Patrick Herregodts

High-performance liquid chromatography with electrochemical detection for the determination of levodopa, catecholamines and their metabolites in rat brain dialysates

A high-performance liquid chromatographic assay with electrochemical detection is described for the simultaneous determination of levodopa, 3-O-methyldopa, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, noradrenaline, adrenaline, 3-methoxy-4-hydroxyphenylethylene glycol and 5-hydroxyindoleacetic acid in rat brain dialysates. Samples are obtained in vivo using the microdialysis technique. Microdialysis probes are placed in the brain area to be studied and neurochemicals are collected by perfusion of the probe with modified Ringers solution. Direct injection of the dialysates allows rapid and reliable results to be obtained.

Changes in cardiocirculatory autonomic function after thoracoscopic upper dorsal sympathicolysis for essential hyperhidrosis

Essential hyperhidrosis (EH) is caused by an unexplained overactivity of the sympathetic fibers which pass through the upper dorsal sympathetic ganglia D2 and D3. Since the D2 and D3 ganglia are also involved in the sympathetic cardiac innervation, cardiocirculatory autonomic function may also be abnormal in EH. In order to study the function of the sympathetic nervous system in EH, and to assess the effects of thoracoscopic sympathiocolysis, cardiocirculatory autonomic function tests were performed in 13 consecutive patients with EH, before (baseline) and 6 weeks after the thoracoscopic intervention. Baseline data were also compared with data obtained from 13 matched healthy volunteers: EH patients showed an increased heart rate at rest, but only in the standing position (94 +/- 18.5 vs 78 +/- 10.9 bpm, P < 0.01), as well as an increased ratio of low to high frequency power of the heart rate variability in the standing position (5.92 +/- 4.4 vs 2.8 +/- 2.5, P < 0.05). Exercise tests were normal in every EH patient. After sympathiocolysis, heart rate at rest (sitting on the cycloergometer) had decreased (75.4 +/- 13 vs 90.4 +/- 16.5 bpm, P < 0.05), as well as heart rate at maximal exercise (165.2 +/- 14.8 vs 180 +/- 10 bpm, P < 0.05). Exercise capacity and the cardiorespiratory responses to exercise were, however, unchanged after sympathicolysis. Resting heart rate in the lying (66 +/- 10 vs 76 +/- 15 bpm, P < 0.05) and standing positions (82 +/- 13.8 vs 94 +/- 18.5 bpm, P < 0.05), and the diastolic blood pressure reaction to a handgrip test (73.6 +/- 8.6 vs 84.7 +/- 11.6 mmHg, P < 0.05) were also lowered after sympathicolysis. In conclusion, patients with EH show an overfunctioning of the sympathetic system which is characterised by an increased reaction to stress (standing, exercise), whereas resting sympathetic tone is unaffected. Thoracoscopic D2-D3 sympathicolysis corrects this hyperfunction and has a partial beta-blocker-like activity, which results in a decrease in heart rate at rest and during maximal exercise, and in the diastolic blood pressure response to the handgrip test. Further studies are needed to assess the long-term consequences of this procedure.

Biotransformation of locally applied L-dopa in the corpus striatum of the hemi-Parkinsonian rat studied with microdialysis

Microdialysis was used to study the biotransformation of l-dopa in intact and denervated striata of rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra. Microdialysis probes were placed in the intact and in the denervated striatum. Observations were then made on freely moving rats. Extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid; HVA) were monitored before, during and after the local administration of l-dopa via the microdialysis probe for 20 min.A dose-dependent increase in extracellular dopamine levels was seen in intact striatum after application of l-dopa in concentrations ranging between 100 nmol/l and 10 μmol/l. In the denervated striatum, the severity of the lesion influenced dopamine formation, so that no dose-effect relation could be established.The effects of the continuous intra striatal infusion of nomifensine, tetrodotoxin or benserazide on the l-dopa-induced dopamine outflow revealed that in the intact striatum this dopamine release is mainly voltage dependent. It was concluded that in the denervated striatum other cells of non-neuronal origin and containing aromatic l-amino acid decarboxylase make a major contribution to the increase in extracellular dopamine levels. Furthermore, l-dopa itself shows no dopamine-releasing properties, at least under the present experimental conditions.

Biotransformation of L-DOPA in striatum and substantia nigra of rats with a unilateral, nigrostriatal lesion : a microdialysis study

SummaryMicrodialysis was used to study the biotransformation of l-DOPA in the striatum and substantia nigra of rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra. The animals were pretreated with carbidopa (50 mg/kg p.o.) for 5 days. They were anaesthetized, and microdialysis probes were implanted into the intact and denervated striatum and into the intact and lesioned substantia nigra. The biotransformation of l-DOPA (5 mg/kg i.p.) in these regions was investigated. These results were compared with those obtained after administration of a much higher dose of l-DOPA (100 mg/kg i.p.). Changes in extracellular l-DOPA, 3-O-methyldopa (3-OMD), dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined by HPLC with electrochemical detection.Although rats with a unilateral nigrostriatal lesion did not show rotational behaviour after 5 mg/kg l-DOPA, DA levels were increased significantly both in the intact and the denervated striatum and in the intact and the lesioned substantia nigra. This increase was most pronounced in the denervated striatum. At 100 mg/kg l-DOPA, the increases in extracellular dopamine in intact and denervated striatum were about twice as high as the increases observed at the lower dose. A similar increase was observed in the intact substantia nigra. However, in the lesioned substantia nigra there was a fourfold increase. l-DOPA, at both doses, was evenly distributed between the brain areas studied and the lesion had no effect on the uptake of the drug at the blood-brain barrier.Our data suggest that l-DOPA in the substantia nigra might play a role additional to that in the striatum in relieving symptoms of Parkinsons disease. Even at a low dose (5 mg/kg i.p.), the drug had an effect on extracellular dopamine in all the brain regions studied.

Distribution of biogenic amines and their catabolites in brains from patients with Alzheimer's disease

Norepinephrine, epinephrine, dopamine, serotonin and their major catabolites were measured in 17 regions of the left hemisphere of two brains obtained from two brothers with Alzheimers disease with very early onset. The clinical diagnosis was confirmed by histological examination of the right hemispheres and brain stems. The quantitative data were compared with our values in normal brains. In the patient suffering from the less severe dementia, there was a severe reduction of the serotonin concentration in all examined neocortical areas and its concentration was even below the detection limit in the nucleus amygdalis, hippocampus, caudate nucleus, putamen, globus pallidus and substantia nigra. In the other patient, who suffered from a more pronounced dementia with myoclonus, the serotonin concentration was below the detection limit in all examined structures. In contrast with these findings, the noradrenergic, adrenergic and dopaminergic systems appeared to be relatively unaffected by the disease process. Focusing our attention on the nuclei wherein the monoamine transmitter systems originate, it appeared that neuronal losses and neurofibrillary tangles clearly predominated in the substantia grisea subependymalis, the nucleus centralis superior and the nucleus raphe dorsalis, origin of the main serotonergic system. The serotonin deficiency sheds light on possible mechanisms of myoclonus in Alzheimers disease.

Monoaminergic neurotransmitters in Alzheimer's disease: An HPLC study comparing presenile familial and sporadic senile cases

Norepinephrine, epinephrine, dopamine, serotonin and their major metabolites were measured in 20 regions of the left hemisphere in 4 presenile familial cases of Alzheimer-type dementia and 4 sporadic senile cases. Both groups were compared to values in normal brains obtained in our laboratory. Quantitative determination of the monoamines was performed by HPLC with electrochemical detection. The clinical diagnosis of Alzheimer-type dementia was confirmed by histological examination of the right hemisphere and brain stem. The serotonergic system was dramatically affected in the familial cases with very low or undetectable serotonin concentrations in most cortical and subcortical areas studied and an important cell loss in the nucleus raphe dorsalis, origin of the main ascending serotonergic system. In the senile demented patients the serotonergic deficit is less important but still clearly present. The noradrenergic, adrenergic and dopaminergic systems were less affected by the disease process in senile sporadic as well as in the presenile familial type of Alzheimers disease.

The Significance of Homovanillic Acid and 3,4-Dihydroxyphenylacetic Acid Concentrations in Human Lumbar Cerebrospinal Fluid

Abstract: The concentrations of the acidic dopamine (DA) catabolites homovanillic acid (HVA) and 3,4‐dihydroxy‐phenylacetic acid (DOPAC) measured in human CSF are supposed to reflect the “turnover” of DA in the brain. The notion of “turnover” is, however, not synonymous with impulse nerve activity in the dopaminergic systems. Significant amounts of DOPAC and HVA could, indeed, be demonstrated in brain structures wherein dopaminergic inner‐vation has not been documented. It must also be noted that DA is not only a neurotransmitter itself, but also a precursor of norepinephrine and epinephrine. Furthermore, in lumbar CSF, levels of biogenic amine catabolites partially reflect metabolism in the spinal cord and may have limited relevance to neurotransmission in the brain. To elucidate these points further, we determined the concentrations of DOPAC and HVA in 22 areas of six human brains and eight levels of six human spinal cords. The data were correlated with the concentration of DA. Quantitative determinations were done using HPLC with electrochemical detection, after solvent and ion‐pair extraction. In this study, significant amounts of both DOPAC and HVA were demonstrated in brain structures not previously associated with dopaminergic innervation. The relatively lower DA concentration in these structures suggests that in these regions, the DOPAC and HVA concentrations are unrelated to dopaminergic neurotransmission. The possible role of capillary walls and glial cells in the catabolism of DA must be further evaluated. The demonstration of DOPAC and HVA in the spinal cord is another argument against the hypothesis that CSF levels of HVA and DOPAC reflect closely the activity of the dopaminergic systems in the brain.

The mesoneocortical dopamine neuron system.

The ascending dopaminergic projections from the midbrain have been subdivided into a nigrostriatal and a mesocorticolimbic ~ornponent.’-~ The neuronal cell bodies are located in the substantia nigra and ventral tegmental area. The axons of the nigrostriatal neurons terminate in the caudate nucleus, putamen, and globus pallidus. A reduction of the dopaminergic neurotransmission in the nigrostriatal pathway gives rise to parkinsonism. A hyperactivity of this system has been implicated in other movement disorders, such as Huntington’s chorea. The term “mesocorticolimbic” is used to designate projections to the nucleus accumbens, amygdala, septal nuclei, olfactory tubercle, and certain cortical areas. It has been hypothesized that an overactivity of this system plays a role in the symptomatology of psychotic disorders, whereas a reduced function might contribute to the cognitive abnormalities found in patients with Parkinson’s disease. Because part of the neurons projecting to the striatum also arise from the ventral tegmental area, some authors prefer the term “mesostriatal” instead of “nigrostriatal,” and the projection to the nucleus accumbens has been included under this system as we112 The mesocorticolimbic system is often referred to as the “mesocortical” system (the nucleus accumbens being excluded).2 Within the mesocortical system, one distinguishes allocortical and neocortical projections. The allocortical axons terminate in phylogenetically old cortical derivates, such as the olfactory tubercle, amygdala, septal nuclei, and piriform cortex, as distinct from the neocortical areas, which this review will cover.

Combined ion-pair extraction and high-performance liquid chromatography for the determination of the biogenic amines and their major metabolites in single brain tissue samples

A combined procedure based on reversed-phase liquid chromatography with electrochemical detection has been developed for the determination in the picomole range of the monoamines dopamine, norepinephrine, epinephrine and serotonin, and their major metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, normetanephrine, metanephrine and 3-methoxytyramine. Sample pretreatment consists of the extraction of the neutral and acidic metabolites with ethyl acetate, followed by the extraction into heptane of the catecholamines with tetraoctylammonium bromide as counter-ion in the presence of diphenylborate. The residual supernatant is directly injected in the chromatographic system for quantification of serotonin, normetanephrine, metanephrine and 3-methoxytyramine.

Continuous intravenous monitoring of levodopa and 3-O-methyldopa by microdialysis and high-performance liquid chromatography with electrochemical detection

Microdialysis, in association with ion-pair reversed-phase high-performance liquid chromatography with electrochemical detection, was applied in vitro for the determination and quantification of levodopa and 3-O-methyldopa in blank spiked plasma and blood. The method presented gave accurate results; the calibration graphs for plasma were linear over the range of the expected values for both compounds. When using a dialysis probe with a membrane length of 1.6 cm at a 5 microliters min-1 perfusion speed, the recovery rate in plasma for levodopa was 30.1% and 68.5% for 3-O-methyldopa. However, less reproducible results were obtained for plasma levodopa levels in the range of 0.5 microliter ml-1 and lower. The microdialysis technique was subsequently successfully applied for the continuous intravenous monitoring of levodopa and 3-O-methyldopa in a levodopa-treated dogs.