Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.

Plasma concentrations of advanced glycation end-products and colorectal cancer risk in the EPIC study.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the non-enzymatic reaction between amino acids and reducing sugars, or dicarbonyls as intermediate compounds. Experimental studies suggest that AGEs may promote colorectal cancer, but prospective epidemiologic studies are inconclusive. We conducted a case-control study nested within a large European cohort. Plasma concentrations of three protein-bound AGEs-Nε-(carboxy-methyl)lysine (CML), Nε-(carboxy-ethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)-were measured by ultra-performance liquid chromatography-tandem mass spectrometry in baseline samples collected from 1378 incident primary colorectal cancer cases and 1378 matched controls. Multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed using conditional logistic regression for colorectal cancer risk associated with CML, CEL, MG-H1, total AGEs, and [CEL+MG-H1: CML] and [CEL:MG-H1] ratios. Inverse colorectal cancer risk associations were observed for CML (OR comparing highest to lowest quintile, ORQ5 versus Q1 = 0.40, 95% CI: 0.27-0.59), MG-H1 (ORQ5 versus Q1 = 0.73, 95% CI: 0.53-1.00) and total AGEs (OR Q5 versus Q1 = 0.52, 95% CI: 0.37-0.73), whereas no association was observed for CEL. A higher [CEL+MG-H1: CML] ratio was associated with colorectal cancer risk (ORQ5 versus Q1 = 1.91, 95% CI: 1.31-2.79). The associations observed did not differ by sex, or by tumour anatomical sub-site. Although individual AGEs concentrations appear to be inversely associated with colorectal cancer risk, a higher ratio of methylglyoxal-derived AGEs versus those derived from glyoxal (calculated by [CEL+MG-H1: CML] ratio) showed a strong positive risk association. Further insight on the metabolism of AGEs and their dicarbonyls precursors, and their roles in colorectal cancer development is needed.

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.

Asymmetric dimethylation and citrullination of proteinic arginine and homoarginine synthesis in human Helicobacter pylori infection.

The importance of L-arginine (Arg) and relatives, including L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA), in humans infected with Helicobacter pylori (Hp) is little understood. ADMA is produced by asymmetric dimethylation of the guanidine group of Arg residues in certain proteins and is released by proteolysis. High concentrations of circulating free ADMA are considered a risk factor for morbidity and mortality in adults. This risk is considered to arise from the inhibition of the synthesis of nitric oxide (NO), which is a potent vasodilator and inhibitor of platelet aggregation. In the present study, we quantified by stable isotope dilution gas chromatography-mass spectrometry (GC-MS) the concentration of free (f) and total (t) ADMA, Arg, hArg, lysine (Lys) and the sum of citrulline (Cit) and ornithine (Orn) (6 M HCl, 20 h, 110 °C) in serum samples of apparently healthy elderly subjects (n = 27; age, 31-105 years) who were tested for Hp infection. Nine subjects (5 males, 4 females) were found to be Hp seropositive (Hp+) and 18 subjects (8 males, 9 females) were found to be Hp seronegative (Hp‒). Proteinic (p) concentrations were determined by difference. fADMA (0.493 ± 0.068 vs 0.466 ± 0.081 µM, P = 0.382), pADMA (113 ± 73 vs 76 ± 59 nM, P = 0.169) and tADMA (0.606 ± 0.126 vs 0.543 ± 0.121 µM, P = 0.280) serum concentrations were found not to differ between the Hp+ and Hp- subjects. Serum concentrations of fArg (162 ± 30 vs 177 ± 36 µM, P = 0.471), fhArg (1.600 ± 0.638 vs 1.831 ± 0.742 µM, P = 0.554), and fLys (388 ± 170 vs 395 ± 149 µM, P = 0.700) also did not differ statistically between Hp+ and Hp- subjects. tArg (12.4 ± 1.49 vs 13.0 ± 1.33 mM, P = 0.190), tLys (23.0 ± 2.65 vs. 23.9 ± 2.66 mM, P = 0.456) and tCit + Orn (2.53 ± 0.76 vs 2.63 ± 0.85 mM, P = 0.817) did not differ between Hp+and Hp‒ subjects as well. phArg concentration was close to the limit of quantitation of the method (Hp+: 30 ± 210 nM; Hp-: 42 ± 205 nM), suggesting that hArg is virtually absent in serum proteins of the investigated subjects. pCit + Orn did not differ between infected and non-infected subjects. Our study suggests that Hp infection is not associated with elevated asymmetric dimethylation and citrullination of Arg proteins present in the serum or with the hArg synthesis from free Arg in elderly subjects. However, asymmetric Arg dimethylation was found to correlate inversely with Arg citrullination in Hp- (r2 = 0.408, P = 0.004) but not in Hp+ (r2 = 0.065, P = 0.506), with Arg citrullination decreasing and Arg asymmetric dimethylation increasing with subjects' age.

Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC-MS/MS method.

Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20 µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, ß-aminoisobutyric acid (ß-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.

Metabolomic profiles and development of metabolic risk during the pubertal transition: a prospective study in the ELEMENT Project.

OBJECTIVES: (1) Examine associations of a branched-chain amino acid (BCAA) metabolite pattern with metabolic risk across adolescence; (2) use Least Absolute Shrinkage and Selection Operator (LASSO) to identify novel metabolites of metabolic risk. METHODS: We used linear regression to examine associations of a BCAA score with change (∆) in metabolic biomarkers over 5-year follow-up in 179 adolescents 8-14 years at baseline. Next, we applied LASSO, a regularized regression technique well suited for reduction of high-dimensional data, to identify metabolite predictors of ∆biomarkers. RESULTS: In boys, the BCAA score corresponded with decreasing C-peptide, C-peptide-based insulin resistance (CP-IR), total cholesterol (TC), and low-density-lipoprotein cholesterol (LDL). In pubertal girls, the BCAA pattern corresponded with increasing C-peptide and leptin. LASSO identified asparagine as a predictor of decreasing C-peptide (ß = -0.33) and CP-IR (ß = -0.012), and acetyl-carnitine (ß = 2.098), 4-hydroxyproline (ß = -0.050), ornithine (ß = -0.353), and α-aminoisobutyric acid (ß = -0.793) as determinants of TC in boys. In girls, histidine was a negative determinant of TC (ß = -0.033). CONCLUSIONS: The BCAA pattern was associated with ∆glycemia and ∆lipids in a sex-specific manner. LASSO identified asparagine, which influences growth hormone secretion, as a determinant of decreasing C-peptide and CP-IR in boys, and metabolites on lipid metabolism pathways as determinants of decreasing cholesterol in both sexes.

Changes in arginine are inversely associated with type 2 diabetes: A case-cohort study in the PREDIMED trial.

The associations between arginine-based metabolites and incident type 2 diabetes (T2D) are unknown. We employed a case-cohort design, nested within the PREDIMED trial, to examine six plasma metabolites (arginine, citrulline, ornithine, asymmetric dimethylarginine [ADMA], symmetric dimethylarginine [SDMA] and N-monomethyl-l-arginine [NMMA]) among 892 individuals (251 cases) for associations with incident T2D and insulin resistance. Weighted Cox models with robust variance were used. The 1-year changes in arginine (adjusted hazard ratio [HR] per SD 0.68, 95% confidence interval [CI] 0.49, 0.95; Q4 vs. Q1 0.46, 95% CI 0.21, 1.04; P trend = 0.02) and arginine/ADMA ratio (adjusted HR per SD 0.73, 95% CI 0.51, 1.04; Q4 vs. Q1 0.52, 95% CI 0.22, 1.25; P trend = 0.04) were associated with a lower risk of T2D. Positive changes of citrulline and ornithine, and negative changes in SDMA and arginine/(ornithine + citrulline) were associated with concurrent 1-year changes in homeostatic model assessment of insulin resistance. Individuals in the low-fat-diet group had a higher risk of T2D for 1-year changes in NMMA than individuals in Mediterranean-diet groups (P interaction = 0.02). We conclude that arginine bioavailability is important in T2D pathophysiology.

Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control.

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Effective therapeutic intervention in metastatic PCa is undermined by our poor understanding of its molecular aetiology. Defining the mechanisms underlying PCa metastasis may lead to insights into how to decrease morbidity and mortality in this disease. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (AP) are AGEs originating from MG-mediated post-translational modification of proteins at arginine residues. AP is involved in the control of epithelial to mesenchymal transition (EMT), a crucial determinant of cancer metastasis and invasion, whose regulation mechanisms in malignant cells are still emerging. Here, we uncover a novel mechanism linking Glo1 to the maintenance of the metastatic phenotype of PCa cells by controlling EMT by engaging the tumour suppressor miR-101, MG-H1-AP and TGF-ß1/Smad signalling. Moreover, circulating levels of Glo1, miR-101, MG-H1-AP and TGF-ß1 in patients with metastatic compared with non-metastatic PCa support our in vitro results, demonstrating their clinical relevance. We suggest that Glo1, together with miR-101, might be potential therapeutic targets for metastatic PCa, possibly by metformin administration.

Effects of linagliptin on endothelial function and postprandial lipids in coronary artery disease patients with early diabetes: a randomized, placebo-controlled, double-blind trial.

BACKGROUND: Early glucose lowering intervention in subjects with type 2 diabetes mellitus was demonstrated to be beneficial in terms of micro- and macrovascular risk reduction. However, most of currently ongoing cardiovascular outcome trials are performed in subjects with manifest atherosclerosis and long-standing diabetes. Therefore, the aim of this study is to investigate the effects of the dipeptidylpeptidase-4 inhibitor linagliptin in subjects with coronary artery disease (CAD) but early type 2 diabetes mellitus (T2DM) on a set of cardiovascular surrogate measurements. METHODS: In this randomized, placebo-controlled, double-blind, single-center study, we included subjects with early diabetes (postchallenge diabetes (2 h glucose > 200 mg/dl) or T2DM treated with diet only or on a stable dose of metformin monotherapy and an HbA1c < 75 mmol/mol) and established CAD. Participants were randomized to receive either linagliptin (5 mg) once daily orally or placebo for 12 weeks. The primary outcome was the change in flow mediated dilatation (FMD). The secondary objective was to investigate the effect of linagliptin treatment on arginine bioavailability ratios [Global arginine bioavailability ratio (GABR) and arginine to ornithine ratio (AOR)]. Arginine, ornithine and citrulline were measured in serum samples with a conventional usual amino acid analysis technique, involving separation of amino acids by ion exchange chromatography followed by postcolumn continuous reaction with ninhydrin. GABR was calculated by L-arginine divided by the sum of (L-ornithine plus L-citrulline). The AOR was calculated by dividing L-arginine by L-ornithine levels. Group comparisons were calculated by using a two-sample t-test with Satterthwaite adjustment for unequal variances. RESULTS: We investigated 43 patients (21% female) with a mean age of 63.3 ± 8.2 years. FMD at baseline was 3.5 ± 3.1% in the linagliptin group vs. 4.0 ± 2.9% in the placebo group. The change in mean FMD in the linagliptin group was not significantly different compared to the change in the placebo group (0.43 ± 4.84% vs. - 0.45 ± 3.01%; p = 0.486). No significant improvements were seen in the arginine bioavailability ratios (GABR; p = 0.608 and AOR; p = 0.549). CONCLUSION: Linagliptin treatment in subjects with CAD and early T2DM did not improve endothelial function or the arginine bioavailability ratios. Trial registration ClinicalTrials.gov, NCT02350478 ( https://clinicaltrials.gov/ct2/show/NCT02350478 ).

Arginine Metabolism Is Altered in Adults with A-ß + Ketosis-Prone Diabetes.

Background: A-ß + ketosis-prone diabetes (KPD) is a subset of type 2 diabetes in which patients have severe but reversible ß cell dysfunction of unknown etiology. Plasma metabolomic analysis indicates that abnormal arginine metabolism may be involved. Objective: The objective of this study was to determine the relation between gut microbiome and arginine metabolism and the relation between arginine availability and ß cell function in KPD patients compared with control participants. Methods: Kinetics of arginine and related metabolites were measured with stable isotope tracers, and insulin secretory responses to arginine and glucose were determined under euglycemic and hyperglycemic conditions in 6 KPD patients and 6 age-, gender-, and body mass index-matched control participants. Glucose potentiation of arginine-induced insulin secretion was performed in a different set of 6 KPD and 3 control participants. Results: Arginine availability was higher in KPD patients during euglycemia [53.5 ± 4.3 (mean ± SEM) compared with 40.3 ± 2.4 µmol · kg lean body mass (LBM)-1 · h-1, P = 0.03] but declined more in response to hyperglycemia (Δ 10.15 ± 2.6 compared with Δ 3.20 ± 1.3 µmol · kg LBM-1 · h-1, P = 0.041). During hyperglycemia, ornithine flux was not different between groups but after an arginine bolus, plasma ornithine AUC trended higher in KPD patients (3360 ± 294 compared with 2584 ± 259 min · µmol · L-1, P = 0.08). In both euglycemia and hyperglycemia, the first-phase insulin responses to glucose stimulation were lower in KPD patients (euglycemic insulin AUC 282 ± 108 compared with 926 ± 257 min · µU · mL-1, P = 0.02; hyperglycemic insulin AUC 358 ± 79 compared with 866 ± 292 min · µU · mL-1, P = 0.05), but exogenous arginine restored first-phase insulin secretion in KPD patients to the level of control participants. Conclusion: Compared with control participants, KPD patients have increased arginine availability in the euglycemic state, indicating a higher requirement. This is compromised during hyperglycemia, with an inadequate supply of arginine to sustain metabolic functions such as insulin secretion. Exogenous arginine administration restores a normal insulin secretory response.

Altered metabolites in newborns with persistent pulmonary hypertension.

BACKGROUND: There is an emerging evidence that pulmonary hypertension is associated with amino acid, carnitine, and thyroid hormone aberrations. We aimed to characterize metabolic profiles measured by the newborn screen (NBS) in infants with persistent pulmonary hypertension of the newborn (PPHN) METHODS: Nested case-control study from population-based database. Cases were infants with ICD-9 code for PPHN receiving mechanical ventilation. Controls receiving mechanical ventilation were matched 2:1 for gestational age, sex, birth weight, parenteral nutrition administration, and age at NBS collection. Infants were divided into derivation and validation datasets. A multivariable logistic regression model was derived from candidate metabolites, and the area under the receiver operator characteristic curve (AUROC) was generated from the validation dataset. RESULTS: We identified 1076 cases and 2152 controls. Four metabolites remained in the final model. Ornithine (OR 0.32, CI 0.26-0.41), tyrosine (OR 0.48, CI 0.40-0.58), and TSH 0.50 (0.45-0.55) were associated with decreased odds of PPHN; phenylalanine was associated with increased odds of PPHN (OR 4.74, CI 3.25-6.90). The AUROC was 0.772 (CI 0.737-0.807). CONCLUSIONS: In a large, population-based dataset, infants with PPHN have distinct, early metabolic profiles. These data provide insight into the pathophysiology of PPHN, identifying potential therapeutic targets and novel biomarkers to assess the response.

Starch synthase IIIa and starch branching enzyme IIb-deficient mutant rice line ameliorates pancreatic insulin secretion in rats: screening and evaluating mutant rice lines with antidiabetic functionalities.

Diabetes mellitus is a metabolic disease spreading worldwide that has been reported to worsen the development and progression of other diseases (cancer, vascular diseases and dementia). To establish functional rice lines with anti-postprandial hyperglycaemic effects, we developed mutant rice lines, which lack one or two gene(s) related to starch synthesis, and evaluated their effects. Powder of mutant rice lines or other grains was loaded to rats fasted overnight (oral grain powder loading test). Incremental area under time-concentration curves (iAUC) were calculated with monitored blood glucose levels. Rice lines with anti-postprandial hyperglycaemic effects were separated by cluster analysis with calculated iAUC. A double mutant rice #4019 (starch synthase IIIa (ss3a)/branching enzyme IIb (be2b)), one of the screened mutant rice lines, was fed to Goto-Kakizaki (GK) rats, an animal model for type 2 diabetes, for 5 weeks. Plasma levels of C-peptide, a marker of pancreatic insulin secretion, were measured with ELISA. For in vitro study, a rat pancreatic cell line was cultured with a medium containing rat serum which was sampled from rats fed #4019 diet for 2 d. After 24-h of incubation, an insulin secretion test was performed. Through the oral rice powder loading test, seven rice lines were identified as antidiabetic rice lines. The intake of #4019 diet increased plasma C-peptide levels of GK rats. This result was also observed in vitro. In rat serum added to cell medium, ornithine was significantly increased by the intake of #4019. In conclusion, the mutant rice #4019 promoted pancreatic insulin secretion via elevation of serum ornithine levels.

Alterations in plasma L-arginine and methylarginines in heart failure and after heart transplantation.

OBJECTIVE: Endothelial function, including the nitric oxide (NO)-pathway, has previously been extensively investigated in heart failure (HF). In contrast, studies are lacking on the NO pathway after heart transplantation (HT). We therefore investigated substances in the NO pathway prior to and after HT in relation to hemodynamic parameters. DESIGN: 12 patients (median age 50.0 yrs, 2 females), heart transplanted between June 2012 and February 2014, evaluated at our hemodynamic lab, at rest, prior to HT, as well as four weeks and six months after HT were included. All patients had normal left ventricular function post-operatively and none had post-operative pulmonary hypertension or acute cellular rejection requiring therapy at the evaluations. Plasma concentrations of ADMA, SDMA, L-Arginine, L-Ornithine and L-Citrulline were analyzed at each evaluation. RESULTS: In comparison to controls, the plasma L-Arginine concentration was low and ADMA high in HF patients, resulting in low L-Arginine/ADMA-ratio pre-HT. Already four weeks after HT L-Arginine was normalized whereas ADMA remained high. Consequently the L-Arginine/ADMA-ratio improved, but did not normalize. The biomarkers remained unchanged at the six-month evaluation and the L-Arginine/ADMA-ratio correlated inversely to pulmonary vascular resistance (PVR) six months post-HT. CONCLUSIONS: Plasma L-Arginine concentrations normalize after HT. However, as ADMA is unchanged, the L-Arginine/ADMA-ratio remained low and correlated inversely to PVR. Together these findings suggest that (i) the L-Arginine/ADMA-ratio may be an indicator of pulmonary vascular tone after HT, and that (ii) NO-dependent endothelial function is partly restored after HT. Considering the good postoperative outcome, the biomarker levels may be considered "normal" after HT.

Identification of an argpyrimidine-modified protein in human red blood cells from schizophrenic patients: A possible biomarker for diseases involving carbonyl stress.

Argpyrimidine (ARP) is an advanced glycation end product thought to be generated from a reaction between methylglyoxal and arginine residues in proteins. In this study, we observed marked accumulation of an approximately 56 kD protein, reactive to anti-ARP antibodies, in the red blood cells (RBCs) of some patients with refractory schizophrenia. This ARP-modified protein was purified from the blood of schizophrenic patients and identified as selenium binding protein 1 (SBP1) by LC-MS/MS. This is the first report of ARP-modified proteins accumulating in RBCs of patients with diseases involving carbonyl stress. We also observed high accumulation of ARP-modified SBP1 in the RBCs of patients with chronic kidney disease. Therefore, this modified protein may be a novel marker of carbonyl stress.

The LargPAD Trial: Phase IIA evaluation of l-arginine infusion in patients with peripheral arterial disease.

OBJECTIVE: Endothelial function is improved by l-arginine (l-arg) supplementation in preclinical and clinical studies of mildly diseased vasculature; however, endothelial function and responsiveness to l-arg in severely diseased arteries is not known. Our objective was to evaluate the acute effects of catheter-directed l-arg delivery in patients with chronic lower extremity ischemia secondary to peripheral arterial disease. METHODS: The study enrolled 22 patients (45% male) with peripheral arterial disease (mean age, 62 years) requiring lower extremity angiography. Endothelium-dependent relaxation of patent but atherosclerotic superficial femoral arteries was measured using a combination of intravascular ultrasound (IVUS) imaging and a Doppler FloWire (Volcano Corporation, Rancho Cordova, Calif) during the infusion of incremental acetylcholine (10-6 to 10-4 molar concentration) doses. Patients received 50 mg (n = 3), 100 mg (n = 10), or 500 mg (n = 9) l-arg intra-arterially, followed by repeat endothelium-dependent relaxation measurement (limb volumetric flow). IVUS-derived virtual histology of the culprit vessel was also obtained. Endothelium-independent relaxation was measured using a nitroglycerin infusion. Levels of nitrogen oxides and arginine metabolites were measured by chemiluminescence and mass spectrometry, respectively. RESULTS: Patients tolerated limb l-arg infusion well. Serum arginine and ornithine levels increased by 43.6% ± 13.0% and 23.2% ± 10.3%, respectively (P < .005), and serum nitrogen oxides increased by 85% (P < .0001) after l-arg infusion. Average vessel area increased by 6.8% ± 1.3% with l-arg infusion (acetylcholine 10-4; P < .0001). Limb volumetric flow increased in all patients and was greater with l-arg supplementation by 130.9 ± 17.6, 136.9 ± 18.6, and 172.1 ± 24.8 mL/min, respectively, for each cohort. Maximal effects were seen with l-arg at 100 mg (32.8%). Arterial smooth muscle responsiveness to nitroglycerin was intact in all vessels (endothelium-independent relaxation, 137% ± 28% volume flow increase). IVUS-derived virtual histology indicated plaque volume was 14 ± 1.3 mm3/cm, and plaque stratification revealed a predominantly fibrous morphology (46.4%; necrotic core, 28.4%; calcium, 17.4%; fibrolipid, 6.6%). Plaque morphology did not correlate with l-arg responsiveness. CONCLUSIONS: Despite extensive atherosclerosis, endothelial function in diseased lower extremity human arteries can be enhanced by l-arg infusion secondary to increased nitric oxide bioactivity. Further studies of l-arg as a therapeutic modality in patients with endothelial dysfunction (ie, acute limb ischemia) are warranted.

Advanced glycation end products in children with type 1 diabetes and early reduced diastolic heart function.

BACKGROUND: Reduced diastolic function is an early sign of diabetes cardiomyopathy in adults and is associated with elevated levels of HbA1c and advanced glycation end products (AGEs). OBJECTIVE: To assess the associations between early reduced diastolic function and elevated levels of HbA1c and AGEs in children and adolescents with type 1 diabetes (T1D). METHODS: One hundred fourty six T1D patients (age 8-18 years) without known diabetic complications were examined with tissue Doppler imaging and stratified into two groups according to diastolic function. A clinical examination and ultrasound of the common carotid arteries were performed. Methylglyoxal-derived hydroimidazolone-1 (MG-H1) was measured by immunoassay. RESULTS: At inclusion, 36 (25%) participants were stratified into a low diastolic function group (E'/A'-ratio < 2.0). Compared to the rest of the T1D children, these participants had higher body mass index (BMI), 22.8 (SD = 4.0) vs. 20.1 (SD = 3.4) kg/m2, p < 0.001, higher systolic blood pressure 104.2 (SD = 8.7) vs. 99.7 (SD = 9.3) mmHg, p = 0.010, and higher diastolic blood pressure, 63.6 (SD = 8.3) vs. 59.9 (SD = 7.9) mmHg, p = 0.016. The distensibility coefficient was lower, 0.035 (SD = 0.010) vs. 0.042 (SD = 0.02) kPa-1, p = 0.013, Young's modulus higher, 429 (SD = 106) vs. 365 (SD = 143), p = 0.009, and MG-H1 higher, 163.9 (SD = 39.2) vs. 150.3 (SD = 33.4) U/ml, p = 0.046. There was no difference in carotid intima-media thickness between the groups. There were no associations between reduced diastolic function and years from diagnosis, HBA1c, mean HBA1c, CRP or calculated glycemic burden. Logistic regression analysis showed that BMI was an independent risk factor for E'/A'-ratio as well as a non-significant, but relatively large effect size for MG-H1, indicating a possible role for AGEs. CONCLUSIONS: Early signs of reduced diastolic function in children and adolescents with T1D had higher BMI, but not higher HbA1c. They also had elevated serum levels of the advanced glycation end product MG-H1, higher blood pressure and increased stiffness of the common carotid artery, but these associations did not reach statistical significance when tested in a logistic regression model.

The Association Between Musculoskeletal Pain and Circulating Ornithine: A Population-Based Study.

Objective: Based on several previous clinical studies, we hypothesized that ornithine levels are different among subjects with persistent musculoskeletal pain compared with other subjects in the population. Design: The study sample consisted of 221 adults with nonpersistent pain, 76 with persistent pain, and 61 with no pain. Concentrations of glutamic acid, ornithine, citrulline, arginine, proline, and spermidine were analyzed using a mass spectrometer. Setting: Lapinlahti municipality in Finland. Results: For the subjects with no pain, nonpersistent pain, and persistent pain, the ornithine concentrations for men were 85.3 µmol/L (SD = 28.9 µmol/L), 98.9 µmol/L (SD = 37.8 µmol/L), and 102.1 µmol/L (SD = 37.1 µmol/L; P = 0.033), respectively. The corresponding concentrations for women were 82.8 µmol/L (SD = 25.2 µmol/L), 83.7 µmol/L (SD = 27.8 µmol/L), and 103.2 µmol/L (SD = 34.9 µmol/L; P = 0.0031). There were no significant differences between the pain groups for any of the other investigated amino acids. Relative sex-specific ornithine concentration adjusted for age, glomerular filtration rate, smoking, body mass index, physical activity, and depressive symptoms was associated with pain ( P = 0.025), the ornithine level being higher in the persistent pain group than in the no pain ( P = 0.006) and nonpersistent pain ( P = 0.032) groups. Conclusion: Ornithine levels are elevated in general population subjects with persistent pain.

High serum level of methylglyoxal-derived AGE, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine, independently relates to renal dysfunction.

BACKGROUND: The dicarbonyl methylglyoxal reacts primarily with arginine residues to form advanced glycation end products, including Nδ-(5-hydro-5-methyl-4 -imidazolone-2-yl)-ornithine (MG-H1), which are risk factors for not only diabetic complications but also lifestyle-related disease including renal dysfunction. However, the data on serum level and clinical significance of this substance in chronic kidney disease are limited. METHODS: Serum levels of MG-H1 and Nε-(carboxymethyl) lysine (CML) in 50 patients with renal dysfunction were measured by liquid chromatography/triple-quadruple mass spectrometry. RESULTS: The median serum MG-H1 levels in patients with estimated glomerular filtration rate (eGFR) of ≥30, 15-30, and <15 ml/min/1.73 m2 was 4.16, 12.58, and 14.66 mmol/mol Lys, respectively (p > 0.05). On the other hand, MG-H1 levels in patients with HbA1c of <6 and ≥6 % was 12.85 and 10.45 mmol/mol Lys, respectively, the difference between which is not significant. In logistic regression analysis, decreased renal function (eGFR <15 ml/min/1.73 m2) significantly associated with high serum levels of MG-H1 [odds ratio: 9.39 (95 % confidence interval 1.528-57.76), p = 0.015; Spearman rank correlation: MG-H1 vs. eGFR, r = -0.691, p < 0.01]. In contrast, the serum level of CML did not correlate with eGFR, but correlated with systolic blood pressure [odds ratio 16.17 (95 % confidence interval 1.973-132.5), p = 0.010; Spearman rank correlation coefficient: CML vs. eGFR, r = 0.454, p < 0.01]. CONCLUSION: These results showed that the serum concentration of MG-H1 was strongly related to renal function rather than to DM.

Decreased Rate of Plasma Arginine Appearance in Murine Malaria May Explain Hypoargininemia in Children With Cerebral Malaria.

BACKGROUND: Plasmodium infection depletes arginine, the substrate for nitric oxide synthesis, and impairs endothelium-dependent vasodilation. Increased conversion of arginine to ornithine by parasites or host arginase is a proposed mechanism of arginine depletion. METHODS: We used high-performance liquid chromatography to measure plasma arginine, ornithine, and citrulline levels in Malawian children with cerebral malaria and in mice infected with Plasmodium berghei ANKA with or without the arginase gene. Heavy isotope-labeled tracers measured by quadrupole time-of-flight liquid chromatography-mass spectrometry were used to quantify the in vivo rate of appearance and interconversion of plasma arginine, ornithine, and citrulline in infected mice. RESULTS: Children with cerebral malaria and P. berghei-infected mice demonstrated depletion of plasma arginine, ornithine, and citrulline. Knock out of Plasmodium arginase did not alter arginine depletion in infected mice. Metabolic tracer analysis demonstrated that plasma arginase flux was unchanged by P. berghei infection. Instead, infected mice exhibited decreased rates of plasma arginine, ornithine, and citrulline appearance and decreased conversion of plasma citrulline to arginine. Notably, plasma arginine use by nitric oxide synthase was decreased in infected mice. CONCLUSIONS: Simultaneous arginine and ornithine depletion in malaria parasite-infected children cannot be fully explained by plasma arginase activity. Our mouse model studies suggest that plasma arginine depletion is driven primarily by a decreased rate of appearance.

Identification of arginine and its "Downstream" molecules as potential markers of breast cancer.

Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide. Arginine is a semiessential amino acid in humans and is essential for several biological pathways in malignant and normal cells, such as ornithine and N1, N12-diacetylspermine (DiAcSpm). This study aimed to determine the role of arginine and these downstream molecules in BC. Plasma arginine, ornithine, and arginine-to-ornithine ratio (AOR) were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples were measured by the colloid gold aggregation to test determination of urinary diAcSpm. A principal component analysis was performed to evaluate the results observed between breast tumor and control characteristics. Differences in individual metabolite concentrations between BC patients and controls were tested by receiver operating characteristics (ROC) analyses. Student's t tests were used to detect the differences between two groups of normally distributed variables, and Wilcoxon sign rank tests were performed for asymmetrically distributed variables. As we analyzed, BC patients had lower plasma arginine and arginine/ornithine level, and higher plasma ornithine and urinary DiAcSpm concentrations as compared with control patients (P = 0.028, 0.020, 0.002, and 0.011, respectively). And the ROC curve was drawn and the area under the curve of the metabolites was calculated to be 0.659 (P = 0.028), 0.645 (P = 0.045), 0.7233 (P = 0.002), 0.683 (P = 0.011), respectively. In addition, our analysis showed that arginine concentrations and AOR had a positive correlation with ER status, while ornithine had a negative correlation with T stage (P = 0.042, 0.023, respectively).In conclusion, arginine and these downstream molecules were biomarkers for BC. More studies are needed to highlight the theoretical strengths. © 2016 IUBMB Life, 68(10):817-822, 2016.