Neuroscience Letters 271 (1999) 3336

Cerebrospinal uid levels of thiamine in patients with Parkinson's disease

Felix Javier Jimenez-Jimenez a,*, Jose Antonio Molina b, Angel Hernanz c, c d Estrella Fernandez-Vivancos , Fernando de Bustos , Beatriz Barcenilla a, Carlos Gomez-Escalonilla b, Martn Zurdo a, Angel Berbel b, Clara Villanueva b
a

Department of Neurology, Hospital Universitario `Prncipe de Asturias', Alcala de Henares, Madrid, Spain b Department of Neurology, Hospital Universitario Doce de Octubre, Madrid, Spain c Department of Biochemistry, Ciudad Sanitaria La Paz, Madrid, Spain d Department of Biochemistry, Hospital Universitario Doce de Octubre, Madrid, Spain Received 16 April 1999; received in revised form 1 June 1999; accepted 7 June 1999

Abstract Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, such as the alpha-ketoglutarate dehydrogenase complex (KGDHC), pyruvate-dehydrogenase complex, and transketolase. The activity of KGDHC is decreased in the substantia nigra or patients with Parkinson's disease (PD). We measured cerebrospinal (CSF) levels of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine, using ion-pair reversed phase high performance liquid chromatography, in 24 PD patients and 40 matched controls. The mean CSF levels of thiamine-derivatives did not differ signicantly from those of controls, with the exception of lower CSF free thiamine levels in the PD-patient group. PD patients under levodopa therapy had signicantly higher CSF thiaminediphosphate and total thiamine than those not treated with this drug. CSF thiamine levels were not correlated with age, age at onset, duration of the disease, scores of the Unied Parkinson Disease Rating Scale of the Hoehn and Yahr staging in the PD group. These results suggest that low CSF free thiamine levels could be related with the risk for PD. q 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Parkinson's disease; Thiamine; Cerebrospinal uid levels

The pathogenesis of the neuronal degeneration of neurons in the pars compacta of the substantia nigra in patients with Parkinson's disease (PD) is unknown. Several studies suggested the presence of oxidative stress in the substantia nigra of PD patients, including decreased enzyme activities of mitochondrial respiratory chain complex I (reviewed in Ref. [10]) and alpha-ketoglutarate dehydrogenase complex (KGDHC) [13]. Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, namely KGDHC, piruvate-dehydrogenase complex, and transketolase [5]. Thiamine deciency or inhibition in experimental models can induce a reduction in the KGDHC complex activity [15], and mitochondrial dysfunction [2].

* Corresponding author. C/ Corregidor Jose de Pasamonte, 24, 3-D., E-28030, Madrid, Spain. Tel.: 134-91-437-6078; fax: 134-91-328-0704. E-mail address: fjimenezj@meditex.es (F.J. Jimenez-Jimenez)

Recently, Gold et al. [7] reported normal plasma thiamine in patients with PD but, to our knowledge, there is no previous information concerning cerebrospinal uid thiamine levels in patients with PD. The aim of this study was to assess the lumbar cerebrospinal uid levels of thiamine and their phosphate esters in patients with PD compared with a control population. We assessed the cerebrospinal uid of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine in 24 patients with PD recruited from outpatients attended in the Neurology Departments of two urban Hospitals. They fullled diagnostic criteria for PD [9] and were evaluated with the Unied Parkinson's Disease Rating Scale (UPDRS) [6] and the Hoehn and Yahr staging [8]. Seven patients were untreated, while the other 17 were treated with antiparkinsonian drugs alone or in combination including levodopa (15 cases), bromocriptine or pergolide (14 cases), and deprenyl (eight cases). The control group comprised 40 `healthy' patients, who

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 51 5- 7

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Table 1 Clinical data and results of PD patient and control groups a PD-patients (n 24) Clinical data Age (years) Female Male Age at onset of PD (years) Duration of PD (years) Scores of the Unied PD Rating scale (UPDRS) Total (items 131) ADL subscale (items 517) Motor subscale (items 1831) Hoehn and Yahr stage Cerebrospinal uid Thiamine levels (nmol/l) Thiamine-diphosphate Thiamine-monophosphate Free thiamine Total thiamine 64.0 ^ 11.4 10 14 58.1 ^ 11.3 5.9 ^ 5.7 Controls (n 40) 63.1 ^ 14.9 24 16

35.3 ^ 17.9 15.3 ^ 9.1 19.6 ^ 8.6 2.7 ^ 1.2

3.9 4.3 0.9 9.1

^ 3.0 ^ 3.3 ^ 1.3 ^ 6.4

3.1 ^ 2.3 4.3 ^ 2.9 1.9 ^ 1.4* 9.3 ^ 5.1

a Data of quantitative variables are expressed as mean ^ SD; PD, Parkinson's disease, ADL, activities of daily living; *P , 0:01.

underwent lumbar puncture because of suspected (but not conrmed) subarachnoid hemorrhage or pseudotumor cerebri, oculomotor palsies or other indications in the usual neurological survey. Routine CSF analysis was normal in each patient or control. Informed consent was obtained in each case. The clinical features of both groups are summarized in Table 1. The following exclusion criteria were applied to both patients and controls: (i) therapy with vitamin supplements in the last 6 months. (ii) Ethanol intake higher than 80 g/day in the last 6 months. (iii) Previous history of chronic hepatopathy, gastrectomy, pancreatic diseases, diseases causing malabsorption, and chronic renal failure. (iv) Atypical dietary habits (diets constituted exclusively by one type of foodstuff, such as vegetables, fruits, meat, or others, special diets because of religious reasons, etc.). (v) Undernutrition. (vi) Previous history of severe systemic disease.

Lumbar CSF sample was taken from each fasted patient or control between 08:00 and 10:00 h. Traumatic spinal punctures were excluded from the study. The CSF and specimens were frozen at 2 308C and protected from light exposure with aluminum foil until analysis. The determinations were performed blindly. The analysis of thiamine in the cerebrospinal uid was done by ion-pair reversed phase high-performance liquid chromatography according to Bettendorff et al. [3]. The derivatization of cerebrospinal uid compounds to yield uorescent thiochromes was carried out adding 50 ml of alkaline ferricyanide solution (4.3 mM potassium ferricyanide in 15% NaOH) to 80 ml of cerebrospinal uid, and then 20 ml were injected on the chromatograph. The mobile phase was composed of 50 mM Na2HPO4, t nM tetrabutylamonium phosphate (PIC A, Waters) and 4% tetrahydrofurane (pH 9.0). The thiamine derivatives were eluted isocratically at 448C from a Shodex RSpak DS-613 C18 column (Showa Denko, 6 m 150 mm) with a Shodex RSpak DS-G precolumn (Showa Denko), with a ow rate of 0.8 ml/min using a Waters Alliance 2690 chromatograph. A Waters 474 scanning uorescence detector was used for detection of thiamine derivatives. The uorescence intensities were measured with excitation at 365 nm and emission at 433 nm. All compounds were quantied with a Waters Millennium 2010 chromatography manager. Fig. 1 represents a chromatogram from a standard mixture of thiamine and their derivatives, and from a patient with PD. The measurements were expressed in nmol/l. The results were expressed as mean ^ SD. The statistical analysis used the Biostatistical Packet of `R-Sigma Data Base' (Horus Hardware) [14], and included the two-tailed Student's t-test, ANOVA, MannWhitney test, and calculation of Pearson's correlation coefcient when appropriate. The results are summarized in Tables 1 and 2. The mean CSF levels of thiamine-diphosphate, thiamine-monophosphate, and total thiamine of PD patients did not differ signicantly from those of controls, although free CSF thiamine levels were signicantly lower in the PD-patient group. PD patients who were treated with levodopa had

Table 2 Inuence of antiparkinsonian treatment on CSF levels of thiamine, measured in nmol/l (mean ^ SD) a Thiamine diphosphate Levodopa Yes (n 15) No (n 9) Dopamine agonist Yes (n 14) No (n 10) Deprenyl Yes (n 8) No (n 16)
a

Thiamine monophosphate

Free thiamine

Total thiamine

5.1 ^ 3.1 2.0 ^ 1.5 4.6 ^ 3.1 3.0 ^ 2.8 4.4 ^ 3.3 3.7 ^ 2.9

5.2 ^ 3.6* 2.8 ^ 2.1 4.3 ^ 3.8 4.4 ^ 2.8 4.1 ^ 2.9 4.4 ^ 3.6

1.1 ^ 1.6 0.4 ^ 0.3 1.0 ^ 1.6 0.7 ^ 0.7 0.7 ^ 0.8 0.9 ^ 1.5

11.5 ^ 6.7* 5.2 ^ 3.7 9.8 ^ 7.0 8.1 ^ 5.7 9.2 ^ 6.1 9.1 ^ 6.7

*P , 0:01.

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signicantly higher CSF thiamine-diphosphate and total thiamine than those not treated with this drug. There was no signicant correlation in PD patients between the CSF thiamine levels (in all their forms) and the following values: age, age at onset of PD, duration of PD, scores of the UPDRS (total and subtotals of Activities of Daily Living and motor examination), and the Hoehn and Yahr staging. Thiamine has an important role in the brain oxidative metabolism. Mitochondrial fractions of rat brain are rich in thiamine derivatives [4]. This vitamin acts as a cofactor of several enzymes, namely the KGDHC, which is probably

the most important and the rate-regulating enzyme in the Krebs' cycle [12]. KGDHC inhibition can induce mitochondrial respiratory chain dysfunction. There have been reported decreased activity of this enzyme [13] in the brain of parkinsonian patients and, more recently, an association between the gene encoding the E2 subunit of KDHGC and PD [11]. In addition, thiamine derivatives, which are present in the human substantia nigra at high concentrations [1], can induce dopamine release in the rat striatum [18]. The data of the present study show that, when compared with controls, PD patients had similar cerebrospinal uid

Fig. 1. Chromatogram obtained from a standard mixture of 250 nmol/l of thiamin monophosphate, thiamin diphosphate, and free thiamine (upper side). Chromatogram of cerebrospinal uid from a patient with Parkinson's disease, showing thiamine monophosphate, thiamin diphosphate, and free thiamine (units in ng/ml; bottom side).

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F Jimenez-Jimenez et al. / Neuroscience Letters 271 (1999) 3336 .J. pair reversed phase high performance liquid chromatography method. Ann. Biochem., 198 (1991) 5259. Bettendorff, L., Wins, P. and Lesourd, M., Subcellular localization and compartmentalization of thiamine derivatives in rat brain. Biochim. Biophys. Acta, 1222 (1994) 16. Blass, J.P., Vitamin and nutritional deciencies. In G.J. Siegel, B.W. Agranoff, R.W. Albers and P.B. Molinoff (Eds.), Basic Neurochemistry, Raven Press, New York, 1994, pp. 749760. Fahn, S., Elton, R.L. and members of the UPDRS Development Committee, Unied Parkinson's Disease Rating Scale, In S. Fahn, C.D. Marsden, M. Goldstein and D.B. Calne (Eds.), Recent Developments in Parkinson's Disease, Vol. 2, MacMillan, Florham Park, NJ, 1987, pp. 153163. Gold, M., Hauser, R.A. and Chen, M.F., Plasma thiamine deciency associated with Alzheimer's disease but not Parkinson's disease. Metab. Brain. Dis., 13 (1998) 4353. Hoehn, M.M. and Yahr, M.D., Parkinsonism: onset, progression, and mortality. Neurology, 17 (1967) 419423. Hughes, A.J., Ben-Shlomo, S.E., Daniel, S.E. and Lees, A.J., What features improve the accuracy of clinical diagnosis in Parkinson's disease: a clinicopathological study. Neurology, 42 (1992) 11421146. Jimenez-Jimenez, F.J., Ort-Pareja, M. and Gasalla-Gonza lez, T., Epidemiologa, etiologa y patogenia de la enferme dad de Parkinson. In F.J. Jimenez-Jimenez, M.R. Luquin and J.A. Molina (Eds.), Tratado de Los Trastornos del Movimiento, IM&C, Madrid, 1998, pp. 223258. Kobayashi, T., Matsumine, H., Matuda, S. and Mizuno, Y., Association between the gene encoding the E2 subunit of the alfa-ketoglutarate dehydrogenase complex and Parkinson's disease. Ann. Neurol., 43 (1998) 120123. Lai, J.C.K., Walsh, J.M., Dennis, S.C. and Clark, J.B., Synaptic and nonsynaptic mitochondria from rat brain: isolation and characterization. J. Neurochem., 28 (1977) 625631. Mizuno, Y., Matuda, S., Yoshino, H., Mori, H., Hattori, N. and Ikebe, S.I., An immunohistochemical study on a-ketoglutarate dehydrogenase complex in Parkinson's disease. Ann. Neurol., 35 (1994) 204210. Moreu, E., Molinero, L.M. and Fernandez, E., R-Sigma: Base de Datos Bioestadstica para un Ordenador Personal, Horus Hardware, Madrid, 1990. Munujos, P., Coll-Canti, J., Beleta, J., Gonzalez-Sastre, F. and Gella, F.J., Brain pyruvate oxidation in experimental thiamin-deciency encephalopathy. Clin. Chim. Acta, 255 (1996) 1325. Pedraza, O.L. and Botez, M.I., Thiamine status in inherited degenerative ataxias. J. Neurol. Neurosurg. Psychiatry, 55 (1992) 136137. Rindi, G., Patrini, C. and Poloni, M., Monophosphate, the only phosphoric ester of thiamin in the cerebro-spinal uid. Experientia, 37 (1981) 975976. Yamashita, H., Zhang, Y.X. and Nakamura, S., The effects of thiamine and its phosphate esters on dopamine release in the rat striatum. Neurosci. Lett., 158 (1993) 229231.

levels of thiamine derivatives, except those of free-thiamine, although the experimental error for free-thiamine is very high, suggesting a high variability for this value. There was no correlation between CSF thiamine levels and the analyzed clinical features of PD. These data do not rule out the possibility that there may be regional deciencies of thiamine or its esters in some areas of the brain. PD patients treated with levodopa showed higher CSF levels of thiamine-diphosphate and total thiamine. The reasons for these ndings are unknown, but the small number of patients under treatment evaluated limits the validity of these observations. The CSF thiamine levels found in this study are very low when compared with those of other previous reports. This is likely to be related with methodological differences. Rindi et al. [17] used an electrophoretic-uorometric method, and they stated that thiamine-monophosphate was the only phosphoric ester of thiamine in the cerebrospinal uid. Pedraza and Botez [16] measured total CSF thiamine levels using a microbiological method with Lactobacillus fermenti. In the present study, we used ion-pair reversed phase high-performance liquid chromatography. This is likely a more sensitive and specic method, that allows the measurement of thiamine-diphosphate, as is shown in Fig. 1. In conclusion, although PD is associated with a KGDHC and complex I deciency in particular areas of the brain, these ndings are unrelated with alterations of CSF levels of thiamine, with the exception of those of free-thiamine, although this value had a high variability. These results suggest that low CSF free thiamine levels could be related with the risk for PD. This work was supported, in part, by a grant of the Comunidad de Madrid (Exp. 08.5/0005/1997) and by the `Funda cion Neurociencias y Envejecimiento'.
[1] Baker, H., Frank, O., Chen, T., Feingold, S., DeAngelis, B. and Baker, E., Vitamin content of some normal human segments. J. Neurosci. Res., 11 (1984) 419435. [2] Bettendorff, L., Goessens, G., Sluse, F., Wins, P., Bureau, M., Laschet, J. and Grisar, T., Thiamine deciency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors. J. Neurochem., 64 (1995) 20132021. [3] Bettendorff, L., Peeters, M., Jouan, C., Wins, P. and Schoffeniels, E., Determination of thiamine and its phosphate esters in cultured neurons and astrocytes using an ion-

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