ALS

JOURNAL OF THE

NEUROLOGICAL SCIENCES ELS EVI E R

Journal of the Neurological Sciences 124 (Suppl.) (1994) 59--41

Pathways of cysteine metabolism in MND/ALS A. P e a n a, G . B . S t e v e n t o n a, R . H . W a r i n g a,,, H. F o s t e r a, S. S t u r m a n b, A . C . W i l l i a m s b

a School of Biochemistr3,, The University of Birmingham, Edgbaston, Birmingham. UK, and b Department of Clinical Neurology, Queen Elizabeth Hospital, Edgbaston. Birmingham. UK

Abstract

Analysis of plasma from MND/ALS patients has shown no significant differences in metabolism of cysteine derivatives, although a sub-set of the population has raised glutamate values. Cysteine dioxygenase was found to have reduced activity in vitro, consistent with previous findings of a high plasma cysteine/sulphate ratio.

Key words: MND/ALS; Cysleine; Glutamate 1. I n t r o d u c t i o n In previous work, we have shown that plasma levels of cysteine are elevated in motor neurone disease (Heafield et al. 1990). Cysteine is now recognised as an excitotoxin which acts on the N M D A sub-type of glutamate receptors, finally leading to cell death. Other metabolites of cysteine, such as cysteine sulphinic acid and homocysteic acid, are also known to be neurotoxic (Minc-Golomb et al. 1989; Cuenod et al. 1991). As an explanation for the differential toxicity to motor neurones in M N D might be found in the relative proportions of cysteine and its oxidised metabolites, we estimated plasma levels of possible toxins. Cysteine in vivo is converted to inorganic sulphate, via cysteine sulphinic acid, which can also be oxidised to taurine. Other, minor, pathways of cysteine metabolism involve formation of cysteamine and cystamine which are converted to hypotaurine (see Fig. 1), and oxidation to cysteic acid. As the concentrations of cysteamine/cystamine are too low to measure accurately, metabolism of the drug ranitidine was used as a 'probe' to estimate this pathway. Both ranitidine and cysteamine are substrates for the microsomal flavin mono-oxygenase enzyme system. 2. M a t e r i a l and m e t h o d s

Patients All patients (n = 26) came from the practice of one neurologist and were on no medication. They were seen shortly after first diagnosis, and were assigned on the * Corresponding author. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0022-510X(94)00073-W

presence of a progressive muscle wasting and fasciculation, confirmed electrophysiologically with signs of upper motor neurone or bulbar muscle dysfunction in the absence of sensory abnormalities. Patients aged less than 30 years or with a positive family history of M N D were excluded. Age-matched controls (n = 30) were recruited from patients with other neurological diseases (multiple sclerosis, benign intracranial hypertension). All patients and controls fasted from midnight the day before the test. At 8.00 am a blood sample (10 ml) was taken. Plasma was spun immediately and the proteins were precipitated with 35% sulphosalicylic acid. The supernatant was neutralised with KOH (10 N) and stored at - 2 0 ° C until analysis. Patients were then given ranitidine (300 rag) (Queen Elizabeth Medical Centre pharmacy). Urine was collected for the next 8 h, the total volume noted and an aliquot (10 ml) frozen at - 20°C for later analysis.

Preparation of samples and HPLC conditions Plasma samples were mixed for 1 rain with o-phthalaldehyde/2-mercaptoethanol derivatising reagent; 10-btl aliquots were then injected onto a Cls 5j,t reverse-phase column (150× 4.6 mm) and fluorescent products were detected with excitation at 330 nm and emission at 408 nm using the method of Hirschberger et al. (1985). Urine samples were analysed for ranitidine and its metabolites by the method of Prueksaritanont et al. (1989). 3. Results

The results of the plasma amino acid analyses are given in Table 1. The values were not significantly different apart from the levels of glutamate. It was found that

A. Pean et al. /Journal of the Neurological Sciences 124 (Suppl.) (1994) 59--61

60 Table 1

Table 2

Plasma amino acid analysis. Results are expressed as nmol/mg plasma protein.

Ranitidine analysis. Excreted metabolites after a 300 mg dose; resuits are given as percentages o f total recovery and are means _+ SD.

Cysteic acid Glutamic acid Serine Glutamine Alanine Taurine Methionine Homocysteic Cysteine sulphinic acid

Controls (n = 30)

M N D (n = 26)

0.0094 + 0.0235 0.3120 + 0.6650 0.7140 + 1.1090 2.1650 + 3.9270 1.2970 + 2.2810 0.1438 + 0.4030 0.4678 + 0.2803 0.00353 +_ 0.009161

0.0034 + 1.4600 + 0.6750 + 6.3000 + 3.4900 + 0.2123 + 0.5779 + 0.0077 +

0.0823 + 0 . 1 7 5 4

0.07730+ 0.2920

0.0081 3.2250 1.1600 17.170 7.4900 4.314 0.3634 0.2714

while most patients with MND have normal plasma glutamate concentrations, a small sub-set (4 out of 26) have much higher values than the rest of the population, suggesting a bimodal distribution. Results of the determinations of metabolites of ranitidine are given in Table 2, where percentage recovery is calculated as total drug plus metabolite recovery as a percentage of the administered drug, and the metabolites are given as a percentage of this total recovery. The major excreted product was the parent drug, and no significant differences were found for any of the metabolites (MannWhitney U-test).

4. Discussion Disorders of glutamate metabolism have been implicated in a variety of neurological conditions, particularly olivopontocerebellar atrophy (Plaitakis et al. 1984), methionine protein

glutathione

\

homocysfeine

/

/

Cysteine ~

I1- mercapto

/ S042-

cysteo,mine cyst~mine

/

cysteine sulphinic ~ acid

pyruvate fl-sulphinyt

pyruvate

hypotaur ine alanine taurine ~_

cysteic acid

SOazS04z-

Fig. 1. Schematic representation of pathways o f cystein¢ metabolism.

Control (n = 35)

MND (n = 24)

Age(yrs)

51.91 _+ 15.47

62.27 _+ 11.70

% Recovery

29.58 + 15.34

32.06 __+_14.07

Des-methyl

4.08 + 1.48

4.25 _+ 0.91

Ranitidine

74.08 _+ 9.41

74.32 _+ 6.08

H-oxide

15.26 + 5.51

16.22 _+ 4.18

S-oxide

6.32 +_ 8.22

5.20 _+ 6.76

where a deficiency of glutamate dehydrogenase was demonstrated (Konagaya et al. 1986). Other workers (Aubby et al. 1988) have suggested that this may be a common feature of some cases of multiple system atrophy, ataxia and Parkinson's disease. High levels of glutamate have been reported in brain from MND/ALS patients (Plaitakis et al. 1988) and in plasma (Plaitakis and Caroscio 1987) and suggested as a causative factor (Plaitakis 1990), as glutamate is an excitatory amino acid. However, other workers (Perry et al. 1990) found normal plasma glutamate concentrations in most MND/ALS cases. Our resuits with 26 patients suggest that a small sub-set exists with high plasma glutamate levels, but the majority of patients have normal values. If this population with increased glutamate can be identified, then it is possible that therapy (such as the branch-chain amino acids designed to alter glutamate dehydrogenase activity) could be effective. However, non-specific intervention seems unlikely to benefit the majority of patients with normal values. Other compounds analysed, although they were part of the pathways of cysteine metabolism, did not show consistent differences from control values. Similarly, analysis of ranitidine excretion in MND/ALS patients shows no differences from controls and the values found are consistent with those reported in the literature (Van Hecker et al. 1982). As the FMO enzymes controlling Nand S-oxidation are the same as those involved in cysteamine metabolism, this strongly suggests that there is no block in this pathway in MND/ALS patients. The formation of desmethyl ranitidine, via a cytochrome P-450 isoform, is also the same in both populations: some P450 isoforms have been reported to have a weak association with neurological disease (Smith et al. 1992) but this metabolic route appears to be normal in the patients in this study. Our results are consistent with the hypothesis that inhibition of cysteine dioxygenase, which converts cysteine to cysteine sulphinic acid, may be a factor in the disease process as in vitro work showed that this hepatic enzyme had reduced activity in patients (Foster et al. 1991). Other parameters, however, do not show any significant variation in MND/ALS.

A. Pean et al. / Journal of the Neurological Sciences 124 (Suppl.) (1994) 59--61

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