Hideaki Iizuka

Quantitative analyses of schizophrenia-associated metabolites in serum: serum D-lactate levels are negatively correlated with gamma-glutamylcysteine in medicated schizophrenia patients.

The serum levels of several metabolites are significantly altered in schizophrenia patients. In this study, we performed a targeted analysis of 34 candidate metabolites in schizophrenia patients (n = 25) and compared them with those in age- and gender-matched healthy subjects (n = 27). Orthogonal partial least square-discriminant analysis revealed that complete separation between controls and patients was achieved based on these metabolites. We found that the levels of γ-glutamylcysteine (γ-GluCys), linoleic acid, arachidonic acid, D-serine, 3-hydroxybutyrate, glutathione (GSH), 5-hydroxytryptamine, threonine, and tyrosine were significantly lower, while D-lactate, tryptophan, kynurenine, and glutamate levels were significantly higher in schizophrenia patients compared to controls. Using receiver operating characteristics (ROC) curve analysis, the sensitivity, specificity, and the area under curve of γ-GluCys, a precursor of GSH, and D-lactate, a terminal metabolite of methylglyoxal, were 88.00%, 81.48%, and 0.8874, and 88.00%, 77.78%, and 0.8415, respectively. In addition, serum levels of D-lactate were negatively correlated with γ-GluCys levels in patients, but not in controls. The present results suggest that oxidative stress-induced damage may be involved in the pathogenesis of schizophrenia.

Fluorescence determination of d- and l-tryptophan concentrations in rat plasma following administration of tryptophan enantiomers using HPLC with pre-column derivatization ☆

Similar to L-tryptophan (L-Trp), D-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life (t(1/2)) between Trp enantiomers was investigated by following the alterations in the plasma concentration of D- or L-Trp after intraperitoneal (i.p.) administration of each enantiomer to male Sprague-Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS). The t(1/2) value of D-Trp was significantly smaller than that of L-Trp, suggesting that D-Trp was eliminated from the plasma more rapidly than L-Trp. In addition, a significant increase in the plasma concentration of L-Trp was observed following administration of D-Trp, whereas no D-Trp was detected after L-Trp administration. Furthermore, the increase in the plasma concentration of L-Trp was significantly suppressed by pretreatment with an inhibitor of D-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of D-Trp to L-Trp in vivo.

Changes in the plasma concentrations of D‐kynurenine and kynurenic acid in rats after intraperitoneal administration of tryptophan enantiomers

An aqueous solution of enantiomerically pure tryptophan (Trp), namely, D-Trp or L-Trp (100 mg/kg), was administered intraperitoneally to male Sprague-Dawley rats. The time-course profiles of the rat plasma concentrations of D-kynurenine (KYN), L-KYN, and kynurenic acid (KYNA), which are metabolites of D- or L-Trp, were investigated using high-performance liquid chromatography (HPLC) systems that were reported in our previous study. The plasma D-KYN concentration increased after the administration of D-Trp, but this increase was not observed after the administration of L-Trp. The plasma L-KYN concentration increased after the administration of L-Trp, but no significant change was observed after the administration of D-Trp. The plasma KYNA concentration drastically increased in the case of rats administered D-Trp and those administered L-Trp. Additionally, an inhibitor of D-amino acid oxidase (DAAO), 5-methylpyrazole-3-carboxylic acid (MPC) (50 mg/kg), was administrated to the rats 30 min before the administration of D-Trp. The preadministration of MPC remarkably increased the D-KYN concentration and suppressed the production of KYNA. These results suggest that DAAO may contribute to the metabolism of D-KYN to KYNA in vivo.

Comparative study on kynurenic acid production in the rat striatum by tryptophan enantiomers: an in vivo microdialysis study.

Kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D-aspartate and α(7) nicotinic acetylcholine receptors, is one of the L-tryptophan (Trp) metabolites. To compare the level of KYNA produced in the striatum of rats after independent administration of L-Trp and D-Trp, rats were intraperitoneally administered L-Trp and/or D-Trp (100 mg/kg), and a microdialysis (MD) probe was implanted in the striatum. The KYNA level in the MD samples was determined using the column-switching high-performance liquid chromatography system. KYNA levels in the MD samples increased by approximately twofold in rats that were administered D-Trp or L-Trp; this result suggests that just as L-Trp, D-Trp was also metabolized to KYNA in the striatum. Additionally, 30 min before the administration of D-Trp, rats were administered 3-methyl pyrazole-5-carboxylic acid (MPC) (50 mg/kg), which is a specific inhibitor of D-amino acid oxidase (DAAO). Pretreatment with MPC suppressed striatal KYNA production; this result suggests that DAAO, encoded by one of the susceptible genes for schizophrenia, may contribute to the production of KYNA from D-Trp in the striatum of rats.

Determination of L-tryptophan and L-kynurenine in Human Serum by using LC-MS after Derivatization with (R)-DBD-PyNCS:

Concentrations of L-tryptophan (L-Trp) and its metabolite, L-kynurenine (L-KYN), in sera of 19 normal subjects (age: 23.6 ± 3.5 y, male: 8, female: 11) were determined by high-performance liquid chromatography with mass-spectrometric detection, following their derivatization with (R)-(–)-4-(N, N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-PyNCS). A significant positive correlation between L-Trp and L-KYN concentrations was observed (r = 0.532, P < 0.05). Serum L-Trp concentration in male subjects (95.65 ± 4.27 μM) was significantly higher than that in female subjects (79.20 ± 3.34 μM; P < 0.05), while no significant differences in L-KYN concentration or the L-KYN:L-Trp ratio were observed between male and female subjects.

Enantiomeric separation of d,l‐tryptophan and d,l‐kynurenine by HPLC using pre‐column fluorescence derivatization with R(−)‐DBD‐PyNCS

The enantiomeric separation of D,L-tryptophan (Trp) and D,L-kynurenine (KYN) was investigated by high-performance liquid chromatography using pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7- (N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS-3 column (250 x 2.0 mm; i.d., 3 μm), four fluorescence peaks of D- and L-Trp as well as D- and L-KYN derivatized with R(-)-DBD-PyNCS were clearly observed, and their chemical structures were confirmed by HPLC-time-of-flight-mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O-CH₃CN (60:40), and the separation factors of D,L-Trp and D,L-KYN derivatized with R(-)-DBD-PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3-0.5 pmol (signal-to-noise ratio of 3).

Determination of Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection

It has been suggested that serum concentrations of polyunsaturated essential fatty acids correlate with the symptoms or severity of various diseases, including depression and Alzheimer-type dementia, and that determination of serum fatty acids might be important for disease diagnosis. Thus, we developed to analyze serum fatty acids in healthy individuals by using a high-performance liquid chromatography method with fluorescence detection, because free fatty acids have a carboxyl group that can be derivatized with a fluorescent reagent, 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. This approach could quantify five types of free fatty acids [α-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA) and oleic acid (OA)] in human serum. The detection limits of the method were in the range of 2.29-4.75 fmol (signal-to-noise ratio 3), and absolute concentrations of ALA, PLA, AA, LA and OA were 8.27 ± 1.04, 18.8 ± 2.95, 49.9 ± 4.03, 230 ± 18.1 and 201 ± 22.1 µM, respectively.

HPLC-fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC-mode column

High-performance liquid chromatography-fluorescence detection using a hydrophilic interaction chromatography-mode column (ZIC®-HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22-38 years and 17 female subjects aged 18-38 years). Serum cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine, derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, were separated on the ZIC®-HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ-glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 μm, respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05).

Simultaneous Determination of 5-Hydroxyindoles and Catecholamines by Hplc with Fluorometric Precolumn Derivatization

We have developed a highly sensitive and simple precolumn derivatization method for simultaneous determination of 5-hydroxyindoles (5HIs) and catecholamines (CAs) with 4-dimethylaminobenzylamine (DMBA). The method was successfully applied to the determination of 5HIs and CAs in plasma and urine.

Potassium Hexacyanoferrate(Iii)-Mediated Fluorometric Determination of Melatonin

This paper describes the development of a novel, simple and sensitive high-performance liquid chromatographic method for the determination of melatonin using precolumn fluorometric derivatization. A strong fluorescence was induced when alkaline solution of melatonin was heated in the presence of potassium hexacyanoferrate (III). This fluorescent reaction was used for the precolumn derivatization of melatonin. Solid phase extraction was used for concentrating melatonin below a level of detection limit.