High Performance Liquid Chromatography And Electrochemical Detection Research Articles

Oxidative damage on nucleobases and Hoehn-Yahr stage in Parkinsons disease

Background: the imbalance between reactive oxygen species (ROS) and antioxidant barrier is a typical condition of Parkinson's disease (PD), and may be the cause of oxidative alterations on DNA. The oxidations on nucleobase guanine have been previously widely studied and the measurements of the hydroxylated form 8-hydroxy-guanine (8-OHGua) and its nucleoside 8-hydroxy-2-deoxy-guanosine (8-OHdG) have been proposed as sensible and reliable markers of this imbalance. Objective: the study was carried out to demonstrate the relations between 8-OHGua, 8-OHdG and the corresponding non-oxidized nucleoside 2-deoxy-guanosine (2-dG) with PD clinical progression. Method: the measurements of 8-OHGua, 8-OHdG and 2-dG were performed in urine samples of 198 PD subjects, ranked according to the Hoehn-Yahr scale (H-Y). The same analysis were also performed in a group of 33 subjects with other parkinsonisms. Results: all markers were analyzed using high performance liquid chromatography and electrochemical detection (HPLC-ED). For 8-OHGua, a new method was specifically optimized; the low limit of detection (LOD 10 ng/L) and good intra- and inter-assay coefficients of variation (CV 2.2% and 3.6%, respectively), allowed reliable measurements of 8-OHGua in a wide range of concentrations (from 0.5 to 2000 ig/L). Compared to the initial stages (H-Y2) and 2-dG halves, both with statistical significance (p

Seizure-induced 5-HT release and chronic impairment of serotonergic function in rats

We analyzed the dynamic concentration change of serotonin (5-HT) and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) within the epileptic hippocampus in rats. Seizure was induced by systemic injection of pilocarpine (320mg/kg, i.p.). Using electroencephalography (EEG) recordings, we found that primary seizure discharge was induced 30min after pilocarpine administration and that recurrent discharge peaked 14d after the onset of status epilepticus (SE). The extracellular fluid in the hippocampus was sampled by microdialysis from conscious animals at various time points before and after SE. The concentrations of 5-HT and 5-HIAA in the samples were measured by high-performance liquid chromatography and electrochemical detection (HPLC-ECD). Interestingly, 5-HT levels in the hippocampus were dramatically increased within the 30min following SE. This reversed to basal level by 4d after SE and continued to drop to 48% at 7d and 28% of basal level 14d after SE. Accordingly, a marked increase of 5-HIAA in the hippocampus appeared at 2d after SE, then gradually declined to levels below baseline. To identify serotonergic neurons in the raphe nuclei (a major source of 5-HT release in the brain), brain sections were immunostained for tryptophan hydroxylase (TPH). The number of TPH positive neurons and the intensity of TPH staining significantly decreased at 28d after SE. These data suggest that pilocarpine induces depletion of 5-HT in the hippocampus and significantly compromise serotonergic neurons in the raphe nuclei. The loss of serotonergic function may play a significant role in the pathophysiology of epilepsy.

Disposition of Protein-bound 3-nitrotyrosine in Rat Plasma Analysed by a Novel Protocol for HPLC-ECD

3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 +/- 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 +/- 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 +/- 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo.

The ability of thiourea to scavenge hydrogen peroxide and hydroxyl radicals during the intra‐coronal bleaching of bloodstained root‐filled teeth

Hydrogen peroxide, an agent used in the intra-coronal bleaching of root-filled teeth for over a century, has been shown to diffuse from the pulp chamber to the outer root surface. Furthermore, it has been demonstrated that destructive hydroxyl radicals, the by-products of the bleaching process, have been detected on the external root surface. The control of such diffusion may be of importance in minimizing the risk of invasive cervical resorption (ICR) which has been linked to intra-coronal bleaching of discoloured root-filled teeth using hydrogen peroxide. The aims of the present in vitro study are to quantify the diffusion of hydrogen peroxide and hydroxyl radicals to the outer root surface following intra-coronal bleaching, and to evaluate the ability of thiourea incorporated into the bleaching protocol to scavenge residual hydrogen peroxide and hydroxyl radicals. Thirty-five single rooted premolar teeth with intact cementum at the cemento-enamel junction were used in this project. Thirty teeth were stained with red blood cells and root-filled with gutta-percha and AH26. The five unstained teeth were root-filled and constituted a negative control (Group 1). The stained teeth were divided equally into the following experimental groups and subjected to various intra-coronal bleaching regimes: Group 2--'walking bleach' with 20 microl 30 per cent w/w hydrogen peroxide; Group 3--20 microl 30 per cent w/w hydrogen peroxide and thermocatalytically activated; Group 4--20 microl acidified thiourea; Group 5--20 microl acidified thiourea and 20 microl 30 per cent w/w hydrogen peroxide; Group 6--20 microl acidified thiourea and 20 microl one per cent sodium hypochlorite; Group 7--20 microl acidified thiourea, 20 microl one per cent sodium hypochlorite and 20 microl 30 per cent w/w hydrogen peroxide. The reaction products of the bleaching process were quantified at the outer root surface using high performance liquid chromatography and electrochemical detection (HPLC-ECD). Results showed that hydrogen peroxide used alone in Groups 2 and 3 was able to be detected at the outer root surface in 100 per cent of the samples, and that the presence of the hydroxyl radical generated in both groups was detected in equal amounts (P < 0.05). When thiourea was incorporated into the bleaching protocols in Groups 5-7, it was shown to scavenge both hydrogen peroxide and hydroxyl radicals to a significant degree (P < 0.05). Acidulated thiourea is an effective scavenger of residual hydrogen peroxide and hydroxyl radicals generated during the intra-coronal bleaching of bloodstained root-filled teeth.

Changes in Spinal Serotonin Turnover Mediate Age-Related Differences in the Behavioral Manifestations of Peripheral Nerve Injury

The Bennett and Xie model of peripheral nerve injury was used to study the effects of aging on the onset and progression of sciatic nerve ligation (SNL)-induced thermal hyperalgesia and tactile-evoked allodynia in young, mature, and aged Fischer 344 FBNF1 male rats (4–6, 14–16, and 24–26 months old, respectively). A plantar analgesia meter and calibrated von Frey pressure filaments were employed as the analgesiometric assays. In the absence of nerve injury, aged rats were found to be more sensitive than younger animals to noxious thermal stimuli. Following the SNL surgery, thermal hyperalgesia was observed in all three age groups within 3 days. On post-SNL day 35, the paw-withdrawal latency values of the young and mature animals returned to presurgical baseline levels, while the aged rats continued to exhibit thermal hyperalgesia. Tactile-evoked allodynia was apparent within 3 days following peripheral nerve injury in the oldest cohort, but was delayed in the younger animals. On post-SNL days 0 (control), 3, 21, and 35, young, mature, and aged rats were sacrificed and high-performance liquid chromatography and electrochemical detection (HPLC/ECD) methods were used for neurochemical analyses of spinal serotonin (5-HT), norepinephrine (NE), and 5-hydroxyindoleacetic acid (5-HIAA). Spinal 5-HT and NE levels were not significantly altered by the aging process, nor were they affected by peripheral nerve injury. However, spinal 5-HT turnover from the aged animals was greater than that detected in spinal tissue from the younger counterparts. Differences in spinal 5-HT turnover may contribute to age-related variability in spinal nociceptive processing.

Injections of N-methyl-D-aspartate into the ventral hippocampus increase extracellular dopamine in the ventral tegmental area and nucleus accumbens.

The nucleus accumbens septi receives inputs from dopaminergic neurons of the ventral tegmental area (VTA) and glutamatergic neurons of the ventral subiculum (VS). The convergence of these inputs in the NAS is important for the normal expression of exploratory locomotion; stimulation of the VS by injection of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) causes dopamine-dependent increases in locomotion. In the present study, in vivo microdialysis in conjunction with high-performance liquid chromatography and electrochemical detection (HPLC-EC) was used to estimate changes in extracellular dopamine in the VTA and NAS in response to intra-VS injections of NMDA (0.074, 0.28, 0.74 microg). NMDA injections caused dose-dependent elevations in extracellular dopamine in each region. Each dose of NMDA clearly increased extracellular dopamine in the NAS, whereas only the two higher doses increased dopamine significantly in the VTA. The highest dose of NMDA elevated extracellular dopamine to approximately 180% of baseline in each region. Whereas elevations in NAS dopamine might be induced by impulse-independent local mechanisms, elevations of dopamine in the VTA are presumed to reflect increased somatodendritic release associated with increased impulse flow through dopamine neurons. Thus, the present study suggests that the modulation of dopaminergic neurotransmission by the ventral subiculum results from a trans-synaptic activation of dopamine cell bodies in the VTA.

Somatosensory afferent inputs release 5-HT and NA from the spinal cord.

Endogenous serotonin (5-HT) and noradrenaline (NA) release by somatosensory afferent inputs was investigated at the level of the spinal cord using in vivo microdialysis technique combined with high performance liquid chromatography and electrochemical detection (HPLC-ECD). Selective stimulation of large myelinated A beta afferent fibers significantly increased 5-HT release to 151.1 +/- 10.1% of the control, but did not affect NA release. However, selective stimulation of small myelinated A delta fibers released NA rather than 5-HT. The NA level enhanced to 128.8 +/- 6.4% of the control after A delta fibers were stimulated with the intensity of 6 times threshold. Stimulation of unmyelinated C fibers unavoidably excited the A beta and A delta afferent fibers, causing both 5-HT and NA release from the spinal cord. The results suggest that both innocuous and noxious information may activate serotonergic descending pathways. The noradrenergic descending pathways are only triggered by noxious inputs transmitted by small afferent fibers.

Serotonin-immunoreactive neurons and endogenous serotonin in the opisthosomal ventral nerve cord of the horseshoe crab, Limulus polyphemus.

It has been suggested that serotonin serves as a neurotransmitter in the horseshoe crab, Limulus polyphemus. While some studies of identified groups of central neurons have been conducted, little is known concerning the neuronal organization in Limulus central ganglia. This study was undertaken to determine the localization of serotoninergic neurons in the opisthosomal ventral nerve cord of Limulus and to construct a basis for further comparative biochemical and pharmacological studies of the specific function of these neurons. Endogenous serotonin was detected in the ventral nerve cord (chain of abdominal ganglia) by high-performance liquid chromatography and electrochemical detection (HPLC-EC). Endogenous serotonin was quantified in the 9th through 13th ganglia, anterior (hemal) nerves, posterior (branchial) nerves, and connectives. The serotonin content in the abdominal ganglia was significantly reduced when the ganglia were incubated for 24 hours in Leibovitz's (L-15) medium containing reserpine or 5,7-dihydroxytryptamine (5,7-DHT), neurotoxins that block the uptake of serotonin into storage vesicles. The distribution of serotonin-immunoreactive neurons in the ventral nerve cord was determined by indirect immunocytochemistry. Treatment of the chain of ganglia with an anti-serotonin antiserum followed by treatment with a fluorescent-labeled antiserum raised against the primary antibody demonstrated specific staining in each ganglion, the ganglionic roots, and connectives. Clusters of serotonin-immunoreactive neurons were observed anteriolaterally and posteriorly in each ganglion. Processes from dense fiber bundles extended from these clusters of neurons to the central region of each ganglion. These results demonstrate that serotonin-immunoreactive neurons are present in the opisthosomal ventral nerve cord of the horseshoe crab and that serotonin may function as a neurotransmitter.

Peripheral nerve stimulation increases serotonin and dopamine metabolites in rat spinal cord

Extracellular serotonin (5-HT), dopamine (DA), and their metabolites, 5-hydroxyin-doleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), were assessed in the rat lumbar spinal cord (L3-4) by in vivo microdialysis with high performance liquid chromatography and electrochemical detection (HPLC-ECD). Under urethane-chloralose anesthesia, basal levels of 5-HT and DA in the dialysates were approximately 1.0–1.2 pg/22 μl sample, 5-HIAA, DOPAC, and HVA were constant at 322.6 ± 14.9, 8.6 ± 0.7, and 10.4 ± 0.4 pg/22 μl sample (mean ± SE), respectively. Local application of 100 mM KCl via the dialysis probe increased the 5-HT and DA. Peripheral nerve stimulation that selectively excited the large (A-beta) or small (A-delta) myelinated fibers increased the metabolites. Excitation of the A-beta fibers increased the levels of 5-HIAA to 138%, DOPAC to 155%, and HVA to 143% of the controls. Stimulation of the A-delta fibers increased 5-HIAA to 121%, DOPAC to 120%, and HVA to 124% of the controls. The results suggest that nonnociceptive peripheral nerve stimulation may activate the descending 5-HT and DA systems in the spinal cord.

The metabolism of dopamine in rat caudate nucleus can be increased by [formula omitted] “dialysis” perfusion cannulae

The development of dialysis cannula techniques coupled with high performance liquid chromatography and electrochemical detection (HPLC-EC) has provided a means to continuously sample extracellular fluid from deep brain structures in vivo . The present studies show that with HPLC-EC analysis of the acid metabolites of dopamine (DA) and 5-hydroxytryptamine (5-HT) in samples from dialysis cannulae implanted in the caudate nucleus of anaesthetized rats, it is possible to determine the time course of the response of dopamine- and 5-HT containing neurones to administration of monoamine oxidase inhibitors and haloperidol. The tissue concentrations of the DA and 5-HT metabolites were also determined at the conclusion of each individual experiment in both the caudate nucleus containing a cannula and in that without a cannula. In perfusion experiments where no drug was administered the content of the DA metabolites, but not that of the 5-HT metabolite, were significantly elevated in the caudate nucleus containing the cannula as compared with the contralateral tissue. These increases occurred whether the cannula was perfused or not, suggesting that the presence of the cannula may have been causing a lesion which altered the activity of the DA neurones. These results emphasize the importance of tissue analysis in conjunction with the dialysis experiments, especially where perfusion sample contents of the monoamine metabolites are being measured as a reflection of the effects of behavioural manipulation or drug treatment on endogenous neuronal activity.