Naoto Suzuki

Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography-tandem mass spectrometry with column-switching technique : sex difference in healthy volunteers and patients with diabetes mellitus

We developed a determination method for human urinary 12-hydroxyeicosatetraenoic acid (12-HETE) using LC-MS-MS. This method, which includes simple extraction and detection in the SRM mode, allows precise and accurate determination of 12-HETE. There was a significant sex difference in urinary 12-HETE levels. Chiral analysis of 12-HETE using LC-MS-MS with column-switching technique revealed that the major enantiomer was 12(S)-HETE. Furthermore, the urinary level in patients with diabetes mellitus (DM) was analyzed. The present in vivo findings indicate that there could be difference in production of 12(S)-HETE between genders and 12(S)-HETE may play a role in the pathogenesis of DM.

Determination of urinary 8-epi-prostaglandin F2α using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.

Increased urinary leukotriene E4 excretion in patients with atopic dermatitis

Background  Synthesis of cysteinyl leukotrienes (LTs) is known to play a part in the pathogenesis of inflammatory diseases.

Functional Characterization of CYP2B6 Allelic Variants in Demethylation of Antimalarial Artemether

Artemether (AM) is one of the most effective antimalarial drugs. The elimination half-life of AM is very short, and it shows large interindividual variability in pharmacokinetic parameters. The aim of this study was to identify cytochrome P450 (P450) isozymes responsible for the demethylation of AM and to evaluate functional differences between 26 CYP2B6 allelic variants in vitro. Of 14 recombinant P450s examined in this study, CYP2B6 and CYP3A4 were primarily responsible for production of the desmethyl metabolite dihydroartemisinin. The intrinsic clearance (Vmax/Km) of CYP2B6 was 6-fold higher than that of CYP3A4. AM demethylation activity was correlated with CYP2B6 protein levels (P = 0.004); however, it was not correlated with CYP3A4 protein levels (P = 0.27) in human liver microsomes. Wild-type CYP2B6.1 and 25 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.21 and CYP2B6.23-CYP2B6.27) were heterologously expressed in COS-7 cells. In vitro analysis revealed no enzymatic activity in 5 variants (CYP2B6.8, CYP2B6.12, CYP2B6.18, CYP2B6.21, and CYP2B6.24), lower activity in 7 variants (CYP2B6.10, CYP2B6.11, CYP2B6.14, CYP2B6.15, CYP2B6.16, CYP2B6.20, and CYP2B6.27), and higher activity in 4 variants (CYP2B6.2, CYP2B6.4, CYP2B6.6, and CYP2B6.19), compared with that of wild-type CYP2B6.1. In kinetic analysis, 3 variants (CYP2B6.2, CYP2B6.4, and CYP2B6.6) exhibited significantly higher Vmax, and 3 variants (CYP2B6.14, CYP2B6.20 and CYP2B6.27) exhibited significantly lower Vmax compared with that of CYP2B6.1. This functional analysis of CYP2B6 variants could provide useful information for individualization of antimalarial drug therapy.

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.

Determination of leukotriene E4 in human urine using liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed for the quantitation of urinary leukotriene E4 (LTE4). LTE4 and its internal standard were extracted by solid-phase extraction and analysed using LC-MS-MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE4 ranged from 97.0% to 108.0% with a mean and SD of 100.6+/-2.4%. We detected LTE4 (63.1+/-18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE4 in biological samples.

Difference in urinary LTE4 and 11-dehydro-TXB2 excretion in asthmatic patients.

Bronchoconstrictor cysteinyl leukotrienes (LT) and thromboxane (TX) A2 have been implicated in the pathogenesis of asthma. Determination of urinary leukotriene E4 (LTE4) and 11-dehydro-TXB2 levels are often used to assess cysteinyl LT and TXA2 production in humans. To define the potential role in the pathogenesis of asthma, we investigated the urinary LTE4 and 11-dehydro-TXB2 levels. LTE4 and 11-dehydro-TXB2 levels were determined using liquid chromatography/tandem mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS), respectively. Urinary LTE4 levels in asthmatic patients (192 +/- 122 pg/mg creatinine, n = 14) were significantly higher (P < 0.005) than those in healthy volunteers (55 +/- 16 pg/mg creatinine, n = 13), but no significant difference in 11-dehydro-TXB2 levels was observed. A significant inverse correlation (r = -0.821, P < 0.005) was found between urinary LTE4 levels and the forced expiratory volume in 1 s (FEV1) but no significant correlation was observed between urinary 11-dehydro-TXB2 levels and FEV1. The present findings suggest that cysteinyl LTs play a more important role in the pathogenesis of asthma than TXA2.

A validated quantitative liquid chromatography–tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20pM, 2nM and 10nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200ng of DNA. Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.

Simultaneous determination of guanidinosuccinic acid and guanidinoacetic acid in urine using high performance liquid chromatography/tandem mass spectrometry

We present a method for the simultaneous determination of guanidinosuccinic acid (GSA) and guanidinoacetic acid (GAA) from urine by protein precipitation and liquid chromatography/tandem mass spectrometry. The chromatographic separation was performed using a cation exchange column with an elution gradient of 0.1 mM and 20 mM ammonium acetate buffers. GSA was detected with the mass spectrometer in negative ion mode monitoring at m/z 174.1, and GAA, creatinine, arginine, and homoarginine were in positive ion mode monitoring at m/z 118.1, 114.1, 175.1, and 189.1, respectively. As an internal standard, L-arginine-(13)C(6) hydrochloride and creatinine-d(3) (methyl-d(3)) were used. The calibration ranges were 0.50-25.0 μg mL(-1), and good linearities were obtained for all compounds (r>0.999). The intra- and inter-assay accuracies (expressed as recoveries) and precisions at three concentration levels (1.00, 5.00 and 25.0 μg mL(-1)) were better than 83.8% and 7.41%, respectively. The analytical performance of the method was evaluated by determination of the compounds in urine from male C57BL/J Iar db/db diabetes mellitus (DM) mice. The values of GSA and GAA corrected by the ratios of the individual compounds to creatinine were significantly increased in DM mice compared with control mice. These results indicated that the newly developed method was useful for determining urinary guanidino compounds and metabolites of arginine.

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.