Using arterial-venous analysis to characterize cancer metabolic consumption in patients.

Understanding tumor metabolism holds the promise of new insights into cancer biology, diagnosis and treatment. To assess human cancer metabolism, here we report a method to collect intra-operative samples of blood from an artery directly upstream and a vein directly downstream of a brain tumor, as well as samples from dorsal pedal veins of the same patients. After performing targeted metabolomic analysis, we characterize the metabolites consumed and produced by gliomas in vivo by comparing the arterial supply and venous drainage. N-acetylornithine, D-glucose, putrescine, and L-acetylcarnitine are consumed in relatively large amounts by gliomas. Conversely, L-glutamine, agmatine, and uridine 5-monophosphate are produced in relatively large amounts by gliomas. Further we verify that D-2-hydroxyglutarate (D-2HG) is high in venous plasma from patients with isocitrate dehydrogenases1 (IDH1) mutations. Through these paired comparisons, we can exclude the interpatient variation that is present in plasma samples usually taken from the cubital vein.

Altered plasma levels of arginine metabolites in depression.

L-Arginine pathway metabolites appear to play differential roles in the pathogenesis of major depressive disorder (MDD). Studies have revealed an antidepressant and anxiolytic effect of agmatine and putrescine. Possible mechanisms of these effects include inhibition of nitric oxide synthase and N-methyl-D-aspartate receptors. The present study sought to determine whether MDD is associated with altered levels of arginine metabolites and whether these metabolites are associated with depression, anxiety and stress severity. Seventy seven MDD patients 21-65 years of age with a minimum score of 18 on the Hamilton Depression Scale, and 27 age and sex matched healthy controls (HC) were included. Patients with uncontrolled physical diseases, abnormal routine lab tests, other psychiatric diagnoses, or under psychotropic medication were excluded. HC subjects were recruited from the community. Rating instruments included Hamilton Depression and Anxiety Scales, Beck Depression and Anxiety Inventory and Perceived Stress Scale. Fasting blood was drawn between 8:30 and 11:00 a.m. and High Performance Liquid Chromatography (HPLC) was used to measure plasma arginine metabolites. ADMA (Asymmetrical dimethylarginine) and putrescine were significantly lower while SDMA (Symmetric dimethylarginine), agmatine and ornithine were significantly higher in MDD patients (p˂0.05). Depression, anxiety and stress severity were negatively correlated with ADMA and putrescine (p˂0.05). Stress was positively correlated with citrulline, NOHA (N-omega-hydroxy-nor-l-arginine), SDMA, agmatine and ornithine (p˂0.05). Lower putrescine levels predicted depression diagnosis (p = 0.039) and depression severity (p = 0.003). Low ADMA level predicted depression severity as well. Arginine pathway metabolites are associated with the pathophysiology of depression. Putrescine may be a biomarker to predict MDD.

Safety and neurochemical profiles of acute and sub-chronic oral treatment with agmatine sulfate.

Agmatine (decarboxylated arginine) exerts numerous central nervous system (CNS) dependent pharmacological effects and may potentially modulate altered neurochemistry seen in neurological disorders. In preclinical studies, injection has been the predominant route of systemic administration. However, a significant translational step would be the use of oral agmatine treatment at therapeutic doses and better understanding of L-arginine metabolic profiles in the CNS post-treatment. The present study systematically investigated the tolerability, safety and brain-plasma neurochemistry following daily oral agmatine sulfate treatment (via gavage) to wild-type (WT) mice up to 900 mg/kg for one week (Experiment 1) or WT and APPswe/PS1ΔE9 transgenic (Tg) mice at 300 mg/kg for fifteen weeks (Experiment 2). Agmatine treatment in both experiments was well tolerated with no marked behavioural impairments, and gross necropsy and organ histology revealed no pathological alterations after 15-week dosing. Moreover, oral treatment increased agmatine levels in the hippocampus and plasma of WT mice (Experiment 1), and in 6 brain regions examined (but not plasma) of WT and Tg mice (Experiment 2), at 30 minutes or 24 hours post-treatment respectively. This study provides fundamental pre-clinical evidence that daily oral delivery of agmatine sulfate to both WT and Tg mice is safe and well tolerated. Exogenous agmatine passes through the blood brain barrier and accumulates in the brain to a greater extent in Tg mice. Furthermore exogenous agmatine has differential actions in the brain and periphery, and its effect on brain putrescine appears to be dependent on the time post-treatment.

Evaluation of plasma agmatine level and its metabolic pathway in patients with bipolar disorder during manic episode and remission period.

Objectives: Agmatine is a cationic amine resulting from the decarboxylation of l-arginine. Agmatine has neuroprotective, anti-inflammatory, anti-stress, and anti-depressant properties. In this study, plasma agmatine, arginine decarboxylase, and agmatinase levels were measured during manic episode and remission period in patients with bipolar disorder. Methods: Thirty healthy volunteers and 30 patients who meet Bipolar Disorder Manic Episode diagnostic criteria were included in the study. Additionally, the changes in the patient group between manic episode and remission period were examined. We evaluated the relationship between levels of l-arginine and arginine decarboxylase in the agmatine synthesis pathway, and level of agmatinase that degrades agmatine. Results: Levels of agmatine and l-arginine were significantly increased than control group during manic episode (p < .01). All parameters were increased during manic episode compared to remission period (p < .05). Agmatinase was significantly decreased both during manic episode (p < .01) and remission period (p < .05) in comparison to the control group. Arginine decarboxylase levels did not show a significant difference between the groups (p > .05). Conclusions: This study indicate that there may be a relationship between bipolar disorder and agmatine and its metabolic pathway. Nonetheless, we believe more comprehensive studies are needed in order to reveal the role of agmatine in etiology of bipolar disorder. Key points Agmantine, agmatinase, l-arginine and arginine decarboxylase levels in BD have not been explored before. Various neuro-chemical mechanisms act to increase agmatine in BD; however, agmatine could have elevated to compensate agmatine deficit prior to the manifestation of the disease as in schizophrenia. Elevated agmatine degradation resulting from excess expression of agmatinase which is suggested to be effective in pathogenesis of mood disorders was compensated by this way. Elevated agmatine may be one of the causes which play a role in mania development. Elevated agmatine levels are also suggested to trigger psychosis and be related with the etiology of manic episode and lead to BD.

Homoarginine and inhibition of human arginase activity: kinetic characterization and biological relevance.

The inhibition of arginase, resulting in higher arginine (ARG) availability for nitric oxide synthesis, may account for the putative protective effect of homoarginine (HOMOARG) against atherosclerosis and cardiovascular disease. However, uncertainty exists regarding the significance of HOMOARG-induced arginase inhibition in vivo. A novel UPLC-MS method, measuring the conversion of ARG to ornithine (ORN), was developed to determine arginase 1 and arginase 2 inhibition by HOMOARG, lysine (LYS), proline (PRO), agmatine (AG), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and NG-Monomethyl-L-arginine (L-NMMA). Plasma HOMOARG, ARG and ORN concentrations were further measured in 50 healthy older adults >65 years (27 males and 23 females). HOMOARG inhibited arginase 1 with IC50 and Ki values of 8.14 ± 0.52 mM and 6.1 ± 0.50 mM, and arginase 2 with IC50 and Ki values of 2.52 ± 0.01 mM and 1.73 ± 0.10 mM, respectively. Both arginase isoforms retained 90% activity vs. control when physiological HOMOARG concentrations (1-10 µM) were used. In partial correlation analysis, plasma HOMOARG was not associated with ARG (P = 0.38) or ARG/ORN ratio (P = 0.73) in older adults. Our results suggest that arginase inhibition is unlikely to play a significant role in the reported cardio-protective effects of HOMOARG.

Decreased plasma agmatine levels in autistic subjects.

Agmatine is a polyamine endogenously synthesized from arginine and is considered to be a new neurotransmitter. Agmatine has been implicated in the pathophysiology of several diseases such as anxiety disorder, depression, and schizophrenia. Agmatine also possesses anticonvulsant, neuroprotective, antiapoptotic, antioxidant, anxiolytic, and antidepressant effects. Furthermore, agmatine inhibits the nitric oxide synthase enzyme and exerts antagonist effects on NMDA, alpha-2, and imidazoline receptors. Considering these characteristics, the present study investigated whether agmatine plays a role in the pathogenesis of autistic spectrum disorders (ASDs). Therefore, plasma agmatine levels were evaluated in 34 patients with ASD and 28 non-ASD controls. Plasma agmatine levels were measured using the HPLC method. The study found remarkably lower agmatine levels in patients with ASD compared with the non-ASD control group (p < 0.001). These findings support the notion that agmatine might contribute to the pathogenesis of ASD and may serve as a new target for treatment.

L-Arginine supplementation prevents allodynia and hyperalgesia in painful diabetic neuropathic rats by normalizing plasma nitric oxide concentration and increasing plasma agmatine concentration.

PURPOSE: Neuropathic pain is a common diabetic complication. It is characterized by symptoms of spontaneous and stimulus-evoked pain including hyperalgesia and allodynia. L-Arginine is a common precursor of many metabolites of biological interest, in particular, nitric oxide (NO), ornithine, and hence polyamines. In central nervous system, NO, glutamate, and polyamines share an N-methyl-D-aspartate (NMDA) receptor-mediated effect. We hypothesized that a variation in arginine metabolism caused by diabetes may contribute to development and maintenance of neuropathic pain and to the worsening of clinical and biological signs of diabetes. METHODS: We examined whether oral L-arginine supplementation (2.58 ± 0.13 g/l in drinking water for 3 weeks) could improve the development of neuropathic pain and the clinical, biological, and metabolic complications of diabetes in streptozocin (STZ)-induced diabetic (D) rats. RESULTS: STZ administration induced classical symptoms of type 1 diabetes. Diabetic rats also displayed mechanical hypersensitivity, tactile, and thermal allodynia. Plasma citrulline and NO levels were increased in diabetic hyperalgesic/allodynic rats. L-Arginine supplementation failed to reduce hyperglycaemia, polyphagia, and weight loss. Moreover, it abolished hyperalgesia and allodynia by normalizing NO plasma concentration and increasing plasma agmatine concentration. CONCLUSIONS: L-Arginine supplementation prevented the development of mechanical hyperalgesia, tactile, and thermal allodynia in painful diabetic neuropathy with concomitant reduction of NO and increased agmatine production, offering new therapeutic opportunities for the management of diabetic neuropathic pain.

[Determination of endogenous agmatine in rat plasma by isotope dilution-gas chromatography-mass spectrometry].

A method for the determination of endogenous agmatine in rat plasma was developed by isotope dilution-gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS). The plasma samples were analyzed after protein precipitation, evaporation, derivatization by hexafluoroacetone (HFAA), and clean-up on a Florisil SPE column. The GC-MS analysis utilized stable isotope d8-agmatine as internal standard. The samples after treatme were tested by negative chemical ionization with selected ion monitoring (SIM) which was set at m/z 492 (molecular ion of agmatine) and m/z 500 (molecular ion of internal standard). The limit of detection (LOD) of agmatine standard solution was 0.005 7 ng/mL. The calibration curve of the agmatine spiked in rat plasma showed a good linear relationship at the range of 1.14-57.0 ng/mL (r = 0.997). The recoveries of agmatine spiked in rat plasma ranged from 92.3% to 109.8%. Inter-day and intra-day precisions were less than 15%. The average concentration level of agmatine in rat plasma was (22 +/- 9) ng/mL, and there was no significant difference between male and female SD rats (p > 0.05). The method is high sensitive and specific, and can be used for the determination of endogenous agmatine in plasma. It provides a strong support for the subsequent research of agmatine.

Increased plasma agmatine levels in patients with schizophrenia.

Agmatine is an endogenous substance, synthesized from l-arginine, and it is proposed to be a new neurotransmitter. Preclinical studies indicated that agmatine may have an important role in the pathophysiology of schizophrenia. This study was organized to investigate plasma agmatine in patients with schizophrenia and in healthy controls. Eighteen patients with schizophrenia and 19 healthy individuals constituted the subjects. Agmatine levels in the plasma were measured using the HPLC method. The S100B protein level, which is a peripheral biomarker for brain damage, was also measured using the ELISA method. While plasma levels of agmatine in patients with schizophrenia were significantly increased (p < 0.0001) compared to those of healthy individuals (control), there were no significant changes in the levels of S100B protein (p = 0.660). An ROC (receiver operating characteristic) curve analysis revealed that measuring plasma agmatine levels as a clinical diagnostic test would significantly differentiate between patients with schizophrenia and those in the control group (predictive value: 0.969; p < 0.0001). The predictive value of S100B measurements was not statistically significant (p > 0.05). A multiple regression analysis revealed that the age of the patient and the severity of the illness, as indicated by the PANSS score, significantly contributed the plasma agmatine levels in patients with schizophrenia. These results support the hypothesis that an excess agmatine release is important in the development of schizophrenia. The findings also imply that the plasma agmatine level may be a potential biomarker of schizophrenia.

Low levels of plasma agmatine in the metabolic syndrome.

BACKGROUND: The biophysiology of the amino acid l-arginine has been a field of active research. Agmatine, which is a metabolite of l-arginine, is known to participate in many biophysical reactions, including those in the cardiovascular system. We sought to investigate plasma agmatine levels in human subjects as a potential biomarker for the metabolic syndrome. METHODS: Agmatine concentration was measured in plasma from 322 elderly participants in the Ansan Geriatric study. The metabolic syndrome was defined according to an Asian modified version of criteria established in the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. We observed that the metabolic syndrome was associated with low levels of plasma agmatine concentration. RESULTS: The mean plasma agmatine level in the metabolic syndrome group was lower than that in the non-metabolic syndrome group (79.42 ng/mL vs. 82.44 ng/mL, P = 0.024). Agmatine remained significant within the regression model after adjustment for different covariates (adjusted odds ratio, 0.962; 95% confidence interval, 0.933-0.993). CONCLUSIONS: We concluded that plasma agmatine levels were lower in subjects with the metabolic syndrome than in those without the metabolic syndrome.

Agmatine levels in the cerebrospinal fluid of normal human volunteers.

Agmatine is an amine formed by the decarboxylation of l-arginine by the enzyme arginine decarboxylase. The fact that exogenous agmatine modulates morphine analgesia and dependence raises the question of whether the biosynthesis of endogenous agmatine is regulated during chronic pain. As a first step to understand the biological role of agmatine in human neurological and psychiatric conditions, this study was aimed to determine the levels of cerebrospinal fluid (CSF) agmatine in normal individuals. The levels of agmatine in the CSF and blood were measured by high-performance liquid chromatography (HPLC) method. Samples of CSF and blood were collected from a total of 10 participants for this study. The CSF agmatine levels ranged from 24.3 to 54.0 ng/mL, whereas the plasma agmatine levels were from 8.4 to 65.1 ng/mL. The mean values with standard error for blood and CSF agmatine were 33.8 +/- 16.6 and 40.4 +/- 9.1, respectively. The statistical analysis of these 10 samples indicated no correlation between blood and CSF samples (r = .29); however, removing one outlier improved the correlation (r = .6). From this study, the authors conclude that human CSF agmatine levels can be measured by HPLC with precision and that a possible correlation exists between plasma and CSF agmatine levels. This study provides basis for future studies in human chronic pain conditions.

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection.

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1x10(-9) M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE-LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5-4.1x10(-7) M.

Exposure of rat isolated stomach and rats in vivo to [(14)C]agmatine: accumulation in the stomach wall and distribution in various tissues.

The aims of the present study were: (i) to investigate the accumulation of radioactivity in the stomach wall after luminal exposure of the rat isolated stomach to[(14)C]agmatine and (ii) to determine the distribution of radioactivity in various tissues after oral administration of this radiolabelled polyamine to rats in vivo. In isolated rat stomach, [(14)C]agmatine was accumulated in part by an energy-dependent uptake process that could be inhibited by phentolamine. These findings correspond to properties of the recently identified specific agmatine transporter in human glioma cells, suggesting that in rat stomach [(14)C]agmatine is taken up by such a carrier. In in vivo experiments, rats received 0.5 microCi [(14)C]agmatine adsorbed to 5 g rat standard chow after a fasting period of 24 h. After oral ingestion of [(14)C]agmatine, radioactivity was recovered in all organs investigated as well as in blood and urine. Radioactivity also seemed to be secreted into the pancreaticobiliary fluid, as it was recovered in the luminal content of distal ileum and sigmoid colon. Accumulation of radioactivity in organs and distal gut luminal content was dose-dependently decreased by simultaneous administration of putrescine. In conclusion, the present data are compatible with the view that agmatine can be absorbed in rat at least from the stomach and probably also from the gut by means of an energy-dependent agmatine transport mechanism. Agmatine itself and/or its degradation products, which also have the potential to be pharmacologically active, are unevenly distributed between the organs. Putative secretion of radioactivity into the pancreaticobiliary fluid suggests the potential for an enterohepatic circulation of agmatine. In view of the high intraluminal concentration of agmatine in the stomach and distal gut and the operation of an agmatine transporter, it is rather likely that agmatine in the chyme of the gut represents an important source for agmatine detected in the tissues of the organism.

Plasma agmatine and platelet imidazoline receptors in depression.

Plasma agmatine concentrations are elevated significantly in depressed patients compared to healthy controls. Treatment with the antidepressant bupropion normalized plasma agmatine levels. Correlational evidence is presented that a change in plasma agmatine levels may lead to similar changes in platelet I1 imidazoline receptors.

Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection.

Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with fluorescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limit of 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999+/-0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.