Liquid chromatography with amperometric detection at a nano crystalline Ce-doped lead dioxide film modified electrode for determination of (R)-Salsolinol, (R)-N-methylsalsolinol and monoamine neurotransmitters in Parkinsonian patients' cerebrospinal fluid.

The fabrication and application of a novel electrochemical detector (ED) with nano crystalline Ce-doped lead dioxide film chemically modified electrode (CME) for liquid chromatography (LC) were described. The Ce-doped PbO(2) film was characterized by X-ray diffractometer (XRD) and scanning tunnel microscope (STM). The electrochemical behaviors of (R)-Salsolinol ((R)-Sal) at the CME were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). It was found that the CME exhibited an efficiently electrocatalytic effect on the current responses of (R)-Sal, (R)-N-methylsalsolinol ((R)-NMSal) and monoamine neurotransmitters. In LC-ED, all (R)-Sal, (R)-NMSal, dopamine (DA), norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) had good and stable current responses at the CME. The linear ranges of the nine analytes were over three orders of magnitude (R(2) > 0.995). The application of this method coupled with microdialysis sampling for the determination of (R)-Sal, (R)-NMSal and monoamine neurotransmitters in Parkinsonian patients' cerebrospinal fluid (CSF) was satisfactory.

Dopamine-derived endogenous N-methyl-(R)-salsolinol: its role in Parkinson's disease.

A dopamine-derived alkaloid, N-methyl-(R)-salsolinol [NM(R)Sal], enantioselectively occurs in human brains and accumulates in the nigrostriatal system. It increases in the cerebrospinal fluid (CSF) of parkinsonian patients and the activity of a neutral (R)-salsolinol [(R)Sal] N-methyltransferase, a key enzyme in the biosynthesis of this toxin, increases in the lymphocytes from parkinsonian patients, suggesting its involvement in the pathogenesis of Parkinson's disease (PD). The studies of animal and cellular models of PD proved that this isoquinoline is selectively cytotoxic to dopamine neurons. Using human dopaminergic SH-SY5Y cells, NM(R)Sal induces apoptosis by the activation of the apoptotic cascade initiated in mitochondria. In this article, we review the recent advance in proving our hypothesis that the dopamine-derived neurotoxin causes the selective depletion of dopamine neurons in PD.

[Pathogenesis of idiopathic Parkinson's disease].

The pathogenesis of idiopathic Parkinson's disease (PD) remains to be elucidated. The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) suggests that neurotoxins in the human brain may cause selective depletion of striatal dopamine neurons, a hallmark of PD. An endogenous isoquinoline, N-methyl(R)salsolinol is a most promising neurotoxin candidate, and it was proved to be selectively toxic to dopamine neurons in the rat brain by in vivo experiments. The level of N-methyl(R)salsolinol in the cerebrospinal fluid obtained from PD patients was significantly higher than control. N-Methyl(R)salsolinol is synthesized by 2 enzymatic reactions from dopamine; condensation of dopamine with acetaldehyde into (R)salsolinol by (R)salsolinol synthase and N-methylation of (R)salsolinol by neutral(R)salsolinol N-methyltransferase. The second enzyme, which catabolizes the N-methylation of (R)salsolinol, was found to determine the level of the neurotoxin in the brain. The activity of neutral(R)salsolinol N-methyltransferase was examined using lymphocytes prepared from PD patients, normal controls and diseased controls as enzyme source. A significant increase in the activity was confirmed in lymphocytes from PD cases compared to normal- and diseased-control. Studies to clarify the environmental and genetic factors determining the activity of the enzyme are now under the way. The cytotoxicity of N-methyl(R)salsolinol was examined using a cultured cell model. N-Methyl(R)salsolinol was found to induce apoptotic cell death in a dose-dependent way. The mechanism of apoptosis was clarified to be mediated by collapse in mitochondrial membrane potential, activation of caspase 3 and fragmentation of nuclear DNA. In addition, propargylamines protected the cells from apoptosis. It was suggested that N-methyl(R)salsolinol and propargylamines have specific binding sites in mitochondria which regulate the death signal transduction. Propargylamines might be applicable as neuroprotective drugs, which can be orally administrated to PD patients.

Involvement of endogenous N-methyl(R)salsolinol in Parkinson's disease: induction of apoptosis and protection by (-)deprenyl.

An endogenous dopamine-derived N-methyl(R)salsolinol has been suggested to be involved in the pathogenesis of Parkinson's disease. In Parkinson's disease, the level of N-methyl(R)salsolinol increased in cerebrospinal fluid and the high activity of a synthesizing enzyme, (R)salsolinol N-methyltransferase, was detected in lymphocytes. This isoquinoline induced apoptotic DNA damage in human dopaminergic neuroblastoma SH-SY5Y cells. Among catechol isoquinolines, only N-methylsalsolinol induced apoptosis in the cells, and the scavengers of hydroxyl radicals and antioxidants suppressed DNA damage, suggesting that reactive oxygen species initiate apoptosis. The isoquinoline activated caspase-3 like proteases and a caspase-3 inhibitor protected the cells from DNA damage. (-)Deprenyl, but neither clorgyline nor pargyline, prevented apoptotic cell death. The mechanism of the protection was due to stabilization of mitochondrial membrane potential reduced by the toxin. In Parkinson's disease apoptosis may be induced in dopamine neurons by this endogenous neurotoxin, and (-)deprenyl may protect them from apoptotic death process.

N-methyl(R)salsolinol and a neutral N-methyltransferase as pathogenic factors in Parkinson's disease.

The pathogenesis of Parkinson's disease is still an enigma. As an endogenous MPTP-like neurotoxin, N-methyl(R)salsolinol was proved to induce parkinsonism in rats and apoptosis in dopaminergic neurons. It is synthesized in the human brain by two enzymes; an (R)salsolinol synthase and an N-methyltransferase, and accumulates in the nigro-striatum in human brains. The activity of a neutral N-methyltransferase in the striatum was found to determine the level of MPP+-like 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion, an oxidation product of N-methyl(R)salsolinol in the substantia nigra. The activity of this N-methyltransferase was found to increase significantly in lymphocytes prepared from parkinsonian patients. In cerebrospinal fluid from untreated parkinsonian patients, N-methyl(R)-salsolinol increases significantly. These results suggest that N-methyl(R)salsolinol and a neutral N-methyltransferase may be endogenous factors in the pathogenesis of Parkinson's disease.

An endogenous MPTP-like dopaminergic neurotoxin, N-methyl(R)salsolinol, in the cerebrospinal fluid decreases with progression of Parkinson's disease.

There have been an increasing number of evidences indicating that dopamine-derived N-methyl(R)salsolinol is an endogenous MPTP-like neurotoxin to cause Parkinson's disease. In the cerebrospinal fluid from newly diagnosed untreated patients with Parkinson's disease, the level of this toxin was found to increase significantly, compared to control and a disease control, multiple system atrophy. The effects of the disease duration and the medication on the level of N-methyl(R)salsolinol were studied from the same patients. After about a 2-year period, the level was significantly reduced. The depletion of dopamine neurons by the disease progression may account for the reduction of the neurotoxin level, whereas L-DOPA therapy did not seem to affect the level of this toxin, even though the enhanced dopamine turnover. The results suggest that N-methyl(R)salsolinol level in the cerebrospinal fluid may indicate remaining dopamine neurons in the parkinsonian brain.

A dopaminergic neurotoxin, (R)-N-methylsalsolinol, increases in Parkinsonian cerebrospinal fluid.

The concentration of (R)-N-methylsalsolinol, which is a dopamine-derived neurotoxin selective to dopamine neurons and induces parkinsonism in rats, was found to be increased significantly in the cerebrospinal fluid of untreated patients with Parkinson's disease. The enantio-specific occurrence of (R)-N-methylsalsolinol in cerebrospinal fluid suggests its enzymatic synthesis in the human brain. The individual differences in the activities of the enzymes determining the metabolism of (R)-N-methylsalsolinol in the brain might be involved in the pathogenesis of Parkinson's disease.

The influence of acute ethanol on the catecholamine system in man as reflected in cerebrospinal fluid and urine. A new condensation product, 1-carboxysalsolinol.

Urine and cerebrospinal fluid (CSF) were collected from 10 healthy male volunteers after ingestion of 120 g ethanol and under similar conditions without ethanol. Dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), 4-hydroxy-3-methoxyphenylglycol (HMPG), 4-hydroxy-3-methoxymandelic acid (HMMA = VMA), 1-carboxysalsolinol (1-CSAL), salsolinol (SAL) and methylated salsolinol (M-SAL) were analyzed with gas chromatography-mass spectrometry. In CSF collected 6 h after ethanol intake the concentration of NE and its metabolite HMPG were significantly elevated (P less than 0.025 and P less than 0.005, respectively) compared to control conditions. The other compounds analyzed did not change significantly. In urine collected during 10 h after ethanol administration the excretion of HMMA was significantly reduced (P less than 0.01) and the HMPG/HMMA ratio was significantly elevated (P less than 0.005) reflecting a change in the peripheral red-ox state during ethanol oxidation. The excretion of DA and its major metabolite HVA did not change. However, the DA-derived condensation products 1-CSAL (from DA and pyruvate) increased (P less than 0.001), while SAL (from DA and acetaldehyde) decreased (P less than 0.005) after ethanol ingestion compared to the control situation. The increased excretion of 1-CSAL indicated that the ethanol metabolism interferes with the glucose metabolism, probably through an acetaldehyde-mediated inhibition of the pyruvate dehydrogenase complex.

Catecholamines and metabolites in cerebrospinal fluid of teetotallers and sober alcoholics.

Dopamine (DA), homovanillic acid (HVA), salsolinol (SAL), methylated salsolinol (M-SAL), norepinephrine (NE) and 4-hydroxy-3-methoxyphenylglycol (HMPG) were measured in cerebrospinal fluid (CSF) from 8 alcoholics who had abstained from ethanol for 3 months and from 8 teetotallers. No significant differences in the concentrations of any of these catecholamines or their metabolites were found between the two groups. In the alcoholics there was a positive correlation between age and DA (P less than 0.005), NE (P less than 0.025) and SAL (P less than 0.005) and also between DA and SAL (P less than 0.01).

Analysis of salsolinol and salsoline in biological samples using deuterium-labelled internal standards and gas chromatography--mass spectrometry.

Salsolinol and salsoline were labelled with deuterium using an acidic exchange reaction in 2HCl--2H2O. Two deuterium atoms were incorporated in both compounds. The deuterium-labelled salsolinol and salsoline were used as internal standards to determine picomol amounts of the corresponding unlabelled compound in the urine, cerebrospinal fluid, brain and liver. The salsolinol was purified on alumina and salsoline collected in the effluent. The compounds were analysed as their pentafluoropropionyl derivatives by gas chromatography on a 1% OV-17 column and were selectively detected with electron-impact mass spectrometry at the molecular ions M+ and M+--15. With human urine the precision of the methods were +/- 4.9% (coefficient of variation, n = 10) for salsolinol and +/- 2.2% for salsoline at a level of 0.100 nmol/ml. Administration of salsolinol to rats intraperitoneally (0.4 mmol/kg) resulted in levels of 1--2 nmol/g in striatum and limbic forebrain after 2 h, whereas the corresponding liver values were about 550 nmol/g. Control animals showed salsolinol values in liver of about 2 nmol/g and in striatum and limbic forebrain 1 nmol/g tissue.

Salsolinol and salsoline in cerebrospinal lumbar fluid of alcoholic patients.

Analyses of salsolinol and salsoline in human lumbar cerebrospinal fluid and urine have been performed by gas chromatography mass spectrometry. Twelve male alcoholic inpatients have been investigated. On the first sampling occasion patients were still intoxicated after a heavy alcohol debauch and on the second sampling occasion they had been inpatients and off alcohol for one week. Urine and lumbar cerebrospinal fluid was collected in a uniform way. In urine salsolinol and salsoline was found in all cases. In cerebrospinal fluid salsolinol was found in totally six subjects--one at the first sampling occasion only, four at both and one subject at the last sampling occasion only. Salsoline was found in nine patients--six in both sampling occasions and three only during intoxication. There was a wide interindividual variation and no statistical significant difference in the levels between the first and second sampling in CSF or urine.