A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

Elevated circulating soluble interleukin-2 receptor (sCD25) level is associated with prefrontal excitatory-inhibitory imbalance in individuals with chronic pain: A proton MRS study.

Aberrant neuronal excitability in the anterior cingulate cortex (ACC) is implicated in cognitive and affective pain processing. Such excitability may be amplified by activated circulating immune cells, including T lymphocytes, that interact with the central nervous system. Here, we conducted a study of individuals with chronic pain using magnetic resonance spectroscopy (MRS) to investigate the clinical evidence for the interaction between peripheral immune activation and prefrontal excitatory-inhibitory imbalance. In thirty individuals with chronic musculoskeletal pain, we assessed markers of peripheral immune activation, including soluble interleukin-2 receptor alpha chain (sCD25) levels, as well as brain metabolites, including Glx (glutamate + glutamine) to GABA+ (γ-aminobutyric acid + macromolecules/homocarnosine) ratio in the ACC. We found that the circulating level of sCD25 was associated with prefrontal Glx/GABA+. Greater prefrontal Glx/GABA+ was associated with higher pain catastrophizing, evaluative pain ratings, and anxiodepressive symptoms. Further, the interaction effect of sCD25 and prefrontal Glx/GABA+ on pain catastrophizing was significant, indicating the joint association of these two markers with pain catastrophizing. Our results provide the first evidence suggesting that peripheral T cellular activation, as reflected by elevated circulating sCD25 levels, may be linked to prefrontal excitatory-inhibitory imbalance in individuals with chronic pain. The interaction between these two systems may play a role as a potential mechanism underlying pain catastrophizing. Further prospective and treatment studies are needed to elucidate the specific role of the immune and brain interaction in pain catastrophizing.

Causal relationship between circulating glutamine levels and idiopathic pulmonary fibrosis: a two-sample mendelian randomization study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and debilitating respiratory disease with a median survival of less than 5 years. In recent years, glutamine has been reported to be involved in the regulation of collagen deposition and cell proliferation in fibroblasts, thereby influencing the progression of IPF. However, the relationships between glutamine and the incidence, progression, and treatment response of IPF remain unclear. Our study aimed to investigate the relationship between circulating glutamine levels and IPF, as well as its potential as a therapeutic target. METHODS: We performed a comprehensive Mendelian Randomization (MR) analysis using the most recent genome-wide association study summary-level data. A total of 32 single nucleotide polymorphisms significantly correlated to glutamine levels were identified as instrumental variables. Eight MR analysis methods, including inverse variance weighted, MR-Egger, weighted median, weighted mode, constrained maximum likelihood, contamination mixture, robust adjusted profile score, and debiased inverse-variance weighted method, were used to assess the relationship between glutamine levels with IPF. RESULTS: The inverse variance weighted analysis revealed a significant inverse correlation between glutamine levels and IPF risk (Odds Ratio = 0.750; 95% Confidence Interval : 0.592-0.951; P = 0.017). Sensitivity analyses, including MR-Egger regression and MR-PRESSO global test, confirmed the robustness of our findings, with no evidence of horizontal pleiotropy or heterogeneity. CONCLUSION: Our study provides novel evidence for a causal relationship between lower circulating glutamine levels and increased risk of IPF. This finding may contribute to the early identification of high-risk individuals for IPF, disease monitoring, and development of targeted therapeutic strategies.

Glucose feeds the TCA cycle via circulating lactate.

Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of 13C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, 13C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours.

Gut Microbiota and Cardiovascular Disease.

Fecal microbial community changes are associated with numerous disease states, including cardiovascular disease (CVD). However, such data are merely associative. A causal contribution for gut microbiota in CVD has been further supported by a multitude of more direct experimental evidence. Indeed, gut microbiota transplantation studies, specific gut microbiota-dependent pathways, and downstream metabolites have all been shown to influence host metabolism and CVD, sometimes through specific identified host receptors. Multiple metaorganismal pathways (involving both microbe and host) both impact CVD in animal models and show striking clinical associations in human studies. For example, trimethylamine N-oxide and, more recently, phenylacetylglutamine are gut microbiota-dependent metabolites whose blood levels are associated with incident CVD risks in large-scale clinical studies. Importantly, a causal link to CVD for these and other specific gut microbial metabolites/pathways has been shown through numerous mechanistic animal model studies. Phenylacetylglutamine, for example, was recently shown to promote adverse cardiovascular phenotypes in the host via interaction with multiple ARs (adrenergic receptors)-a class of key receptors that regulate cardiovascular homeostasis. In this review, we summarize recent advances of microbiome research in CVD and related cardiometabolic phenotypes that have helped to move the field forward from associative to causative results. We focus on microbiota and metaorganismal compounds/pathways, with specific attention paid to short-chain fatty acids, secondary bile acids, trimethylamine N-oxide, and phenylacetylglutamine. We also discuss novel therapeutic strategies for directly targeting the gut microbiome to improve cardiovascular outcomes.

Potential causal role of l-glutamine in sickle cell disease painful crises: A Mendelian randomization analysis.

In a recent clinical trial, the metabolite l-glutamine was shown to reduce painful crises in sickle cell disease (SCD) patients. To support this observation and identify other metabolites implicated in SCD clinical heterogeneity, we profiled 129 metabolites in the plasma of 705 SCD patients. We tested correlations between metabolite levels and six SCD-related complications (painful crises, cholecystectomy, retinopathy, leg ulcer, priapism, aseptic necrosis) or estimated glomerular filtration rate (eGFR), and used Mendelian randomization (MR) to assess causality. We found a potential causal relationship between l-glutamine levels and painful crises (N = 1278, odds ratio (OR) [95% confidence interval] = 0.68 [0.52-0.89], P = 0.0048). In two smaller SCD cohorts (N = 299 and 406), the protective effect of l-glutamine was observed (OR = 0.82 [0.50-1.34]), although the MR result was not significant (P = 0.44). We identified 66 significant correlations between the levels of other metabolites and SCD-related complications or eGFR. We tested these correlations for causality using MR analyses and found no significant causal relationship. The baseline levels of quinolinic acid were associated with prospectively ascertained survival in SCD patients, and this effect was dependent on eGFR. Metabolomics provide a promising approach to prioritize small molecules that may serve as biomarkers or drug targets in SCD.

Plasma glutamine status at intensive care unit admission: an independent risk factor for mortality in critical illness.

BACKGROUND: A plasma glutamine concentration outside the normal range at Intensive Care Unit (ICU) admission has been reported to be associated with an increased mortality rate. Whereas hypoglutaminemia has been frequently reported, the number of patients with hyperglutaminemia has so far been quite few. Therefore, the association between hyperglutaminemia and mortality outcomes was studied in a prospective, observational study. PATIENTS AND METHODS: Consecutive admissions to a mixed general ICU were eligible. Exclusion criteria were < 18 years of age, readmissions, no informed consent, or a 'do not resuscitate' order at admission. A blood sample was saved within one hour from admission to be analysed by high-pressure liquid chromatography for glutamine concentration. Conventional risk scoring (Simplified Acute Physiology Score and Sequential Organ Failure Assessment) at admission, and mortality outcomes were recorded for all included patients. RESULTS: Out of 269 included patients, 26 were hyperglutaminemic (≥ 930 µmol/L) at admission. The six-month mortality rate for this subgroup was 46%, compared to 18% for patients with a plasma glutamine concentration < 930 µmol/L (P = 0.002). A regression analysis showed that hyperglutaminemia was an independent mortality predictor that added prediction value to conventional admission risk scoring and age. CONCLUSION: Hyperglutaminemia in critical illness at ICU admission was an independent mortality predictor, often but not always, associated with an acute liver condition. The mechanism behind a plasma glutamine concentration outside normal range, as well as the prognostic value of repeated measurements of plasma glutamine during ICU stay, remains to be investigated.

Effects of sevoflurane anesthesia and abdominal surgery on the systemic metabolome: a prospective observational study.

BACKGROUND: Metabolic status can be impacted by general anesthesia and surgery. However, the exact effects of general anesthesia and surgery on systemic metabolome remain unclear, which might contribute to postoperative outcomes. METHODS: Five hundred patients who underwent abdominal surgery were included. General anesthesia was mainly maintained with sevoflurane. The end-tidal sevoflurane concentration (ETsevo) was adjusted to maintain BIS (Bispectral index) value between 40 and 60. The mean ETsevo from 20 min after endotracheal intubation to 2 h after the beginning of surgery was calculated for each patient. The patients were further divided into low ETsevo group (mean - SD) and high ETsevo group (mean + SD) to investigate the possible metabolic changes relevant to the amount of sevoflurane exposure. RESULTS: The mean ETsevo of the 500 patients was 1.60% ± 0.34%. Patients with low ETsevo (n = 55) and high ETsevo (n = 59) were selected for metabolomic analysis (1.06% ± 0.13% vs. 2.17% ± 0.16%, P < 0.001). Sevoflurane and abdominal surgery disturbed the tricarboxylic acid cycle as identified by increased citrate and cis-aconitate levels and impacted glycometabolism as identified by increased sucrose and D-glucose levels in these 114 patients. Glutamate metabolism was also impacted by sevoflurane and abdominal surgery in all the patients. In the patients with high ETsevo, levels of L-glutamine, pyroglutamic acid, sphinganine and L-selenocysteine after sevoflurane anesthesia and abdominal surgery were significantly higher than those of the patients with low ETsevo, suggesting that these metabolic changes might be relevant to the amount of sevoflurane exposure. CONCLUSIONS: Sevoflurane anesthesia and abdominal surgery can impact principal metabolic pathways in clinical patients including tricarboxylic acid cycle, glycometabolism and glutamate metabolism. This study may provide a resource data for future studies about metabolism relevant to general anaesthesia and surgeries. TRIAL REGISTRATION: www.chictr.org.cn . identifier: ChiCTR1800014327 .

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

High Plasma Glutamate and a Low Glutamine-to-Glutamate Ratio Are Associated with Increased Risk of Heart Failure but Not Atrial Fibrillation in the Prevención con Dieta Mediterránea (PREDIMED) Study.

BACKGROUND: Although the association between glutamate and glutamine in relation to cardiometabolic disorders has been evaluated, the role of these metabolites in the development of atrial fibrillation (AF) and heart failure (HF) remains unknown. OBJECTIVES: We examined associations of glutamate, glutamine, and the glutamine-to-glutamate ratio with AF and HF incidence in a Mediterranean population at high cardiovascular disease (CVD) risk. METHODS: The present study used 2 nested case-control studies within the PREDIMED (Prevención con Dieta Mediterránea) study. During ∼10 y of follow-up, there were 509 AF incident cases matched to 618 controls and 326 HF incident cases matched to 426 controls. Plasma concentrations of glutamate and glutamine were semiquantitatively profiled with LC-tandem MS. ORs were estimated with multivariable conditional logistic regression models. RESULTS: In fully adjusted models, per 1-SD increment, glutamate was associated with a 29% (95% CI: 1.08, 1.54) increased risk of HF and glutamine-to-glutamate ratio with a 20% (95% CI: 0.67, 0.94) decreased risk. Glutamine-to-glutamate ratio was also inversely associated with HF risk (OR per 1-SD increment: 0.80; 95% CI: 0.67, 0.94) when comparing extreme quartiles. Higher glutamate concentrations were associated with a worse cardiometabolic risk profile, whereas a higher glutamine-to-glutamate ratio was associated with a better cardiometabolic risk profile. No associations between the concentrations of these metabolites and AF were observed. CONCLUSIONS: Our findings suggest that high plasma glutamate concentrations possibly resulting from alterations in the glutamate-glutamine cycle may contribute to the development of HF in Mediterranean individuals at high CVD risk.This trial was registered at www.isrctn.com as ISRCTN35739639.

Metabolomic changes of blood plasma associated with two phases of rat OIR.

Although acute hyperoxia/hypoxia alternation can shift sharply physiological processes of vessel development, e.g. oxygen induced retinopathy (OIR), very little is known of metabolic products resulted from the neovascularization disorder. In this study, the influence of abnormal oxygen exposures on the plasma metabolomic profiles of rats with OIR was investigated by the gas chromatography mass spectrometry (GC-MS). Rat pups were divided into four groups, each with 12 individuals: (i) reared in room air and sampled at P12 (CT1); (ii) exposed to high oxygen for 5 days and sampled at P12 (HO1, simulating the vaso-obeliteration process (phase I)); (iii) reared in room air and sampled at P17 (CT2); (iv) exposed to high oxygen for 5 days then followed by room air for 5 days and sampled at P17 (HO2, simulating the neovasculization one (phase II)). Plasma samples were analyzed with GC-MS, resulted in 122 metabolite species. Distinct differences in the plasma metabolome were found between groups of CT1 vs. HO1, and HO1 vs. HO2, by using univariate and multivariate analyses. Alternating hyperoxia/hypoxia conditions induced significant changes of richness of proline, ornithine and glutamine, that were important components of 'arginine and proline metabolism' pathway. These metabolites contributed largely to plasma sample classification, determined with receiver operating characteristic curve analysis and were involved profoundly in the proline-dependent production of reactive oxygen species (ROS) related to the cellular redox reactions. Our results from the rat OIR model suggest proline and 'arginine and proline metabolism' pathway as the potential biomarkers for human retinopathy of prematurity (ROP) diagnosis.

The IN-PANCIA Study: Clinical Evaluation of Gastrointestinal Dysfunction and Failure, Multiple Organ Failure, and Levels of Citrulline in Critically Ill Patients.

PURPOSE: Gastrointestinal dysfunction and failure (GID and GIF) in critically ill patients are a common, relevant, and underestimated complications in ICU patients. The aims of this study were (1) to determine plasmatic levels of citrulline, glutamine, and arginine as markers of GID/GIF in critically ill patients with or without GID/GIF with or without multiple organ failure (MOF) and (2) to assess the role of intra-abdominal hypertension in these patient groups. MATERIALS AND METHODS: This is a 1-year, monocentric (Italian hospital), prospective observational study. Inclusion criteria were adult patients with GID/GIF, with or without MOF. The GIF score was daily evaluated in 39 critically ill patients. Amino acids were measured at the time of GID or GIF. RESULTS: We enrolled 39 patients. Nine patients developed GID and 7 GIF; 6 of patients with GID/GIF developed MOF. Citrulline was lower (P < .001) in patients with GID/GIF (11.3 [4.4] µmol/L), compared to patients without GID/GIF (22.4 [6.8] µmol/L); likewise, glutamine was lower in patients with GID/GIF, whereas arginine was nonstatistically different between the 2 groups. Intra-abdominal pressure was higher in patients affected by MOF (13.0 [2.2] mm Hg) than in patients with GIF/GID without MOF (9.6 [2.6] mm Hg) and compared to patients without GID/GIF (7.2 [2.1] mm Hg). CONCLUSIONS: Both GID and GIF in critically ill patients are associated with low levels of citrulline and glutamine, which could be considered as markers of small bowel dysfunction. The higher the GIF score, the lower the citrulline levels. Patients affected by MOF had higher levels of intra-abdominal pressure.

Kinetic and Cross-Sectional Studies on the Genesis of Hypoargininemia in Severe Pediatric Plasmodium falciparum Malaria.

The low bioavailability of nitric oxide (NO) and its precursor, arginine, contributes to the microvascular pathophysiology of severe falciparum malaria. To better characterize the mechanisms underlying hypoargininemia in severe malaria, we measured the plasma concentrations of amino acids involved in de novo arginine synthesis in children with uncomplicated falciparum malaria (UM; n = 61), children with cerebral falciparum malaria (CM; n = 45), and healthy children (HC; n = 109). We also administered primed infusions of l-arginine uniformly labeled with 13C6 and 15N4 to 8 children with severe falciparum malaria (SM; age range, 4 to 9 years) and 7 healthy children (HC; age range, 4 to 8 years) to measure the metabolic flux of arginine, hypothesizing that arginine flux is increased in SM. Using two different tandem mass spectrometric methods, we measured the isotopic enrichment of arginine in plasma obtained at 0, 60, 90, 120, 150, and 180 min during the infusion. The plasma concentrations of glutamine, glutamate, proline, ornithine, citrulline, and arginine were significantly lower in UM and CM than in HC (P ≤ 0.04 for all pairwise comparisons). Of these, glutamine concentrations were the most markedly decreased: median, 457 µM (interquartile range [IQR], 400 to 508 µM) in HC, 300 µM (IQR, 256 to 365 µM) in UM, and 257 µM (IQR, 195 to 320 µM) in CM. Arginine flux during steady state was not significantly different in SM than in HC by the respective mass spectrometric methods: 93.2 µmol/h/kg of body weight (IQR, 84.4 to 129.3 µmol/h/kg) versus 88.0 µmol/h/kg (IQR, 73.0 to 102.2 µmol/h/kg) (P = 0.247) by the two mass spectrometric methods in SM and 93.7 µmol/h/kg (IQR, 79.1 to 117.8 µmol/h/kg) versus 81.0 µmol/h/kg (IQR, 75.9 to 88.6 µmol/h/kg) (P = 0.165) by the two mass spectrometric methods in HC. A limited supply of amino acid precursors for arginine synthesis likely contributes to the hypoargininemia and NO insufficiency in falciparum malaria in children.

Validation of a Metabolite Panel for a More Accurate Estimation of Glomerular Filtration Rate Using Quantitative LC-MS/MS.

BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.

Alteration in NMDAR-related amino acids in first episode psychosis.

The aim of the present study was to explore the role of N-methyl-D-aspartate receptor (NMDAR) related amino acids in drug-naive first episode psychosis (FEP) patients. The medication naïve patients with FEP (n = 40) and healthy volunteers with no family history of schizophrenia (n = 35) were recruited to the study and followed up for 10 weeks. Liquid chromatography-mass spectrometry method was used to measure plasma levels of the amino acids. The plasma glutamine, glutamic acid, proline, serine, asparagine, and hydroxyproline levels were significantly higher in the FEP patients compared to healthy controls (p values < .0001). The glutamine/glutamic acid ratio in FEP patients was not different from the healthy controls (p > .05). After the antipsychotic treatment, plasma glutamic acid, proline, and hydroxyproline levels were significantly increased (p values < .05) while the asparagine level and glutamine/glutamic acid ratio were decreased (p values < .05). The serine and glutamine levels did not show any differences with the treatment (p > .05). The initial plasma glutamine levels were negatively correlated with the initial Scale for the Assessment of Positive Symptoms (SAPS) score (r = -.45, p = .003). The initial plasma proline levels were negatively correlated with the initial and follow-up SAPS scores (r = -.51 and -.39, p values < .05). The initial plasma proline and hydroxyproline levels were both negatively correlated with the initial Brief Psychiatric Rating Scale score (r = -.37, p = .017 and r = -.33, p = .033, respectively). Increase in NMDAR-related amino acid levels during the FEP may be a compensatory response to glutamatergic hypofunction. Their plasma levels were significantly correlated with several psychotic symptoms before and after 10-week treatment. Antipsychotic treatment has differential effects on the plasma levels of these amino acids.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Quantitative characterization of glutaminolysis in human plasma using liquid chromatography-tandem mass spectrometry.

Glutaminolysis is the metabolic pathway that lyses glutamine to glutamate, alanine, citrate, aspartate, and so on. As partially recruiting reaction steps from the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle, glutaminolysis takes essential place in physiological and pathological situations. We herein developed a sensitive, rapid, and reproducible liquid chromatography-tandem mass spectrometry method to determine the perturbation of glutaminolysis in human plasma by quantifying 13 involved metabolites in a single 20-min run. A pHILIC column with a gradient elution system consisting of acetonitrile-5 mM ammonium acetate was used for separation, while an electrospray ionization source (ESI) operated in negative mode with multiple reaction monitoring was employed for detection. The method was fully validated according to FDA's guidelines, and it generally provided good results in terms of linearity (the correlation coefficient no less than 0.9911 within the range of 0.05-800 µg/mL), intra- and inter-day precision (less than 18.38%) and accuracy (relative standard deviation between 89.24 and 113.4%), with lower limits of quantification between 0.05 and 10 µg/mL. The new analytical approach was successfully applied to analyze the plasma samples from 38 healthy volunteers and 34 patients with type 2 diabetes (T2D). Based on the great sensitivity and comprehensive capacity, the targeted analysis revealed the imperceptible abnormalities in the concentrations of key intermediates, such as iso-citrate and cis-aconitate, thus allowing us to obtain a thorough understanding of glutaminolysis disorder during T2D. Graphical abstract ᅟ.

High plasma glutamate and low glutamine-to-glutamate ratio are associated with type 2 diabetes: Case-cohort study within the PREDIMED trial.

BACKGROUND AND AIMS: Glutamate, glutamine are involved in energy metabolism, and have been related to cardiometabolic disorders. However, their roles in the development of type-2 diabetes (T2D) remain unclear. The aim of this study was to examine the effects of Mediterranean diet on associations between glutamine, glutamate, glutamine-to-glutamate ratio, and risk of new-onset T2D in a Spanish population at high risk for cardiovascular disease (CVD). METHODS AND RESULTS: The present study was built within the PREDIMED trial using a case-cohort design including 892 participants with 251 incident T2D cases and 641 non-cases. Participants (mean age 66.3 years; female 62.8%) were non diabetic and at high risk for CVD at baseline. Plasma levels of glutamine and glutamate were measured at baseline and after 1-year of intervention. Higher glutamate levels at baseline were associated with increased risk of T2D with a hazard ratio (HR) of 2.78 (95% CI, 1.43-5.41, P for trend = 0.0002). In contrast, baseline levels of glutamine (HR: 0.64, 95% CI, 0.36-1.12; P for trend = 0.04) and glutamine-to-glutamate ratio (HR: 0.31, 95% CI, 0.16-0.57; P for trend = 0.0001) were inversely associated with T2D risk when comparing extreme quartiles. The two Mediterranean diets (MedDiet + EVOO and MedDiet + mixed nuts) did not alter levels of glutamine and glutamate after intervention for 1 year. However, MedDiet mitigated the positive association between higher baseline plasma glutamate and T2D risk (P for interaction = 0.01). CONCLUSION: Higher levels of glutamate and lower levels of glutamine were associated with increased risk of T2D in a Spanish population at high risk for CVD. Mediterranean diet might mitigate the association between the imbalance of glutamine and glutamate and T2D risk. This trial is registered at http://www.controlled-trials.com, ISRCTN35739639.