Sensitive and Reproducible Mass Spectrometry-Compatible RP-UHPLC Analysis of Tricarboxylic Acid Cycle and Related Metabolites in Biological Fluids: Application to Human Urine.

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

Urinary organic acids as biomarkers in the assessment of pulmonary function in children with asthma.

Childhood asthma prevalence continues to rise despite advancements in prevention and medical management strategies. The purpose of this study was to investigate correlations between urinary organic acids and pulmonary diagnostic tests, asthma control in Greek asthmatic children. We hypothesized that urinary organic acids are positively associated with poor pulmonary function in children with asthma. Seventy-two children, 5 to 12 years old with asthma were recruited from a pediatric asthma clinic in Athens, Greece. Pulmonary function was assessed using spirometry and exhaled nitric oxide analysis. Asthma control was measured qualitatively using the Asthma Control Questionnaire. Targeted metabolomic analysis of 34 urinary organic acids in children was conducted by gas chromatography-mass spectrometry. A statistically significant difference between girls and boys was found for asthma control score (P = .02), lactic acid (P = .03), but not for any other organic acids (P > .05). Statistically significant correlations were found between lactic acid and Forced Expiratory Volume in 1 second (FEV1) (P = .02), Forced Vital Capacity (FVC) (P = .03); 4- hydroxyphenylacetic acid and FEV1 (P = .01), FVC (P = .01); 5-hydroxyindoleacetic acid and FEV1/FVC (P = .03), eNO (P = .05); glycolic acid with Peak Expiratory Flow (PEF) (P = .03); and malic acid with asthma control (P = .02). In conclusion, metabolomics was used to determine correlations between urinary organic acids and conventional pulmonary diagnostic tests in Greek asthmatic children. Metabolomics could be a promising approach for asthma research and in detection of novel biomarkers for asthma monitoring and therapeutic targets for childhood asthma. This study contributes towards a better understanding of the biochemical pathways involved in asthma.

Coconut Water: An Unexpected Source of Urinary Citrate.

PURPOSE: Coconut water has long been touted for its medicinal qualities including natural hydration. We sought to determine whether its consumption would induce changes to urinary lithogenic factors beyond changes in urine volume. MATERIALS AND METHODS: After Institutional Review Board approval, volunteers with no prior history of nephrolithiasis were recruited. Each participant was randomized initially to either the coconut water or the water phase of the study. Participants kept meticulous food and fluid intake logs during the first phase of the study and were asked to replicate that diet for the second phase. For each phase the participant consumed 2L of either Taste of Nirvana® pure coconut water or tap water daily for four days. Participants were not restricted to consume additional fluid of their choice during their assigned study phase. During days 3 and 4 of each phase the participant collected a 24-hour urine specimen. Coconut water citrate and malate content were measured and were used along with the beverage pH to calculate the total alkali content of the coconut water. Supersaturation levels were calculated using Equil2. Nonparametric paired analysis using the Wilcoxon test was performed for statistical analysis. RESULTS: There were 4 adult male and 4 adult female participants. Each individual's 24-hour urine collection had a creatinine excretion within 20% of the mean for each subject's four samples corroborating that all samples were collected properly. The two samples from each phase for each individual were averaged. The coconut water itself was also analyzed and it was calculated to have a total alkali content of 13.8 mEq/L. Consumption of coconut water significantly increased urinary citrate (29%, p=0.02), urinary potassium (130%, p=0.01), and urinary chloride (37%, p=0.03), without affecting urine pH (p=0.16) or volume beyond that of tap water (p=1.00). CONCLUSIONS: Coconut water consumption increases urinary potassium, chloride, and citrate in nonstone forming individuals.

Urine Tricarboxylic Acid Cycle Metabolites Predict Progressive Chronic Kidney Disease in Type 2 Diabetes.

Context: Metabolites in the tricarboxylic acid (TCA) cycle are not only involved in energy metabolism but also play important roles in non-energy production activities. Objective: To study whether baseline urine key TCA cycle metabolites (lactate, pyruvate, citrate, α-ketoglutaric acid, succinate, fumarate, and malate) independently predict risk of chronic kidney disease (CKD) progression [fast estimated glomerular filtration rate (eGFR) decline] in individuals with type 2 diabetes mellitus (T2DM). Design: One discovery and one validation nested case-control studies in two independent T2DM cohorts. Setting and Participants: Subjects with T2DM were recruited and followed in a regional hospital and at a primary care facility. Main Outcome Measures: eGFR trajectory (slope) was estimated by linear regression. Progressive CKD was defined as eGFR decline of ≥5 mL/min/1.73 m2 per year. Results: As compared with those with stable renal function (n = 271), participants who experienced progressive CKD (n = 116) had a lower level of urine citrate but significantly higher levels of lactate, fumarate, and malate levels at baseline. Both fumarate and malate predicted progressive CKD independent of traditional cardio-renal risk factors, including eGFR and albuminuria. Fumarate interacted with sex (P for interaction = 0.03) and independently predicted progressive CKD in male but not female participants. All these findings were reproducible in a validation study (case n = 96, control n = 402). Exploratory analysis suggested that fumarate might partially mediate the effect of oxidative stress on CKD progression. Conclusions: Key TCA cycle metabolites, especially fumarate, may be involved in the pathophysiologic pathway independent of traditional cardio-renal risk factors, leading to CKD progression in patients with T2DM.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.

Anaplerotic treatment of long-chain fat oxidation disorders with triheptanoin: Review of 15 years Experience.

BACKGROUND: The treatment of long-chain mitochondrial ß-oxidation disorders (LC-FOD) with a low fat-high carbohydrate diet, a diet rich in medium-even-chain triglycerides (MCT), or a combination of both has been associated with high morbidity and mortality for decades. The pathological tableau appears to be caused by energy deficiency resulting from reduced availability of citric acid cycle (CAC) intermediates required for optimal oxidation of acetyl-CoA. This hypothesis was investigated by diet therapy with carnitine and anaplerotic triheptanoin (TH). METHODS: Fifty-two documented LC-FOD patients were studied in this investigation (age range: birth to 51 years). Safety monitoring included serial quantitative measurements of routine blood chemistries, blood levels of carnitine and acylcarnitines, and urinary organic acids. RESULTS: The average frequency of serious clinical complications were reduced from ~60% with conventional diet therapy to 10% with TH and carnitine treatment and mortality decreased from ~65% with conventional diet therapy to 3.8%. Carnitine supplementation was uncomplicated. CONCLUSION: The energy deficiency in LC-FOD patients was corrected safely and more effectively with the triheptanoin diet and carnitine supplement than with conventional diet therapy. Safe intervention in neonates and infants will permit earlier intervention following pre-natal diagnosis or diagnosis by expanded newborn screening.

Combined D2-/L2-hydroxyglutaric aciduria (SLC25A1 deficiency): clinical course and effects of citrate treatment.

Combined D,L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182) is a rare neurometabolic disorder clinically characterized by muscular hypotonia, severe neurodevelopmental dysfunction, and intractable seizures associated with respiratory distress. Biochemically, DL-2HGA patients excrete increased amounts of D- and L-2-hydroxyglutarate (D2HG and L2HG, respectively), with predominance of D2HG, and α-ketoglutarate, and show a decrease in urinary citrate. Impaired function of the mitochondrial citrate carrier (CIC) due to pathogenic mutations within the SLC25A1 gene has been identified as the underlying molecular cause of the disease. CIC mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Thus, depletion of cytosolic citrate as well as accumulation of citrate inside mitochondria have been considered to play a role in the pathophysiology of DL-2HGA. Here, we report for the first time on a patient with a genetically confirmed diagnosis of DL-2HGA and treatment with either malate or citrate. During malate treatment, urinary malate concentration increased, but beyond that, neither biochemical nor clinical alterations were observed. In contrast, treatment with citrate led to an increased urinary excretion of TCA cycle intermediates malate and succinate, and by trend to an increased concentration of urinary citrate. Furthermore, excretion of D2HG and L2HG was reduced during citrate treatment. Clinically, the patient showed stabilization with regard to frequency and severity of seizures. Treating DL-2HGA with citrate should be considered in other DL-2HGA patients, and its effects should be studied systematically.

GC/TOFMS analysis of metabolites in serum and urine reveals metabolic perturbation of TCA cycle in db/db mice involved in diabetic nephropathy.

Early diagnosis of diabetic nephropathy (DN) is difficult although it is of crucial importance to prevent its development. To probe potential markers and the underlying mechanism of DN, an animal model of DN, the db/db mice, was used and serum and urine metabolites were profiled using gas chromatography/time-of-flight mass spectrometry. Metabolic patterns were evaluated based on serum and urine data. Principal component analysis of the data revealed an obvious metabonomic difference between db/db mice and controls, and db/db mice showed distinctly different metabolic patterns during the progression from diabetes to early, medium, and later DN. The identified metabolites discriminating between db/db mice and controls suggested that db/db mice have perturbations in the tricarboxylic acid cycle (TCA, citrate, malate, succinate, and aconitate), lipid metabolism, glycolysis, and amino acid turnover. The db/db mice were characterized by acidic urine, high TCA intermediates in serum at week 6 and a sharp decline thereafter, and gradual elevation of free fatty acids in the serum. The sharp drop of serum TCA intermediates from week 6 to 8 indicated the downregulated glycolysis and insulin resistance. However, urinary TCA intermediates did not decrease in parallel with those in the serum from week 6 to 10, and an increased portion of TCA intermediates in the serum was excreted into the urine at 8, 10, and 12 wk than at 6 wk, indicating kidney dysfunction occurred. The relative abundances of TCA intermediates in urine relative to those in serum were suggested as an index of renal damage.

Increased urinary excretion of analogs of Krebs cycle metabolites and arabinose in two brothers with autistic features.

A marked increase in analogs of Krebs cycle metabolites was found in the urine of two brothers with autistic features. These metabolites included citramalic, tartaric (3-OH-malic), and 3-oxoglutaric acids and compounds tentatively identified as a citric acid analog and partially identified as a phenylcarboxylic acid by the fragmentation pattern of the trimethylsilyl (TMS) derivatives of the compounds and mass shifts of the same compounds derivatized with perdeuterated N,O-bis(trimethylsilyl)trifluoroacetamide. The molecular mass of the TMS derivative of the tentatively identified citric acid analog was 596 Da, based on a finding of a significant M - 15 ion at m/z 581. The citric acid analog was excreted in quantities as high as 137 mmol/mol creatinine, based on the response factor of citric acid as a surrogate calibrator. A carbohydrate with a retention time and mass spectrum identical to arabinose was also found in high concentrations in the urine of these brothers.

Organic aciduria in rats fed high amounts of xylitol or sorbitol.

The acidification of urine during polyol feeding was investigated with 27 Long-Evans male rats (aged 12 weeks) which were fed a xylitol diet (X), a sorbitol diet (S), or a basal diet for 4 weeks. The amount of polyols in the diet was increased from 5% to the final 20% level within 3 weeks. The polyol-fed animals showed reduced weight gain, lowered urine pH (from 6.5 to 5.6), and a 4-fold increase in the titratable acid excretion. X and S increased the daily urine volumes by 49 and 63%, respectively, but did not affect the wet weight or the pH values of the feces. as chromatographic-mass spectrometric analyses of organic acids revealed highly increased amounts of methylmalonic acid (13- to 20-fold) and 2-oxoglutaric acid (4- to 5-fold) in the urine of polyol-fed rats. The urinary excretion of citric acid and malic acid was also increased significantly (2- to 4-fold). The acidity of urine was not reflected in the blood acid-base balance of the animals. The increases in the levels of urinary organic acids in the polyol-fed rats were explained in terms of impaired mitochondrial oxidation of these acids and of impaired conversion of methylmalonic acid to succinic acid.

[Occurrence of alpha-alkyl-substituted malic acids, and beta-hydroxy-beta-alkyl-substituted dicarboxylic and tricarboxylic acid derivatives in normal urine (author's transl)]. / Uber das auftreten alpha-alkyl-substituierter Apfelsäuren und beta-hydroxy-beta-alkyl-substituierter Dicarbon-und Tricarbonsäurederivate als normale Stoffwechselprodukte.

Urine contains a number of alpha-hydroxy acids so far unknown to occur in biological liquids. Besides the already as urine constituent known methylmalic acid, also the ethyl, isopropyl and butyl derivatives of malic acid were found. Further metabolites in urine are a beta-propyl-substituted beta-hydrosyglutaric acid, a beta-hydroxy-beta-[methyl-carbomethoxy]-adipinic acid and two isomeric alpha-methylcitric acids.

Citric, orotic, and other organic acids in rats injected with active or inactive urease.

Male rats were fed laboratory chow or a purified L-amino acid diet containing 11.2 or 5.6 g arginine/kg. Hyperammonemia was produced by injection of crystalline jackbean urease. Control animals were injected with saline or inactivated urease. Rats injected with 55 U urease activity/kg body wt (an LD50 dose) exhibited acute signs of hyperammonemia and elevated orotate and citrate in their urine. Plasma glucose, lactate, citrate, and alpha-ketoglutarate concentrations were also markedly elevated. Three injections of active urease (10 U/kg body wt) given at intervals of about 10 h produced hyperammonemia, which persisted for 25 h after the first injection. Blood glucose and ammonia concentrations were increased 2.6- and 22-fold, respectively, when compared with controls. Total urinary citrate excretion for 25 h was 371 mueq for active urease-injected rats compared with 62 mueq for rats injected with inactivated urease. Rats fed a purified amino acid diet containing 5.6 g arginine/kg excreted greater quantities of urea, citrate, and orotic acid than rats fed 11.2 g arginine/kg of diet. Injection of active urease increased citrate excretion by rats fed either concentration of dietary arginine. Changes produced with active urease were not observed if inactivated urease was injected.