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Cancer‐associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor‐cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse‐Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.

Solid-phase extraction technologies are widely used for sample pretreatment in bioanalysis. Monolithic silica disk-packed spin columns modified with phenylboronate moieties have been developed for the selective extraction of cis-diol compounds such as catecholamines. However, in our preliminary studies, serotonin was found to also be extracted in this treatment, along with catecholamines. In this study, the interaction between serotonin-related compounds (serotonin, tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid) and phenylboronate moieties was investigated. We found that only serotonin was extracted with phenylboronate-modified monolithic silica, whereas tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid were not. Hydrophobic interactions rather than ionic interactions were the primary factor for the adsorption of serotonin to phenylboronate. Finally, the selective pretreatment procedure for catecholamines was improved: thus, the method could be applied for the pretreatment of bio-samples.

The synthesized sheet-like polypyrrole (ppy) nanowires were used as solid phase extraction materials, followed by gas chromatography-mass spectrometry (GC–MS) for the detection of traces residues of pyrethroid pesticides in human plasma. A multiresidue method was developed and verified for the determination of trace pyrethroid residues (transfluthrin, allethrin, resmethrin, fenpropathrin, etofenprox, fenvalerate) in human plasma. In this study, using the cationic surfactant cetyltrimethylammonium bromide (CTAB) as a soft template, ppy nanowires with regular morphology were prepared by oxidative polymerization. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and other techniques were employed for characterization. Molecular dynamics analyses were used to simulate the adsorption mechanism of each pyrethroid and ppy nanowires. Based on density analysis, molecular recognition analysis, and binding energy, the van der Waals force was considered as an important driving force for the adsorption of pyrethroids and ppy nanowires. The limits of detection (LOD) of six pyrethroids were 0.008–0051 ng mL−1, and the limits of quantification (LOQ) were 0.028–0.162 ng mL−1. The relative standard deviations of ppy nanowires were 2.12–5.09%, and the recoveries of six pyrethroids ranged from 76.9 to 110.4%. The enrichment factors were within the range of 47.09–51.30. The experimental results showed that the method could be an efficient detection method for trace residue analysis of pyrethroid pesticides in complex biological samples. It would be advantageous for clinical monitoring and toxicological studies of pyrethroids.

There is an increasing need for new analytical methods that can handle a large number of analytes in complex matrices. Hydrophilic interaction chromatography (HILIC) has recently been demonstrated as an important supplement to reversed-phase liquid chromatography for polar analytes, particularly endogenous compounds. With the increasing popularity of HILIC, progressively more polar phases with diverse functional groups have been developed. In addition, the coupling of HILIC to mass spectrometry offers the advantages of improved sensitivity by employing an organic-rich mobile phase. This article reviews recent applications of HILIC for the analysis of endogenous and pharmaceutical compounds in plasma samples. Furthermore, based on recent studies, we provide a discussion of column selection, sample pretreatment for HILIC analysis, and detection sensitivity.

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.

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

Background/Aims: The plasma concentration of catecholamines and their metabolites generated by catechol-O-methyl transferase (COMT) were measured and their correlation with the progress of renal dysfunction was investigated in two distinctive animal models: a 5/6 nephrectomized Sprague-Dawley rat model and a 1/2 nephrectomized diabetic fatty Zucker rat model. Methods: A highly sensitive, high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection was employed to obtain values for the ratio [NMN]/([NE] + [NMN]), where [NE] represents the plasma concentration of norepinephrine and [NMN] represents the plasma concentration of normetanephrine. Results: The [NMN]/([NE] + [NMN]) ratio correlated with both the increase in blood urea nitrogen concentration and the decrease in creatinine clearance. Conclusion: The [NMN]/([NE] + [NMN]) ratio represents a quantitative indicator of the progress of renal dysfunction in the animal models. Regulation of COMT activity seemed to relate with the progress of renal dysfunction.

Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of NG,NG-dimethyl- l -arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) – 140 μM (1.0 nmol per injection) using NG-monomethyl- l -arginine ( l -NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82 ± 0.05 μM (n = 4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.

Background Plasma catecholamines (CAs) are widely used as an index of sympathetic nervous system activity. In addition, CAs are known to be metabolized by catechol- O -methyltransferase (COMT) to produce their 3- O -methyl metabolites. We previously established a sensitive determination method of CAs and their 3- O -methyl metabolites using HPLC-peroxyoxalate chemiluminescence (POCL) reaction detection system. In this study, a microcolumn (100 × 1.0 mm I.D.) was used for separation to obtain higher sensitivity and shorter analysis time. Methods The system included automated precolumn ion-exchange extraction of amines, followed by separation on an ODS column, coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally POCL reaction detection. Results The detection limits for CAs and their 3- O -methyl metabolites were 0.3–2.0 fmol. The analysis time was about 35 min, about half that of previously reported results. The method developed was used in monitoring changes in CAs and 3- O -methyl metabolite concentrations in human plasma during exercise. Conclusion The simultaneous determination method for concentrations of CAs and their 3- O -methyl metabolites in human plasma was developed using micro LC-peroxyoxalate chemiluminescence detection. We were successful in quantitating the changes in plasma CAs and their 3- O -methyl metabolites during exercise.

Catecholamines (CAs) are important hormones in regulating blood pressure both in centrally and peripheral sympathetic nerve endings. Production of CAs, release and inactivation are three components to regulate CAs level. We have reported that the inactivation of CAs by catechol-O-methyltransferase (COMT) in the liver is important in high blood pressure in spontaneously hypertensive rats (SHR). In the present study, we investigated central role of COMT in hypertension. We investigated COMT activities in cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus of SHR and Wistar-Kyoto (WKY) rats. COMT activities were assessed by measuring normetanephrine with the use of norepinephrine as an endogenous substrate. Membrane-bound COMT activities in cerebral cortex were significantly reduced in SHR (19.1+/-1.8 pmol/min/mg protein) compared with WKY rats (25.0+/-3.3 pmol/min/mg protein). The ratio of concentrations of normetanephrine/norepinephrine in cerebral cortex was also lower in SHR than in WKY rats. Our results suggest that there is an association between MB-COMT in cerebral cortex and blood pressure regulation.

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.

Background Catechol- O-methyltransferase (COMT) catalyses the inactivation of catecholamines. It is widely distributed in most tissues in soluble (S-COMT) and membrane-bound (MB-COMT) forms. Recently, we used a new assay for COMT activity and demonstrated that COMT plays an important role in blood pressure regulation in spontaneously hypertensive rats. In order to investigate whether this is true for human hypertension, we have evaluated the erythrocyte COMT assay in humans. Method The assay procedure included the use of norepinephrine (NE) as a natural substrate and the quantification of the reaction product, normetanephrine, followed by high-performance liquid chromatography separation and fluorescence or chemiluminescence detection. Results After evaluation of the method, the optimum conditions were obtained for the assay of human erythrocyte COMT. The S- and MB-COMT activities obtained were 50·6 (24·5) and 329·8 (179·4) fmol/min/mg protein, respectively [mean (standard deviation); n = 54]. The Km values for NE were 91·3 (14·1) and 11·7 (1·1) μmol/L for S- and MB-COMT, respectively ( n=6). Conclusion The established assay method used to assess S- and MB-COMT activities in human erythrocytes could be useful to elucidate catecholamine metabolism in the normal physiological state as well as in the pathology of certain diseases.

A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol-O-methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3-O-methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. Km and Vmax values for COMT obtained with the system were 503 µM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day. Copyright © 2002 John Wiley & Sons, Ltd.

An analytical method for catecholamines and related compounds using hydrophilic interaction liquid chromatography (HILIC) with native fluorescence detection has been developed. We found that ZIC-cHILIC with phosphorylcholine was suitable for the separation of catechol compounds with good peak shapes among six different HILIC columns (Inertsil SIL, Inertsil Amide, Inertsil Diol, TSKgel NH2-100, ZIC-HILIC, and ZIC-cHILIC). Using ZIC-cHILIC, eight catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelic acid, and internal standard 3,4-dihydroxybenzylamine) were separated within 15 min. The limit of detection at a signal to noise ratio of 3 was 3–28 nM. An improved sensitivity was obtained as compared to that of reversed-phase liquid chromatography. This was partly attributed to the increase in the fluorescence intensity of the catechol compounds in the acetonitrile-rich mobile phase. Solid phase extraction using a monolithic silica disk-packed spin column with phenylboronate moieties, which have affinity to catechol compounds, was performed for the selective extraction of catechol compounds from mouse urine. Dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were successfully quantified in mouse urine.

Background l -dopa ( l -3,4-dihydroxyphenylalanine) is commonly used for treating Parkinson's disease (PD). However, regardless of its prominent effect, therapeutic range of l -dopa narrows down with disease progression, which leads to development of motor complications including wearing off and dyskinesias. In addition, intestinal absorption of l -dopa is inversely correlated with the amount of oral protein intake, and shows intra- and inter-day variability. Hence, frequent monitoring of plasma l -dopa concentrations is beneficial, but frequent venipuncture imposes physical and psychological burdens on patients with PD. Methods We investigated the usefulness of sweat samples instead of plasma samples for monitoring l -dopa concentrations. With a monolithic silica disk-packed spin column and the high-performance liquid chromatography-electrochemical detection system, l -dopa in sweat samples was successfully quantified and analyzed in 23 PD patients. Results We found that the Pearson's correlation coefficient of the plasma and sweat l -dopa concentrations was 0.678. Although the disease durations and severities were not correlated with the deviation of the actual sweat l -dopa concentrations from the fitted line, acquisition of the sweat samples under a stable condition was technically difficult in severely affected patients. The deviations may also be partly accounted for by skin permeability of l -dopa. Conclusions Measuring l -dopa concentrations in sweat is suitable to get further insights into the l -dopa metabolism.

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.

We have previously demonstrated that the release of plasma norepinephrine (NE) in response to blood pressure reduction induced by calcium antagonist was substantially decreased in spontaneously hypertensive rats (SHR), as compared to the age-matched normotensive Wistar Kyoto (WKY) rats. Here we examined extraneuronal methylation of NE to normetanephrine (NMN) by catechol-O-methyltransferase (COMT). In the face of an acute hypotension by benidipine, the ratio of the increase of NMN to the increase of NE in plasma was lower in SHR than in WKY rat. The finding suggests that the extraneuronal inactivation of the released NE is blunted in SHR, namely, COMT is deactivated in this hypertensive rats. To define the role of NE methylation in hypertension, S-adenosyl-L-methionine (SAMe; 1.0mg/kg, iv.), a coenzyme of COMT, was administered to SHR. The treatment induced a greater antihypertensive effect, along with a greater decrease of NE and a larger increase of NMN in SHR. The data suggest that SAMe may be used as a possible remedy for hypertension.

Metabolism by catechol-O-methyltransferase (COMT) is one of the inactivation pathways of catecholamines (CAs), which are important hormones in regulating blood pressure both in central and in peripheral sympathetic nerve endings. We have reported the rapid determination method of COMT activity using high-performance liquid chromatography (HPLC)-fluorescence detection. In the present study, we applied the method to brain tissues, cerebral cortex, cerebellum, hypophysis and hypothalamus. COMT activities were assessed by measuring normetanephrine with the use of norepinephrine as an endogenous substrate. We examined the COMT activities in the brains of Dahl salt-sensitive (DS) rats given normal-salt or high-salt diet for 13 weeks, and found that membrane-bound COMT activities in the cerebral cortex were significantly reduced in high-salt loaded DS rats compared with normal-salt loaded DS rats.

In this study, the separation efficiency of a core–shell particle column was compared with particle-packed and monolithic silica columns, which showed that the core–shell particle column had a smaller theoretical plate height and that its separation efficiency was not affected significantly by the increase in flow rate. A fast HPLC method using a core–shell particle column was developed for the determination of amino acids. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used as a fluorescence derivatization reagent for amino acids, followed by separation on a core–shell Kinetex C18 column. The analysis time for 21 NBD-amino acids was within 7 min, which was faster than that in our previous studies with conventional particle-packed columns or monolithic silica columns. The linearities of the calibration curves for all the amino acids were found to be good over a range of injection amounts from 40 fmol to 40 pmol. The accuracies for the amino acid determinations were 90.9–107%. The method was proved to have potential for the fast determination of amino acids in biological samples.

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase family member, which induces apoptosis in various cells through JNK and p38 MAP kinase cascades. In addition to apoptosis signaling, a number of recent in vitro studies have suggested that ASK1 may play roles in neural function. However, the behavioral significance of ASK1 has remained unclear. Here, we subjected ASK1 (-/-) mice to a battery of behavioral tests and found that they displayed temporary hyperactivity in an open-field test. Activities in the familiar field were normal, indicating that the hyperactivity observed was specific to the novel environment. ASK1 (-/-) mice also exhibited impairment of novelty preference 24h after training and superior performance on the rotarod test. Brain tissue contents of dopamine and 4-dihydroxyphenylacetic acid (DOPAC) were elevated in ASK1 (-/-) mice. Our findings thus demonstrate novel behavioral functions of ASK1, including regulation of locomotor activity, novelty preference, and motor coordination with dopaminergic transmission.

Catecholamines, namely, dopamine, norepinephrine and epinephrine, play important roles in higher animals as neurotransmitters or hormones, and are metabolized by catechol-O-methyltransferase (COMT). To elucidate the role of COMT in blood pressure regulation, we have developed simultaneous determination methods for catecholamines and their 3-O-methyl metabolites using high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection. Using the developed method, we have found that inactivation of catecholamines by COMT is attenuated in hypertensive rats compared to normotensive rats. Furthermore, both the activities and the amounts of membrane-bound (MB-)COMT in the liver were found to be lower in hypertensive rats than in normotensive rats, which indicated that liver MB-COMT may be a relevant factor in blood pressure regulation.

6-Nitronorepinephrine (nitroNE) and 6-nitroepinephrine (nitroE) are reaction products of nitric oxide and norepinephrine and epinephrine, respectively. The authors have previously reported a method for determination of nitroNE and nitroE in rat brain using high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection. In this study, the effect of oxidative stress on nitroNE and nitroE concentrations in rat brain was examined using this method. After kainic acid administration in rats for 2 days, the concentrations of both nitroNE and nitroE in rat brains were found to have increased by 400-600%, which was partly suppressed by the co-administration of a superoxide dismutase mimetic. This indicates that oxidative stress might increase nitroNE and nitroE concentrations in rat brains.

We have previously established a rapid catechol-O-methyltransferase (COMT) assay using norepinephrine (NE) as a natural substrate and flow-injection analysis. In this study, the method is improved for screening of COMT inhibitors or activators using reversed-phase high-performance liquid chromatographic separation with fluorescence detection. The excess substrate, NE, was removed by the addition of borate in the eluent for HPLC to make an ionic complex with NE, which was eluted faster than the enzymatic product, normetanephrine. The method had good precision and accuracy, and was able to assay one sample in 5 min, showing the usability for screening of COMT inhibitors or activators.

NG-Monomethyl- l -arginine ( l -NMMA), NG,NG-dimethyl- l -arginine (ADMA), and NG,NG′-dimethyl- l -arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of l -NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nω-Propyl- l -arginine was used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for l -NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for l -NMMA, ADMA and SDMA were linear within the range of 50–5000 fmol. The method was applied to the quantitative determination of l -NMMA, ADMA and SDMA in 200 μl of rat plasma. The concentrations of l -NMMA, ADMA and SDMA in rat plasma were 0.16 ± 0.03, 0.80 ± 0.25 and 0.40 ± 0.21 μM, respectively (n = 5).

An automated analyzer for vancomycin in rat plasma by column-switching high-performance liquid chromatography (HPLC) with UV detection was developed. The method includes in-line extraction of vancomycin by ion-exchange cartridge column and a separation on a reversed-phase column with UV detection at 215 nm. Plasma samples were diluted by mobile phase solution and directly injected to HPLC. Vancomycin was quantitatively recovered from rat plasma samples. The separation was completed within 15 min. The calibration curve was linear over the range from 0.5 to 100 microg/mL with the detection and quantification limits of 0.5 microg/mL (2.5 ng on column; signal-to-noise ratio = 3). The values of precision in intra- and inter-day assays (n = 3) were less than 1.92 and 3.69%, respectively. This method does not require time-consuming pre-treatment and is suitable for the routine assay of plasma samples.

A natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) was administered intravenously to male Sprague-Dawley rats and the plasma concentration of gamma-CEHC along with urinary sodium (Na+) excretion was investigated. The plasma gamma-CEHC concentrations were fluorimetrically determined by a column-switching HPLC method consisting of both phenyl and octadecyl silica columns, following a pre-column fluorescence derivatization with a fluorescence reagent, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). In rats fed with a high-NaCl (8.0%) diet, plasma gamma-CEHC concentrations rapidly decreased by 20% in 15-45 min after the administration of gamma-CEHC, while Na+ excretion gradually increased with time. Considering these results, the Na+ excretion effect appeared not to be associated with plasma gamma-CEHC concentration. In addition, attempts were made to examine a main urinary metabolite of gamma-CEHC, a large amount of 6-O-sulfated gamma-CEHC found to be present in the urine using an HPLC-tandem mass spectrometry. Thus, it is plausible that gamma-CEHC was easily metabolized to 6-O-sulfated metabolite and excreted into urine in rats.

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.

The determination of catecholamines and their 3-O-methyl metabolites in a single mouse plasma is necessary to understand the role of the sympathetic nervous activity, while the inactivation of catecholamines by catechol-O-methyltransferase indicates the activity of blood pressure regulation in animals. Here we report the basal catecholamines and their 3-O-methyl metabolite concentrations obtained from 15 µL of mouse plasma utilizing semi-microcolumn high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence detection system. The concentrations were 6.63 ± 1.37 pmol/mL plasma, 0.49 ± 0.10 pmol/mL plasma, 5.25 ± 2.30 pmol/mL plasma, 3.23 ± 0.84 pmol/mL plasma, 0.44 ± 0.11 pmol/mL plasma, and 3.39 ± 1.67 pmol/mL plasma for norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively (n = 5–7). Further, when blood pressure was reduced by minoxidil, plasma catecholamines were found to be significantly increased by the baroreflex-mediated response in mouse. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: CA catecholamine COMT catehol-O-methyltransferase DA dopamine E epinephrine MN metarephrines 3-MT, 3-methoxytyramine NE norepinephrine NMN normetanephrine SHR spontaneously hypertensive rats. WKR Wystar Kyoto rats.

Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to regulate feeding behavior and energy homeostasis. This system is referred to as the gut-brain axis. Here we show that both brush cells and type II taste cells are eliminated in the gastrointestinal tract of transcription factor Skn-1 knockout (KO) mice. Despite unaltered food intake, Skn-1 KO mice have reduced body weight with lower body fat due to increased energy expenditure. In this model, 24-h urinary excretion of catecholamines was significantly elevated, accompanied by increased fatty acid β-oxidation and fuel dissipation in skeletal muscle and impaired insulin secretion driven by glucose. These results suggest the existence of brain-mediated energy homeostatic pathways originating from brush cells and type II taste cells in the gastrointestinal tract and ending in peripheral tissues, including the adrenal glands. The discovery of food-derived factors that regulate these cells may open new avenues the treatment of obesity and diabetes. Research Context Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to regulate feeding behavior and energy homeostasis along the gut-brain axis. We propose the concept that taste-receiving cells in the oral cavity and/or food-borne chemicals-receiving brush cells in the gut are involved in regulation of the body weight and adiposity via the brain. The discovery of food-derived factors that regulate these cells may open new avenues for the treatment of obesity and diabetes., Highlights • Skn-1a is a crucial transcription factor for generating brush cells and type II taste cells in the gastrointestinal tract. • Despite unaltered food intake, Skn-1 KO mice have reduced body weight with lower body fat due to elevated energy expenditure. • Urinary excretion of catecholamines was elevated in Skn-1 KO mice, suggesting brain-mediated energy homeostatic pathways.