Neuroscience Letters, 157 (1993) 103 106 103 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

NSL 09663

Serum levels offl-carotene and other carotenoids in Parkinson's disease

F61ix Javier J im6nez-J im6nez a, Jos6 A n t o n i o Mol ina b, Pilar Fernfindez-Cal le o, A n t o n i o Vfizquez c, F ranc i sco Cabrera-Vald iv ia a, Mar i a Jos6 Catal~in c, Es teban Garc ia -Albea a, F61ix Bermejo b

and R o s a C o d o c e o d

Departments of Neurology of Hospital Universitario a'Principe de Asturias'-Alcaldt de Henares, bDoce de Octubre, ' Universitario San Carlos and dDepartment of Biochemistry of Ciudad Sanitaria, La Paz, Madrid (Spain)

(Received 19 February 1993; Revised version received 16 April 1993; Accepted 16 April 1993)

Key words: Parkinson's disease; fl-Carotene; a-Carotene; Lycopene; Retinol; Provitamin A; Vitamin A; Serum level

To elucidate the possible role of carotenoids in the risk for developing Parkinson's disease (PD), we compared serum levels of fl-carotene, a-carotene and lycopene, measured by high performance liquid chromatography, of 61 PD patients using their spouses as the control group. The serum levels of these 3 carotenoids did not differ significantly between PD patients and control groups. There was no influence of antiparkinsonian therapy on serum carotenoids levels, and these did not correlate with age, age at onset, scores of the Unified Parkinson Disease Rating Scale or the Hoehn and Yahr staging in the PD group. These results show that serum carotenoids concentrations are apparently unrelated to the risk for developing PD.

The pathogenesis of the neuronal degeneration of neu- rons in the pars compacta of the substantia nigra in pa- tients with Parkinson's disease (PD) might be related to an excessive lipid peroxidation promoted by free radicals and reactive species [19, 29]. Increased lipid peroxidation has been reported in the substantia nigra [6, 7], plasma [27], erythrocytes [27] and platelets [5], although the sig- nification of these findings is unclear [31, 33, 41, 47]. Several authors reported a postmortem decrease in the catalase, peroxidase [1], and glutathion-peroxidase activ- ities [28], and reduced glutathione [37, 43] in the substan- tia nigra of patients with PD, suggesting the possible contribution of a metabolic failure in antioxidant mecha- nisms to the chemical processes that lead to lipid peroxi- dation. The concentrations of some vitamins with anti- oxidant properties in substantia nigra [8, 43] and in serum [11, 12, 25] are normal.

fl- and a-carotene (precursors of vitamin A), and ly- copene are carotenoids that are present mainly in fruits and vegetables [15, 16, 32]. fl-Carotene protects lipids by interfering with their oxidation, and behaves as a reduc- ing agent by trapping free radicals [3, 14, 15, 20, 46]. a-Carotene and lycopene may have, to a lesser extent, antioxidant actions [3]. In addition, vitamin A inhibits lipid peroxidation in rat brain [26]. Therefore, carote-

Correspondence: F.J. Jim~nez-Jim6nez, C/ Corregidor Jos6 de Pasamonte, 24, 3-D, E-28030, Madrid, Spain.

noids themselves or after their transformation to vitamin A might act as a protective mechanism against lipid per- oxidation of substantia nigra.

The aim of this study was to assess the serum levels of some carotenoids in patients with PD compared to a con- trol population, in order to elucidate whether low serum levels of these antioxidants could play a role as risk fac- tor for PD.

We assessed the serum levels of fl-carotene, a-caro- tene, and lycopene, and the retinol/fl-carotene ratio in 61 patients with diagnostic criteria for PD [52], recruited from outpatients attended in the neurology departments of several urban hospitals, and their respective spouses as the control group. PD patients were evaluated with the Unified PD Rating Scale (UPDRS) [10] and the Hoehn and Yahr staging [21]. Clinical data of parkin- sonian and control groups are summarized in Table 1. Nine patients were untreated (the blood was taken at the first visit), while the other 52 were treated with antipark- insonian drugs alone or in combination with levodopa (51 cases), bromocriptine or pergolide (35 cases), deprenyl (12 cases), and anticholinergics (3 cases).

Exclusion criteria for patients and controls were the following: (i) ethanol intake higher than 80 g/day in the last 6 months; (ii) previous history of chronic hepatopa- thy, diseases causing malabsorption or severe systemic disease; (iii) atypical dietary habits (diets constituted ex- clusively by one type of foodstuff, such as vegetables, meat, or others; special diets because of religious rea-

104

sons, etc); (iv) intake of drugs which modify lipid absorp- tion; (v) therapy with vitamins in the last 6 months.

A 2-cc blood sample from each patient and the respec- tive control was taken, simultaneously, during fasting using a syrinx with a vacuum system (Venoject, Torumo, Belgium), collected on ice and centrifuged. The serum was frozen immediately at -20°C and protected from light exposure to avoid photo-oxidation, with aluminium foil, until analysis. The determinations were performed blindly 1 to 3 months after blood extraction.

fl-Carotene, a-carotene, and lycopene serum levels were assessed by high performance liquid chromatogra- phy (HPLC) according to a modification of the method of Ito et al. [22, 23]. One-hundred/21 of serum was mixed with 0.5 ml of water and 0.5 ml of ethanol in a brown glass vial and filled with liquid nitrogen. Tubes were mixed in a stirrer plate during 2 min. 2.5 ml of n-hexane was added to the nitrogen filled vial and mixed during 25 min at room temperature. Tubes were centrifuged at 1500 rpm during 10 min. Two ml of the upper layer was extracted and dried with nitrogen. Dried carotenoids were dissolved in 100/21 of ethanol and 50/21 was injected into the HPLC system. The operational conditions for the HPLC (Waters, Division of Millipore, MA, USA)

method were as follows: column, Nova-Pack Cartridge C-18 (4 /2m, 8 x 10 cm. Waters): solvent: acetoni- trile:dichloromethane:methanol (HPLC grade) = 7:2:6: wavelength, 436 nm; flow rate: 0.8 ml/min. Calibration was done by using an internal standard: ethil fl-apo-8'- carotenoate (trans). Standard substances, lycopene, a- carotene, fl-carotene and the internal standard were purchased from Sigma (Sigma Co, Ltd). The respective retention times were 3.55, 6.36, 9.65 and 10.3 min. Intra- batch coefficients of variation were 8.3, 7.6 and 4.5%, and interbatch coefficients of variation 21, 16 and 9%, respectively, for lycopene, a-carotene, and fl-carotene. The measurements of serum carotenoid [eves were ex- pressed as/2g/ml.

The results are expressed as means + S.E.M. For sta- tistical analysis the biostatistical packet 'Sigma Data Base' (Horus Hardware) [13] was used, and included the two- tailed student's t-test (carotenoids values in serum were normally distributed in both groups), analysis of variance (ANOVAL and calculation of Pearson's corre- lation coefficient when appropriate.

The mean serum levels of the carotenoids that we stud- ied did not differ significantly from those of controls (Table 1 ), and they did not correlate in PD patients with

T A B L E I

C L I N I C A L DATA A N D R E S U L T S O F H E A L T H Y A N D P A R K I N S O N I A N PATIENT G R O U P S

PD, Parkinson's disease: ADL, activities of daily living. Data of quantitative variables are expressed as mean _+ S.E.M. In parentheses are expressed the values of the 52 treated patients. Type 11 error means the probability that the absence of significant differences between the study groups should be a false negative result.

PD-patients Controls Total(n = 61) In = 61)

Treated (n = 52)

Type I1 error

Age (years) 66.0 -+ 0.96

Female 29

Male 32

Age at onset of PD (years) 60.5 +_ 1.29

Duration of PD (years) 5.5 _+ 0.67

Scores of the Unified PD Rating Scale Total (items I 31) 29.0 _+ 1.68

A D L subscale (items 5 17) 12.0 +_ 0.77

Motor subscale (items 18 31) 14.6 + 0.83

Hoehn and Yahr stage 2.5 _+ 0.09

,6-Carotene (,ug/dl) 0.87 _+ 0.08

(0.86 + 0.08)

~-Carotene (j2g/dl) 0.092 +_ 0.012

(0.093 + 0.012}

Lycopene (J2g/dll 1.10 + 0.11

(1.10 _+ 0.11)

Retinol (J2g/dl) 0.69 + 0.03 (0.65 _+ 0.02)

Retinol/fl-carotene ratio 1.35 + 0.16 (1.32 _+0.17)

65.4 _+ 1.06

32

29

1.01 + 0.12

0.094 _+ 0.014

1.21 + 0.14

(i).64 _+ 0.02

1.19 _+ 0.13

0.42

0.42

0.07

0.06

0.29

0.29

0.35

0.17

0.33

0.29

age, age at onset and dura t ion o f PD, scores o f the

U P D R S (total and subtotals o f activities o f daily living

and m o t o r examination), and the Hoehn and Yahr stag- ing. An A N O V A showed that these values were not in-

fluenced significantly by ant iparkinsonian drugs in pa- tients.

In the last years there has been an increasing interest

on the possible role o f oxidative mechanisms in the pa- thogenesis o f PD. I f this hypothesis o f 'oxidative stress'

[19, 29] is true, intake o f ant ioxidant vitamins may be protective. Some authors suggested that PD patients in early life may have eaten foods with lower vi tamin E content than did controls [17, 18, 49, 50]. This hypothesis

has also led to begin several trials with 'ant ioxidant ther-

apy ' a t tempt ing to delay the natural evolution o f this disease, involving vitamins E [9, 36] and C [9]. The possi- ble protective effect o f these vitamins against MPTP- or

6-hydroxy-dopamine- induced parkinsonism is contradic- tory [2, 4, 24, 30, 38-40, 44, 45, 51, 53]. However, the

concentra t ions o f vitamins C and E in substantia nigra [8, 43] and o f vitamins C, E and A in serum [11, 12, 25] o f PD patients are similar to those o f control groups.

f l -Carotene [3, 14, 15, 20, 46], retinol [26], and proba- bly other carotenoids [3] have an ant ioxidant action, which seems to be synergistic with those o f vitamin E [3,

14, 45]. Pretreatment with fl-carotene showed partial protect ion against MPTP- induced neurotoxici ty experi- ment in mice [38, 53], but this effect was not evident in marmosets [39] and in cultured dopaminergic neurons

f rom fetal rat mesencephalon [44]. In contras t with other antioxidants, f l-carotene has no t been tried as 'preven- tive' therapy in PD.

Our results showed that the serum levels off l -carotene, s -carotene, lycopene and the retinol/fl-carotene ratio were non-significantly different in PD patients than in controls, and the same values did not correlate with the analysed clinical features o f PD. Serum levels of f l -caro-

tene and other carotenoids reflect only recent weeks or months intake, while adipose tissue and liver concentra-

tions are likely to reflect m a n y years o f comsumpt ion patterns o f fruits and vegetables [35]. Therefore, a more accurate measure o f carotenoid status should require bi- opsy techniques.

The normal i ty o f serum carotenoid levels does not rule out the possibility that there may be regional deficiencies that were not expressed in serum, and does not mean that utilization binding as redox-state is normal. To our knowledge CSF concentra t ions o f carotenoids have not been yet measured, and brainstem levels off l -carotene in humans are below the detection limits [48]. However, in animals fed with diets supplemented with fl-carotene, brain carotenoid levels are a round 1/6 and 1/200 of those in adipose tissue and liver, respectively [42]. Despite

105

these observations, our results show that serum carote-

noid concentra t ions are apparent ly unrelated to the risk

for PD.

Concepci6n Ramirez and Isabel Garcia (nurses at the Depar tments o f Neuro logy o f Hospitals Universi tario

San Carlos and 'Principe de Asturias ' , respectively) col-

laborated in the collection o f samples. This work was supported in par t by the Gran t FIS-90 0775.

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