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Imatinib has been reported to induce heart failure and/or QTc prolongation. To better understand their underlying mechanisms, we assessed its effects on cardiohemodynamic, electrocardiographic and echocardiographic variables along with biomarkers of myocardial damage. Imatinib mesylate in doses of 1 and 10 mg/kg was intravenously administered to the halothane-anesthetized beagle dogs (n = 4). Effects of imatinib on each phase of isovolumetric contraction, ejection, isovolumetric relaxation and filling were studied, whereas its electrophysiological effects on early and late repolarization were analyzed by measuring J-Tpeakand Tpeak-Tend, respectively. The low and high doses of imatinib provided peak plasma concentrations of 3.23 and 17.39 μg/mL, reflecting clinically-relevant and supratherapeutic concentrations, respectively. Neither lethal ventricular tachyarrhythmia nor cardiohemodynamic collapse was observed. Imatinib decreased amplitude of peak −dP/dt, indicating suppression of isovolumetric relaxation, whereas no significant change was detected in the other phases. Imatinib prolonged QTc and J-Tpeakc without altering Tpeak-Tend, indicating increase of net inward current, which leads to intracellular Ca2+overload. Thus, imatinib suppressed ventricular active relaxation and early repolarization, which may suggest the association of mitochondrial dysfunction-associated inhibition of ATP production. Since those findings were also reported for dasatinib, sunitinib and lapatinib, they could be common cardiac phenotype of tyrosine kinase inhibitors in vivo.

d-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-d-kynurenine (MeS-d-KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, λex= 364 nm, λem= 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R2= 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-d-KYN is feasible. MeS-d-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2-benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-d-KYN as the substrate.

IMPORTANCE: Reports on dermatomyositis (DM) sine dermatitis (DMSD) are scarce, and the concept of the disease has not been widely accepted. OBJECTIVE: To confirm the existence of DMSD, determine its prevalence, and characterize its serologic features. DESIGN, SETTING, AND PARTICIPANTS: This is a cohort study that reviewed clinical information, laboratory data, and muscle pathology slides from January 2009 to August 2019. We further assessed the follow-up data of 14 patients with DMSD. The median (interquartile range) follow-up period was 34 (16-64) months.Muscle biopsy samples, along with clinical information and laboratory data, were sent to a referral center for muscle diseases in Japan for diagnosis.Of patients whose myopathologic diagnosis was made at the National Center of Neurology and Psychiatry between January 2009 and August 2019, 199 patients were eligible for inclusion. These patients underwent full investigation for DM-specific autoantibodies (against transcriptional intermediary factor γ, Mi-2, melanoma differentiation–associated gene 5, nuclear matrix protein 2 [NXP-2], and small ubiquitin-like modifier activating enzyme ); however, 17 patients were excluded because their muscle fibers did not express myxovirus resistance protein A, a sensitive and specific marker of DM muscle pathology. MAIN OUTCOMES AND MEASURES: Diagnosis of DMSD was based on the absence of a skin rash at the time of muscle biopsy. RESULTS: Of the 182 patients, 93 were women (51%) and 46 were children (25%) (<18 years). Fourteen patients (8%) had DMSD and none were clinically diagnosed with DM. Among the 14 patients with DMSD, 12 (86%) were positive for anti-NXP-2 autoantibodies, while the remaining 2 were positive for anti–transcriptional intermediary factor γ and anti-Mi-2 autoantibodies, respectively. Only 28% of patients (47 of 168) with a skin rash were positive for anti-NXP-2 autoantibodies, indicating a significant association between anti-NXP-2 autoantibodies and DMSD (86% [12 of 14] vs 28% [47 of 168]; P < .001). This association was also supported by multivariable models adjusted for disease duration (odds ratio, 126.47; 95% CI, 11.42-1400.64; P < .001). CONCLUSIONS AND RELEVANCE: Dermatomyositis sine dermatitis does exist and accounts for 8% of patients with DM confirmed with muscle biopsy. Dermatomyositis sine dermatitis is significantly associated with anti-NXP-2 autoantibodies, which contrasts with anti-MDA5 DM, which is typically clinically amyopathic in presentation. It is essential to distinguish DMSD from other types of myositis because DM-specific therapies that are currently under development, including Janus kinase inhibitors, may be effective for DMSD.

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. Chirality, 2010. © 2010 Wiley‐Liss, Inc.

A solution of optically pure kynurenine (KYN), i.e., D‐KYN or L‐KYN, was administered intravenously to male Sprague‐Dawley rats (10 mg kg−1ml−1). The time‐course of changes in the concentrations of urinary monoamines and their metabolites such as 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindole acetic acid (5‐HIAA), dopamine, and 3‐methoxytyramine were investigated by reversed‐phase high‐performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D‐KYN, not L‐KYN, caused a significant increase in urinary 5‐HT levels within 30 min after its administration. With regard to the metabolites, urinary 3‐MT level was increased by D‐KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D‐KYN or L‐KYN. These results suggest that D‐KYN could affect the activity of neuroactive amines, especially 5‐HT, in vivo. Chirality, 2010. © 2009 Wiley‐Liss, Inc.

L‐Kynurenine (KYN), a tryptophan metabolite, is metabolized to kynurenic acid (KYNA), which is an antagonist of N‐methyl‐D‐aspartate and α7 nicotinic acetylcholine receptors, by kynurenine aminotransferase (KAT) I and KAT II. In this study, optically pure KYN, namely L‐KYN or D‐KYN, was administered intraperitoneally to male Sprague‐Dawley rats (16.3 μmol kg−1), and the change in plasma KYNA was investigated by using column‐switching high‐performance liquid chromatography (HPLC) with fluorescence detection. Unexpectedly, no remarkable alteration in the plasma KYNA was observed when a natural isomer, L‐KYN, was administered, whereas plasma KYNA concentration was unequivocally increased when an unnatural isomer, D‐KYN, was administered. Serum protein bindings of L‐KYN and D‐KYN were also studied, and the protein binding of L‐KYN (∼65%) in rat serum was larger than that of D‐KYN (∼12%), suggesting that D‐KYN may be easily incorporated and metabolized in tissues during blood circulation to generate KYNA in mammals. In addition, the increase in plasma KYNA by the administration of D‐KYN was suppressed in rats pretreated with a selective inhibitor of D‐amino acid oxidase (DAAO), 5‐methylpyrazole‐3‐carboxylic acid (80 mg/kg). These results suggest that DAAO might be responsible for the production of KYNA from D‐KYN in vivo. Chirality, 2009. © 2008 Wiley‐Liss, Inc.

2R-γ-Tocotrienol (γ-T3) is currently receiving attention because it has beneficial effects not observed with α-tocopherol. To achieve the effective delivery of γ-T3, we synthesized three kinds of ester derivatives of γ-T3 and evaluated their use as hydrophilic prodrugs for γ-T3 in vitro and in vivo. 2R-γ-Tocotrienyl N,N-dimethylamino-acetate hydrochloride (compound 3) was a solid compound, with high solubility and stability in water, and was converted to γ-T3 by esterases in rat and human liver. Intravenous administration of 3 in rats led to a rapid increase in the plasma, liver, heart, and kidney levels of γ-T3. The bioavailability (plasma level) after intravenous administration was 82.5 ± 13.4% and 100 ± 11.3% for 3 and γ-T3 in surfactant, respectively, and the availability in liver was 213 ± 47.6% and 100 ± 4.8% for 3 and γ-T3 in surfactant, respectively. Furthermore, the systemic availability of 2,7,8-trimethyl-2S-(β-carboxyethyl)-6-hydroxychroman (S-γ-CEHC), a metabolite of γ-T3, was 78.6% for compound 3, 47.1% for γ-T3 in surfactant, and 100% for racemic γ-CEHC. Based on these results, we identified compound 3 as the most promising water-soluble prodrug of γ-T3 and two-step prodrug of S-γ-CEHC.

2R-γ-Tocotrienol (γ-T3) is currently receiving attention because it has beneficial effects not observed with α-tocopherol. To achieve the effective delivery of γ-T3, we synthesized three kinds of ester derivatives of γ-T3 and evaluated their use as hydrophilic prodrugs for γ-T3 in vitro and in vivo. 2R-γ-Tocotrienyl N,N-dimethylamino-acetate hydrochloride (compound 3) was a solid compound, with high solubility and stability in water, and was converted to γ-T3 by esterases in rat and human liver. Intravenous administration of 3 in rats led to a rapid increase in the plasma, liver, heart, and kidney levels of γ-T3. The bioavailability (plasma level) after intravenous administration was 82.5 ± 13.4% and 100 ± 11.3% for 3 and γ-T3 in surfactant, respectively, and the availability in liver was 213 ± 47.6% and 100 ± 4.8% for 3 and γ-T3 in surfactant, respectively. Furthermore, the systemic availability of 2,7,8-trimethyl-2S-({szligbeta}-carboxyethyl)-6-hydroxychroman (S-γ-CEHC), a metabolite of γ-T3, was 78.6% for compound 3, 47.1% for γ-T3 in surfactant, and 100% for racemic γ-CEHC. Based on these results, we identified compound 3 as the most promising water-soluble prodrug of γ-T3 and two-step prodrug of S-γ-CEHC.

Abstract: To improve the marine corrosion resistance of stainless steel coatings fabricated by high-velocity oxyfuel (HVOF) spraying with a gas shroud attachment, the molybdenum (Mo) content of stainless steel was increased to form coatings with a chemical composition of Fe balance-18mass%Cr-22mass%Ni-2∼8mass%Mo. These coatings were highly dense, with <0.1 vol.% in porosity, and less oxidized, with 0.5 mass% in oxygen content at most. The corrosion mechanism and resistance of the coatings were investigated by electrochemical measurement, chemical analysis, and statistical processing. The general corrosion resistance of the coatings in 0.5 mol/dm3 sulfuric acid was improved with increases in Mo content, and the corrosion rate could be decreased to 8.8 10−2 mg/cm2 per hour (∼1 mm/year) at 8 mass% Mo. The pitting corrosion resistance of the coatings in artificial seawater was improved with increases in Mo content and was superior to that of the 316L stainless steel coating. The crevice corrosion resistance of the coatings in artificial seawater was improved and the number of rust spots at 4 mass% Mo was decreased to 38% of that for the 316L coating. Accordingly, Mo is highly effective in improving the corrosion resistance of the stainless steel coatings by HVOF spraying.

Both fluoxetine (FLX) and its N-demethylated metabolite, norfluoxetine (NFLX), have been reported to be potent serotonin-reuptake inhibitors. A sensitive and reliable method that allows simultaneous quantification of their plasma levels would be valuable and was developed in this work. The procedure included extraction of FLX and NFLX from plasma, fluorescence derivatization with 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl), separation of the derivatives on an octadecylsilica column with acetonitrile–water (55:45,v/v) as mobile phase and fluorescence detection with emission at 537 nm and excitation at 478 nm. The calibration curves were linear for FLX and NFLX concentration over the range of 10–1000 nM (r = 0.9992 and r = 0.9997) and the limits of quantitation were 10 nM in 100 µL of plasma. Precision of intra- and inter-day RSD of less than 12% and accuracy of intra- and inter-day RE within −6.0–13% were achieved. The method described was applied to analysis of the plasma samples from rats treated with FLX hydrochloride and to the pharmacokinetic study. Copyright © 2002 John Wiley & Sons, Ltd.

Both fluoxetine (FLX) and its N‐demethylated metabolite, norfluoxetine (NFLX), have been reported to be potent serotonin‐reuptake inhibitors. A sensitive and reliable method that allows simultaneous quantification of their plasma levels would be valuable and was developed in this work. The procedure included extraction of FLX and NFLX from plasma, fluorescence derivatization with 4‐(N‐chloroformylmethyl‐N‐methyl) amino‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐COCl), separation of the derivatives on an octadecylsilica column with acetonitrile–water (55:45,v/v) as mobile phase and fluorescence detection with emission at 537 nm and excitation at 478 nm. The calibration curves were linear for FLX and NFLX concentration over the range of 10–1000 nM(r= 0.9992 and r= 0.9997) and the limits of quantitation were 10 nMin 100 µL of plasma. Precision of intra‐ and inter‐day RSD of less than 12% and accuracy of intra‐ and inter‐day RE within −6.0–13% were achieved. The method described was applied to analysis of the plasma samples from rats treated with FLX hydrochloride and to the pharmacokinetic study. Copyright © 2002 John Wiley & Sons, Ltd.

In this study, we examined the affinities of many (21) compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor (ER) by ER binding assay using fluorescence polarization (FP). This method is based on the competitive binding assay using fluorescein-labeled estradiol (F–E2), in which the polarization values decreased with the addition of the compounds (competitors). The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50% inhibition (IC₅₀) and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.

In this study, we examined the affinities of many 21 compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor ER by ER binding assay using fluorescence polarization FP. This method is based on the competitive binding assay using fluorescein-labeled estradiol F–E2, in which the polarization values decreased with the addition of the compounds competitors. The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50 inhibition IC50 and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.

d-gamma-Tocopherol (gamma-Toc) and its major metabolite, 2, 7, 8-trimethyl-2S-(beta-carboxyethyl)-6-hydroxychroman (S-gamma-CEHC), are currently receiving attention concerning their unique pharmacological activities. In order to achieve the efficient delivery of gamma-Toc and S-gamma-CEHC in vivo, we synthesized d-gamma-tocopheryl N,N-dimethylglycinate hydrochloride (gamma-TDMG) as a water-soluble prodrug of gamma-Toc and a two-step prodrug of S-gamma-CEHC. gamma-TDMG is a solid (mp 161-163 degrees C) and is quite soluble in water over 50 mM. The hydrolysis of gamma-TDMG was effectively catalyzed by esterases in rat and human liver microsomes. The disposition of gamma-TDMG after iv administration in rats was compared with that of gamma-Toc solubilized with the surfactant, polyoxyethylene hydrogenated castor oil. The plasma and liver levels of gamma-Toc rapidly increased after the iv administration of the gamma-TDMG. The liver availability of gamma-Toc after the administration of gamma-TDMG was two times higher than that of the gamma-Toc administration. The relative systemic availability of S-gamma-CEHC after the gamma-TDMG administration was an equivalent value (102%), and the mean residence time of S-gamma-CEHC was eight times longer than the racemic gamma-CEHC administration. Based on these results, gamma-TDMG was identified as the most promising water-soluble prodrug of gamma-Toc and the two-step prodrug of S-gamma-CEHC.

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (α) and resolution (R_s) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6–112.7%) and precision (1.47–10.60%) were satisfactory in the range 10–1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5–92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

Bradykinin (BK) in rat urine was determined by coupled-column HPLC with precolumn fluorogenic derivatization with a water-soluble reagent, 3-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (m-BS-ABD-F). The derivatization of BK with m-BS-ABD-F was completed at 70°C for 100 min and gave only a single peak of BK derivative in addition to the peaks of the blank. The hydrophilicity of the derivatization reagent effectively prevented the adsorption of BK during the sample pretreatment and improved the recovery of BK. Good linearity was shown between the amount of BK spiked in urine (0–10 pmol) and the peak area of the BK derivatives (correlation coefficients >0.999), and the detection limits of the BK derivative were 35 fmol (S/N = 3). The precisions (cv, %) of intra- and interday assay were not more than 5.5% and the accuracies were in the range of 95.3–111% (1 and 5 pmol of BK in urine, n = 3). Although the peak regarded as that of the BK derivative rapidly decreased after incubation at 37°C, addition of urinary kininase inhibitors to the urine samples drastically suppressed the decrease of this peak, confirming that the identified peak was that of the BK derivative. The urinary kinin excretion in male SD rats (9–11 weeks old) determined by the present method was 56.0 ± 22.1 pg/min/kg (mean ± SE, n = 5). Copyright 2001 Academic Press.

Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes. Copyright © 2001 John Wiley & Sons, Ltd.

Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3‐benzoxadiazole) skeleton such as 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐N,N‐dimethylaminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), 4‐aminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole (ABD‐F), ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulfonate (SBD‐F), 4‐hydrazino‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐H), 4‐N,N‐dimethylaminosulfonyl‐7‐hydrazino‐2,1,3‐benzoxadiazole (DBD‐H), 4‐nitro‐7‐N‐piperazino‐2,1,3‐benzoxadiazole (NBD‐PZ), 4‐N,N‐dimethylaminosulfonyl‐7‐N‐piperazino‐2,1,3‐benzoxadiazole (DBD‐PZ), 4‐(N‐chloroformylmethyl‐N‐methyl)amino‐7‐N,N‐dimethylaminosulfonyl‐2,1,3‐benzoxadiazole (DBD‐COCl) and 7‐N,N‐dimethylaminosulfonyl‐4‐(2,1,3‐benzoxadiazolyl) isothiocyanate (DBD‐NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes. Copyright © 2001 John Wiley & Sons, Ltd.

A chiral stationary phase (CSP 1) derived from an (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine showed excellent enantiomeric separation for amino acid derivatives with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), in high-performance liquid chromatography (HPLC). We compared elution profiles (separation factor and elution order) of NBD-amino acids and their analogs on HPLC, to determine the diastereomeric complex between the chiral moiety of CSP 1 and NBD-amino acid, which is responsible for the chiral recognition. ¹H-NMR studies of a mixture of model compound of CSP 1 and NBD-Ala suggest that the diastereomeric complex is composed of two hydrogen bonding sites at the amino proton and oxygen atom, and a π–π interaction by the benzofurazan structure (2,1,3-benzoxadiazole) of NBD-amino acid. Furthermore CSP 1 was able to separate esters, amides and α-methyl amino acids derivatized with NBD-F. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: ABD-F 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole APCI atmospheric chemical ionization CSP chiral stationary phase; DCC N,N'-dicyclohexylcarbodiimide; DMAP 4-dimethylamino pyridine DBD-F 4-(N,N'-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole DNM [3,5-dinitrophenylamido-(s)-1-naphthylglycine] HBD-F 4-fluoro-2,1,3-benzoxadiazole NBD-CL 4-chloro-7-nitro-2,1,3-benzoxadiazole NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole TFA trifluoroacetic acid

A chiral stationary phase (CSP 1) derived from an (S)‐N‐3,5‐dinitrobenzoyl‐1‐naphthylglycine showed excellent enantiomeric separation for amino acid derivatives with a fluorogenic reagent, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), in high‐performance liquid chromatography (HPLC). We compared elution profiles (separation factor and elution order) of NBD‐amino acids and their analogs on HPLC, to determine the diastereomeric complex between the chiral moiety of CSP 1 and NBD‐amino acid, which is responsible for the chiral recognition. 1H‐NMR studies of a mixture of model compound of CSP 1 and NBD‐Ala suggest that the diastereomeric complex is composed of two hydrogen bonding sites at the amino proton and oxygen atom, and a π–π interaction by the benzofurazan structure (2,1,3‐benzoxadiazole) of NBD‐amino acid. Furthermore CSP 1 was able to separate esters, amides and α‐methyl amino acids derivatized with NBD‐F. Copyright © 2001 John Wiley & Sons, Ltd.

A highly sensitive method for the determination of D-lactic acid and 3-hydroxybutyric acid (3-HB) in rat plasma was developed using high-performance liquid chromatography with octadecylsilica (ODS) connected to a chiral column. At first, (D + L)-lactic acid and 3-HB in the plasma were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on the ODS column and determined fluorimetrically at 547 nm with 491 nm of excitation wavelength. During the separation step on the ODS, the peak fraction of (D + L)-lactate derivative was introduced directly to a phenylcarbamoylated β-cyclodextrin chiral column by changing the flow of the eluent via a six-port valve. Then, D-lactate derivative was separated enantiomerically from the L-lactate derivative, and the enantiomeric ratio was determined from the chromatogram. Intra- and inter-day accuracy values for the determination of D-lactic acid in 10 µL of rat plasma were 97.8–109.2 and 98.4–109.9%, and those for 3-HB were 99.8–108.4 and 99.8–103.8%, respectively. The intra- and inter-day precision values were within 4.6 and 5.1% for D-lactic acid, and 2.7 and 2.4% for 3-HB, respectively. The detection limits for D-lactic acid and 3-HB were approximately 2.0 and 0.04 µM, respectively (signal-to-noise ratio 3). The proposed method was applied to the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D-lactic acid and 3-HB concentrations were observed in the diabetic rats as compared to the normal rats. Copyright © 2001 John Wiley & Sons, Ltd.

A highly sensitive method for the determination of D‐lactic acid and 3‐hydroxybutyric acid (3‐HB) in rat plasma was developed using high‐performance liquid chromatography with octadecylsilica (ODS) connected to a chiral column. At first, (D+ L)‐lactic acid and 3‐HB in the plasma were derivatized with a fluorescent reagent, 4‐nitro‐7‐piperazino‐2,1,3‐benzoxadiazole (NBD‐PZ), separated on the ODS column and determined fluorimetrically at 547 nm with 491 nm of excitation wavelength. During the separation step on the ODS, the peak fraction of (D+ L)‐lactate derivative was introduced directly to a phenylcarbamoylated β‐cyclodextrin chiral column by changing the flow of the eluent via a six‐port valve. Then, D‐lactate derivative was separated enantiomerically from the L‐lactate derivative, and the enantiomeric ratio was determined from the chromatogram. Intra‐ and inter‐day accuracy values for the determination of D‐lactic acid in 10 µL of rat plasma were 97.8–109.2 and 98.4–109.9%, and those for 3‐HB were 99.8–108.4 and 99.8–103.8%, respectively. The intra‐ and inter‐day precision values were within 4.6 and 5.1% for D‐lactic acid, and 2.7 and 2.4% for 3‐HB, respectively. The detection limits for D‐lactic acid and 3‐HB were approximately 2.0 and 0.04 µM, respectively (signal‐to‐noise ratio 3). The proposed method was applied to the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D‐lactic acid and 3‐HB concentrations were observed in the diabetic rats as compared to the normal rats. Copyright © 2001 John Wiley & Sons, Ltd.

A method for the enantiomeric determination of 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman (LLU-α, γ-CEHC) in rat plasma was developed using high-performance liquid chromatography (HPLC) with a fluorimetric derivatization with 4-N, N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) followed by O-acetylation with acetyl chloride. The proposed HPLC system used two non-chiral columns (phenyl and octadecylsilica) and a chiral column (a modified cellulose type), which were connected via two column-switching valves. A derivatized sample prepared from rat plasma was first separated on the phenyl column, and the fraction including LLU-α derivative was introduced to the octadecylsilica column to quantify the concentration of the mixture of S- and R-LLU-α. Finally, the LLU-α derivative was directly injected into the chiral column to obtain the ratio of the enantiomers. The proposed HPLC system was applied to the enantiomeric determination of LLU-α in plasma after intravenous administration of racemic LLU-α. S-LLU-α was eliminated faster than R-LLU-α, and its concentration in plasma decreased to one-third at 2 min after dosing. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: DBD-PZ 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole DPDS 2,2'-dipyridinyl disulfide LLU-α, γ-CEHC 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman TFA trifluoroacetic acid TPP triphenylphosphine

A method for the enantiomeric determination of 2,7,8‐trimethyl‐2‐(β‐carboxyethyl)‐6‐hydroxy chroman (LLU‐α, γ‐CEHC) in rat plasma was developed using high‐performance liquid chromatography (HPLC) with a fluorimetric derivatization with 4‐N, N‐dimethylaminosulfonyl‐7‐piperazino‐2,1,3‐benzoxadiazole (DBD‐PZ) followed by O‐acetylation with acetyl chloride. The proposed HPLC system used two non‐chiral columns (phenyl and octadecylsilica) and a chiral column (a modified cellulose type), which were connected via two column‐switching valves. A derivatized sample prepared from rat plasma was first separated on the phenyl column, and the fraction including LLU‐α derivative was introduced to the octadecylsilica column to quantify the concentration of the mixture of S‐ and R‐LLU‐α. Finally, the LLU‐α derivative was directly injected into the chiral column to obtain the ratio of the enantiomers. The proposed HPLC system was applied to the enantiomeric determination of LLU‐α in plasma after intravenous administration of racemic LLU‐α. S‐LLU‐α was eliminated faster than R‐LLU‐α, and its concentration in plasma decreased to one‐third at 2 min after dosing. Copyright © 2001 John Wiley & Sons, Ltd.

Water-soluble fluorogenic reagents having a 2,1,3-benzoxadiazole (benzofurazan) structure, 2-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)ethanesulfonic acid (ES-ABD-F), 4-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (p-BS-ABD-F) and 3-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (m-BS-ABD-F), were synthesized and evaluated for their fluorescence characteristics and reactivities with peptides. Although these reagents showed no fluorescence, their derivatives with valine exhibited fluorescence at maximum wavelengths from 562 to 566 nm with excitation from 424 to 426 nm. The derivatives with ES-ABD-F and m-BS-ABD-F showed a similar sensitivity to that of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), a previously developed fluorogenic reagent for amino compounds. The fluorescence intensity obtained with the p-BS-ABD-F derivative was about one-third of that of the others. Angiotensin II (ANG II), an octapeptide, was successfully derivatized with ES-ABD-F and m-BS-ABD-F and determined by using high performance liquid chromatography (HPLC) with fluorescence detection. Good linearity was obtained between the amount of peptide (1–200 pmol) and the peak area of their derivatives (correlation coefficients >0.999). The detection limits of the ANG II derivatives with ES-ABD-F and m-BS-ABD-F were 210 and 270 fmol, respectively. m-BS-ABD-F, however, showed some disadvantages in the simultaneous determination of peptides: a possible interference with the separation of derivatized peptides due to some blank peaks, and its complicated and changeable pH-reactivity profile. From these results, ES-ABD-F was recommended for the sensitive and simultaneous determination of peptides. The derivatization reaction with ES-ABD-F commonly reached a plateau at 60 °C and pH > 8.5 for several hours. The peptide derivatives of ES-ABD-F (ANG I, ANG II, ANG III, bradykinin, and substance P) were well separated by reversed phase HPLC. In addition, the amyloid β-peptide (1–40) [Aβ(1–40)] derivative with ES-ABD-F was successfully eluted from a YMC-pack C4 (150 × 4.6 mm id, 5 μm particle size) under conditions such that underivatized Aβ(1–40) was not eluted, suggesting that the derivatization with ES-ABD-F decreased the adsorption of hydrophobic peptides to the HPLC stationary phase.

A fluorimetric determination method for N‐arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled‐column high‐performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4‐N‐chloroformylmethyl‐N‐methylamino‐7‐N,N‐dimethylaminosulfonyl‐2,1,3‐benzoxadiazole (DBD‐COCl), purified by a solid‐phase extraction (Empore™), and assayed by the coupled‐column HPLC. The HPLC consisted of phenyl (100 × 4.6 mm i.d.) and octadecylsilica columns (250 × 4.6 mm i.d.), both connected by a six‐port valve. The concentration of anandamide in rat brain was 3.37 ± 0.73 pmol/g with 6.47 and 3.57% of intra‐ and inter‐day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16‐fold higher than that of rats without PMSF (p< 0.01). Copyright © 2000 John Wiley & Sons, Ltd.

A fluorimetric determination method for N-arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled-column high-performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4-N-chloroformylmethyl-N-methylamino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Empore™), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 × 4.6 mm i.d.) and octadecylsilica columns (250 × 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3.37 ± 0.73 pmol/g with 6.47 and 3.57% of intra- and inter-day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16-fold higher than that of rats without PMSF (p &lt; 0.01). Copyright © 2000 John Wiley & Sons, Ltd.

A highly sensitive method, based on fluorescence derivatization with 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), was developed for the determination of D- and L-lactic acid. High-performance liquid chromatographic separation of the lactic acid derivative was achieved using an octadecylsilica column followed by enantiomeric separation on a phenylcarbamoylated β-cyclodextrin chiral column. The separation factor for D,L-lactic acid derivatives was 1.30 using MeOH/H2O (80/20) as a mobile phase, and the detection limits were approximately 360 and 300 fmol on column for D- and L-lactic acid derivative, respectively. The proposed method was applied to determine D- and L-lactate in a wine and a lactic drink. Both D- and L-lactate could be determined simultaneously, with the precisions ranging from 3.50 to 5.78% for intra-day and 4.28–9.92% for inter-day determinations. The relative recovery was in the range of 91.6–100%. Copyright © 1999 John Wiley & Sons, Ltd.

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)-β-cyclodextrin(TM-β-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-β-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. Copyright © 1999 John Wiley & Sons, Ltd.

A method is described for the quantitative determination ofd- andl-lactate in 10 μl of rat serum, which includes fluorescence derivatization ofd- andl-lactate with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3- benzoxadiazole (DBD-PZ) followed byO-acetylation. The derivatives are separated by HPLC on an octadecylsilica, and, via column switching, on a cellulose-type chiral column. Levulinic acid was used as the internal standard. The enantiomers of lactate were separated with the separation factor (α) of 1.27 and the resolution (Rs) of 2.72, while the linearity for the detection was over the range of 10 nmol/ml to 20 μmol/ml (r= 0.999). Interday precision values ford-lactate in rat serum were 5.8, 5.3, and 4.1% for 10, 100, and 1000 nmol/ml, and accuracy values were 109.6, 98.2, and 103.1%, respectively (n= 5). The reduction ofd-lactate concentration in rat serum by fasting was observed with the method.

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)‐β‐cyclodextrin(TM‐β‐CD) was most effective for enantiomeric separation of NBD‐D,L‐Asp with optimum conditions of 30 mMTM‐β‐CD in 50 mMphosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nMfor 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. Copyright © 1999 John Wiley & Sons, Ltd.

A method for amino acid sequence andd/lconfiguration identification of peptides by using fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying theird/lconfigurations. Combination of the two HPLC systems, the amino acid sequence andd/lconfiguration of peptides could be determined. This method will be useful for searchingd-amino-acid-containing peptides in animals.

A highly sensitive method, based on fluorescence derivatization with 4‐N,N‐dimethylaminosulfonyl‐7‐piperazino‐2,1,3‐benzoxadiazole (DBD‐PZ), was developed for the determination of D‐ and L‐lactic acid. High‐performance liquid chromatographic separation of the lactic acid derivative was achieved using an octadecylsilica column followed by enantiomeric separation on a phenylcarbamoylated β‐cyclodextrin chiral column. The separation factor for D,L‐lactic acid derivatives was 1.30 using MeOH/H2O (80/20) as a mobile phase, and the detection limits were approximately 360 and 300 fmol on column for D‐ and L‐lactic acid derivative, respectively. The proposed method was applied to determine D‐ and L‐lactate in a wine and a lactic drink. Both D‐ and L‐lactate could be determined simultaneously, with the precisions ranging from 3.50 to 5.78% for intra‐day and 4.28–9.92% for inter‐day determinations. The relative recovery was in the range of 91.6–100%. Copyright © 1999 John Wiley & Sons, Ltd.

Nicardipine, a dihydropyridine type calcium channel blocker, was infused at two flow-rates into spontaneously hypertensive (SH) and control normotensive Wistar–Kyoto (WKY) rats (young, 6-week-old and adult, 23-week-old, n = 5) under pentobarbital anesthesia, to cause hypotension. Mean arterial blood pressure and the concentrations of plasma amino acids and norepinephrine (NE) were measured before infusion and at each step of the infusion. The reduction in blood pressure caused by nicardipine induced a decrease in plasma L-arginine concentration in both young and adult SH rats, this effect being larger in adult rats. There was no significant change in plasma levels of L-arginine in age-matched WKY rats. The concentration of other amino acids did not change in both rat strains. On the contrary, there was an increase in plasma NE concentration in both SH and WKY rats after infusion with nicardipine. Plasma L-arginine concentration showed a good inverse correlation with the logarithm of plasma NE concentration in SH and WKY rats and the correlation was expressed as Y = −αlog(X) + m (Y, plasma L-arginine concentration (nmol/mL); X, plasma NE concentration (pmol/mL); α, a slope; and m, an intercept). α, 43.0 and 4.35 for 23-week-old SH and WKY rats, respectively, and 17.0 and 4.0 for 6-week-old SH and WKY rats, respectively. The present data together with previous data suggest a direct noradrenergic stimulation of the synthesis of nitric oxide (NO) from L-arginine. The findings also indicate an impairment of the L-arginine metabolism or pools in SH rats compared with WKY rats. The deficiency of L-arginine increases with the age of SH rats and could be related to the development and maintenance of hypertension due to inefficient production of NO. Copyright © 1999 John Wiley & Sons, Ltd.

4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-Pro), 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylamino-sulphonyl)-2,1,3-benzoxadiazole DBD-Pro), 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole NBD-N-Me-Ala), 4-(N-1-carboxyethyl-N-methyl) amino-7-(N,N-dimethylamino-2,1,3-benzoxadiazole. (DBD-N-Me-Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at λ ex = 469, λ em = 569 for DBD-moeity and λ ex = 469, λ em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed-phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were DL when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N-methyl alanine as the chiral selector. DBD-Pro and NBD-Pro seem to give better separation as compared to DBD-N-Me-Ala and NBD-N-Me-Ala. © 1998 John Wiley & Sons, Ltd.

In this communication, we demonstrate that d‐aspartate (d‐Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of d‐Asp in mammalian cells. Synthesis of d‐Asp was demonstrated by its time‐dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. d‐Asp in PC12 cells was identified by (i) co‐elution with authentic d‐Asp on two different HPLC columns, an octadesyl silica column and a Pirkle‐type chiral column, (ii) reversed elution order of d‐Asp and l‐Asp on another Pirkle‐type chiral column with an opposite configuration, and (iii) sensitivity to d‐Asp oxidase. In the cells the amount of d‐Asp was approx. 12–14% of total Asp and no other investigated d‐amino acid was detected. The amount of d‐Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB‐1 cells. Immunocytochemical staining with anti‐d‐Asp antiserum demonstrated that d‐Asp synthesized is present in the cytoplasm of the cells.

Specific polyclonal antibody was raised against D-aspartate (D-Asp) which had been conjugated to glutaraldehyde and was purified by affinity chromatography. Immunohistochemical staining of rat pineal gland with the antibody demonstrated the presence of D-Asp in the cytoplasm of pinealocytes, the predominant cell type in this gland. D-Asp immunoreactivity was more evident in the distal region than in the proximal region of the gland. Pinealocytes in the distal region are presumably involved in the synthesis and secretion of the pineal hormone, melatonin, and the results of staining may indicate some yet unknown role of D-Asp in the regulation of melatonin secretion.

Developmental changes in cellular localization of D-aspartic acid (D-Asp) were investigated in rat adrenal gland with polyclonal anti-D-Asp antibody. At 1 and 3 weeks of age, immunoreactivity (IR) toward this amino acid was intense in the cytoplasm of cells in the zona fasciculata (ZF) and zona reticularis (ZR) of the adrenal cortex but was less so in the zona glomerulosa (ZG). Conversely at 8 weeks of age, intense IR was observed in the ZG and less in the ZF and ZR. In the adrenal medulla, IR was evident in large clusters of cells which were identified as adrenaline-storing cells. The emergence of D-Asp in specific types of cells at distinct periods of development of rat adrenal gland suggests that this amino acid may have a physiological role in the maturation of the organ.

In this communication, we demonstrate that d-aspartate ( d-Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of d-Asp in mammalian cells. Synthesis of d-Asp was demonstrated by its time-dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. d-Asp in PC12 cells was identified by (i) co-elution with authentic d-Asp on two different HPLC columns, an octadesyl silica column and a Pirkle-type chiral column, (ii) reversed elution order of d-Asp and l-Asp on another Pirkle-type chiral column with an opposite configuration, and (iii) sensitivity to d-Asp oxidase. In the cells the amount of d-Asp was approx. 12–14% of total Asp and no other investigated d-amino acid was detected. The amount of d-Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB-1 cells. Immunocytochemical staining with anti- d-Asp antiserum demonstrated that d-Asp synthesized is present in the cytoplasm of the cells.

Significant amounts of D-aspartate (Asp) are found in mammalian tissues and D-Asp is presumed to play some significant, but as yet undefined physiological role. However, it is not known whether D-Asp is synthesized in mammals. In this study, we addressed this issue in cultured rat pinealocytes, parenchymal cells of the pineal gland, which contain significant amounts of D-Asp. Biosynthesis of D-Asp was found to be minimal to non-existent in cultured rat pinealocytes. We then investigated the mechanism of uptake of D-Asp into these cells and its consequent effect on cell function. D-Asp was efficiently taken up into cells, in a time- and dose-dependent manner. Interestingly, the L-Asp levels in the cells and media decreased concomitantly with the uptake of D-Asp. This decrease was not due to D-Asp cytotoxicity, since the cellular levels of othernted.D-Serine and D-alanine were not taken up efficiently into the cells and the cellular levels of L-serine and L-alanine were unchanged. Also, immunocytochemical staining with anti-D-Asp antibody showed that D-Asp, which had been taken up into the cells, was dispersed throughout the cytoplasm. In response to norepinephrine stimulation, pinealocytes, which had been pretreated with D-Asp released D-Asp as well as L-Asp. In these cells, norepinephrine-induced secretion of melatonin, a pineal hormone, was suppressed. The mechanism of this suppression is discussed here.

1‐(5‐Dimethylamino‐1‐naphthalenesulphonyl)‐(S)‐3‐aminopyrrolidine (DNS‐Apy) has been synthesized for the separation of carboxylic acid enantiomers by high‐performance liquid chromatography (HPLC) and sensitive detection. The reagent reacts with carboxylic acids at room temperature in the presence of activation agents 2,2′‐dipyridyl disulphide (DPDS) and triphenylphosphine (TPP). The maximum emission of the diastereomeric amide derived from (S)‐phenylpropionic acid and ketoprofen derivatives of DNS‐Apy was at 530 nm with excitation at 340 nm. The emission wavelength shifted towards the blue and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The diastereomers derived from anti‐inflammatory drugs were efficiently resolved with a reverse‐phase column using water:acetonitrile mixture as mobile phase. All of the racemate of arylpropionic acid derivatives gave equal fluorescence intensity of the two enantiomers with the exception of ketoprofen derivatives where the intensity of the first eluting enantiomer was half that of the second. © 1997 John Wiley & Sons, Ltd.

Nicardipine, a dihydropyridine type calcium channel blocker, was infused at two flow‐rates into spontaneously hypertensive (SH) and control normotensive Wistar–Kyoto (WKY) rats (young, 6‐week‐old and adult, 23‐week‐old, n= 5) under pentobarbital anesthesia, to cause hypotension. Mean arterial blood pressure and the concentrations of plasma amino acids and norepinephrine (NE) were measured before infusion and at each step of the infusion. The reduction in blood pressure caused by nicardipine induced a decrease in plasma L‐arginine concentration in both young and adult SH rats, this effect being larger in adult rats. There was no significant change in plasma levels of L‐arginine in age‐matched WKY rats. The concentration of other amino acids did not change in both rat strains. On the contrary, there was an increase in plasma NE concentration in both SH and WKY rats after infusion with nicardipine. Plasma L‐arginine concentration showed a good inverse correlation with the logarithm of plasma NE concentration in SH and WKY rats and the correlation was expressed as Y= −αlog(X) + m(Y,plasma L‐arginine concentration (nmol/mL); X,plasma NE concentration (pmol/mL); α, a slope; and m,an intercept). α, 43.0 and 4.35 for 23‐week‐old SH and WKY rats, respectively, and 17.0 and 4.0 for 6‐week‐old SH and WKY rats, respectively. The present data together with previous data suggest a direct noradrenergic stimulation of the synthesis of nitric oxide (NO) from L‐arginine. The findings also indicate an impairment of the L‐arginine metabolism or pools in SH rats compared with WKY rats. The deficiency of L‐arginine increases with the age of SH rats and could be related to the development and maintenance of hypertension due to inefficient production of NO. Copyright © 1999 John Wiley & Sons, Ltd.

4‐(2‐carboxypyrrolidin‐1‐yl)‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐Pro), 4‐(2‐carboxypyrrolidin‐1‐yl)‐7‐(N,N‐dimethylamino‐sulphonyl)‐2,1,3‐benzoxadiazole DBD‐Pro), 4‐(N‐1‐carboxyethyl‐N‐methyl)amino‐7‐nitro‐2,1,3‐benzoxadiazole NBD‐N‐Me‐Ala), 4‐(N‐1‐carboxyethyl‐N‐methyl) amino‐7‐(N,N‐dimethylamino‐2,1,3‐benzoxadiazole. (DBD‐N‐Me‐Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2′‐dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at λ ex = 469, λ em = 569 for DBD‐moeity and λ ex = 469, λ em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed‐phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were DL when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N‐methyl alanine as the chiral selector. DBD‐Pro and NBD‐Pro seem to give better separation as compared to DBD‐N‐Me‐Ala and NBD‐N‐Me‐Ala. © 1998 John Wiley & Sons, Ltd.

It is shown that an electron-withdrawing or -donating group at the para-position of aromatic isothiocyanate significantly affects the racemization of 2,1,3-benzoxadiazolylthiazolinone (TZ) derivatives of amino acids, derivatized with newly synthesized benzoxadiazolyl isothiocyanates in Edman sequence analysis. A linear relationship between the logarithms of the TZ-amino acid enantiomer ratio and the para-substituent constants (ς_p) for the isothiocyanate moiety was obtained, and the D/L configuration of the amino acid residue was retained with an isothiocyanate containing an electron-donating group at the para-position. The para-substitution effect on the racemization of phenylthiohydantoin (PTH) amino acids was also confirmed by several para-substituted phenylisothiocyanate (PITC) reagents, including nitro-PITC, chloro-PITC, PITC, methyl-PITC and methoxy-PITC. The relationship between the fluorescence intensity of the 2,1,3-benzoxadiazolyl TZ amino acid and ς_p was also demonstrated. When the isothiocyanate containing an electron-donating group was used, the fluorescence intensity of the TZ-amino acid decreased while retaining the D/L configuration of the amino acid residues.

In the current study, localization ofd-aspartic acid (d-Asp) in rat testis was studied by immunohistochemical and biochemical techniques. Immunohistochemical staining of this tissue using specific polyclonal antibody tod-Asp revealedd-Asp immunoreactivity (IR) in the cytoplasm of germ cells, especially around the region rich in elongate spermatids, the most mature of the germ cells. Weak IR was also noted in cytoplasm of spermatocytes and round spermatids; however, it was negligible in interstitial cells and Sertoli cells. The intensity of immunostaining in each seminiferous tubule differed according to its distinct germ cell composition. In testis of young rats, seminiferous tubules lack elongated spermatids, andd-Asp was found to be localized in spermatocytes, the most mature population of germ cells at that age. We used various toxicants to destroy specific testicular cell populations and to confirm the localization ofd-Asp in rat testis. Administration of ethane dimethane sulfonate induced a selective destruction of all Leydig cells in this tissue. This resulted in a significant decrease in thed-Asp level, which was probably due to a drop in testosterone brought about by this treatment, and this was followed by a modulation of spermatogenesis. Three days after treatment with methoxyacetic acid (MAA), many seminiferous tubules were found to lack or to have severe depletions of pachytene spermatocytes, but not of elongate spermatids. This caused reductions in protein content and in the total amount ofl-Asp, but not that ofd-Asp. Twenty days after treatment with MAA, the depleted population of germ cells progressed through the spermatogenic cycle from pachytene spermatocytes to elongate spermatids. At this time, the level ofd-Asp decreased significantly, as did that ofl-Asp and protein, consistent withd-Asp localization in elongate spermatids. This decrease in thed-Asp level was also seen with immunostaining.

To develop new fluorogenic reagents having the benzofurazan structure, we investigated the effects of the substituent groups at the 4- and 7-positions of the benzofurazan skeleton on the fluorescence characteristics (fluorescence intensity, maximum excitation wavelength and maximum emission wavelength). Seventy benzofurazan compounds substituted at the 4- and 7-positions were obtained for this purpose. The Hammett substituent constant (σp) was adopted as a parameter for electronic effects by substituent groups. The study using the sum and the difference of the Hammett substituent constants (σp) at the 4- and 7-positions revealed that the highly fluorescent benzofurazan compounds were classified into two groups and that singlet excitation energies, calculated by the maximum excitation and emission wavelengths, of the benzofurazan compounds were different between these two groups. The fluorescence characteristics of benzofurazan compounds substituted at the 4- and 7-positions were empirically predictable by these relationships and σp values. A new fluorogenic reagent, 4-phenylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole, for amines was developed based on this method and applied to the amino acids analysis.

1-(5-Dimethylamino-1-naphthalenesulphonyl)-(S)-3-aminopyrrolidine (DNS-Apy) has been synthesized for the separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC) and sensitive detection. The reagent reacts with carboxylic acids at room temperature in the presence of activation agents 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP). The maximum emission of the diastereomeric amide derived from (S)-phenylpropionic acid and ketoprofen derivatives of DNS-Apy was at 530 nm with excitation at 340 nm. The emission wavelength shifted towards the blue and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The diastereomers derived from anti-inflammatory drugs were efficiently resolved with a reverse-phase column using water:acetonitrile mixture as mobile phase. All of the racemate of arylpropionic acid derivatives gave equal fluorescence intensity of the two enantiomers with the exception of ketoprofen derivatives where the intensity of the first eluting enantiomer was half that of the second. © 1997 John Wiley & Sons, Ltd.

By use of a Crithidiabioassay, the production of a 6-(l-erythrodihydroxypropyl)pterin (biopterin)-like substance was observed during the cultivation of Chinese hamster ovary cell line (CHO K1) and mouse neuroblastoma cell line (C-1300). Further studies were carried out with the CHO K1 cells using various suspected precursors. In a cell culture [2-14C]folic acid was converted to 2-amino-4-hydroxypteridine (pterin), 6-hydroxymethylpterin, and 6-carboxypterin, but not to biopterin. When [2-14C]guanine was added to the culture, radioactive biopterin and pterin were obtained. However, when [2-14C]guanine was replaced by [8-14C]guanine, little or no radioactivity was incorporated into biopterin and other pterins, indicating that the purine undergoes ring opening and elimination of carbon atom 8. When the cells were cultured in a biopterin-free medium with [2-14C]guanine, the specific radioactivities of the isolated biopterin and GMP from RNA were in close agreement. A similar experiment with [U-14C]-guanosine showed that only about 10% of the label found in the GMP of RNA appeared in the ribosyl moiety. Therefore, using this approach the possible involvement of the ribosyl moiety of GMP in biopterin biosynthesis could not be unequivocally determined. In addition, the results suggested that the radioactivity found in the other nucleotides of RNA was distributed among the ribose moiety. When [6-14C]glucose was used most of the radioactivity found in biopterin was localized in the side chain and in carbon atom 5 of the ribose moiety of GMP in RNA. The results indicate that guanosine or one of its phosphorylated derivatives is the precursor of biopterin and that the side chain is of ribose origin. The addition of 6-lactyl-7,8-dihydropterin (sepiapterin) to a cell culture with [2-14C]guanine increased the amount of biopterin isolated and prevented the incorporation of radioactivity into the biopterin. This result indicates the possible intermediary role of sepiapterin. Based upon these observations, the metabolic relationship of pterins in CHO cell culture is proposed.