Elevated levels of urine isocitrate, hydroxymethylglutarate, and formiminoglutamate are associated with arterial stiffness in Korean adults.

Recent evidence suggests that cellular perturbations play an important role in the pathogenesis of cardiovascular diseases. Therefore, we analyzed the association between the levels of urinary metabolites and arterial stiffness. Our cross-sectional study included 330 Korean men and women. The brachial-ankle pulse wave velocity was measured as a marker of arterial stiffness. Urinary metabolites were evaluated using a high-performance liquid chromatograph-mass spectrometer. The brachial-ankle pulse wave velocity was found to be positively correlated with L-lactate, citrate, isocitrate, succinate, malate, hydroxymethylglutarate, α-ketoisovalerate, α-keto-ß-methylvalerate, methylmalonate, and formiminoglutamate among men. Whereas, among women, the brachial-ankle pulse wave velocity was positively correlated with cis-aconitate, isocitrate, hydroxymethylglutarate, and formiminoglutamate. In the multivariable regression models adjusted for conventional cardiovascular risk factors, three metabolite concentrations (urine isocitrate, hydroxymethylglutarate, and formiminoglutamate) were independently and positively associated with brachial-ankle pulse wave velocity. Increased urine isocitrate, hydroxymethylglutarate, and formiminoglutamate concentrations were associated with brachial-ankle pulse wave velocity and independent of conventional cardiovascular risk factors. Our findings suggest that metabolic disturbances in cells may be related to arterial stiffness.

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.

A metabolomics approach to the identification of biomarkers of sugar-sweetened beverage intake.

BACKGROUND: The association between sugar-sweetened beverages (SSBs) and health risks remains controversial. To clarify proposed links, reliable and accurate dietary assessment methods of food intakes are essential. OBJECTIVE: The aim of this present work was to use a metabolomics approach to identify a panel of urinary biomarkers indicative of SSB consumption from a national food consumption survey and subsequently validate this panel in an acute intervention study. DESIGN: Heat map analysis was performed to identify correlations between ¹H nuclear magnetic resonance (NMR) spectral regions and SSB intakes in participants of the National Adult Nutrition Survey (n = 565). Metabolites were identified and receiver operating characteristic (ROC) analysis was performed to assess sensitivity and specificity of biomarkers. The panel of biomarkers was validated in an acute study (n = 10). A fasting first-void urine sample and postprandial samples (2, 4, 6 h) were collected after SSB consumption. After NMR spectroscopic profiling of the urine samples, multivariate data analysis was applied. RESULTS: A panel of 4 biomarkers-formate, citrulline, taurine, and isocitrate-were identified as markers of SSB intake. This panel of biomarkers had an area under the curve of 0.8 for ROC analysis and a sensitivity and specificity of 0.7 and 0.8, respectively. All 4 biomarkers were identified in the SSB sample. After acute consumption of an SSB drink, all 4 metabolites increased in the urine. CONCLUSIONS: The present metabolomics-based strategy proved to be successful in the identification of SSB biomarkers. Future work will ascertain how to translate this panel of markers for use in nutrition epidemiology.

Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies: an underlying mechanism to reduce lipid accretion by whey protein ingestion?

Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice.

The automated enzymic assay of D-isocitrate in human urine.

A simple, automated, enzymic assay for the analysis of D-isocitrate in urine is described. The method detects D-isocitrate in the range 0.05-1.00 mmol/L, producing within-batch and between-batch coefficients of variation of 1.8-3.2% and 2.7-3.0% respectively. The assay was also shown to be robust, tolerating extremes of sample pH. Of a wide range of substances tested, only Fe3+ ions interfered, but only at levels considerably in excess of those encountered in urine. D-isocitrate was stable in urine for up to one week at room temperature or at 4 degrees C, with or without preservatives.

Isolation and identification of some urinary inhibitors of calcium phosphate formation.

Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.

The estimation of D-isocitric acid in urine using isocitrate dehydrogenase.

The amount of D-isocitric acid in urine has been estimated using the enzyme isocitrate dehydrogenase. The technique is rapid and easy to perform. Reproducibility of results was good and results from recovery experiments were excellent. The concentration range for early morning urine samples from normal adults was 0.08-0.65 mmol/l with a mean of 0.29 mmol/l.