Effect of branched-chain amino acids on glutamate metabolism in amyotrophic lateral sclerosis

JOURNAL OF THE

NEUROLOGICAL SCIENCES

ELSEVIER

Journal of the Neurological Sciences 129 (1995) 40-43

Effect of branched-chain amino acids on glutamate metabolism in amyotrophic lateral sclerosis Ole Gredal Department

of Biochemistry

*, Svend Erik Mgller

and Clinical Pharmacology, Research Institute of Biological Psychiatry, St Hans Hospital, DK-4000 Roskilde, Denmark

Received 30 March 1994; revised 27 September 1994; accepted 7 October 1994

Abstract Data from the literature about glutamate metabolism remain controversial. To refine such analysis we have studied plasma glutamate and aspartate levels after glutamate loading (60 mg/kg) in 6 fasting controls and ALS patients, before and after at

least 2 weeks treatment with branched-chain amino acids. ALS patients showed no difference from age-matched controls in basal plasma glutamate or aspartate levels, but significantly elevated levels of glutamate and aspartate at 30 and 4.5min after loading,

and an increased area under the curve in plasma for glutamate following oral glutamate loading. Two weeks BCAAs treatment did not affect plasma glutamate metabolism in ALS patients. Keywords:

Amyotrophic

lateral sclerosis; Glutamate

metabolism;

1. Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative human disease characterized by degeneration of upper and lower motor neurons. The cause of the disease remains unknown, and no effective treatment is yet available. However, the diffuse and selective involvement of motor neurons in the disease suggests the possibility of a generalized metabolic defect in central motor systems or interaction of such a defect with an unrecognized systemic metabolic disorder or intoxication. Recent studies suggest that exitotoxic mechanisms might be involved in the pathogenesis of the disease, since measurements of plasma glutamate have shown significantly increased concentrations in ALS patients compared to controls (Plaitakis and Caroscio, 1987; Perry et al., 1990; Iwasaki et al., 1992). Moreover, following oral glutamate loading, Plaitakis and Caroscio (1987) found significantly greater elevations of plasma glutamate and aspartate levels in ALS patients relative to controls.

* Corresponding author. Tel.: (45) 4633 4968; Fax (45) 4633 4355. 0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0022-510X(94)00243-6

Branched-chain

amino acids

Glutamate is metabolized in part by the enzyme glutamate dehydrogenase that interconverts glutamate and a-ketoglutarate. The activity of the enzyme has been reported as normal (Plaitakis and Caroscio, 1987) or reduced (Hugon et al., 1989) in ALS-leukocytes, and elevated in ALS dorsal horns of spinal cord (Malessa et al., 1988). Because glutamate dehydrogenase can be activated in vitro by the branched-chain amino acids (BCAAs) L-leucine and L-isoleucine (Yielding and Tomkins, 19611, BCAAs have been used for treatment of ALS. In a pilot study (Plaitakis et al., 1988) BCAAs significantly benefitted ALS patients in terms of maintenance of extremity muscle strength and continued ability to walk, but in two other studies (Testa et al., 1989; Beghi et al., 1993) no statistically significant differences were found in the clinical outcome between the patients treated with BCAAs and the control groups. As far as we know only the effects of BCAAs on mortality and clinical deterioration have been measured, whereas the effect of BCAAs on glutamate metabolism has not been investigated. The aim of this study was to evaluate the effect of subchronic treatment with BCAAs on glutamate metabolism in ALS. Plasma glutamate and aspartate levels were measured

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at basal conditions and after an oral load of glutamate in patients with ALS before and after at least two weeks’ treatment with BCAAs.

2. Material

and methods

2.1. Patients and control subjects

Six ALS patients and 6 healthy controls from the hospital staff (2 males and 4 females in both groups) with mean ages 59 and 54 years, ranges 43-73 and 47-59 years, respectively, were studied. The study was approved by the Scientific-Ethical Committee for the Municipalities of Copenhagen and Frederiksberg. In addition, basal plasma glutamate levels were measured in 28 fasting, healthy control subjects with mean age 25 years, range 20-27 years. The ALS diagnosis was established by clinical criteria (signs of both upper and lower motor neuron symptoms and history of progression for at least 3 months) and electromyographical criteria (evidence of acute and chronic denervation involving limb and axial musculature). The mean disease duration was 14 months, range 3-24 months. Two patients showed bulbar involvement at onset of the disease and all patients had symptoms from the arms and legs when they entered the study. 2.2. Design After an overnight fast, patients and controls received an oral load of sodium L-glutamate, 60 mg/kg, dissolved in 200 ml water, as used by Plaitakis and Caroscio (1987). ALS patients were subsequently treated for at least 2 weeks with 4 daily doses (half an hour before meals) of a mixture of 3 g L-leucine, 2 g L-isoleucine, and 1.6 g L-valine, as prescribed by Plaitakis et al. (19881, then the loading was repeated.

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2.3. Amino acids analysis

Amino acids were analyzed according to Moller (1993). Venous blood samples were collected in EDTA tubes before and every 15 min after the load until 3 h. After centrifugation at 2000 X g for 15 min at 4°C the plasma samples were stored at -80°C until analysis. Samples were protein precipitated by adding a cooled solution of sulfosalicylic acid. Plasma glutamate and aspartate were assayed by ion exchange chromatography using o-phthaldialdehyde postcolumn derivatization and fluorometric detection. 2.4. Statistical analysis

Basal levels were evaluated by Student’s two-tailed t-test, whereas the plasma levels during the three hours after loading were evaluated by analysis of variance (ANOVA) and further more by Duncan’s multiplerange test. p values above 0.05 were regarded as not significant (ns).

3. Results 3.1. Plasma glutamate

ALS patients, age-matched controls and young controls showed comparable basal plasma glutamate levels (Table 1). Fig. 1 shows plasma glutamate level (mean f SEM) versus time following oral glutamate loading for controls, and for ALS patients before and during treatment with BCAAs. In ALS patients relative to controls there was a significant difference in the plasma levels during the three hours (p < 0.001) with increased plasma glutamate levels at 30 and 45 min (p < 0.01). The area under the curve in plasma for glutamate was increased in ALS patients compared with controls (p = 0.026; Table 11,whereas the peak concentration for

Table 1 Plasma glutamate. Results are presented as mean f SD. The inconsistencies between the plasma peak levels of glutamate presented in the table and Fig. 1 are due to different time points for the maximum levels. Controls(age-matched) ALS patients pretreatment ALS patients intreatment Controls (young) (age-matched) (n = 6) (n=6) (n = 28) (n = 6) Basal (FM) Peak (PM) Ratio (peak/basal) AUC (mM x h)

25*a a -

52&26 250f 126 4.9* 1.6 5.8k3.2

AUC = area under the curve for plasma glutamate. ap = 0.0503 relative to “ALS age-matched” controls, Student’s t-test. bp < 0.03 relative to controls, Student’s r-test.

so*22 506 f 284 10.9+ 6.1 15.0f 8.0 b

54+17 360 f 129 7.0 + 2.7 11.6+6.3

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Sciences 129 (1995) 40-43

there was a significant difference in the plasma levels during the three hours (p < 0.051, and plasma aspartate concentration was significantly increased at 30 and 45 min (p < 0.01). Comparing pretreatment and intreatment courses, ALS patients showed no significant effect of BCAAs treatment for plasma aspartate (p = 0.08).

400 300

100 2000 1

4. Discussion time [min] Fig. 1. Plasma glutamate level (mean+SEM) versus time following oral glutamate loading (60 mg/kg) for controls (open bars) and for ALS patients, before (solid bars) and during treatment with BCAAs (hatched bars). ALS compared to controls (ANOVA): treatment F[1,126] = 11.7, p < 0.001; time F[12,126] = 13.9, p < 0.001. ALS before compared to AJS during treatment with BCAAs (ANOVA): treatment F[1,126] = 0.9, p = 0.36 (ns); time F[12,126] = 13.0, p < 0.001. * p < 0.01 (Duncan’s multiple-range test).

glutamate and the ratio plasma peak level: plasma basal Ieve did not differ between ALS and controls. Comparing pretreatment and intreatment courses, ALS patients showed no significant effect of BCAAs treatment for plasma glutamate (p = 0.36). 3.2. Plasma asparta te

No significant difference was found in basal plasma aspartate levels between age-matched controls and ALS patients (4.0 * 1.4 and 4.3 + 2.2 nmol/ml, respectively). The effect of glutamate loading on plasma aspartate is shown in Fig. 2. In ALS patients relative to controls

r;i r!z a 9z n

30 20

10 0 0

30

60

90

120

150

180

time [min] Fig. 2. Plasma aspartate level (meanf SEMI versus time following oral glutamate loading (60 mg/kg) for controls (open bars) and for ALS patients, before (solid bars) and during treatment with BCAAs (hatched bars). ALS compared to controls (ANOVA): treatment F[1,123] = 5.4, p < 0.05; time F[12,123] = 11.9, p < 0.001. ALS before compared to ALS during treatment with BCAAs (ANOVA): treatment F[1,128] = 3.1, p = 0.08 (ns); time F[12,128]= 14.2, p < 0.001. * p < 0.01 (Duncan’s multiple-range test).

This study showed no difference between young and elderly controls in basal plasma glutamate levels. But we find that age-matching of the populations is important since Perry et al. (1990), have showed a positive correlation between plasma glutamate and age. We found no significant difference in basal glutamate levels between ALS patients and age-matched controls, consistent with some studies (Patten et al., 1978; Testa et al., 1989; Camu et al., 1993) but not others (Plaitakis and Caroscio, 1987; Perry et al., 1990; Iwasaki et al., 1992). Age and duration of illness may account for the discrepancies, as well as diagnostic classification (Camu et al., 19931, since subtype analysis showed a 25% increase of the glutamate level in ALS with spinal onset and a 30% decrease in ALS with bulbar onset compared to control levels. Administration of glutamate to fasting control subjects is known to cause a dose-dependent increase in plasma glutamate levels, however, when glutamate was administered along with a meal no significant increase in plasma glutamate was found (Ghezzi et al., 1980,1985X In our design monosodium glutamate was dissolved in water and the ratio (plasma peak level: plasma basal level) was approximately 2 times higher in control subjects compared to the ratio values found by Ghezzi et al. (1983), who administered glutamate dissolved in bouillon solutions. Following glutamate loading, we found significantly increased plasma levels of glutamate and aspartate, and area under the curve in plasma for glutamate, in ALS patients relative to controls. These results accord with other findings (Plaitakis and Caroscio, 1987) and suggest a deranged metabolism of glutamate and aspartate in ALS under conditions, where monosodium glutamate is dissolved in water and administrated to fasting individuals. In our experimental design subchronic BCAAs treatment did not significantly affect plasma glutamate levels in ALS patients at basal conditions or during glutamate loading. To further evaluate the effect of BCAAs on glutamate metabolism, we have decided to evaluate the acute effect of BCAAs on plasma levels of glutamate and other amino acids in ALS (in preparation).

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Acknowledgements

This work was supported by grants from P. Carl Petersens Foundation, Danish Hospital Foundation for Medical Research, Foundation for experimental research in Neurology. References Beghi, E., Fiordelli, E., Mora, G., Mazzmi, L., Poloni, M., Pine& P., Testa, D., Cornelio, F., Leone, M., Bottacchi, E., Chio, A., Schiffer, D., Magi, S., Sabatelli, M., Tonali, P., Formica, A., Berardelli, A., Manfredi, M., Cattich, N., Ferrannini, E., Livrea, P., Coretti, A., Huber, R., Musicco, M. (1993) Branched-chain amino acids and amyotrophic lateral sclerosis - a treatment failure. Neurology, 43: 2466-2470. Camu, W., Billiard, M., Baldy-Moulinier, M. (1993) Fasting plasma and CSF amino acid levels in amyotrophic lateral sclerosis: a subtype analysis. Acta Neural. Stand., 88: 51-55. Ghezzi, P., Salmona, M., Recchia, M., Dagnino, G., Garattini, S. (1980) Monosodium glutamate kinetic studies in human volunteers. Toxicol. Lett., 5: 417-421. Ghezzi, P., Garattini, S., Caccia, S., Salmona, M., Zanini, M.G. (1983) Plasma pyroglutamic acid levels after oral administration of monosodium glutamate to human volunteers. Toxicol. Lett., 15: 123-129. Ghezzi, P., Bianchi, M., Gianera, L., Salmona, M., Garattini, S. (1985) Kinetics of monosodium glutamate in human volunteers under different experimental conditions. Food Chem. Toxicol., 23: 975-978.

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