to training. Future studies should focus on the validation of these findings and the development of metabolic models to identify professional football players at risk of developing muscular injuries.

Objective: To determine the pattern of various inherited metabolic disorders specifically through plasma amino acid and urine organic acid testing in high-risk paediatric population.., Methods: The cross-sectional retrospective study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, and comprised data from April 2015 to March 2018 of children referred to the Department of Chemical Pathology and Endocrinology for work-up of suspected inherited metabolic disorders. Complete clinical history, baseline biochemical investigations, plasma amino acid and urine organic acid profiles, where indicated, were collected. Quantitative plasma amino acid and analysis was carried out by Ion Exchange Chromatography on Biochrome 30+ amino acid analyser, and urine organic acid analysis by Gas Chromatography-Mass Spectrometry. Findings were linked to the identified disorders. SPSS 21 was used for data analysis., Results: Of the 805 cases reviewed, 49(6%) had an inherited metabolic disorder. Male:Female ratio of the cases was 1.5:1, and the median age was 240 days (interquartile range: 1-15695 days). The most common presenting symptom was seizures 316(39.3%) followed by lethargy 283(35.2%). Of the diagnosed cases, aminoacidopathies were 28(57%) and in them, non-ketotic hyperglycaemia accounted for 7(25%.). There were 12(24.5%) cases of organic acidurias followed by 9(18.4%) that were other than the two diagnoses., Conclusions: The cases of inherited metabolic disorder detected indicated significant prevalence. Non-ketotic hyperglycinemia was the commonest disorder diagnosed.

Objective: Alzheimer's disease (AD) is a common neurodegenerative disorder with the symptoms of cognitive impairment and decreased learning and memory abilities. Metabolomics can reflect the related functional status and physiological and pathological changes in the process of AD. Moxibustion is a unique method in traditional Chinese medicine, which has been used in the treatment and prevention of diseases for thousands of years., Methods: A total of 32 APP/PS1 mice were randomly divided into the model group, moxibustion group, moxa smoke group and smoke-free moxibustion group (n=8/group), using the random number table method, while eight C57BL/6 mice were used as the control group. The five groups were measured for 20 min/day, 6 days/week, for 4 weeks. After 4 weeks' experiment, all the mice were placed in metabolic cages to collect urine continuously for 24 hours, for UPLC-MS analysis., Results: Principal component analysis (PCA) was used to identify the different metabolites among the five groups, and partial least squares discriminant analysis (PLS-DA) was performed to reveal the effects on the metabolic variance. Sixteen potential biomarkers were identified among the five groups, primarily related to amino acid metabolism, starch metabolism, sucrose metabolism, interconversion of pentose and glucuronate, and aminoacyl biosynthesis. There were 17 differences in the potential metabolites between the control and model groups, involving the metabolism of amino acid, purine, pyrimidine, nicotinic acid and nicotinamide, and biosynthesis of pantothenate and coenzyme A. Fifteen potential biomarkers were identified between the model and moxibustion groups, related to starch metabolism, sucrose metabolism, interconversion of pentose and glucuronate, glyoxylate, dicarboxylate anions and some amino acid metabolism., Conclusion: Moxibustion can regulate the metabolism of substance and energy by improving the synthesis and decomposition of carbohydrates and amino acids in APP/PS1 transgenic AD model mice., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cri du Chat or 5p minus (5p-) syndrome is characterized by a deletion located on the chromosome 5 short (-p) arm and has an incidence rate of 1 in 50,000 individuals worldwide. This disease manifests in disturbances across a range of systems biochemicals. Therefore, a targeted metabolomics analysis was evaluated in patients with 5p- syndrome to help unravel the biochemical changes that occur in this disease. Urine samples were collected from people of both sexes aged 1-38 years old and analyzed by ultra-performance liquid chromatography coupled to mass spectrometry. Student' statistical test, metabolomic pathway analysis and metabolite set enrichment analysis were applied to the data. Alterations of some amino acids and amine biogenics levels were found in Cri du Chat Syndrome individuals. The alteration of most of these metabolites is associated with energy recuperation and glycolysis. In general, we found the catabolism of some metabolic pathways to be affected in 5p- patients., (© 2019 John Wiley & Sons, Ltd.)

Objective: establishing the types and frequency of disembriogenetic stigma in children with joint hypermobility given the clinical and laboratory features, genetic component and endocrine regulation of these disorders in a late period upon the accident., Materials and Methods: Children (n = 109) inhabiting the radiologically contaminated territories and having the connective tissue dysplasia (CTD) signs were involved in the study. Diseases in family history, ossalgia complaints, fractures in a personal history, bone disembriogenetic stigma, joint hypermobility, type of somatic diseases, blood serum biochemical parameters (namely calcium, alkaline phosphatase, total protein, cholesterol, creatinine, iron, ferritin content), serum cortisol, free thyroxine, pituitary thyroid-stimulating hormone (TSH) levels, free amino acid composition in urine and radiation dose were considered., Results: Radiation doses in children having the CTD ranged from (0.37 ± 0.11) mSv to (0.56 ± 0.10) mSv with no difference from that in those without CTD. Joint hypermobility (JHM) correlated with cancer in family history (rs = 0.53) and lower extremity varicose vein disease (rs = 0.40) (p < 0.05). Incidence of ossalgia, easy fatigability, and bone fractures was higher in children with CTD. Anomalies of the dentofacial system were first in line (38.5 %) in these children. Proportion of children with grade II JHM and platypodia was lower (rs = 0.42), but with lower extremity deformations was higher (rs = 0.68) (p < 0.05) vs. in the control group. Iron and ferritin deficiencies both with lymphocytosis were more common in children with CTD than in the comparison group (p < 0.05). The increased content of oxyproline, lysine, proline both with glycine deficiency were detected in children having the CTD, i.e. an imbalance of amino acids from the collagen content was observed featuring a predominance of catabolic processes over anabolic ones. There was a direct correlation between the TSH level and the JHM grade (rs = 0.49), although the values of hormone concentration in these children did not exceed the reference range (maximum values were 3.3 μIU/ml)., Conclusions: The revealed abnormalities in amino acid content, ferrokinetics, and thyroid function indices can affect the collagen formation, organic matrix structure of bone tissue and significantly deregulate the hemato- poiesis. The later can underlie the pathways of haematologic malignancy development., (V. G. Bebeshko, K. M. Bruslova, T. T. Volodina, L. O. Lyashenko, N. M. Tsvetkova, I. V. Trychlib, T. O. Chernysh, V. G. Boyarskiy, L. O. Gonchar, N. V. Kavardakova.)