Published papers

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7,012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine-N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.

Cellular metabolism is dynamic, but quantifying non-steady metabolic fluxes by stable isotope tracers presents unique computational challenges. Here, we developed an efficient 13C-tracer dynamic metabolic flux analysis (13C-DMFA) framework for modeling central carbon fluxes that vary over time. We used B-splines to generalize the flux parameterization system and to improve the stability of the optimization algorithm. As proof of concept, we investigated how 3T3-L1 cultured adipocytes acutely metabolize glucose in response to insulin. Insulin rapidly stimulates glucose uptake, but intracellular pathways responded with differing speeds and magnitudes. Fluxes in lower glycolysis increased faster than those in upper glycolysis. Glycolysis fluxes rose disproportionally larger and faster than the tricarboxylic acid cycle, with lactate a primary glucose end product. The uncovered array of flux dynamics suggests that glucose catabolism is additionally regulated beyond uptake to help shunt glucose into appropriate pathways. This work demonstrates the value of using dynamic intracellular fluxes to understand metabolic function and pathway regulation.

A rapid and easy method for the determination of halides, oxyhalides and metal oxoacids by capillary electrophoresis was developed. This is the first paper to report their simultaneous analysis. Electroosmotic flow was suppressed more than 40-fold by using a poly(ethyleneglycol)-coated capillary, compared with that in a bare fused-silica capillary under similar conditions. Using this capillary, the separation of eleven anions was accomplished. Anion migration was dependent only on their mobility and detection was carried out directly with a diode-array detector. The R.S.D.s were better than 0.6% for migration time and between 1.0% and 3.4% for peak area. Calibration graphs for all the anions were linear, with correlation coefficients better than 0.9995. Using stacking sample preconcentration, the achievable lower detection limits were found to be in the range 14–260 μg 1−1. This represents a 33–43-fold increase in sensitivity over the results obtained by using 200 mbar·s pressure injection.

An automated method of analysis of catecholamines using a new packing material, internal surface boronic acid gel, was developed. The new support is designed with a carboxymethylcellulose-bonded external surface in order to be non-adsorptive to proteins and with a phenylboronic acid-bonded internal surface to retain only catecholamines. This packing support displayed an affinity for basic or neutral catecholic compounds with no protein adsorption and enabled on-line sample pretreatment of catecholamines in urine and deproteinized plasma. The catecholamines were selectively adsorbed on the new material and separated on a reversed-phase or a cation-exchange column. These compounds were then detected electrochemically. The limits of quantitation were 1.5–3.0 ng/ml in urine and 10–15 pg/ml in plasma, at a signal-to-noise ratio of 5.

An HPLC method for the determination of carbohydrates using a postcolumn pH adjustment technique with lithium hydroxide solution followed by pulsed amperometric detection was developed. In conjunction with hydrophilic interaction chromatography, the technique was used to determine reducing sugars, non-reducing sugars and sugar alcohols. Detection limits from 0.7 to 2.7 ng with a signal-to-noise ratio of 3 were achieved. The relative standard deviation for peak was better than 2.7%. The postcolumn pH adjustment technique enabled gradient elution to be used.

A rapid, easy, and reproducible method for the determination of inorganic and organic anions in beer and wort by capillary electrophoresis was developed. Three inorganic anions (chloride, sulfate, and phosphate) and nine organic acids (oxalate, formate, malate, citrate, succinate, pyruvate, acetate, lactate, and pyroglutamate) were separated and quantified in less than 7 min. Sample preparation was simple and consisted only of degassing the beer by sonication and 1:5 dilution with Milli Q water prior to injection. Satisfactory reproducibility was obtained, and the concentrations of anions were in good agreement with those found by conventional techniques.

A capillary zone electrophoresis method was developed with indirect UV detection for the analysis of inorganic and organic anions using a background electrolyte (BGE), 2,6-pyridinedicarboxylic acid (PDC). The parameters which influence the separation of these anions, such as electrolyte pH, concentration of PDC and applied voltage, were investigated. Three inorganic anions and nine organic acids were determined simulataneously in less than 7 min. The R.S.D. (n66) values of the method were around 0.1% for migration time and better than 2.6% for peak area. This method is rapid, sensitive and quantitative and could be readily applied to analysis of inorganic and organic anions in beer. In the present paper, the effect of the complexing ability of the BGE on the analysis of organic acids is also discussed.

A rapid, easy, and reproducible capillary electrophoretic method for the simultaneous determination of acidic, neutral, and amino sugars and sugar alcohols was developed. Underivatized mannuronic acid, glucuronic acid, galacturonic acid, N-glycolylneuraminic acid, N-acetylneuraminic acid, glucosamine, galactosamine, mannose, xylose, glucose, galactose, fucose, ribose, mannitol, sorbitol, xylitol, and inositol were simultaneously determined with indirect UV detection using 2,6-pyridinedicarboxylic acid as a background electrolyte. A highly alkaline pH condition was used in order to charge carbohydrates negatively and to promote migration toward the anode. Electroosmotic flow was reversed to the direction of the anode by adding cetyltrimethylammonium bromide to the electrolyte. The separation of the carbohydrates was investigated by optimizing the operating pH value and satisfactory resolution was obtained at pH 12.1. The relative standard deviations of the method for carbohydrates were between 0.02 and 0.33% for migration times and were greater than 2.7% for peak areas (n = 6). The minimum detectable level ranged from 23 to 71 microM with a 6-nl injection at a signal-to-noise ratio of 3. This method was applied to the composition analysis of monosaccharides in glycoprotein. After acid hydrolysis of fetuin under each optimum condition, sialo, neutral, and amino sugars were quantified under the same electrophoretic condition.

A capillary electrophoresis method for the simultaneous analysis of inorganic anions, organic acids and metal cations was developed. 2,6-Pyridinedicarboxylic acid (PDC) was used as the background electrolyte to permit indirect UV detection of anions. The PDC also worked to confer a negative charge on metal cations via PDC-complex formation. In order to detect both anions and metal–PDC complexes simultaneously, the electroosmotic flow was reversed by adding cetyltrimethylammonium hydroxide. In the method detection of anions was carried out with indirect UV detection, whereas metal cations were detected directly. Thirteen anions and three metal cations were completely separated at the operating buffer, pH 5.7. The R.S.D. (n=6) values of the method were better than 0.2% for migration time and between 1.1% and 3.9% for peak area. Calibration graphs were linear with correlation coefficients better than 0.9992. The minimum detectable levels ranged from 0.2 mg l−1 to 1.9 mg l−1 with 300 mbar pressure injection (6 nl) at a signal-to-noise ratio of 3. This method is simple, rapid and reproducible and could be readily applied to the simultaneous determination of anions and metal cations in a plating bath sample.

A capillary zone electrophoretic method for the simultaneous analysis of inorganic anions, organic acids, amino acids and carbohydrates was developed with indirect UV detection using 2,6-pyridine dicarboxylic acid as the background electrolyte. This is the first paper to report their simultaneous analysis. Highly alkaline conditions were used in order to confer a negative charge not only on inorganic and organic anions but also on amino acids and carbohydrates, and to promote their migration towards the anode. Electroosmotic flow was reversed in the direction of the anode by adding cetyltrimethylammonium hydroxide to the electrolyte. Outstanding separations were obtained and electrophoretic mobilities of 82 compounds including nine inorganic anions, 23 organic acids, 18 amino acids and 32 carbohydrates were determined by the method. Under the optimized conditions 43 compounds were well separated in a single run. The relative standard deviations (n=5) of the method were better than 0.5% for migration times and between 0.8% and 4.9% for peak areas except for a few compounds. The detection limits for anions and amino acids were in the range from 6 to 12 mg/l and for carbohydrates from 23 to 35 mg/l with 300 mbar·s pressure injection (6 nl) at a signal-to-noise ratio of three. This method could be readily applied to the simultaneous determination of inorganic anions, organic acids, amino acids and carbohydrates in soy sauce, nutrient tonic and pineapple.

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE−ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol−water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 μmol/L and for acidic and low molecular weight amino acids were less than 6.0 μmol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.

Chiral resolution of native dl-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-β-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-β-cyclodextrin with an effective voltage of −30 kV at 16°C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, d- and l-lactic acids in food products were analyzed successfully.

An easy and reproducible capillary electrophoretic method with indirect UV detection for the determination of carbohydrates in food samples was described. A highly alkaline pH condition was used in order to charge every carbohydrate negatively. Electroosmotic flow (EOF) was reversed to detect sugar alcohols and to improve the separation of neutral and amino sugars. Under optimized conditions 28 carbohydrates including mono- and disaccharides, uronic acids, sialic acids, amino sugars and sugar alcohols were separated in less than 25 min. The detection limits for fructose, glucose and sucrose were in the range from 12 to 16 mg l−1 with pressure injection of 50 mbar for 6 s (ca. 6 nl) at a signal to noise ratio of 3. The calibration curves were linear over the range from 50 to 10 000 mg l−1 with correlation coefficients better than 0.9999. This method could be readily applied to the analysis of carbohydrates in several foods and beverages. Sample preparation was minimal and consisted either of dilution or dilution with filtration.

Chiral resolution of native DL-malic acid was achieved by ligand-exchange capillary electrophoresis using copper(II)-L-tartrate as a chiral selector. Factors affecting chiral resolution, migration time, and peak area of malic acid were studied. The running conditions for optimum separation of malic acid were found to be 1 mM copper(II) sulfate-1 mM L-tartrate (pH 5.1) with an effective voltage of -20 kV at 30 degrees C, using direct detection at 280 nm, and resolution (Rs) of racemic malic acid was approximately 4. With this system, D- and L-malic acids in apple juice were analyzed successfully.

A previously developed capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of inorganic and organic anions, amino acids and carbohydrates using 20 mM 2,6‐pyridinedicarboxylic acid (PDC) as the background electrolyte was extended to allow determination of 206 anions including those above – mentioned and physiological amino acids, nucleotides, aromatic acids, haloacetic acids, alcohols, phosphorylated saccharides, oxyhalides, metal oxoacids, metal‐ethylenediaminetetraacetic acid (EDTA) complexes, forensic anions, Good's buffers and herbicides. Every compound could be analyzed and their electrophoretic mobility determined simply by selecting detection wavelength. This method is simple and universal for anion analysis, and could be readily applied to the simultaneous determination of anionic compounds. In this work, it was used to identify and quantify important anions in sea urchin and sake.

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.