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Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine &beta, synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

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.

Type 2 diabetes mellitus (T2DM) has become a major disease threatening human health worldwide. At present, the treatment of T2DM cannot cure diabetes and is prone to many side effects. Psidium guajava L. leaves have been reported to possess hypoglycemic activity, and they have been widely used in diabetes treatment in the folk. However, the antidiabetic mechanism has not been clearly explained. Also, the change in amino acid profile can reflect a metabolic disorder and provide insights into system-wide changes in response to physiological challenges or disease processes. The study found that P. guajava L. leaves can decrease fasting blood glucose and lipid levels in type 2 diabetic rats induced by streptozotocin. Through the analysis of amino acid profiling following 20 days of gavage administration, the concentration data were modeled by principal component analysis and orthogonal partial least squares discriminant analysis to find the different metabolites and related metabolic pathways (including cysteine and methionine metabolism, valine, leucine, and isoleucine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis) for the explanation of the hypoglycemic mechanism of P. guajava L., which provides an experimental and theoretical basis for diabetes prediction and for the development of new drugs for the treatment of diabetes.

Summary At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

Carboxyl-bearing low-molecular-weight compounds such as keto acids, fatty acids, and other organic acids are involved in a myriad of metabolic pathways owing to their high polarity and solubility in biological fluids. Various disease areas such as cancer, myeloid leukemia, heart disease, liver disease, and lifestyle diseases (obesity and diabetes) were found to be related to certain metabolic pathways and changes in the concentrations of the compounds involved in those pathways. Therefore, the quantification of such compounds provides useful information pertaining to diagnosis, pathological conditions, and disease mechanisms, spurring the development of numerous analytical methods for this purpose. This review article addresses analytical methods for the quantification of carboxylic acids, which were classified into fatty acids, tricarboxylic acid cycle and glycolysis-related compounds, amino acid metabolites, perfluorinated carboxylic acids, α-keto acids and their metabolites, thiazole-containing carboxylic acids, and miscellaneous, in biological samples from 2000 to date. Methods involving liquid chromatography coupled with ultraviolet, fluorescence, mass spectrometry, and electrochemical detection were summarized.

Background There has been much discussion recently about the occurrence of neuropsychological complications during the perioperative period. Diabetes is known to be one of the metabolic risk factors. Although the number of patients with diabetes mellitus (DM) has been increasing, the pathophysiology of postoperative neuropsychological dysfunction in DM patients is still unclear. Recently, a deficiency of neurotransmitters, such as monoamines, was reported to be associated with mental disorders. Therefore, we investigated the effects of surgical stress on behavioral activity and hippocampal noradrenaline (NA) level in type 2 diabetes mellitus model (T2DM) mice. Methods Eighty-four 6-week-old male C57BL/6J mice were divided into four groups (non-diabetes, non-diabetes with surgery, T2DM, and T2DM with surgery groups). T2DM mice were established by feeding a high-fat diet (HFD) for 8 weeks. At 14 weeks of age, fifteen mice in each group underwent a series of behavioral tests including an open field (OF) test, a novel object recognition (NOR) test and a light–dark (LD) test. In the surgery groups, open abdominal surgery with manipulation of the intestine was performed 24 h before the behavioral tests as a surgical stress. Hippocampal noradrenaline (NA) concentration was examined in six mice in each group by high-performance liquid chromatography. The data were analyzed by the Mann–Whitney U test, and p values less than 0.05 were considered significant. Results The T2DM group showed significantly increased explorative activity in the NOR test (P = 0.0016) and significantly increased frequency of transition in the LD test (P = 0.043) compared with those in the non-diabetic group before surgery. In T2DM mice, surgical stress resulted in decreased total distance in the OF test, decreased explorative activity in the NOR test, and decreased frequency of transition in the LD test (OF: P = 0.015, NOR: P = 0.009, LD: P = 0.007) and decreased hippocampal NA (P = 0.015), but such differences were not observed in the non-diabetic mice. Conclusions Mice with T2DM induced by feeding an HFD showed increased behavioral activities, and surgical stress in T2DM mice caused postoperative hypoactivity and reduction of the hippocampal NA level.

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.

Reprogrammed cellular metabolism is a common characteristic observed in various cancers. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukaemia (CML) in humans and in mouse models of CML. BCAT1 is upregulated during progression of CML and promotes BCAA production in leukaemia cells by aminating the branched-chain keto acids. Blocking BCAT1 gene expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML both in vitro and in vivo. Stable-isotope tracer experiments combined with nuclear magnetic resonance-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function through BCAA production in blast crisis CML cells. Importantly, BCAT1 expression not only is activated in human blast crisis CML and de novo acute myeloid leukaemia, but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for blast crisis CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukaemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukaemia.

Background Noninvasive biomarkers for Parkinson's disease (PD) are currently unavailable. Objective To search for a biomarker unique to PD in sweat and serum. Methods Sweat samples in 42 PD patients and 16 controls were analyzed using liquid chromatography/mass spectrometry (LC/MS). The principal component analysis (PCA) and the orthogonal projections to latent structures (OPLS) analysis were employed. Serum Phe and Tyr levels were determined using the HPLC-fluorescence detection system in 28 de novo PD patients, 52 L-Dopa-treated PD patients, and 27 controls. Results PCA and OPLS analyses of LC/MS of sweat samples revealed that Tyr, Phe, Leu (Ile), and Asp have high effect sizes to differentiate PD and controls. As Phe and Tyr are precursors of dopamine, we quantified the serum Phe and Tyr levels in de novo and treated PD patients, as well as in controls. Phe was high in de novo patients, but not in treated patients. In contrast, Tyr tended to be low in treated patients, but not in de novo patients. Tyr/Phe ratios were lower in both de novo and treated patients than in controls. The Tyr/Phe ratios were all higher than 0.82 in controls, whereas 49% of the de novo and treated patients had Tyr/Phe ratios less than 0.82. The low Tyr/Phe ratios were associated with male patients and low doses of entacapone. However, Tyr/Phe ratios were not different between male and female patients, and between patients with and without entacapone. Conclusions The low serum Tyr/Phe ratio differentiates PD from controls with sensitivity = 0.49, specificity = 1.00, positive predictive value = 1.00, and negative predictive value = 0.40.

Incorporation profiles of d-Trp and l-Trp into the striatum following intraperitoneal (i.p.) administration of d-Trp or l-Trp in male Sprague-Dawley rats (100mg/kg) were investigated by using a brain microdialysis technique. Alterations in the extracellular dopamine (DA) concentration in the rat striatum were also examined. Incorporation profiles of d-Trp and l-Trp were almost identical; however, transient DA release was only observed 0-30 min following d-Trp administration. Pretreatment with 3-methylpyrazole-5-carboxylic acid, an inhibitor of d-amino acid oxidase (DAAO), significantly suppressed the DA release induced by d-Trp. These findings suggest that d-Trp-induced DA release may be mediated by certain d-Trp metabolites produced by DAAO.

Aim: Catecholamines, which are physiologically important neurotransmitters and hormones, apparently decrease in the brain and plasma as some species age. Because this observation has engendered controversy, we used mice to investigate whether age-related changes occur in adrenal catecholamine levels and in the expression of catecholamine synthetic enzymes. Methods: Adrenal glands were collected from male C57BL/6NCr mice at the ages of 6, 12 and 24 months. Catecholamines, such as dopamine (DA), noradrenaline (NA) and adrenaline (AD) from those glands, were measured by using a highly sensitive liquid chromatographic method with peroxyoxalate chemiluminescence reaction detection. Tyrosine hydroxylase (TH), dopa decarboxylase, dopamine beta hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression levels were measured by quantitative real-time polymerase chain reaction. Results: Although DA levels in the adrenals of 24-month-old mice were higher than in 6- and 12-month-old mice, the AD content decreased with age. In such mice, the ratio of DA to NA at 24 months was lower than at 12 months, and the ratio of NA to AD content at 24 months was significantly lower than at 6 months. The mRNA expression ratios in TH, DBH and PNMT in 24-month-old mice were all lower than in 12-month-old mice. Conclusions: These results strongly suggest that catecholamine synthesis, in general, declines with aging in the adrenal glands of mice and that AD, in particular, undergoes a significant decrease with advancing age. Geriatr Gerontol Int 2013; 13: 490–496.

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.

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.

Neuronal function and innervation density is regulated by target organ-derived neurotrophic factors. Although cardiac hypertrophy drastically alternates the expression of various growth factors such as endothelin-1, angiotensin II, and leukemia inhibitory factor, little is known about nerve growth factor expression and its effect on the cardiac sympathetic nerves. This study investigated the impact of pressure overload-induced cardiac hypertrophy on the innervation density and cellular function of cardiac sympathetic nerves, including kinetics of norepinephrine synthesis and reuptake, and neuronal gene expression. Right ventricular hypertrophy was induced by monocrotaline treatment in Wistar rats. Newly developed cardiac sympathetic nerves expressing β 3 -tubulin (axonal marker), GAP43 (growth-associated cone marker), and tyrosine hydroxylase were markedly increased only in the right ventricle, in parallel with nerve growth factor upregulation. However, norepinephrine and dopamine content was paradoxically attenuated, and the protein and kinase activity of tyrosine hydroxylase were markedly downregulated in the right ventricle. The reuptake of [ 125 I]-metaiodobenzylguanidine and [ 3 H]-norepinephrine were also significantly diminished in the right ventricle, indicating functional downregulation in cardiac sympathetic nerves. Interestingly, we found cardiac sympathetic nerves in hypertrophic right ventricles strongly expressed highly polysialylated neural cell adhesion molecule (PSA-NCAM) (an immature neuron marker) as well as neonatal heart. Taken together, pressure overload induced anatomical sympathetic hyperinnervation but simultaneously caused deterioration of neuronal cellular function. This phenomenon was explained by the rejuvenation of cardiac sympathetic nerves as well as the hypertrophic cardiomyocytes, which also showed the fetal form gene expression.

Catecholamines have been reported to be involved in the development of salt-sensitive hypertension. We investigated the relation between catechol-O-methyltransferase (COMT) and salt-sensitivity. In the first experiment, Dahl salt-sensitive (DS) rats were given a normal-salt (NS), high-salt (HS), or HS+hydralazine (80 mg/l water) diet for 4 or 13 weeks, and Dahl salt-resistant (DR) rats were given a NS or HS diet. COMT activities in both the kidneys and liver and urinary norepinephrine (NE) and dopamine (DA) excretion were measured. In the second experiment, HepG2 cells were used to investigate the role of NE in regulating COMT activity. In the third experiment, we investigated the reactivity of pre- and postsynaptic alpha(2)-adrenoceptor (AR) in DS rats. HS loading significantly suppressed the activities of membrane-bound COMT (MB-COMT) and, consistent with this finding, increased the urinary NE level in DS rats, but not in DR rats. Hydralazine did not restore the MB-COMT activities, which suggested that HS loading rather than elevated blood pressure suppressed the MB-COMT activities. The in vitro experiment using HepG2 cells revealed that NE increased the MB-COMT activity via the alpha(2)-AR. However, both the pre- and postsynaptic alpha(2)-AR reactivity was decreased by HS loading in DS rats. In conclusion, HS intake suppresses the MB-COMT activities in DS rats, presumably by blunting alpha(2)-AR signaling. The suppression of MB-COMT activities, consequent decrease in degradation of NE, and increase in NE release by blunting of alpha(2)-AR function may be involved in the development of salt-sensitive hypertension in DS rats, in whom DA-dependent natriuresis may be suppressed.

Plasma concentration of three methylated arginines, endogenous nitric oxide synthase inhibitors, is not studied in schizophrenic patients. The purpose of this study was to determine plasma concentrations of N(G)-monomethyl-L-arginine (l-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), N(G),N(G')-dimethyl-L-arginine (SDMA), and l-arginine in 56 male and 45 female schizophrenic patients undergoing antipsychotic drug treatment versus those of 39 male and 24 female healthy controls. Plasma concentrations of methylated arginines and l-arginine were measured using newly developed high performance liquid chromatography with fluorescence detection which we previously reported. Methylated arginine levels were slightly but significantly higher in schizophrenic patients. L-Arginine levels and the l-arginine/(ADMA+l-NMMA) ratio were higher in schizophrenic patients than in healthy controls. It is considered that pharmacological treatment of schizophrenic patients may lower methylated arginine levels that are increased by the disease, and increase L-arginine levels, eliciting an improvement in nitric oxide (NO) bioavailability.

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.

We have previously reported that methylation of catecholamines by catechol-O-methyltransferase (COMT) was attenuated in spontaneously hypertensive rats (SHR) with acute hypotension as compared with that of Wistar-Kyoto (WKY) rats. Here we examined the soluble (S-) and membrane-bound (MB-) COMT activities and COMT protein in the liver, kidney, and erythrocytes in both strains. Both the activities and the amounts of MB-COMT in the liver were lower in SHR than in WKY rats, but no such trend was found in the kidney or erythrocytes. Nor was such a trend observed in any of these three tissues for S-COMT. These results indicate that liver MB-COMT may be a relevant factor in blood pressure regulation in rats.

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.

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.

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.

The effect of an AA deficiency on catecholamine biosynthesis in adult mice in vivo is unknown. Therefore, we quantified catecholamine and the expression of catecholamine synthetic enzymes in the adrenal glands of senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice placed in an AA-deficient state. At 30 days of age, mice were divided into the following 4 groups: AA (−) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (−) wild type (WT), and AA (+) WT. The AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (−) groups received water without AA until the experiment ended. In addition, all mice were fed an AA-depleted diet. Catecholamine levels were measured by a liquid chromatographic method. Tyrosine hydroxylase, dopa decarboxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase mRNA expression levels were measured with the quantitative real-time polymerase chain reaction (qPCR). Tyrosine hydroxylase and dopamine β-hydroxylase protein levels were quantified by Western blot analysis. In the adrenals of AA (−) SMP30/GNL KO mice, noradrenaline and adrenaline levels decreased significantly compared to other three groups of mice, although there were no significant differences in dopamine β-hydroxylase or phenylethanolamine N-methyltransferase mRNA content. Moreover, there was no significant difference in their dopamine β-hydroxylase protein levels. On the other hand, AA depletion did not affect dopamine levels in adrenal glands of mice. An AA deficiency decreases the noradrenaline and adrenaline levels in adrenal glands of mice in vivo.

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.

A liquid chromatography–chemiluminescence detection method was developed and validated for the determination of catecholamines (norepinephrine, epinephrine, and dopamine) in mouse brains. Chromatography was performed on a strong cation exchange column (150 × 2.0-mm id) using an isocratic mobile phase of 65 mM potassium acetate/75 mM potassium phosphate (95:5, pH 3.5) at a flow rate of 0.2 mL/min following post-column fluorescence derivatization of catecholamines with ethylenediamine and peroxyoxalate chemiluminescence reaction detection. The recovery of catecholamines added to mouse brain samples was more than 95.0%, while intra- and inter-day precision of the assay were

The complex formation of mercury(II) with glycine(HL) and β-alanine(HL) was investigated under physiological conditions (at 37°C in 0.15 mol dm−3 NaCl aqueous solution) by glass-electrode potentiometry. From the analysis of the emf data in the two systems by use of computer program MIQUV, it was concluded that the species formed in both the systems are [HgL]+ and [HgL2] and their formation constants in the glycine system are larger than the corresponding ones in the β-alanine system, notwithstanding the lower basicities of the amino and carboxylate groups within glycine than those within β-alanine. The formation constants in the glycine system measured under the present experimental conditions (no data in the β-alanine system is available in the literature) are extremely less than those observed in different ionic media such as KNO3, probably because chloride is bound to mercury(II) with high stability.

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.

The measurement of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is useful for an index of dopamine turnover. We developed a simultaneous determination method for dopamine and DOPAC with high-performance liquid chromatography-fluorescence detection. Mixed-mode reversed-phase and cation-exchange column (CAPCELL PAK CR column) which contained C18 silica particles and sulfonic acid cation-exchange particles was used for the separation of 3,4-dihydroxy-l-phenylalanine, catecholamines (norepinephrine, epinephrine, and dopamine) and DOPAC. The mobile phase was optimized for factors such as pH and counter ion concentration. With a mobile phase of 15 mM sodium acetate buffer (pH 4.5), separation was achieved within 22 min. The developed method was applicable to the determination of dopamine and DOPAC in mouse striatum. The concentrations of dopamine and DOPAC in mouse striatum were 4.98+/-0.66 and 1.00+/-0.11 microM, respectively (n=10).

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.

The unique structural features of mesoporous silica nanoparticles (MSN) have made them very useful in biological applications, such as gene therapy and drug delivery. Flow cytometry, confocal microscopy, and electron microscopy have been used for observing the endocytosis of MSN. However, flow cytometry cannot directly observe the process of endocytosis. Confocal microscopy requires fluorescence labeling of the cells. Electron microscopy can only utilize fixed cells. In the present work, we demonstrate for the first time that differential interference contrast (DIC) microscopy can be used to observe the entire endocytosis process of MSN into living human lung cancer cells (A549) without fluorescence staining. There are three physical observables that characterize the locations of MSN and the stages of the endocytosis process: motion, shape, and vertical position. When it was outside the cell, the MSN underwent significant Brownian motion in the cell growth medium. When it was trapped on the cell membrane, the motion of the MSN was greatly limited. After the MSN had entered the cell, it resumed motion at a much slower speed because the cytoplasm is more viscous than the cell growth medium and the cellular cytoskeleton networks act as obstacles. Moreover, there were shape changes around the MSN due to the formation of a vesicle after the MSN had been trapped on the cell membrane and prior to entry into the cell. Finally, by coupling a motorized vertical stage to the DIC microscope, we recorded the location of the MSN in three dimensions. Such accurate 3D particle tracking ability in living cells is essential for studies of selectively targeted drug delivery based on endocytosis.

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.

Catecholamines, for example epinephrine, norepinephrine, and dopamine, are widely distributed and are important neurotransmitters and hormones in mammalian species. Several methods have been developed for analysis of catecholamines and related compounds. Determination of catecholamines in biological fluids has enabled us to clarify the physiological role played by these amines. Catecholamine levels in plasma and/or urine are also useful for diagnosis of several diseases, for example hypertension, pheochromocytoma, and neuroblastoma. This review covers reports from 2000 to the present of methods for the analysis of catecholamines and their metabolites.

Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

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.

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 method has been developed for measurement of catechol-O-methyltransferase (COMT) activity in the rat adrenal gland. Epinephrine, synthesized in the adrenal gland, was used as substrate, and its enzymatic product, metanephrine, was quantified by high-performance liquid chromatography (HPLC) with fluorescence detection. The method has sufficient precision and accuracy. Soluble (S) and membrane-bound (MB) COMT activity in Wistar-Kyoto (WKY) rats was 20.7 +/- 3.5 and 18.6 +/- 3.4 pmol min(-1) mg(-1) protein (n = 5), respectively. To clarify the role of adrenal COMT in blood-pressure regulation, S and MB COMT activity in spontaneously hypertensive rats were determined. Respective activity was 18.6 +/- 3.4 and 17.0 +/- 1.1 pmol min(-1) mg(-1) (n = 5), which is similar to that in WKY rats. This finding suggests that COMT in the adrenal gland might not be related to blood pressure regulation.

A method for the determination of S-d-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 microl of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn fluorescence derivatization with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity (r20.999) over the range of 20-100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97-104% (20-100 pmol SLG spiked in rat blood sample, N = 4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra- and interday coefficients of variance were 4.63% (N = 5) and 9.98% (N = 5), respectively. The concentrations of SLG in whole blood from male Wistar-Kyoto rats (12 weeks old) were 3.48+/-0.78 microM (N = 4, mean+/-SE). In streptozotocin-induced diabetic rats, the concentration of SLG was significantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic flux of the glyoxalase system increases in red blood cells during hyperglycemia.

A method to measure catechol- O-methyltransferase (COMT) activity using high performance liquid chromatography-fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3-8 times lower than S-COMT activities. However, considering Vmax/ Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.

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.

L-DOPA (L-3,4-dihydroxyphenylalanine) is commonly used in the treatment of Parkinson's disease. Monitoring the concentration of L-DOPA in human plasma will enable dose optimization, but is rarely performed because current quantification methods are tedious and time-consuming. In this study, a simple method for the determination of L-DOPA in the plasma of patients with Parkinson's disease was developed. A monolithic silica disk-packed spin column with a phenylboronate moiety, which forms stable anionic complexes with the cis-hydroxyl groups of L-DOPA, was used to extract L-DOPA from plasma with extraction recoveries exceeding 90%. The extracted L-DOPA was then separated on a reversed-phase column and detected electrochemically with a boron-doped diamond electrode. The method, which has a limit of detection of 10 fmol, was then successfully applied for the determination of L-DOPA in the plasma of healthy volunteers and patients with Parkinson's disease.