Urinary Amino Acids Research Articles

An analysis of urine and serum amino acids in critically ill patients upon admission by means of targeted LC\u2013MS/MS: a preliminary study

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.

Clinical features and amino acid profiles of dogs with hepatocutaneous syndrome or hepatocutaneous-associated hepatopathy.

BackgroundSuperficial necrolytic dermatitis (SND), hepatocutaneous‐associated hepatopathy (HCH), aminoaciduria, and hypoaminoacidemia define hepatocutaneous syndrome (HCS) in dogs. Dogs without SND but that possess all other syndrome components are not well described.Hypothesis/objectivesTo define an inclusive syndrome, aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) for dogs with HCH or HCS. Compare clinical features, salient clinicopathologic variables, and plasma and urine amino acid (AA) profiles among ACHES cases by skin lesion status.AnimalsDogs of various breeds and ages diagnosed with ACHES (n = 41). A control (CON) cohort (n = 12) provided AA profile data.MethodsRetrospective case series. Available medical records of previously identified cases were reviewed for salient clinical features and clinical pathology data. Plasma and urine AA profiles were performed. Cutaneous lesion status was classified as none, mild, or fulminant.ResultsThirty cases (73%) developed SND at some time. Dogs with fulminant skin lesions at diagnosis (n = 22/41, 54%) had significantly lower hematocrit (P = .05) and mean corpuscular volume (P = .01) than dogs without SND. Principal component analysis of plasma AA profiles identified distinct clustering of CON from ACHES dogs, but not by skin lesion status. Plasma 1‐methylhistidine (<7 nmol/mL) and cystathionine (<7.5 nmol/mL) were robust ACHES biomarkers. Urine lysine (>344 nmol/mg creatinine) and methionine (>68 nmol/mg creatinine) also were useful ACHES biomarkers.Conclusions and Clinical ImportanceSpecific AA biomarkers provide additional diagnostic utility in ACHES. Data suggests that HCH is an early stage, and SND a later stage manifestation of ACHES.

Abstract P165: Urinary Amino Acid Profile And Cardiovascular Parameters In Overweight And Obese Brazilian Children

Despite being thoroughly studied, there isn’t complete elucidation of mechanisms underlying obesity, and especially regarding overweight in children. Small molecule metabolites are promising candidates for understanding obesity′s phenotypes. Recent metabolic studies have advanced in discovery of biomarkers for better knowledge of mechanisms related to obesity and its comorbidities. In this cross-sectional study it was evaluated the profile of urinary amino acids of 110 children that were classified in into groups of nutritional status according to their BMI/A (Body Mass Index/age), as normal weight (NW) (n=45), overweight (OW) (n=21) and obese (OB) (n=44). The 24h urine samples were submitted to LC-MS/MS for evaluation of 47 amino acids using Amino Acids Analysis Kit (Zivak®, Turkey), corrected by creatinine concentration. Biochemical profile, cardiovascular parameters and anthropometric measurements were assessed following standard protocols. Girls showed a higher heart rate (G: 82bpm vs B:79bpm, p=0,01 ) than boys. Increased weight presented higher values of systolic blood pressure ( NW: 102.0, OW: 109.0, OB: 110.0; NW vs OW, p = 0.0001; NW vs OB, p = 0.0001 ) and diastolic blood pressure ( NW: 60.0, OW: 67.0, OB: 66.75, NW vs OW, p &lt; 0.0001; NW vs OB, p &lt; 0.0001 ). The following amino acids (expressed in nmol/mg of creatinine) were found in higher concentrations in children’s urine with overweight or obesity, when compared with NW group, glutamine ( NW: 1024.3 vs OW: 1309.2, p=0.05), tryptophan (NW: 85.65, OW:113.55, OB:109.36; NW vs OW p=0.01; OW vs OB p=0.004), phenylalanine (NW: 43.04, OW:49.66, OB:72.08; NW vs OB p=0.01; OW vs OB p=0.04), and Tyrosine (NW: 85.07, OW: 109.74, OB: 140.54; NW vs OB p&lt;0.01; OW vs OB p=0.04). In contrast, sarcosine had reduced concentrations for groups with weight excess (OW: 78.7 vs OB: 62.3, p=0.04). Urine levels of glutamine and aromatic amino acids were higher in OW and OB groups. Tyrosine also showed a positive association with systolic blood pressure values. Thus, there was a positive correlation between levels of glutamine and aromatics amino acids and excess weight, suggesting that they may be candidates to biomarkers for obesity in children. Furthermore, obesity was directly related to increase in blood pressure values.

Acquired transient Fanconi syndrome after feeding jerky treats in dogs - A case series

Between April 2018 and August 2019, 6 dogs with laboratory diagnostic evidence of Fanconi syndrome were presented to the Tierklinik Hofheim. The presumptive diagnosis was confirmed via urine amino acid analysis in all dogs. In 5 of the 6 dogs an alimentary origin was suspected, in 3 dogs the course of the disease could be followed. Supportive therapeutic measures and elimination of jerky treats from the dogs' diet improved the clinical symptoms in all dogs with known follow-up. This is the first case series of dogs with alimentary acquired Fanconi syndrome in Germany. The frequency of occurrence of the disease in that short period of time in only one small animal clinic suggests a significantly higher incidence than currently assumed.

Profile of urinary amino acids and their post-translational modifications (PTM) including advanced glycation end-products (AGEs) of lysine, arginine and cysteine in lean and obese ZSF1 rats

Heart failure with preserved ejection fraction (HFpEF) is associated with high mortality and has an increasing prevalence associated with the demographic change and limited therapeutic options. Underlying mechanisms are largely elusive and need to be explored to identify specific biomarkers and new targets, which mirror disease progression and intervention success. Obese ZSF1 (O-ZSF1) rats are a useful animal model, as they spontaneously develop hypertension, hyperlipidemia and glucose intolerance and finally HFpEF. The urinary profile of amino acids and their metabolites of post-translational modifications (PTM), including the advanced glycation end-products (AGEs) of lysine, arginine and cysteine, are poorly investigated in HFpEF and ZSF1 rats. The aim of the present study was to characterize the status of free amino acids and their metabolites of PTM and glycation in lean ZSF1 (L-ZSF1) and O-ZSF1 rats in urine aiming to find possible effects of glucose on the excretion of native and modified amino acids. In the urine of twelve L-ZSF1 and twelve O-ZFS1 rats collected at the age of 20 weeks, we measured the concentration of native and modified amino acids by reliable previously validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) approaches. Serum glucose was 1.39-fold higher in the O-ZSF1 rats, while urinary creatinine concentration was 2.5-fold lower in the O-ZSF1 rats. We observed many differences in urinary amino acids excretion between L-ZSF1 and O-ZSF1 rats. The creatinine-corrected homoarginine excretion was twofold lower in the O-ZSF1 rats. We also observed distinct associations between the concentrations of serum glucose and urinary amino acids including their PTM and AGE metabolites in the L-ZSF1 and O-ZSF1 rats. Our study shows that PTM metabolites and AGEs are consistently lower in the L-ZSF1 than in the O-ZSF1 rats. Serum malondialdehyde (MDA) concentration was higher in the O-ZSF1 rats. These results suggest that hyperglycemia, hyperlipidemia and elevated oxidative stress in the O-ZSF1 rats favor PTM methylation of arginine and lysine and the glycation of lysine and cysteine. The area under the receiver operation characteristic (ROC) curve values were 0.996 for serum glucose, 0.951 for urinary creatinine, 0.939 for serum MDA, 0.885 for Nε-carboxyethyl-lysine, 0.830 for carboxyethyl-cysteine, and 0.792 for monomethyl-lysine. Non-invasive measurement of methylation and glycation products of arginine, lysine and cysteine residues in proteins in urine of L-ZSF1 and O-ZSF1 rats may be useful in studying pathophysiology and pharmacology of HFpEF.

Plasma and Urinary Amino Acid Profile in Children with Infantile Tremor Syndrome.

Infantile Tremor Syndrome (ITS) is a disorder of infancy, and characterized by developmental delay and/or regression, pallor, skin hyperpigmentation and hypopigmented hair. It is commonly seen in infants in whom exclusive breastfeeding is given inappropriately for longer durations than recommended. ITS is predominantly reported from the Indian subcontinent and in children from a lower socioeconomic background. It is a clinical diagnosis and vitamin B12 deficiency is the most commonly accepted etiology of this entity. The primary objectives of study were to compare the plasma and urine amino acid levels among children with ITS spectrum with those of healthy children. The secondary objectives were to compare the plasma and urine amino acid levels among children with ITS and Pre-ITS. This cross-sectional, observational study was carried out at a tertiary care hospital in North India. A total of 50 children aged < 36 months with ITS/Pre-ITS were enrolled. Children with Pre-ITS and ITS were compared with healthy age-matched study subjects. Thirty-nine (78%) cases and twelve (24%) healthy children had low serum vitamin B12 levels. Folate levels were normal in all the controls, while only one case had folate deficiency. There were significant differences (p < 0.05) in the values of 32 amino acids in plasma. Among 44 urinary amino acids, levels of 30 amino acids were significantly different in the cases compared with the controls (p < 0.05). Several changes in amino acids in the children suffering from ITS were observed. These changes may be a reflection of the metabolic derangements in ITS.

Amino Acid Profile in 18 Patients with Rheumatic Diseases Treated with Glucocorticoids and BCAAs.

The administration of glucocorticoids to patients with rheumatic diseases often results in glucocorticoid-induced myopathy. We previously found that administration of branched-chain amino acids (BCAA) to such patients improves the loss of skeletal muscle, however, their individual differences were often observed. The present study, therefore, aims to identify specific parameters associated with BCAA-induced increases in skeletal muscle mass. Eighteen patients with rheumatic diseases treated with prednisolone were randomly assigned to receive additional BCAAs for 12 wk. Serum biochemistry, plasma fibroblast growth factor (FGF) 19 and 21, and plasma and urinary amino acid concentrations were assessed. The relationship between these parameters and the cross-sectional area (CSA) of the biceps femoris (slow-twitch muscle) and rectus femoris (fast-twitch muscle) was assessed using computed tomography. BCAA supplementation increased serum levels of creatinine and albumin and decreased ammonia and urinary 3-methylhistidine levels. With or without BCAA supplementation, each plasma amino acid concentration decreased during the study period, but the decrease was lower in patients receiving BCAA. Interestingly, a positive correlation was observed between plasma isoleucine, aspartate, and glutamate concentrations and improvement in the biceps femoris muscle atrophy. Plasma amino acid concentrations in patients with rheumatic diseases treated with glucocorticoids decreased despite tapering the dose of glucocorticoids, with a smaller decrease in the BCAA-treated group. Plasma BCAA, aspartic acid, and glutamate concentrations correlated positively with the rate of improvement in biceps femoris muscle atrophy, suggesting that these amino acids are associated with the BCAA-induced increase in muscle mass.

186-OR: Risk Prediction of Early Decline in Renal Function in Diabetic Kidney Disease with Algorithm Including Fractional Excretion of Glycated Amino Acids

Diabetic kidney disease occurs in ca. 40% patients with diabetes. Approximately 1 in 5 patients with type 1 diabetes mellitus (T1DM) develop early decline in renal function (EDRF), requiring renal dialysis after 5 - 20 years. Currently, it is uncertain which patients are at high risk of EDRF. With Joslin Kidney Study investigators, we found patients with T1DM with and without microalbuminuria who later develop EDRF (Decliners) have higher fractional excretion (FE) of 6 glycated amino acids - fructosyl-lysine and 5 advanced glycation endproducts (AGEs), compared to patients with stable renal function (Non-decliners) (Perkins et al., Sci Rep 10, 12709, 2020). No individual FE could classify patients. In this study, we applied artificial intelligence machine learning to develop diagnostic algorithms to classify Decliners and Non-decliners, exploring A1C, log(ACR), 14 glycated and oxidized amino acids in plasma, urine, related FEs and other features. Data from patients with T1DM with stable renal function (n = 52) and EDRF (n = 33) were available. Algorithms were trained and tested on 90%/10% data split, repeated 1000 times, using Extreme Gradient Boosting. Optimum classification required measurement of 3 AGEs: Nꞷ-carboxymethylarginine (CMA) and glyoxal-derived hydroimidazolone (G-H1) in plasma and urine, and N-carboxymethyl-lysine (CML) in plasma; measured in 15 min by liquid chromatography-tandem mass spectrometry. Optimum algorithm features were: A1C, log[ACR], FECMA, FEG-H1 and plasma CML free adduct. The diagnostic performance for risk prediction of future EDRF was (mean ± SD): sensitivity 0.74 ± 0.09, specificity 0.91 ± 0.04 and accuracy 87 ± 4%. The positive likelihood ratio LR+ of 11.0 indicates that this method gives strong, often conclusive evidence of future EDRF in patients with T1DM. With further validation, including in patients with type 2 diabetes mellitus, this method may markedly improve risk prediction of EDRF. Disclosure P. Thornalley: None. A. De la fuente: None. N. Rabbani: None. Funding Qatar Foundation; Qatar University

Chronological Metabolic Response to Intensive Phase TB Therapy in Patients with Cured and Failed Treatment Outcomes.

Despite the arguable success of the standardized tuberculosis (TB) treatment regime, a significant number of patients still present with treatment failure. To improve on current TB treatment strategies, we sought to gain a better understanding of the hosts' response to TB therapy. A targeted metabolomics approach was used to compare the urinary acylcarnitine and amino acid profiles of eventually cured TB patients with those of patients presenting with a failed treatment outcome, comparing these patient groups at the time of diagnosis and at weeks 1, 2, and 4 of treatment. Among the significant metabolites identified were histidine, isoleucine, leucine, methionine, valine, proline, tyrosine, alanine, serine, and γ-aminobutyric acid. In general, metabolite fluctuations in time followed a similar pattern for both groups for most compounds but with a delayed onset or shift of the pattern in the successfully treated patient group. These time-trends detected in both groups could potentially be ascribed to a vitamin B6 deficiency and fluctuations in the oxidative stress levels and urea cycle intermediates, linked to the drug-induced inhibition and stimulation of various enzymes. The earlier onset of observed trends in the failed patients is proposed to relate to genotypic and phenotypic variations in drug metabolizing enzymes, subsequently leading to a poor treatment efficiency either due to the rise of adverse drug reactions or to insufficient concentrations of the active drug metabolites. This study emphasizes the need for a more personalized TB treatment approach, by including enzyme phenotyping and the monitoring of oxidative stress and vitamin B6 levels, for example.

Differential Metabolites in Chinese Autistic Children: A Multi-Center Study Based on Urinary 1H-NMR Metabolomics Analysis.

Background: Autism spectrum disorder (ASD) is a group of early-onset neurodevelopmental disorders. However, there is no valuable biomarker for the early diagnosis of ASD. Our large-scale and multi-center study aims to identify metabolic variations between ASD and healthy children and to investigate differential metabolites and associated pathogenic mechanisms.Methods: One hundred and seventeen autistic children and 119 healthy children were recruited from research centers of 7 cities. Urine samples were assayed by 1H-NMR metabolomics analysis to detect metabolic variations. Multivariate statistical analysis, including principal component analysis (PCA), and orthogonal projection to latent structure discriminant analysis (OPLS-DA), as well as univariate analysis were used to assess differential metabolites between the ASD and control groups. The differential metabolites were further analyzed by receiver operating characteristics (ROC) curve analysis and metabolic pathways analysis.Results: Compared with the control group, the ASD group showed higher levels of glycine, guanidinoacetic acid, creatine, hydroxyphenylacetylglycine, phenylacetylglycine, and formate and lower levels of 3-aminoisobutanoic acid, alanine, taurine, creatinine, hypoxanthine, and N-methylnicotinamide. ROC curve showed relatively significant diagnostic values for hypoxanthine [area under the curve (AUC) = 0.657, 95% CI 0.588 to 0.726], creatinine (AUC = 0.639, 95% CI 0.569 to 0.709), creatine (AUC = 0.623, 95% CI 0.552 to 0.694), N-methylnicotinamide (AUC = 0.595, 95% CI 0.523 to 0.668), and guanidinoacetic acid (AUC = 0.574, 95% CI 0.501 to 0.647) in the ASD group. Combining the metabolites creatine, creatinine and hypoxanthine, the AUC of the ROC curve reached 0.720 (95% CI 0.659 to 0.777). Significantly altered metabolite pathways associated with differential metabolites were glycine, serine and threonine metabolism, arginine and proline metabolism, and taurine and hypotaurine metabolism.Conclusions: Urinary amino acid metabolites were significantly altered in children with ASD. Amino acid metabolic pathways might play important roles in the pathogenic mechanisms of ASD.

Is Spontaneous Hypoglycemia a Feature of XLID Type Nascimento?

Introduction: X-linked intellectual disability (XLID) type Nascimento is a rare, under-diagnosed genetic disorder caused by intragenic mutations in the UBE2A gene. The syndrome was first described in 2006 by Nascimento et el. It is characterized by intellectual disability, dysmorphic facial features, skin anomalies, hypotonia, impaired speech, and seizures. To date, there is no literature to suggest that it affects glucose homeostasis.Case: A 63-year-old male with a history of XLID (mutation in UBE2A pL112P variant), seizures, dementia, stage II CKD, polycystic kidneys, and hyperkalemia presented to the emergency department with altered mental status and blood glucose (BG) of 22 mg/dL. Mental status improved after correction of hypoglycemia.He had multiple admissions for hypoglycemia, however, no history of diabetes mellitus, or use of hypoglycemia inducing medications. Home medications included Patiromer for hyperkalemia. Family history was remarkable for XLID type Nascimento in his brother and sister’s son. Imaging and laboratory studies conducted on prior admissions were inconclusive. Abdominal CT scan showed a 6mm cyst in the head of the pancreas, but no mass. A 72-hour fast after holding dextrose infusion did not yield a clear biochemical differential. BG 51 mg/dL, Insulin <0.5 (3.0–25 mU/L), C-peptide 0.6 (1.1–4.4 ng/mL), pro-insulin 3.2 (0.0–10.0 pmol/L), β-hydroxybutyrate 0.2 (<0.6 mmol/L). BG increased to greater than 25 mg/dL with 1 mg IV glucagon. Insulin antibody was negative (<5 uU/ml). IGF-1, IGF-2 were low at 17 (49–188 ng/mL) and 142 (333-967ng/mL) respectively. Thyroid function and ACTH stimulation test were within normal limits. Aldosterone <0.1(0.0–30 ng/dL), renin 0.195 (0.167–5.38 ng/mL/hr). Urine metabolic screen, urine amino acid screen, plasma amino acid screen and white cell lysosomal enzymes were normal for the nephew and not performed in our patient. The patient’s family requested not pursuing a repeat 72-hour fast, additional imaging or invasive procedures. Dexamethasone 0.5mg orally at bedtime was started which improved blood sugars and patient was discharged. He presented 2 weeks later with recurrent hypoglycemia. Dexamethasone was titrated to 1.5 mg with no improvement. Diazoxide 3mg/kg/day was subsequently started while dexamethasone was tapered and discontinued. Hypoglycemia did not recur.Conclusion: This is the first case report describing spontaneous hypoglycemia in an individual with XLID type Nascimento. Our patient also had hyporeninemic hypoaldosteronism which raises the question of endocrine abnormalities as a manifestation of XLID type Nascimento and if hypoglycemia is an inborn error of metabolism in this patient.

Amino Acid Signature in Urine is Associated With Cardiovascular Risk in Brazilian Children

Childhood obesity is a complex multifactorial disease that contributes to higher cardiometabolic risk. Other important parameter is the pattern of fat distribution, even among normal weight children, those with central fat deposition show higher cardiovascular risk. The waist to height ratio (WHtR) is a useful and practical tool to assess central obesity and cardiovascular risk. Childhood obesity is associated with metabolic abnormalities which are mechanisms underlying cardiovascular diseases. The metabolomics can detect biomarkers for exposure, disease progression and susceptibility contributing to early intervention strategies. In this cross-sectional study it was evaluated the profile of amino acids in urine of children with different cardiovascular risk status according to WHtR. The 110 participants aged from 9 to 10 years were classified according to WHtR into the groups: high cardiovascular risk (n = 48) and normal cardiovascular risk (n = 62). The 24 hours urine samples were collected, and aliquots submitted to LC–MS/MS for evaluation of 47 amino acids using the Amino Acids Analysis Kit (Zivak®, Turkey), values were corrected by creatinine. Biochemical profile, cardiovascular parameters and anthropometric measurements were assessed following standard protocols. The following amino acids were found in higher concentrations in urine of children from high cardiovascular risk group, cystathionine (25 vs 21 nmol/mg of creatinine, p = 0.03), Lys (212 vs 160, p < 0.01) and the aromatic amino acids Tyr (131 vs 90, p < 0.01), Trp (109 vs 95, p =0.02) and Phe (65 vs 46, p = 0.04). Linear regression identified that cystathionine, Lys, Tyr, Trp and Phe presented weak but significant positive correlation with anthropometric measurements. Cystathionine, Trp and Lys negatively correlates with HDL while Phe and Tyr positively correlated with triglycerides. Also, Lys and Tyr were positively associated with higher systolic blood pressure. Findings corroborate to literature and indicate that there is a signature profile of amino acids in urine of children with increased cardiovascular risk associated to central obesity. The augmented Tyr and Lys may be the most significant markers once they were also associated to higher SBP.

Analysis and correlation of urinary amino acids and nutritional status in Brazilian children

Impairment in metabolic pathways is associated with cardiometabolic diseases such as obesity, diabetes and cardiovascular diseases. Obesity, for instance, is a multifactorial disease that is linked to physical, social, psychological and economic burdens being characterized as a health public problem. Childhood obesity is tightly related with obesity and cardiovascular problems in adulthood. Inadequate diet and lifestyle in early childhood can contribute to alterations in the metabolites levels and development of childhood obesity and associated diseases. In this cross-sectional study it was evaluated the profile of amino acids in urine of 110 children that were classified in into three groups of nutritional status according to their BMI (Body Mass Index), as normal weight (NW) (n=46), overweight (OW) (n=22) and obese (OB) (n=45). The 24 hours urine samples were submitted to LC–MS/MS for evaluation of 47 amino acids using the Amino Acids Analysis Kit (Zivak®, Turkey), values were corrected by creatinine concentration. Biochemical profile, cardiovascular parameters and anthropometric measurements were assessed following standard protocols. The following amino acids were found in higher concentrations in urine of children with overweight or obesity, when compared with normal weight group, glutamine (NW: 1024.3 vs OW: 1309.2 nmol/mg of creatinine, p=0.05), tryptophan (NW: 85.65, OW:113.55, OB:109.36 nmol/mg of creatinine; NW vs OW p=0.01; OW vs OB p=0.004), phenylalanine (NW: 43.04, OW:49.66, OB:72.08 nmol/mg of creatinine; NW vs OB p=0.01; OW vs OB p=0.04), Tyrosine (NW: 85.07, OW: 109.74, OB: 140.54 nmol/mg of creatinine; NW vs OB p<0.01; OW vs OB p=0.04).In addition, sarcosine showed reduced concentrations for obese participants (OW: 78.7 vs OB: 62.3, p=0.04). The urine levels of glutamine and of the aromatic amino acids were influenced by nutritional status being higher in OW and OB children. Interestingly, the tryptophan showed a positive correlation with the BMI of the participant's mother. Also, tryptophan andtyrosine were negatively correlated with HDL, while phenylalanine and tyrosine hada positive correlation with triglycerides levels. Tyrosine also showed a positive association with systolic blood pressure values. In our results, the correlation between the concentrations of the aromatic amino acids and the increase in children's obesity levels stands out, and this corroborates with the literature data and can represent markers of obesity.

Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC-MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts.

Analysis of amino acids by gas chromatography-mass spectrometry (GC–MS) requires at least one derivatization step to enable solubility in GC–MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC–MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC–MS using in situ prepared deuterium-labelled methyl ester as internal standard.

Determination of seventeen free amino acids in human urine and plasma samples using quadruple isotope dilution mass spectrometry combined with hydrophilic interaction liquid chromatography – Tandem mass spectrometry

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.

Effects of long-term antibiotic treatment on mice urinary aromatic amino acid profiles.

The gut microbiota–host co-metabolites are good indicators for representing the cross-talk between host and gut microbiota in a bi-direct manner. There is increasing evidence that levels of aromatic amino acids (AAAs) are associated with the alteration of intestinal microbial community though the effects of long-term microbial disturbance remain unclear. Here we monitored the gut microbiota composition and host–microbiota co-metabolites AAA profiles of mice after gentamicin and ceftriaxone treatments for nearly 4 months since their weaning to reveal the relationship between host and microbiome in long- term microbial disturbances. The study was performed employing targeted LC-MS measurement of AAA-related metabolites and 16S RNA sequence of mice cecal contents. The results showed obvious decreased gut microbial diversity and decreased Firmicutes/Bacteroidetes ratio in the cecal contents after long-term antibiotics treatment. The accumulated AAA (tyrosine, phenylalanine and tryptophan) and re-distribution of their downstreaming metabolites that produced under the existence of intestinal flora were found in mice treated with antibiotics for 4 months. Our results suggested that the long-term antibiotic treatment significantly changed the composition of the gut microbiota and destroyed the homeostasis in the intestinal metabolism. And the urinary AAA could be an indicator for exploring interactions between host and gut microbiota.

Diagnostic Approach in Cystinuria: A Case Report

Cystinuria is an, inherited metabolic disorder progressing with recurrent kidney stones due to impaired reabsorption of dibasic amino acids and arises from mutations in the SLC3A1 and SLC7A9 on chromosome 2. Cystine crystals were detected in the urinalysis of a 17-year-old male patient who was investigated for recurrent kidney stones. Because of demonstration of cystine excretion in the urinary amino acid analysis and having positive family history, we suspected Cystinuria Type B and initiated supportive therapy. However, based on the results of molecular analyses his diagnosis was changed as Cystinuria Type A. In conclusion, our final diagnosis was changed according to the molecular analyses but our treatment approach did not change. Therefore we would like to emphasize that, prominent physical examination findings and supportive laboratory test results will be sufficient for the diagnosis of cystinuria.

NAT8 Variants, N-Acetylated Amino Acids, and Progression of CKD.

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. 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 BioMe, 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). 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. 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.

Effects of Aging, Long-Term and Lifelong Exercise on the Urinary Metabolic Footprint of Rats.

Life expectancy has risen in the past decades, resulting in an increase in the number of aged individuals. Exercise remains one of the most cost-effective treatments against disease and the physical consequences of aging. The purpose of this research was to investigate the effects of aging, long-term and lifelong exercise on the rat urinary metabolome. Thirty-six male Wistar rats were divided into four equal groups: exercise from 3 to 12 months of age (A), lifelong exercise from 3 to 21 months of age (B), no exercise (C), and exercise from 12 to 21 months of age (D). Exercise consisted in swimming for 20 min/day, 5 days/week. Urine samples collection was performed at 3, 12 and 21 months of life and their analysis was conducted by liquid chromatography-mass spectrometry. Multivariate analysis of the metabolite data did not show any discrimination between groups at any of the three aforementioned ages. However, multivariate analysis discriminated the three ages clearly when the groups were treated as one. Univariate analysis showed that training increased the levels of urinary amino acids and possibly protected against sarcopenia, as evidenced by the higher levels of creatine in the exercising groups. Aging was accompanied by decreased levels of urinary amino acids and signs of increased glycolysis. Concluding, both aging and, to a lesser degree, exercise affected the rat urinary metabolome, including metabolites related to energy metabolism, with exercise showing a potential to mitigate the consequences of aging.

Abstract 14928: The Factors Impacting on Resting Energy Expenditure in Refractory Heart Failure Patients and the Probability of Urine Amino Acids Analysis as an Indicator for Calorie Intake Insufficiency

Introduction: Cachexia is a critical issue in patients with refractory heart failure (HF). Although the elevation of resting energy expenditure (REE) can progress cachexia through relatively insufficient calorie intake, the detail of REE in HF patients remains uncertain and no useful marker for predicting insufficient calorie intake has been established. This study aimed to assess the REE of HF patients and to evaluate the detectability of serum and urine laboratory markers for insufficient calorie intake. Methods: We measured REE using indirect calorimetry in 69 HF patients (NYHA class II-IV) in our hospital. In 25 out of 69 patients, serum albumin, amino acids, and 24-hour urine amino acids were measured. The calorie intake insufficiency was determined based on measured REE multiplied by activity index Results: As compared to the predicted values by Harris-Benedict equation, 43 patients (63%) had an elevated REE and the mean REE was approximately 4% elevated. There was no significant correlation between the REE and parameters indicating HF severity, including cardiac index on right heart catheterization and BNP level. Only body mass index (BMI) negatively correlated with the REE (r=-0,66, p&lt;0.01). Of 25 patients, 16 (64%) had calorie intake insufficiency. The excreted amount of urine essential amino acids was lower and mostly under normal range, especially significantly decreased in Histidine and Phenylalanine levels in the patients with calorie intake insufficiency compared to those without calorie intake insufficiency, whereas serum levels of albumin and essential amino acids were kept within a normal range in the both. Conclusions: Our findings suggest that the patients with refractory HF, especially having low BMI, are likely to be under inadequate calorie intake due to elevated REE. The calorie intake insufficiency could not be identified with serum levels of albumin nor essential amino acids. Instead, urine essential amino acids would decrease first prior to plasma levels, indicating this measurement would be a useful marker to detect calorie intake insufficiency.