OBJECTIVE To determine the concentrations of β-phenylethylamine (PEA) in CSF in patients with Parkinson’s disease, and to evaluate the relation between concentration of PEA in CSF and severity of Parkinson’s disease.
METHODS Using gas chromatography-chemical ionisation mass spectrometry, CSF concentrations of PEA were measured in 23 patients with Parkinson’s disease (mean age, 64.0 (SD 8.2) years), of whom three were at Hoehn and Yahr stage II, 11 were at stage III, and nine were at stage IV. Comparison was made with eight patients with neuropathy (mean age, 57.0 (SD 19.2) years) and 12 controls without neurological disease (mean age, 57.6 (SD 4.8) years).
RESULTS Concentrations of PEA in CSF in Parkinson’s disease were significantly lower (mean 205 (SD 131) pg/ml) than in patients with peripheral neuropathy (433 (SD 254) pg/ml) and controls (387 (SD 194) pg/ml). The concentrations of PEA in CSF correlated negatively with Hoehn and Yahr stage (P<0.01).
CONCLUSIONS There are decreased CSF concentrations of PEA in patients with Parkinson’s disease.
- cerebrospinal fluid
- Parkinson’s disease
- Hoehn and Yahr stage
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Parkinson’s disease is a relatively common progressive nigrostriatal neuronal degeneration. Recently alterations of several neurotransmitters related to dopaminergic neurons have been emphasised in Parkinson’s disease.1 2 Some studies have indicated that β-phenylethylamine (PEA) acts as a neurotransmitter or neuromodulator in the nigrostriatal dopaminergic pathway. PEA, which has been implicated in the pathogenesis of certain psychiatric and neurological diseases,3-5 was identified as a biogenic trace amine in mammalian tissues including brain in the 1960s.6 7 Since then, PEA has been investigated extensively in neurochemical, neuropharmacological, and neurophysiological contexts.8-12
The synthesis of PEA is by decarboxylation of phenylalanine13 and it is metabolised either first to phenylacetaldehyde by monoamine oxidase type B (MAO-B) and subsquently to phenylacetic acid (PAA) by aldehyde dehydrogenase, or alternatively to phenylethanolamine by dopamine-β-hydroxylase (fig1).14 Acting on presynaptic dopaminergic nerve terminals, PEA stimulates dopamine release.13 15 PEA also has an indirect sympathomimetic effect.16 Although some authors have reported altered PEA concentrations in psychiatric diseases,17-19 PEA concentrations in patients with Parkinson’s disease have not been reported. We measured PEA in the CSF of patients with Parkinson’s disease as the best reflection of its status in the CNS in vivo and examined relations between CSF concentration of PEA and severity of disease.
Patients and methods
CHEMICALS AND REAGENTS
Deuterium-labelled PEA (D4-PEA) was purchased from MSD Isotopes (Quebec, Canada). Pentafluoropropionic anhydride was obtained from TCI (Tokyo, Japan). PEA and other chemicals were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan).
Specimens of CSF were obtained from 23 patients with Parkinson’s disease (11 men, 12 women; mean age, 64.2 (SD 8.2) years; mean duration of disease, 6.4 (SD 2.9) years, range 2 to 14 years). Specimens were also obtained from eight non-parkinsonian patients with peripheral neuropathy (five men, three women; mean age, 57.0 (SD 19.2) years), and from 12 controls without neurological disease, including eight orthopaedic patients, three urological patients, and one gynaecological patient (six men, six women; mean age 57.6 (SD 4.8) years). Severity of Parkinson’s disease was rated according to Hoehn and Yahr stage.20 All patients were receiving antiparkinsonian drugs including levodopa-benserazide and bromocriptine, but not the MAO-B inhibitor deprenyl, which affects concentrations of PEA in CSF. The amount of levodopa given was 200 to 400 mg per day, except in three patients (patients 1, 7, and 19 in the table). For patients 7 and 19, L-threo-DOPS was administered.21 Ten patients showed depression; some received antidepressant drugs. With the informed consent of the patients, lumbar puncture was performed between 8 30 and 11 00 am. No CSF samples visibly contaminated with blood were used. The first ml of CSF obtained was immediately frozen and stored at −80°C until assay.
EXTRACTION AND DERIVATISATION
The extraction of PEA from CSF was performed by a method described previously.22 Samples of CSF (0.5 ml) were placed in silicon coated tubes. Then 5 ng D4-PEA were added to each tube as an internal standard. In addition, 5 ng D4-PEA was added to each of four tubes with 0.5 ml Ringer’s solution. Then, 30 pg, 100 pg, 300 pg, or 1 ng PEA were each added to one of these four tubes as standards. All samples were kept at 0°C for 20 minutes to equilibrate. After the addition of 50 μl 6 N NaOH, CSF was applied to an Extrelut column (2 cm×2.5 cm, Merck, Darmstadt, Germany) left for five minutes, and eluted with 7 ml ethyl acetate. The eluate was collected in a tube which contained 100 μl 1 mM HCL to prevent loss of PEA during evaporation in a concentrator (Savant, Farmingdale, NY). The residue was derivatised with 50 μl pentafluoropropionate (PFP) anhydride at 100°C for 20 minutes. The solvent was evaporated to dryness with dry nitrogen gas, and the sample was reconstituted with 20 μl ethyl acetate.
GAS CHROMATOGRAPHY-CHEMICAL IONISATION MASS SPECTOMETRY (GC/CI /MS)
Sample volumes of 2 μl were injected into a GC-CI mass spectrometer. The double-focused mass spectrometer was interfaced with a data aquisition system (Hitachi M-80B, Hitachi, Japan). Isobutane was used as the reagent gas (source pressure, 3×10-6 torr) and helium was the GC carrier gas (40ml/ min). The GS/MS interface oven and transfer line were set at 260°C. A Megabore column (0.56 mm internal diameter, 15 m long, J and W Scientific Co, Folsom, CA, USA) coated with DB-1 was used. The oven temperature was maintained isothermally at 100°C. The mass numbers used for quantitative analysis were m/z 268 corresponding to PEA-PFP and m/z 272 corresponding to D4-PEA-PFP. Peak area measurement was used for the estimation of ion currents. The calibration curve was highly correlated in standard samples (r=0.970) in the range of 30-1000 pg /ml PEA (fig 2).
MEASUREMENT OF HOMOVANILLIC ACID (HVA)
Specimens of CSF from 19 patients with Parkinson’s disease and 12 controls were ultracentrifuged for five minutes at 15 000 g at 4°C. HVA was determined in 20 μl supernatant by high performance liquid chromatography (Gilson Model-231, Germany; Eicom ECD-100, Japan). Details have been reported previously.23
Data were expressed as mean (SD) and analysed with analysis of variance (ANOVA), and followed by Scheffe’s F test or Student’s t test (two tailed) to evaluate the significance of differences between each groups. The relation between severity of disease in Parkinson’s disease and CSF PEA concentration was analysed with Pearson’s correlation. A P value >0.05 was considered significant.
The table shows the results of assays for PEA and HVA together with clinical characteristics of the patients with Parkinson’s disease. No significant difference was evident for sex or age distribution between control and neuropathy patients and patients with Parkinson’s disease. Mean concentrations of PEA in patients with Parkinson’s disease (n=23), patients with peripheral neuropathy (n=8), and controls (n=12) were 205 (SD 131), 433 (SD 254), and 387 (SD 194) pg/ml, respectively, with the concentration in patients with Parkinson’s disease being significantly lower than those in the neuropathy and control groups (P<0.001). No significant difference was noted between the peripheral neuropathy and control groups (fig 3). Concentrations of PEA in CSF from patients with Parkinson’s disease with depression (163 (SD 72.8) pg/ml) were lower than those in patients with Parkinson’s disease without depression (244 (SD 242) pg/ml), but not significantly (P=0.07). The present study did not show a significant relation between concentration of PEA in CSF and dose of levodopa, or between the concentrations and duration of Parkinson’s disease. The concentration of PEA in CSF showed a significant negative correlation with Hoehn and Yahr stage (P<0.001, fig 4).
The CSF concentrations of HVA did not differ significantly between Parkinson’s disease (35.3 (SD 17.9) ng/ml, n=19) and control groups (39.5 (SD 15.4) ng/ml, n=12). No significant correlation was found between CSF concentrations of PEA and HVA.
Our results showed lower CSF concentrations of PEA than those previously reported by Lauber and Waldmeier (mean 17.3 (SD 3.3) ng/ml, range 3-45 ng/ml, n = 15).24 PEA readily permeates the blood brain barrier and plasma concentrations of PEA have been reported as ranging from 100 to 1000 pg/ml,22 25 26 which is similar to the presently obtained concentrations in CSF. Experimental studies have found brain tissue concentrations of PEA in rats to be<10 ng/g.3 11 25 26 The present study is the first to show decreased CSF concentrations of PEA in patients with Parkinson’s disease compared with neurological controls (patients with peripheral neuropathy) and with neurologically normal controls of comparable ages.
Three reasons could account for decreased CSF concentrations of PEA in patients with Parkinson’s disease. (1) PEA may be decreased due to neuronal degeneration, as the amine may be synthesised by the nigral dopaminergic neurons which diminish in this disease.11 27 28 Greenshaw et al 29 have found degeneration involving the substantia nigra to result in both depletion of dopamine (DA) and a decrease in the rate of PEA accumulation in deprenyl pretreated rats. (2) Postsynaptic PEA catabolism may be increased in Parkinson’s disease. Changes in MAO-B activity are capable of altering catabolism of PEA. However, the activity of MAO-B in patients with Parkinson’s disease has been a matter of controversy.30 31 In a recent report, more 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTiQ), which is formed from PEA and its metabolite phenylacetaldehyde, was found in the CSF of patients with Parkinson’s disease than in the CSF of controls. Increased formation of 1BnTiQ in Parkinson’s disease may result in reduction of CSF PEA concentrations.32 (3) Increased presynaptic DA concentrations are known to inhibit PEA synthesis at the decarboxylation step,11 a phenomenon which may be relevant in patients with Parkinson’s disease under treatment with levodopa (fig 1). Thus the low concentration of PEA in Parkinson’s disease may be caused by antiparkinsonian drugs used for treatment. However, we found no correlation between CSF concentration of PEA and the dose of levodopa taken by each patient in our study.
Some researchers have suggested that the concentration of PEA or its metabolite phenylacetic acid (PAA) may be a useful biochemical marker in psychiatric and behavioural research.19 33 This may also be true in neurodegenerative disease, as we found a significant negative correlation between CSF concentration of PEA and severity of Parkinson’s disease (Hoehn and Yahr stage). We think that CSF PEA concentrations reflect either the severity of nigrostriatal degeneration in patients with Parkinson’s disease or a reduced modulatory effect of PEA on a postsynaptic receptor response to DA.
Reduced CSF concentrations of HVA in unmedicated patients with Parkinson’s disease have been reported.34 LeWitt et al have reported that concentrations of DA metabolites do not correlate with clinical severity of Parkinson’s disease because HVA is known to be influenced by therapy with levodopa and other antiparkinsinian drugs.35 We found no significant difference between the CSF concentration of HVA in Parkinson’s disease and control groups, possibly because even patients at Hoehn and Yahr stage IV in our study were receiving comparatively small amounts of levodopa.
PEA may affect behaviour and even be important in psychiatric disease, as it is similar in structure to amphetamine and is able to evoke stereotyped behaviours. Decreased urinary concentrations of PEA have been reported in patients with depression,18 Depression is a common symptom in patients with Parkinson’s disease,36and we found a marginally significant difference in concentration of PEA in CSF between patients with Parkinson’s disease with depression and those without depression (P=0.07). The possibility remains that reduced PEA concentrations might participate in the pathogenesis of depression in Parkinson’s disease.
We thank Professor Toshiharu Nagatsu, Department of Neurochemistry, Institute for Comprehensive Medical Science, Fujita Health University, for valuable comments; and Miss Yoko Kuroiwa and Miss Seiko Umezaki for their assistance in manuscript preparation.
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