High-performance Liquid Chromatography With Electrochemical Detection Research Articles

Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical

Nitroxyl radicals can trap fatty acid allyl radicals on ferric-lipoxygenases at lower oxygen content, which are an intermediate in the lipoxygenase reaction. In the present study, we examined whether nitroxyl radical-trapping of fatty acid allyl radicals on the enzyme proceeds in biological fluids with abundant antioxidants. The fatty acid allyl radical-nitroxyl radical adducts were quantified by HPLC with electrochemical detection (HPLC-ECD); the adducts in eluate degraded into nitroxyl radical by passing through heating coil at 100 degrees C, and then nitroxyl radical was detected by electrochemical detector. Soybean 15-lipoxygenase and nitroxyl radical (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl, CmDeltaP) were mixed with rat serum prepared from fresh venous blood, and the solution was stood at 37 degrees C for 1 h. One volume of the solution was mixed with 5 vols of cold acetonitrile. After centrifugation, the supernatant was subjected to HPLC-ECD. Arachidonate allyl radical-CmDeltaP adducts as well as linoleate allyl radical-CmDeltaP adducts were detected in the solution, and the content of these adducts remarkably increased in the presence of phospholipase A(2). It is proved for the first time that nitroxyl radical traps fatty acid allyl radicals generated in the lipoxygenase reaction in biological fluid without competition from endogenous antioxidants.

Buprenorphine Modulates Methamphetamine-Induced Dopamine Dynamics in the Rat Caudate Nucleus

Methamphetamine (METH) abuse and addiction present a major problem in the United States and globally. Oxidative stress associated with exposure to METH mediates to the large extent METH-evoked neurotoxicity. While there are currently no medications approved for treating METH addiction, its pharmacology provides opportunities for potential pharmacotherapeutic adjuncts to behavioral therapy in the treatment of METH addiction. Opioid receptor agonists can modulate the activity of dopamine neurons and could, therefore, modify the pharmacodynamic effects of METH in the dopaminergic system. Efficacy of the adjunctive medication with buprenorphine has been demonstrated in the treatment of cocaine addiction extending beyond opiate addiction. We investigated the interactions of morphine (10mg/kg) and buprenorphine (0.01 and 10mg/kg) with METH (2mg/kg) affecting striatal dopaminergic transmission. The extracellular concentration of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined using brain microdialysis coupled with high performance liquid chromatography with electrochemical detection (HPLC-ED) in the caudate nucleus of adult, awake, male Sprague-Dawley rats. Compared to METH alone, extracellular DA release was prolonged for 140min without changes in DA peak-effect by combined treatment with morphine/METH. Morphine did not change DOPAC efflux evoked by METH. On the other hand, both buprenorphine doses attenuated the METH-induced DA peak-effect. However, whereas high buprenorphine dose extended DA outflow for 190min, the low-dose abbreviated DA release. High buprenorphine dose also shortened METH-induced decrease in DOPAC efflux. Data confirm that opiates modulate dopaminergic neurotransmission evoked by METH. Alteration of dopaminergic response to METH challenge under buprenorphine may suggest effectiveness of buprenorphine treatment in METH addiction.

Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection

A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g(-1) for danofloxacin, from 25 to 100 ng g(-1) for sarafloxacin and from 50 to 315 ng g(-1) for difloxacin, respectively. The method presents detection limits under 10 ng g(-1) and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques.

Effects of Serotonin on Acid Secretion in Isolated Rat Stomach: the Role of 5-HT3 Receptors

Serotonin (5-hydroxytryptamin; 5-HT) content has been measured using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The distributions of 5-HT-containing cells and 5-HT3 receptors have been determined with specific antibodies against 5-HT and 5-HT3 receptors, respectively. The effect of serotonin on acid secretion has been studied using an isolated rat stomach model. It has been shown that 5-HT concentrations in the fundus, mucosal layers of the corpus, remaining layer of the corpus and antrum are approximately 152, 498, 1494 and 972 nmol/mg protein, respectively. The distribution of 5-HT-containing cells is concentrated in the enteric plexus and enterochromaffin (EC) cells in the deep mucosal layer. Immunoreactivity to 5-HT3 receptors is localized in numerous neurons of the myenteric and submucosal plexus and concentrated in the neuronal plasma membrane, submucosa, endocrine cells and lamina propria. In the present study, the effect of 5-HT on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. 5-HT at 1-100 microM reduced acid secretion stimulated by oxotremorine while 10 microM 5-HT did not modify the basal secretion of gastric acid. We further showed that 10 microM 5-HT reduced acid secretion and pepsin output stimulated by oxotremorine, histamine and pentagastrin; among the 5-HT receptors agonists tested, 2-methyl-5-HT (1-10 microM), a 5-HT3 receptor agonist, inhibited oxotremorine-, histamine- and pentagastrin-stimulated acid secretions, and this inhibitory effect was blocked by 1 microM MDL 72222, a specific 5-HT3 receptor antagonist. These results suggest that 5-HT is released from serotoninergic neurons, their processes and EC cells. The effect of 5-HT mediated by 5-HT3 receptors involves distinct neuronal and non-neuronal pathways which modulate gastric acid secretion.

Determination of serum cholestanol by semi‐micro high‐performance liquid chromatography with electrochemical detection

A simple and sensitive method that dose not require derivatization for determining cholestanol has been developed using HPLC with electrochemical detection (HPLC-ECD). The current peak height was linearly related to the amount of cholestanol injected, ranging from 1 to 200 muM (r = 0.999). The detection limit (S/N = 3) of cholestanol was 0.23 muM (1.2 pmol). Total cholestanol in control human and mouse serum was determined by the present method with a recovery rate of more than 90% and an RSD (n = 5) of less than 7.3%. Further, this method was successfully applied to monitor experimental hypercholestanolemia in mice fed a high-cholestanol diet, an animal model of cerebrotendinous xanthomatosis (CTX). In conclusion, we found this method to be both simple and useful for the determination of cholestanol in serum, helping in the diagnosis of CTX.

Determination of Short-chain Fatty Acids in Rat and Human Feces by High-Performance Liquid Chromatography with Electrochemical Detection

A simple method for determining short-chain fatty acids (SCFAs) in rat and human feces was developed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). A two-channel HPLC-ECD system was fabricated using an ion exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solutions of 0.1 mM HClO(4) and of ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and a quinone solution, respectively. Peak areas for lactic, acetic, propionic, butyric, isovaleric, and valeric acids at a detection potential of -0.9 V vs. an Ag/AgCl electrode showed a linear relationship with the acid amount in the range 0.1 to 40 nmol. Standard acids at 4 nmol were determined ten times with relative standard deviations (RSD) of less than 2.0%. The analytical results of healthy human feces were measured within 35 min. RSD (n = 5) in all SCFAs were less than 2.7%, and recoveries of SCFAs were more than 92%. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes, and it has the potential for clinical and biomedical applications.

An LC method for monitoring medium-chain fatty acid permeation through CaCo-2 cell monolayers

A simple method was developed for monitoring the permeation of medium-chain fatty acids of C8 (octanoic acid) and C10 (decanoic acid) through CaCo-2 cell monolayers by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The detection was made based on the electrochemical reduction prepeak of quinone caused by acids, requiring the fabrication of a two-channel HPLC-ECD system. In one channel, acetonitrile-water (7:3, v/v) was used as a mobile phase to separate acids by a C30 column. In the other channel, acetonitrile-water (7:3, v/v) containing 6 mmol/L 3,5-di-t-butyl-1,2-benzoquinone and 20 mmol/L LiClO(4) was used as a quinone solution to detect acids by an electrochemical cell with a glassy carbon working electrode. In this HPLC-ECD system, eluted acids were mixed with the quinone solution in a post column fashion to obtain current signals caused by acids. The peak area was found to be linearly related to the acid amount ranging from 25 to 1,000 pmol (r > 0.992). The detection limits of octanoic acid and decanoic acid were 7.5 and 8.8 pmol, respectively. Octanoic acid and decanoic acid spiked into cell culture media samples were extracted with acetonitrile and their recoveries were more than 89.5% with an RSD of less than 8.2%. This method was applied to the permeation experiment of octanoic acid and decanoic acid with CaCo-2 cell monolayers formed on the Transwell system.

Selection of the Optimal Solvent Grade for the Mobile Phase in HPLC with Electrochemical Detection Based on FIMI Theory

The optimum conditions of the mobile phase for HPLC with electrochemical detection (HPLC-ECD) were selected from among solvents of different grades with the standard deviation (SD) of area measurements based on FUMI (function of mutual information) theory as a criterion. In HPLC-ECD for determining baicalin and baicalein using a phosphoric acid-methanol-water mixture (0.5:60:40, v/v/v) as a mobile phase, the effects of three grades of phosphoric acid, of five grades of methanol, and of seven grades of water on the measurement precision were examined. The mixture, if characterized by the minimum SD of measurements, was selected as the optimal mobile phase. This selection method saves considerable amounts of chemicals and experimental time, and would be a useful exploration technique for the routine check and troubleshooting of HPLC-ECD.

Determination of arbutin, rutin, total content of phenols and antioxidant capacity in fruits and leaves of lingonberry, Vaccinium vitis-idaea L. (Ericaceae)

The aim of this study was to determine arbutin, rutin, total phenols content (TCP) and total antioxidant capacity (AC) in the fruits and leaves of lingonberry, Vaccinium vitis-idaea L. (Ericaceae). Dry fruits and leaves of lingonberry were collected in Bosnia. For determination of AC was used Oxygen Radical Absorbance Capacity (ORAC) assay with trolox, a water soluble analogue of vitamin E, as a standard. The TCP was estimated by photometric method with Folin-Ciocalteau reagent at 765nm, with gallic acid as a standard. Arbutin and rutin determined using High Pressure Liquid Chromatography with electrochemical detection (HPLC-ED). As mobile phase it was used a mixture of water, methanol, acetonitrile, formic acid and isopropyl alcohol in proper proportion: 75.5: 12:6:5:1.5. Performance was done at 25° C with flow rate of 0.5ml/min and potential of 0.750 V. The TCP, expressed in milligramms of phenols per gramme of dry weight (mg/gd.w.) was 12.7 in fruits, and 164.4 in leaves. The content of arbutin was 0.51mg/gd.w. in leaves, and 0.04mg/gd.w. in fruits. The content of rutin was 2.54mg/gd.w. in leaves, and 0.06mg/gd.w. in fruits. The values of AC, expressed as mmol trolox equivalents per gramme of dry weight (mMTE/gd.w.), were 46.45 mMTE/gd.w. in fruits, and 235.23 mMTE/gd.w. in leaves. The analysis has shown the higher content of phenols, arbutin and rutin in the leaves of lingonberry in comparison with fruits and higher AC in leaves. The higher AC in leaves correspond to the higher values of phenolic compounds.

Interlaboratory comparison of methodologies for the measurement of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine

Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is widely used as a marker of oxidative stress. Here we report the comparison of two, distinct chromatographic assays with an enzyme-linked immunosorbent assay (ELISA). The chromatographic assays displayed good agreement (r =:0.89, p < 0.0001), whereas there was markedly worse, albeit still significant, agreement with the ELISA (high-pressure liquid chromatography followed by gas chromatography (HPLC-GC/MS), r = 0.43; HPLC with electrochemical detection (HPLC-EC), r = 0.56; p < 0.0001). Mean values differed significantly between the chromatographic assays and the ELISA (HPLC-GC/MS 3.86, HPLC-EC 4.20, ELISA 18.70 ng mg−1 creatinine; p < 0.0001). While it is reassuring to note good agreement between chromatographic assays, this study reveals significant short-comings in the ELISA, which brings into question its continued use in its present form.

Dietary supplementations of amino acids: evidence for enhanced serotonergic functions following haloperidol withdrawal in rat medial prefrontal cortex.

To investigate the effects of orally supplemented amino acids L-Tryptophan (Trp) and L-Valine (Val) in rats repeatedly injected with haloperidol following one week of drug withdrawal, with particular reference to extrapyramidal symptoms (EPS) and serotonin (5-hydroxytryptamine; 5-HT) metabolism in medial prefrontal cortex (mPFC). Experimental study. Department of Biochemistry, University of Karachi from December 2007 to February 2008. The study was conducted on thirty six locally bred male Albino Wistar rats. Freshly prepared amino acids (Val and Trp) were added in the drinking water of rats on alternate days and haloperidol at doses of 5.0 mg/kg or saline were injected twice daily for three weeks following one week of withdrawal. Locomotor/ exploratory activities were scored in activity boxes and open field apparatuses. Catalepsy was monitored on an inclined surface. The animals tested for locomotor activity and catalepsy for two weeks follow-up post-injections plus one week of drug withdrawal were decapitated to collect mPFC regions of rat brain for neurochemical analysis by high performance liquid chromatography with electrochemical detection (HPLC-EC). There was significant increase (p<0.01) in locomotor activity in rats orally supplemented with Val and Trp following one week of drug withdrawal from repeated administration. Marked reduction in cataleptogenic effects of the drug was also observed. Significant (p<0.01) increases in the brain Trp and mPFC 5-HT metabolism in Val and Trp supplemented animals were also noticed. These findings help to demonstrate the effect of dietary amino acids, in particular, Trp to potentiate mPFC serotonergic modulation of neuroleptic activity.

Impairment in pain perception in adult rats treated with N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) as neonates

To examine the impact of a central noradrenergic lesion on antinociceptive effects of morphine, paracetamol and nefopam, we compared intact male rats with rats in which noradrenergic nerve terminals were largely destroyed with the neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine; 50 mg/kg, sc ×2] shortly after birth, on the 1st and 3rd day of postnatal life. When the rats attained 10 weeks of age, painful reactions were assessed by means of the tail immersion test (thermal stimulus) and the paw pressure test (mechanical stimulus). In addition, monoamine levels in some parts of the brain were estimated using the high pressure liquid chromatography with electrochemical detection (HPLC/ED) method. In the tail immersion test, we showed that there were no differences in the antinociceptive effect evoked by morphine (5.0 mg/kg, sc) and paracetamol (100 mg/kg, ip) between control and DSP-4 rats. Nefopam (20 mg/kg, ip) elicited only slight analgesia in control rats (~17%), and this effect was not observed in the DSP-4 treated group; differences were statistically significant at 90 and 120 min of this test. In the paw pressure test we demonstrated that morphine produced lower analgesia in DSP-4 rats in comparison to the control, and the effect was significant at 60, 90 and 120 min of the test. The antinociceptive effect of paracetamol was also greatly diminished in the DSP-4 group and significant in all tested intervals. Nefopam produced only slight analgesia in both tested groups. In biochemical studies we showed that in DSP-4 treated rats there was a marked decrease in NA level in the prefrontal cortex (to 10.4%, p < 0.005), the thalamus with the hypothalamus (to 54.4%, p < 0.005) and the spinal cord (to 12.3%, p < 0.005) in comparison to the control group. Conversely, in the cerebellum and brain stem of rats with DSP-4 lesions there was a significant increase in the NA content vs. control (to 171.2% and 123.5% of NA, respectively, with p < 0.005 and p < 0.05, respectively). In the striatum we did not observe any changes in NA level between the examined groups. The levels of 5-HT and its metabolite 5-HIAA were also not altered by DSP-4 treatment in all tested structures with the exception of the spinal cord (approx. 40% decrease) and the level of DOPAC (also 40% reduction). In conclusion, obtained results showed that neonatal DSP-4 treatment alters the antinociceptive effects of tested drugs (each of them with a different mechanism of action). These data lead to the proposal that perhaps there is a need to adjust the doses of analgesics applied to patients with noradrenergic system dysfunction (e.g., depression and/or anxiety disorders).

The influence of catecholamine on the migration of gonadotropin-releasing hormone-producing neurons in the rat foetuses

Catecholamines (CA) play an important role in the regulation of GnRH neurons in adults, and it is probable that they control GnRH-neuron development. Migration of GnRH neurons was evaluated in male and female rats at the 17th embryonic day (E17) and E21, following the daily treatment of their pregnant mothers from the 11th to the 16th and 20th day of gestation with alpha-methyl-para-tyrosine (alphaMPT), an inhibitor of catecholamine synthesis. High-performance liquid chromatography with electrochemical detection (HPLC-ED) was used to specify the alphaMPT-induced CA depletion. There was a 50-70% decrease in dopamine and noradrenaline content in the nose and in the brain of alphaMPT-treated foetuses, proving the efficacy of this pharmacological model. Immunohistochemistry was used to evaluate the percentage (%) of GnRH neurons along their migration pathway from the vomeronasal organ (VNO) in the nose to the septo-preoptic area in the forebrain which is considered as an index of neuron migration. Special attention was paid to the topographic relationships of GnRH neurons with catecholaminergic fibres. These were observed in apposition with GnRH neurons in the entrance to the forebrain. In CA-deficient foetuses, the percentage of GnRH neurons located in the rostral regions extending from the VNO to the septum was greater than in controls. However, no statistically significant difference was found in the forebrain which extended from the septum to the retrochiasmatic area. In conclusion, these data suggest that endogenous catecholamines stimulate the GnRH neuron migration in ontogenesis.

Impact of regional 5-HT depletion on the cognitive enhancing effects of a typical 5-ht6 receptor antagonist, Ro 04-6790, in the Novel Object Discrimination task

Selective 5-ht(6) receptor antagonists like Ro 04-6790 prolong memory in many rodent preclinical paradigms, possibly by blocking tonic 5-HT-evoked GABA release and allowing disinhibition of cortico-limbic glutamatergic and cholinergic neurones. If this is the case, behavioural responses to Ro 04-6790 should be abolished by depletion of endogenous 5-HT, and selective lesions of dorsal raphé (DR) or median raphé (MR) 5-HT pathways would allow the neuroanatomical substrates underlying the cognitive effects of 5-ht(6) receptor antagonists to be elucidated. This study compared the effect of Ro 04-6790 on novel object discrimination (NOD) before and after sham or 5,7-dihydroxytryptamine (5,7-DHT)-induced lesions produced by injection into the lateral ventricles (LV), DR or MR. NOD tests used a 4 h inter-trial interval (ITI) and Ro 04-6790 (10 mg kg(-1) i.p.) was administered 20 min before the familiarization trial. Brain region-specific 5-HT depletion was assessed by high performance liquid chromatography with electrochemical detection (HPLC-ED). Widespread LV or selective MR, but not DR lesions, abolished the ability of Ro 04-6790 to delay natural forgetting. Successful performance of all lesioned rats in subsequent 'drug-free' NOD tests using a 1 h ITI excluded the possibility of any confounding effects on visual acuity or motivation. The ability of Ro 04-6790 to prolong object recognition memory requires blockade of MR 5-HT function. Because DR lesions did not produce the expected depletion of striatal 5-HT an additional contribution of DR inputs to this region cannot be completely excluded.

Changes in the levels of serotonin and dopamine in the central nervous system and ovary, and their possible roles in the ovarian development in the giant freshwater prawn, Macrobrachium rosenbergii

Serotonin or 5-hydroxytryptamine (5-HT) and dopamine (DA) are the two key neurotransmitters that control gonadal development in decapod crustaceans. This study investigated changes in the levels of 5-HT and DA in the CNS and ovary during different phases of the ovarian cycle of the freshwater prawn, Macrobrachium rosenbergii. The levels of 5-HT and DA were quantified by using High Performance Liquid Chromatography with electrochemical detection (HPLC-ECD). Moreover, changes of vitellogenin (Vg) concentrations in the hemolymph after treatment with 5-HT and DA (at doses of 2.5 × 10 −6 and 2.5 × 10 −7 mol per prawn) were also examined. 5-HT exhibited a gradual increase in concentration in the brain and thoracic ganglia from ovarian stage I (0.12 ± 0.01 nmol/mg, 0.22 ± 0.01 nmol/mg, respectively) to reach a maximum (0.66 ± 0.03 nmol/mg, 1.48 ± 0.03 nmol/mg, respectively) at ovarian stage IV. In contrast, DA in the brain and thoracic ganglia showed the highest concentrations at ovarian stage II (0.20 ± 0.01 nmol/mg, 1.27 ± 0.06 nmol/mg, respectively) and then decreased to the lowest concentrations (0.06 ± 0.01 nmol/mg, 0.28 ± 0.04 nmol/mg, respectively) at ovarian stage IV. The ovarian concentration of 5-HT was 0.53 ± 0.11 nmol/mg at ovarian stage I and gradually increased to 1.63 ± 0.16 nmol/mg at ovarian stage IV. In contrast, the concentration of DA was highest at ovarian stage I (29.05 ± 1.31 nmol/mg), and lowest at the ovarian stage IV (11.43 ± 0.74 nmol/mg). Injecting 5-HT into prawns significantly increased Vg concentration in the hemolymph at ovarian stage IV compared to control groups, and injecting DA into prawns had the opposite effect. The inverse relationship between 5-HT and DA levels in neural ganglia and ovaries, and their opposing effects on hemolymph Vg levels suggest that these two transmitters play opposite regulatory roles in controlling ovarian maturation and oocyte development in this species.

Plasma proinflammatory and anti-inflammatory cytokine and catecholamine concentrations as predictors of neurological outcome in acute stroke patients

Proinflammatory and anti-inflammatory cytokines may play a pivotal role in cerebral inflammation, which is implicated in the development of brain injury. Systemic cytokine release is mediated by the sympathetic nervous system and catecholamines. The aim of this study was to investigate which parameters, among plasma levels of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) and the levels of the catecholamines, epinephrine and norepinephrine, contribute to the clinical outcome in acute stroke patients. Thirty-seven acute stroke patients (ischemic, n = 19; hemorrhagic, n = 18) were enrolled. All of them were admitted to our hospital within 8 h after stroke onset. Neurological status was evaluated by a modified National Institute of Health Stroke Scale (mNIHSS) on admission and by a modified Rankin Scale (mRS) at 1 month. An mRS score of 3 or more at 1 month was considered to indicate poor outcome. Serum samples for the cytokine and catecholamine measurements were collected on admission. Plasma levels of IL-1beta, IL-6, IL-10, and TNF-alpha were determined by an enzyme-linked immunosorbent assay (ELISA) method and epinephrine and norepinephrine concentrations were determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC). In the ischemic stroke patients, poor outcome was noted in 9 (47%). There were no significant differences in cytokine or catecholamine concentrations between patients with poor and good outcomes, and there was no association between clinical outcome and cytokine and catecholamine concentrations. In the hemorrhagic stroke patients, poor outcome was noted in 10 (56%). IL-6 and IL-10 levels were higher in patients with poor outcome. On logistic regression analysis, higher values of IL-6 were significantly associated with clinical outcome at 1 month (odds ratio [OR], 1.25; 95% confidence interval [CI], 1.02-1.54). In ischemic stroke, plasma cytokines and catecholamines were not predictors of neurological outcome at 1 month. In hemorrhagic stroke, high levels of IL-6 in the early phase indicated a poor neurological outcome.

Comparison of free plasma metanephrines enzyme immunoassay with 131I-MIBG scan in diagnosis of pheochromocytoma

Measurement of free plasma metanephrines (metanephrine and normetanephrine), usually performed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), has been recommended as the single biochemical test of choice for the diagnosis of pheochromocytoma. Alternatively, a widely available, simple means to measure these biomarkers with enzyme immunoassay (EIA) needs to be studied. The aim of this study was to investigate the diagnostic efficacy of such a method in comparison with (131)I-metaiodobenzylguanidine (MIBG) whole body scan (WBS) in patients with pheochromocytoma. We enrolled patients undergoing (131)I-MIBG WBS due to clinical findings suggestive of pheochromocytoma (n = 45), and patients with primary hypertension (n = 36). All subjects had blood tests for free plasma metanephrine (MN) and normetanephrine (NM) with a commercially available EIA kit. WBS was positive in 30 pheochromocytoma patients and negative in 15 refuted ones, with 100% accuracy. The sensitivity, specificity and accuracy of MN and NM in combination (either or both positive) were 96.7%, 86.3% and 90.1%, showing comparable diagnostic performance both to (131)I-MIBG WBS (all p > 0.1), and also to the same markers measured with HPLC-ECD reported in the literature. These results showed that the EIA method may be eligible as an alternative to HPLC-ECD for plasma metanephrine determination in the identification of pheochromocytoma.

Localization of N‐methyl‐norsalsolinol within rodent and human brain

The isoquinoline derivative N-methyl-6,7-dihydroxytetrahydroisoquinoline (N-methyl-norsalsolinol) is present in normal human brain and has been identified in the cerebrospinal fluid of patients with Parkinson's disease (PD). Endogenously, N-methyl-norsalsolinol may be derived from dopamine by condensation with aldehydes or alpha-ketoacids. In vitro experiments suggest that N-methyl-norsalsolinol is neurotoxic. In this study, high-performance liquid chromatography with electrochemical detection (HPLC-EC) was used to determine N-methyl-norsalsolinol concentrations in mouse, rat, normal human, and PD brain. In addition, a monoclonal antibody was generated against N-methyl-norsalsolinol and used to determine the cellular localization of N-methyl-norsalsolinol in brain. With HPLC-EC, N-methyl-norsalsolinol was detected in all regions of rodent and human brain subjected to analysis. In rodent brains, N-methyl-norsalsolinol tissue concentrations were similar among frontal cortex, ventral midbrain, striatum, hippocampus, and cerebellum. Conversely, in normal human control brains, N-methyl-norsalsolinol was concentrated in the substantia nigra and striatum. In comparison to normal human controls, N-methyl-norsalsolinol levels were significantly lower in the substantia nigra and caudate nuclei from PD patients, a finding possibly related to the death of nigrostriatal dopaminergic neurons. N-methyl-norsalsolinol immunoreactivity colocalized with a general neuronal marker (neuron-specific enolase) and a monoaminergic marker (tyrosine hydroxylase) but not with a glial marker (glial fibrillary acidic protein). The widespread neuronal localization of N-methyl-norsalsolinol in several mammalian species suggests that, in isolation, this compound is a "weak" neurotoxin. However, endogeneously derived N-methyl-norsalsolinol could contribute to the pathobiology of PD in genetically predisposed individuals after years of accumulation in dopaminergic neurons.

High Susceptibility to Experimental Myopia in a Mouse Model with a Retinal ON Pathway Defect

Nob mice share the same mutation in the Nyx gene that is found in humans with complete congenital stationary night blindness (CSNB1). Nob mutant mice were studied to determine whether this defect resulted in myopia, as it does in humans. Refractive development was measured in unmanipulated wild-type C57BL/6J (WT) and nob mice from 4 to 12 weeks of age by using an infrared photorefractor. The right eye was form deprived by means of a skull-mounted goggling apparatus at 4 weeks of age. Refractive errors were recorded every 2 weeks after goggling. The content of dopamine and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection (HPLC-ECD) in retinas of nob and WT mice under light- and dark-adapted conditions. The nob mice had greater hyperopic refractive errors than did the WT mice under normal visual conditions, until 12 weeks of age when both strains had similar refractions. At 6 weeks of age, refractions became less hyperopic in the nob mice but continued to become more hyperopic in the WT mice. After 2 weeks of form deprivation (6 weeks of age), the nob mice displayed a significant myopic shift (~4 D) in refractive error relative to the opposite and control eyes, whereas WT mice required 6 weeks of goggling to elicit a similar response. As expected with loss of ON pathway transmission, light exposure did not alter DOPAC levels in the nob mice. However, dopamine and DOPAC levels were significantly lower in the nob mice compared with WT. Under normal laboratory visual conditions, only minor differences in refractive development were observed between the nob and WT mice. The largest myopic shift in the nob mice resulted after form deprivation, suggesting that visual pathways dependent on nyctalopin and/or abnormally low dopaminergic activity play a role in regulating refractive development. These findings demonstrate an interaction of genetics and environment in refractive development.

Inhibition by oxytocin of methamphetamine-induced hyperactivity related to dopamine turnover in the mesolimbic region in mice

Accumulated data have shown the neuroactive properties of oxytocin (OT), a neurohypophyseal neuropeptide, and its capability of reducing the abuse potential of drugs. The present study investigated the effect of OT on methamphetamine (MAP)-induced hyperactivity in mice and its possible mechanism of action. Locomotor activity was measured after administered with MAP using an infrared sensor. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) was used to detect the content of monoamines and their metabolites in the striatum and accumbens and prefrontal cortex in mice after the behavioral test. OT (0.1, 0.5, and 2.5 microg/mouse, i.c.v.) had no effect on locomotor activity in naïve mice, but inhibited, in a dose-dependent manner, the hyperactivity induced by acute administration of MAP. Atosiban (Ato) (2.0 microg/mouse, i.c.v.), the selective inhibitor of OT receptor, attenuated the inhibitory effect of OT on MAP. A marked reduction of the ratios of 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) to dopamine (DA) was observed in the striatum and accumbens of mice after acute administration of MAP. OT (2.5 microg, i.c.v.) significantly inhibited the reduction of DOPAC/DA and HVA/DA ratios. However, Ato decreased the ratio of DOPAC/DA significantly in mice compared with OT (2.5 microg) in combination with MAP. There was no significant change in serotonin (5-HT) metabolism in mice after a single administration of MAP. These results suggested that OT inhibited the MAP-induced hyperactivity by altering the DA turnover in the mesolimbic region of mice.