Kazuki Akira

1H NMR-based metabonomic analysis of urine from young spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) and their substrains are a useful model for studying essential hypertension which is a complex, polygenic, and multifactorial disorder. Their genetic and metabolic features are of great interest because they may provide insights into the mechanism of blood pressure regulation. We have compared urinary metabolic profiles of young SHR with those of their age-matched normotensive controls, Wistar Kyoto rats, using (1)H NMR-based metabonomics. Principal components analysis was applied to the NMR spectral data after data-reduced and normalized by the total integral or the creatinine integral. Consequently, a clear separation of urine samples between the two strains was observed in the principal components scores plot. The loadings plot from the data normalized by the creatinine integral showed that many metabolites such as citrate, alpha-ketoglutarate, and hippurate contributed to the separation, and the urinary levels of most metabolites used in this study, including these three, were lower in SHR than in Wistar Kyoto rats. These metabolic changes may be concerned with blood pressure regulation in SHR, although a relation to other strain differences cannot be ruled out. The present study suggests the usefulness of a (1)H NMR-based metabonomic approach using SHR in the field of hypertension research.

Determination of urinary glyoxal and methylglyoxal by high-performance liquid chromatography.

Abstract Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase highperformance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100–120 mOsm/kg H2O) under acidic conditions (pH 4.5) at 60°C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within-and between-day relative standard deviations of less than 5%. Urinary levels (mean±standard deviation, n=16) of glyoxal and methylglyoxal in healthy subjects were 4.7±1.35 μg/mg creatinine, 2.2±0.65 μg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.

Application of radioluminography to off-line counting of radioactivity in high-performance liquid chromatographic eluates

Abstract An off-line counting method for the determination of carbon-14 in HPLC eluates was developed using radioluminography (RLG). A succession of aliquots of the eluate were collected in the flat-bottomed wells of a polystyrene microplate, evaporated to dryness and contacted with an imaging plate, and radioactivity was determined with a Bio-image analyser. The limit of detection was 0.35 Bq per injection. The inter- assay relative standard deviation was less than 3% over the range 2–20 Bq. The RLG off-line counting method was utilized to determine [ 14 C]eicosapentaenoic acid metabolites formed by rat hepatic microsomes. The results were compared with those obtained with an off- line liquid scintillation counting method and an on-line counting method.

A metabonomic study of biochemical changes characteristic of genetically hypertensive rats based on 1 H NMR spectroscopic urinalysis

Spontaneously hypertensive rats (SHR) provide a simple model for studying essential hypertension. Their genetic and metabolic features are of great interest because they may provide insights into the pathophysiological processes underlying essential hypertension. We have thus investigated the metabolic characteristics of SHR at various ages, covering the prehypertensive stage and the developmental phase of hypertension, using a 1H nuclear magnetic resonance (NMR)-based metabonomic approach. Twenty-four-hour urine samples from the SHR and their age-matched normotensive control, Wistar–Kyoto rats, were analyzed using 1H NMR spectroscopy, and the spectral data were subjected to principal components analysis (PCA) to find metabolic differences between the two strains. Consequently, it was possible to separate the urine samples between the two strains at any age ranging from 4 to 20 weeks in the principal component scores plots. The major spectral regions and signals (metabolites) contributing to the separation were picked up based on the loadings. Subsequently, the urinary excreted levels of metabolites highlighted by the PCA were compared based on the signal intensities corrected by urine volume and body weight. These investigations revealed the major metabolic changes characteristic of the SHR, which included differences in citrate, α-ketoglutarate, succinate, hippurate, phenylacetylglycine, p-cresol glucuronide, creatine, taurine, medium-chain dicarboxylates (tentative), unknown (δ 3.11), and the regions at 3.60, 3.64, 3.68 and 3.88 p.p.m. The results supported the occurrence of metabolic acidosis in the SHR in the period of prehypertension as well as rapidly rising blood pressure. In addition, the intestinal microfloral populations in the SHR were suggested to be altered in the developmental phase of hypertension.

LC-NMR identification of a novel taurine-related metabolite observed in 1H NMR-based metabonomics of genetically hypertensive rats

This paper describes the LC-NMR spectroscopic identification of a novel urinary endogenous metabolite responsible for the signals, which were found as major contributors to the separation between genetically hypertensive rats (SHRSP) and normotensive control rats (WKY) in previous NMR-based metabonomic studies. Urine samples from 26-week-old normotensive rats were analyzed by an LC-NMR system equipped with a reversed-phase column having high retention ability for polar compounds. (1)H NMR spectra were continuously obtained in the on-flow mode, and the retention times of the unassigned signals in question were determined. Various two-dimensional spectra were subsequently measured for the fraction containing the unassigned signals under the stop-flow mode, which enables for a long accumulation resulting in the enhancement of signal-to-noise ratios. The candidate compound obtained from these LC-NMR data was synthesized, and the NMR and mass spectra were compared with those of the LC-NMR fraction. The unknown metabolite was identified as succinyltaurine from these experiments together with standard addition experiments. This novel metabolite, which is characteristic of the normotensive rats, is very interesting because it is structurally related to hypotensive taurine, and not substantially detected in the genetically hypertensive rats, which excreted more taurine than the normotensive rats. The biological and pathophysiological significances of succinyltaurine remain to be investigated.

Investigations into Biochemical Changes of Genetic Hypertensive Rats Using ^1H Nuclear Magnetic Resonance-Based Metabonomics

Genetically hypertensive rats provide a simple and accessible model for studying essential hypertension, which is a polygenic, heterogenous and multifactorial disease. Their genetic and metabolic features are of great interest because they may provide insight into the pathophysiological processes underlying essential hypertension. We have investigated the genetic influence on metabolic balance and metabolite excretion patterns in stroke-prone spontaneously hypertensive rats (SHRSP) with established hypertension using 1H NMR-based metabonomics. Urinary metabolite profiles for SHRSP and their age-matched normotensive controls, Wistar Kyoto rats, were acquired using 1H NMR spectroscopy. Principal components analysis was applied to these complex NMR data to facilitate differentiation and determine metabolic differences between urine samples collected from the hypertensive and normotensive rats. Consequently, it was possible to distinguish urine samples between the two strains in the principal components scores plot. The loadings plot showed that taurine, creatine and some unidentified metabolites resonating at around δ 2.48, 3.10 and 3.58 predominantly contributed to the separation. In SHRSP, the urinary levels of taurine and creatine were found to be higher and the intensities of the unknown signals much lower than those in the Wistar Kyoto rats. Although the pathophysiological significance of these components remains to be elucidated, this study suggests that 1H NMR-based metabonomics is a promising approach to provide new information on metabolic changes related to the pathophysiological processes of the genetically hypertensive rats.

Direct detection of the internal acyl migration reactions of benzoic acid 1-O-acylglucuronide by 13C-labeling and nuclear magnetic resonance spectroscopy

1-O-Acyl-beta-D-glucopyranuronates can undergo irreversible binding to proteins mainly through internal acyl migration reactions, which may have toxicological significance. A new method based on the 13C-labeling and nuclear magnetic resonance (NMR) spectroscopy has been developed to study the reactivity of the 1-O-acyl-beta-D-glucopyranuronate of benzoic acid. In phosphate buffer (pH 7.4) solution at 37 degrees C, the glucuronide showed apparent first-order degradation kinetics (T1/2, 125 min), and concurrent and sequential appearance of 2-, 3- and 4-O-acyl isomers as both alpha- and beta-anomers was observed. The isomeric glucuronides were identified by two-dimensional NMR of the reaction mixture. The direct approach using 13C-labeling and NMR could also provide insights into the reactivities of other labile drug acylglucuronides and their isomeric glucuronides.

Direct determination of diastereomeric 13C-labeled ketoprofen glucuronides in human urine by nuclear magnetic resonance spectroscopy

Abstract A new method based on the 13 C-labeling and nuclear magnetic resonance (NMR) spectroscopy was developed to study the human urinary excretion of diastereomeric acylglucuronides after the oral administration of 100 mg racemic [3- 13 C]ketoprofen (KP). The urinary excretion of diastereomeric [3- 13 C]KP glucuronides formed from [3- 13 C]KP was followed by proton-decoupled 13 C NMR spectroscopy (9.4 T, 10min accumulation time) without any separation procedures such as extraction and chromatography. The C3 resonances due to the diastereomers were satisfactorily separated from each other by the addition of methyl-β-cyclodextrin as an achiral shift reagent through the formation of inclusion complexes. The C3 resonances were detected with an acceptable signal-to-noise ratio over 8 h at a therapeutic dosage. The concentrations of [ 13 C]KP glucuronides were calculated from the inverse gated decoupling and proton-decoupling experiments using [2- 13 C]glycine as an internal standard. The urinary excretion of diastereomeric [3- 13 C]KP glucuronides over 8 h amounted to 29% (downfield position) and 25% (upfield position) of the administered dose, respectively, and the former was presumed to have S -configuration. The elimination rate constants of both glucuronides were virtually the same ( K = 0.19 h −1 ). In addition, the susceptibility of the glucuronides to acyl migration as well as hydrolysis was suggested under the physiological conditions (37 °C, pH 7.3). The present direct approach is simple and convenient, and has the potential for wide application to the analysis of the labile acylglucuronides of 2-arylpropionic acids in biofluids.

Metabonomic study on the biochemical response of spontaneously hypertensive rats to chronic taurine supplementation using 1H NMR spectroscopic urinalysis

There is a wealth of experimental information and some clinical evidence available in the literature suggesting that taurine exerts preventive effects on cardiovascular diseases. In particular, taurine has been shown to reduce blood pressure in not only hypertensive patients but also in a number of hypertensive rodent models such as spontaneously hypertensive rats (SHR). However, the molecular basis of the efficacy and toxicity of the compound has not been fully characterized. We have investigated the effects of taurine supplementation to urinary low-molecular-weight endogenous metabolites in SHR using a (1)H NMR-based urinary metabonomic approach. The SHR were chronically treated with 3% taurine in drinking water from four to 14 weeks of age, and 24-h urine samples were analyzed using (1)H NMR spectroscopy. Metabolic information was extracted from the NMR data by principal components analysis as well as visual inspection. Consequently, the metabolite profile started to change with considerable interindividual variation from six weeks of age. The extent of change became increasingly remarkable with the duration of treatment, with the concurrent observation of the hypotensive effect. The metabolic changes included a decreased urinary output of tricarboxylic acid cycle intermediates (citrate, α-ketoglutarate, and succinate) and an increased output of phenylacetylglycine and p-cresol sulfate. The results suggest that chronic taurine supplementation to the SHR resulted in an acceleration of metabolic acidosis with perturbation in the tricarboxylic acid cycle and the modulation of the intestinal microbial metabolism.

Profiling of arachidonic acid metabolites in rabbit platelets by radio gas chromatography

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane,O-methylhydroxylamine and dimethylisopropylsilylimidazole. The recovery of total14C-radioactivity was 93.1±7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.