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Abnormal glutamic acid metabolism in multiple sclerosis
Journal of the Neurological Sciences, 1980,47:353-364
353
© Elsevier/North-HollandBiomedicalPress
A B N O R M A L G L U T A M I C ACID M E T A B O L I S M IN M U L T I P L E SCLEROSIS
FRED C. WESTALL,ANGELA HAWKINS, GEORGE W. ELLISONand LAWRENCEW. MYERS The Salk Institute, San Diego, CA and the Multiple Sclerosis Research Clinic, Department of Neurology, UCLA School of Medicine, Los Angeles, CA (U.S.A.)
(Received 18 February, 1980) (Accepted 16 April, 1980)
SUMMARY We have found extensive amino acid abnormalities in multiple sclerosis sera. The most consistent abnormality is an elevation in serum glutamate, which is most striking during relapses. The increase in glutamate in the patients does not occur sharply during the onset of the relapse. Instead it appears to rise gradually within a month or two prior to the onset of the clinical relapse, to reach a peak during the relapse and then to slowly decline.
INTRODUCTION For the past 7 years we have been searching for metabolic abnormalities in patients with multiple sclerosis (MS). Initially we compared urinary amine compositions from MS patients with those of controls (Robinson and Westall 1974). We found that concentrations of tryptophan, glutamic acid and glycine were elevated and that ornithine and leucine were lowered in the MS population. We have further examined tryptophan metabolism and found (unpublished) that the urinary 5hydroxy indole acetic acid (5-HIAA) tryptophan ratio was significantly depressed as MS patients developed relapses. Others have found (Sonninen et al. 1973; Claveria et al. 1974; Johansson and Roos 1976) low 5-HIAA concentrations in the cerebral spinal fluid (CSF) from MS patients. Gottesfeld et al. (1976) discovered that concentrations of gamma-amino butyric acid and glutamic acid dehydrogenase (GAD), but not glutamate, were reduced in the lumbar cord of guinea pigs with hind leg paralysis, due to experimental allergic encephalomyelitis (EAE). The This work was supported by NIH grants NS-12391 and NS-08711. Address reprint requests to : Dr. F.C. Westall, Salk Institute Post Office Box 85800, San Diego, CA 92138, U.S.A.
354 decrease in G A D preceded the onset of motor dysfunction. There was no change in G A D in other parts of the CNS or in animals which were sensitized with allergic encephalitogenic antigens but which did not develop clinical signs. In this study we report that MS sera contain elevated concentrations of glutamic acid, glycine, and tryptophan. Of the abnormalities we have observed that the glutamic acid elevation is the most prominent. Even when clinical change is minimal, glutamic acid metabolism is markedly affected. However, the abnormality is exaggerated when patients are in a clinically active state. While observing a series of patients examined periodically over a 3-year period, we noted glutamic acid varied as their clinical status changed. Patients All subjects used in this study were from the U C L A Multiple Sclerosis Research Clinic. All patients were classified as "'definite" multiple sclerosis by two neurologists ( G W E and LWM). The patients were also assigned a "phase" according to the definitions of the Schumacher Committee (Schumacher et al. 1965). Thus, a patient was classified, e.g., as definite, relapsing type, relapse phase. At each visit the patients were classified as to the degree of disability according to the Kurtzke Disability Status Scale and Functional Systems Scores (Kurtzke 1961). The controls consisted of 25 males and 25 females. Eight of each sex were employees of the U C L A Neurology Department. Seventeen males and 9 females were spouses of the MS patients. The remaining females were genetically related to a MS patient. The two groups were matched by sex and race. In our previous studies (Robinson and Westall 1974; Robinson et al. 1974) we did not find that age significantly affected the data but differences in age were kept minimal. Individual serum samples were collected randomly over a period of 6 months from 99 different MS patients of varying disability (see Table 1) and the 50 controls. No attempt was made to control lbr dietary variations. All samples were coded and analyzed "blind" in a random fashion. Throughout the experiment MS and control sera were treated in an identical fashion. The medical record of each patient was carefully reviewed to ensure that the results described in this study were not due to patient medication. Over a period of 3 years we examined sera samples collected periodically from 24 patients with clinically definite multiple sclerosis and a relapsing-type course, who had experienced at least one relapse in the preceding year. Patients' visits were scheduled at approximately every 2 months, irrespective of their clinical status. Eight of the patients have been eliminated from the current analysis for the following reason : (l) no relapses during the study - 1 patient (2) insufficient number of samples - 2 patients (3) number of relapses per year were greater than three - 5 patients This last restriction was imposed on the study to ensure a sufficiently long nonrelapse period before the onset of the relapse in order that a proper comparison could made.
355 TABLE 1 CLASSIFICATION OF PATIENTS PROVIDING SERUM Type
Phase b
Number of patients 1-5a
Relapsing Relapsing Relapsing Relapsing Relapsing and progressive Relapsing and progressive Relapsing and progressive Progressive
total stationary relapse c remission total stationary progressive total
6-9a
Total
F
M
F
M
F
M
31 15 10 6 2 2 0 0
11 7 4 0 1 1 0 1
8 3 5 0 17 9 8 3
7 3 4 0 13 3 10 5
39 18 15 6 19 11 8 3
18 10 8 0 14 4 10 6
a Disability status according to Kurtzke (1961). Definitions of phase are those adopted by the Schumacher Committee (Schumacher et al. 1965). c Includes 17 patients: 11 females (8 with disability 1-5 and 3 with disability 6-9) and 6 males (3 with disability 1-5 and 3 with disability 6 9) from whom samples were obtained and tested in this study when they were also in stationary phase. E = female, M = male.
Blood studies A t each clinic visit b l o o d was collected, s e r u m s e p a r a t e d a n d s t o r e d at - 4 0 to - 80 °C. A m i n o a c i d c o n c e n t r a t i o n s were d e t e r m i n e d b y m e a n s o f a B e c k m a n 121 a u t o m a t e d a m i n o a c i d a n a l y z e r ( M o o r e a n d Stein 1951). T h e a m o u n t o f each a m i n o a c i d in each s a m p l e was n o r m a l i z e d , i.e. the c o n c e n t r a t i o n o f each a m i n o a c i d was d i v i d e d by the t o t a l c o n c e n t r a t i o n o f the a m i n o a c i d s used in n o r m a l i z a t i o n . T h o s e p e a k s t h a t have a r e a s g r e a t e r t h a n 5~o o f the t o t a l a r e a o f all the p e a k s for a n y single analysis were o m i t t e d f r o m the n o r m a l i z a t i o n . This p r e v e n t e d a n y single p e a k f r o m d o m i n a t i n g the n o r m a l i z a t i o n . W e have used n o r m a l i z e d values in several o f the T a b l e s i n s t e a d o f a c t u a l c o n c e n t r a t i o n s o f the a m i n o acids. W e believe this is a m o r e a c c u r a t e p r o c e d u r e . N o r m a l i z a t i o n e l i m i n a t e s e r r o r s due to v a r i a t i o n s in w a t e r c o n t e n t , in s a m p l e d i l u t i o n a n d c o l u m n a b s o r p t i o n . In an e x p e r i m e n t o f this m a g n i t u d e in which a n a l y s e s were p e r f o r m e d over a n e x t e n d e d p e r i o d these sources o f e r r o r can be significant. In o r d e r to c o m p a r e results o b t a i n e d f r o m sera f r o m M S p a t i e n t s with sera f r o m n o r m a l s a r a t i o o f the m e a n s o f the n o r m a l i z e d values in n o r m a l s e r u m was calculated. T h e M a n n W h i t n e y U test (Siegel 1956) was used to e v a l u a t e the statistical significance o f the c o m p a r i s o n o f the a m i n o a c i d d a t a ( o b t a i n e d f r o m M S p a t i e n t s a n d n o r m a l s ) . This test was e m p l o y e d i n s t e a d o f the t-test since the shape o f the d i s t r i b u t i o n curve o f the c o n c e n t r a t i o n s o f each a m i n o a c i d is u n k n o w n (Siegel 1956).
356
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357 RESULTS
Table 2 displays the ratio of the means of the normalized values of various amino acids in MS sera versus the means to their normalized values in normal sera. Values for all other naturally occurring amino acids* not given in Table 2 did not show any significant amino acid differences in the MS sera. Glutamic acid levels ranged from a ratio of 1.35 (i.e. 35~o above normal) for relapsing-type patients in stationary phase to a ratio of 1.89 (89~ above) for relapsing-type patients in relapse. However, in the other classifications glutamic acid ranged between 29~o and 55~o higher. Even in stationary phase glutamic acid metabolism was markedly affected. However, the abnormality was exaggerated when the patients were in relapse phase. The high ratios of glutamic acid are not due to deamidation of glutamine during processing of the samples since the control samples were collected simultaneously with the MS samples. Glycine and tryptophan values appeared to parallel each other. They were higher than normal in the lower disability states but decreased with greater disability. However, in comparing the relapsing-progressive and progressive MS types, the glycine and tryptophan results do not agree. In these cases the glycine MS results are not statistically different from the normals, while the tryptophan values are lower for the relapsing-progressive type and higher for the other two MS types than the normals. Phenylalanine values appeared to increase at higher disability which consequently changes the tyr/phe ratio. The aberrations appear to be due to the presence of MS and not to general muscle degeneration, since in neuromuscular diseases such as Duchenne dystrophy we have not observed significant changes in individual amino acid concentrations. Furthermore we have not seen this pattern in any of the following neurological diseases: amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease or myotonic dystrophy. However, it is certainly premature and probably incorrect to state that this serum amino acid pattern is characteristic only of MS. Tables 3A and 3B indicate normalized glutamic acid values from serum from 16 MS patients collected over a 3-year span. Figure 1 graphically depicts these data. Fifteen of these patients were of a relapsing type throughout the study. One of these (patient 16) went into a progressive phase of the illness during the study. Listed with each patient is the range of disability status scales (DSS) experienced by each patient during the study. The 16 patients range from DSS 1 to 7. Some show great DSS change, e.g. patients 4, 10, and 16, while others do not. Concentrations of no amino acid, other than glutamic acid, correlated with clinical status and values for these other amino acids are therefore not included in the tables. In individual patients the greatest glutamate values occur during a period of relapse. The increase in serum glutamate shown by all the patients does not occur sharply during the
* Arginine, lysine, histidine, aspartic acid, serine, threonine, glutamine, asparagine, proline, valine, cystine, isoleucine, methionine, leucine, and alanine.
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359 TABLE 3B GLUTAMIC ACID VALUES FROM 2 MS PATIENTS OF RELAPSING-PROGRESSIVE TYPES Relapse onset
Sample day
Sample value
Patient 15 (5, 6) a Relapsing-progressive
12/ 3/73
Sample day
Sample value
Patient 16 (3-7) a
Progressive after 7/4/74
29/ 7/74
0.0916 --0.0854
21/10 6/ 1/75 20/ 1 3/ 3 14/ 4 27/ 5 18/ 8 29/ 9 11/11 22/12
0.0660 0.0674 0.0729 0.0562 0.0892 0.1644 0.1688 0.0505 0.0488 0.0597
26/ 3/74 17/ 6/74
Relapse onset
15/ 1/73
0.1408
25/ 4/73 21/ 5 18/10
0.0929 0.1346 0.0692
26/ 8/74 18/11 12/ 5/75 4/ 8 26/ 8 15/ 9 27/10
0.1331 0.1263 0.1688 0.1703 0.1331 0.1456 0.1346
26/ 1/73
11/ 9/74 10/12/73 28/12/73 7/ 4/74
a Disability status.
onset of the relapse but gradually rises within a month or two prior to the onset of the relapse, to reach a peak during the relapse followed by slow decline*. There are only 3 instances where a relapse value is preceded by a higher value - 29/5/75 sample of patient 5 and samples 13/2/75 and 8/9/75 from patient 13. In no instance is the value following the relapse higher than the relapse value. In 7 instances a rise in glutamate value was not associated with a clinical relapse. This could represent subclinical disease activity or may indicate that the glutamate value may fluctuate unrelated to disease activity. As mentioned above (Table 3B) patient 16 possesses a relapsing and progressive type of MS. The disease in patient 16 became progressive only after mid 1974. With this change in status the patient's glutamic acid values increased markedly. Table 4 compares the averaged glutamic acid values obtained during the relapses for each patient with those collected during stationary phase MS. The average relapse values are a composite of all those values obtained beginning one month prior to the onset of the clinical relapse and extending to the next stationary p h a s e . P a t i e n t 16 is e x c l u d e d f r o m t h e t a b l e s i n c e h e d o e s n o t h a v e s t r i c t l y r e l a p s i n g
* In 8/11 instances in which a sample was obtained before, during and after a relapse the value during the relapse was higher than the value preceding or following the relapse.
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362 TABLE 4 COMPARISON OF NORMALIZED GLUTAMIC ACID VALUES OBTAINED DURING RELAPSE AND STATIONARY PHASES
Average normalized glutamic acid valuc¢' Patient
relapsc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.1675 0.1562 0.2153 0.1484 0.1686 0.1636 0.1579 0.1346 0.1099 0.1337 0.1548 0.1541 0.1339 0.1801 0.0810
stationary (3) h (3) (3) (6) (3) (3) (2) (2) ( 1) (2) (2) (3) (6) (3) (3)
0.1038 0.0994 0.1792 0.1094 0.1333 0.1514 0.0897 0.1289 0.0843 0.1173 0.1062 0.1106 0.1316 (I.1315 0.0861
(101 h (6) (9) (7) (5) (3) (14) (2) (4) (3) (5) (11 (9) (4) (9)
"o difference~ 61 57 20 36 26 8 76 4 30 14 46 39 2
37 -9 Av. 33
" Average values are composite of all those values obtained beginning one month prior to onset of
relapse and lasting until next stationary phase. Number of samples in ( ). ~" Average relapse/average stationary - 1 × 100 = ",, difference. h
MS. Otherwise, for every patient except No. 15 the averaged relapse value is higher than the corresponding stationary phase value. The difference ranges from 2'~ to 76~i with an average of 33 °,~,. DISCUSSION
There are several possible sources of the high serum glutamic acid levels found. The normal ratio of concentrations of glutamic acid in brain compared to blood plasma is approximately 150 to 1 (McIlwain 1966). It is maintained by an active transport system. Therefore, by disruption of the blood brain barrier the higher glutamic acid values could be due to a change in this normally observed glutamic acid gradient. However, this is unlikely. We have not found any of the other amino acids which are at higher concentrations in the brain than in blood (such as aspartate) to be elevated either in serum or urine (Robinson and Westall 1974; Robinson et al. 1974). Therefore, unless the glutamic acid transport system is selectively disrupted, the high concentrations of glutamic acid in sera are not due to disruption of the blood-brain barrier.
363 It is also highly unlikely that the changes are due to alterations in the life-style of the patients during relapses. The glutamate values increase a substantial time prior to the patient's awareness of a clinical exacerbation. We have attempted to determine which metabolic pathways involving glutamic acid are affected during disease. In the serum samples tested, none of the values for the amino acids other than the ones listed in Table 2 were either statistically elevated or depressed. Therefore, the pathways involving production of glutamine (via L-glutaminase), transamination (via L-glutamate transaminase) or histidine degradation (via histidine-deaminase) apparently are not specifically affected by the glutamate abnormality. Cerkez et al. (1962) have shown that ~-ketoglutaric acid levels are normal in MS patients. However, during glucose tolerance tests ~-ketoglutarate concentrations were higher than expected in their patients' sera. This is not surprising since ~-ketoglutaric acid is utilized to form glutamic acid by both glutamate dehydrogenase and glutamate transaminase. Glutamic acid-7-decarboxylase converts glutamic acid to gamma-aminobutyric acid (GABA). Since the GABA concentration is normally extremely low in serum, we have not attempted to measure it. However, recently GABA concentrations have been reported in MS cerebrospinal fluid (Achar et al. 1976). Detectable GABA appeared related to spinal cord lesions in multiple sclerosis. Therefore, the glutamate-GABA pathway may be selectively affected in MS. However, because of the extent of the abnormality it is unlikely that it is entirely due to disturbance of this one pathway. Conceivably, hormonal aberrations due to the disease process might affect the serum glutamate. Whether this abnormality is part of the process which initiates clinical MS or is just a secondary effect of a relapse is not known. It is known that in MS plaque margins there is an increase in oxidative enzymes, possibly involving oligodendroglia (Ibrahim and Adams 1963). One would expect this change to begin before the onset of observable clinical changes. Our data support this assumption. Currently there are no objective methods for following disease activity in patients with MS. Clinical measurements are the most commonly used but it is well recognized that these are inadequate because of subclinical disease activity (Rose 1974). Dowling and Cook (1976) have suggested that measurement of the serum acute phase reactants, C-reactive protein, C~, proactivator and orosomucoid, may be of value in assessing disease activity but longitudinal studies have not yet been reported. Following the level of myelin basic protein in the spinal fluid with a radioimmunoassay may provide a means of measuring disease activity (Cohen et al. 1976; Whitaker 1977) but because of the difficulty of performing routine serial lumbar punctures this does not appear to be practical except for special research protocols. Serial determination of serum glutamic acid levels offers a more practical measurement. Certainly it warrants further study to determine its potential to predict relapses.
364 ACKNOWLEDGEMENTS W e w o u l d like t o t h a n k R a c h e l C o r o n a d o
and Cheryl Look for their valuable
a s s i s t a n c e . M a r i e H o e v e t a s s i s t e d in s a m p l e c o l l e c t i o n , s t o r a g e , a n d in d a t a p r o c e s sing, w h i l e A l i c e V i g i l a s s i s t e d in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t . REFERENCES Achar, V. S., K. M. A. Welch, E. Chabi, K. Bartosh and J.S. Meyer (1976) Cerebrospinal fluid gammaaminobutyric acid in neurologic disease, Neurology (Minneap.), 26:777 780. Cerkez, C., J. M. Chandler and D. Chandler (1962) Glucose metabolism in multiple sclerosis, Dis. Nerv. Syst., 23:377 382. Claveria, L. E., G. Lurzon, M. S. G. Harrison, et al. (1974) Amine metabolites in the cerebrospinal fluid of patients with disseminated sclerosis, J. Neurol. Neurosurg. Psychiat., 37 : 715 718. Cohen, S. R., R. M. Herndon and G. M. McKhann (1976) Radioimmunoassay of myelin basic protein in spinal fluid, New Engl. J. Med., 295 (26): 1455 1459. Dowling, P. C. and S. D. Cook (1976) Disease markers in acute multiple sclerosis, Arch. Neurol. (Chic.), 33 (10): 668 670. Gottesfeld, Z., D. Teitelbaum, C. Webb and R. Arnon (1976) Changes in the GABA system in experimental allergic encephalomyelitis-induced paralysis, J. Neurochem., 27 (3) : 695-699. Ibrahim, M.Z. and C. W. M. Adams (1963) The relationship between enzyme activity and neurologia in early plaques of multiple sclerosis, J. Neurol. Neurosurg. Psychiat., 26:101 -110. Johansson, B. and B.F. Roos (1976) 5-HIAA and homovanillic acid in cerebrospinal fluid of patients with neurological diseases, Europ. Neurol., 11 : 3745. Kurtzke, J.F. (1961) On the evaluation of disability in multiple sclerosis, Neurology (Minneap.), 11: 686694. McIlwain, H., Biochemist O' and the Central Nervous System, Longman, New York, NY, 1966, p. 78. Moore, S. and N. M. Stein (1951) Chromatography of amino acids on sulfonated polystyrene resins, J. biol. Chem., 192:663 681. Robinson, A.B. and F.W. Westall (1974) The use of urinary amine measurements for orthomolecular diagnosis of multiple sclerosis, J. Orthomol. Psych., 3:1 10. Robinson, A. B., F. C. Westall and G. Ellison (1974) Multiple sclerosis Urinary amine measurements for orthomolecular diagnosis, L(/b Sci., 14:1747 1753. Rose, A. S. (1974) Multiple sclerosis A clinical and theoretical review, J. Neurosurg., 41 : 279 284. Schumacher, G.A., G. Beebe, R.F. Kibler, et al. (1965) Problems of experimental trails of therapy in multiple sclerosis, N. Y. Aead. Sci., 122: 552-568. Siegel, S. (1956) Non-Parametric Statistics, McGraw-Hill, New York, NY, pp. 117 126. Sonninen, V., P. Riekkinen and U. K. Rinne (1973) Acid monoamine metabolities in cerebrospinal fluid in multiple sclerosis, Neurology (Minneap.), 23: 760-763. Whitaker, J. N. (1977) Myelin encephalitogenic protein fragments in cerehralspinal fluid in persons with multiple sclerosis, Neurology (Minneap.), 27 (10): 911-920.
353
© Elsevier/North-HollandBiomedicalPress
A B N O R M A L G L U T A M I C ACID M E T A B O L I S M IN M U L T I P L E SCLEROSIS
FRED C. WESTALL,ANGELA HAWKINS, GEORGE W. ELLISONand LAWRENCEW. MYERS The Salk Institute, San Diego, CA and the Multiple Sclerosis Research Clinic, Department of Neurology, UCLA School of Medicine, Los Angeles, CA (U.S.A.)
(Received 18 February, 1980) (Accepted 16 April, 1980)
SUMMARY We have found extensive amino acid abnormalities in multiple sclerosis sera. The most consistent abnormality is an elevation in serum glutamate, which is most striking during relapses. The increase in glutamate in the patients does not occur sharply during the onset of the relapse. Instead it appears to rise gradually within a month or two prior to the onset of the clinical relapse, to reach a peak during the relapse and then to slowly decline.
INTRODUCTION For the past 7 years we have been searching for metabolic abnormalities in patients with multiple sclerosis (MS). Initially we compared urinary amine compositions from MS patients with those of controls (Robinson and Westall 1974). We found that concentrations of tryptophan, glutamic acid and glycine were elevated and that ornithine and leucine were lowered in the MS population. We have further examined tryptophan metabolism and found (unpublished) that the urinary 5hydroxy indole acetic acid (5-HIAA) tryptophan ratio was significantly depressed as MS patients developed relapses. Others have found (Sonninen et al. 1973; Claveria et al. 1974; Johansson and Roos 1976) low 5-HIAA concentrations in the cerebral spinal fluid (CSF) from MS patients. Gottesfeld et al. (1976) discovered that concentrations of gamma-amino butyric acid and glutamic acid dehydrogenase (GAD), but not glutamate, were reduced in the lumbar cord of guinea pigs with hind leg paralysis, due to experimental allergic encephalomyelitis (EAE). The This work was supported by NIH grants NS-12391 and NS-08711. Address reprint requests to : Dr. F.C. Westall, Salk Institute Post Office Box 85800, San Diego, CA 92138, U.S.A.
354 decrease in G A D preceded the onset of motor dysfunction. There was no change in G A D in other parts of the CNS or in animals which were sensitized with allergic encephalitogenic antigens but which did not develop clinical signs. In this study we report that MS sera contain elevated concentrations of glutamic acid, glycine, and tryptophan. Of the abnormalities we have observed that the glutamic acid elevation is the most prominent. Even when clinical change is minimal, glutamic acid metabolism is markedly affected. However, the abnormality is exaggerated when patients are in a clinically active state. While observing a series of patients examined periodically over a 3-year period, we noted glutamic acid varied as their clinical status changed. Patients All subjects used in this study were from the U C L A Multiple Sclerosis Research Clinic. All patients were classified as "'definite" multiple sclerosis by two neurologists ( G W E and LWM). The patients were also assigned a "phase" according to the definitions of the Schumacher Committee (Schumacher et al. 1965). Thus, a patient was classified, e.g., as definite, relapsing type, relapse phase. At each visit the patients were classified as to the degree of disability according to the Kurtzke Disability Status Scale and Functional Systems Scores (Kurtzke 1961). The controls consisted of 25 males and 25 females. Eight of each sex were employees of the U C L A Neurology Department. Seventeen males and 9 females were spouses of the MS patients. The remaining females were genetically related to a MS patient. The two groups were matched by sex and race. In our previous studies (Robinson and Westall 1974; Robinson et al. 1974) we did not find that age significantly affected the data but differences in age were kept minimal. Individual serum samples were collected randomly over a period of 6 months from 99 different MS patients of varying disability (see Table 1) and the 50 controls. No attempt was made to control lbr dietary variations. All samples were coded and analyzed "blind" in a random fashion. Throughout the experiment MS and control sera were treated in an identical fashion. The medical record of each patient was carefully reviewed to ensure that the results described in this study were not due to patient medication. Over a period of 3 years we examined sera samples collected periodically from 24 patients with clinically definite multiple sclerosis and a relapsing-type course, who had experienced at least one relapse in the preceding year. Patients' visits were scheduled at approximately every 2 months, irrespective of their clinical status. Eight of the patients have been eliminated from the current analysis for the following reason : (l) no relapses during the study - 1 patient (2) insufficient number of samples - 2 patients (3) number of relapses per year were greater than three - 5 patients This last restriction was imposed on the study to ensure a sufficiently long nonrelapse period before the onset of the relapse in order that a proper comparison could made.
355 TABLE 1 CLASSIFICATION OF PATIENTS PROVIDING SERUM Type
Phase b
Number of patients 1-5a
Relapsing Relapsing Relapsing Relapsing Relapsing and progressive Relapsing and progressive Relapsing and progressive Progressive
total stationary relapse c remission total stationary progressive total
6-9a
Total
F
M
F
M
F
M
31 15 10 6 2 2 0 0
11 7 4 0 1 1 0 1
8 3 5 0 17 9 8 3
7 3 4 0 13 3 10 5
39 18 15 6 19 11 8 3
18 10 8 0 14 4 10 6
a Disability status according to Kurtzke (1961). Definitions of phase are those adopted by the Schumacher Committee (Schumacher et al. 1965). c Includes 17 patients: 11 females (8 with disability 1-5 and 3 with disability 6-9) and 6 males (3 with disability 1-5 and 3 with disability 6 9) from whom samples were obtained and tested in this study when they were also in stationary phase. E = female, M = male.
Blood studies A t each clinic visit b l o o d was collected, s e r u m s e p a r a t e d a n d s t o r e d at - 4 0 to - 80 °C. A m i n o a c i d c o n c e n t r a t i o n s were d e t e r m i n e d b y m e a n s o f a B e c k m a n 121 a u t o m a t e d a m i n o a c i d a n a l y z e r ( M o o r e a n d Stein 1951). T h e a m o u n t o f each a m i n o a c i d in each s a m p l e was n o r m a l i z e d , i.e. the c o n c e n t r a t i o n o f each a m i n o a c i d was d i v i d e d by the t o t a l c o n c e n t r a t i o n o f the a m i n o a c i d s used in n o r m a l i z a t i o n . T h o s e p e a k s t h a t have a r e a s g r e a t e r t h a n 5~o o f the t o t a l a r e a o f all the p e a k s for a n y single analysis were o m i t t e d f r o m the n o r m a l i z a t i o n . This p r e v e n t e d a n y single p e a k f r o m d o m i n a t i n g the n o r m a l i z a t i o n . W e have used n o r m a l i z e d values in several o f the T a b l e s i n s t e a d o f a c t u a l c o n c e n t r a t i o n s o f the a m i n o acids. W e believe this is a m o r e a c c u r a t e p r o c e d u r e . N o r m a l i z a t i o n e l i m i n a t e s e r r o r s due to v a r i a t i o n s in w a t e r c o n t e n t , in s a m p l e d i l u t i o n a n d c o l u m n a b s o r p t i o n . In an e x p e r i m e n t o f this m a g n i t u d e in which a n a l y s e s were p e r f o r m e d over a n e x t e n d e d p e r i o d these sources o f e r r o r can be significant. In o r d e r to c o m p a r e results o b t a i n e d f r o m sera f r o m M S p a t i e n t s with sera f r o m n o r m a l s a r a t i o o f the m e a n s o f the n o r m a l i z e d values in n o r m a l s e r u m was calculated. T h e M a n n W h i t n e y U test (Siegel 1956) was used to e v a l u a t e the statistical significance o f the c o m p a r i s o n o f the a m i n o a c i d d a t a ( o b t a i n e d f r o m M S p a t i e n t s a n d n o r m a l s ) . This test was e m p l o y e d i n s t e a d o f the t-test since the shape o f the d i s t r i b u t i o n curve o f the c o n c e n t r a t i o n s o f each a m i n o a c i d is u n k n o w n (Siegel 1956).
356
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357 RESULTS
Table 2 displays the ratio of the means of the normalized values of various amino acids in MS sera versus the means to their normalized values in normal sera. Values for all other naturally occurring amino acids* not given in Table 2 did not show any significant amino acid differences in the MS sera. Glutamic acid levels ranged from a ratio of 1.35 (i.e. 35~o above normal) for relapsing-type patients in stationary phase to a ratio of 1.89 (89~ above) for relapsing-type patients in relapse. However, in the other classifications glutamic acid ranged between 29~o and 55~o higher. Even in stationary phase glutamic acid metabolism was markedly affected. However, the abnormality was exaggerated when the patients were in relapse phase. The high ratios of glutamic acid are not due to deamidation of glutamine during processing of the samples since the control samples were collected simultaneously with the MS samples. Glycine and tryptophan values appeared to parallel each other. They were higher than normal in the lower disability states but decreased with greater disability. However, in comparing the relapsing-progressive and progressive MS types, the glycine and tryptophan results do not agree. In these cases the glycine MS results are not statistically different from the normals, while the tryptophan values are lower for the relapsing-progressive type and higher for the other two MS types than the normals. Phenylalanine values appeared to increase at higher disability which consequently changes the tyr/phe ratio. The aberrations appear to be due to the presence of MS and not to general muscle degeneration, since in neuromuscular diseases such as Duchenne dystrophy we have not observed significant changes in individual amino acid concentrations. Furthermore we have not seen this pattern in any of the following neurological diseases: amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease or myotonic dystrophy. However, it is certainly premature and probably incorrect to state that this serum amino acid pattern is characteristic only of MS. Tables 3A and 3B indicate normalized glutamic acid values from serum from 16 MS patients collected over a 3-year span. Figure 1 graphically depicts these data. Fifteen of these patients were of a relapsing type throughout the study. One of these (patient 16) went into a progressive phase of the illness during the study. Listed with each patient is the range of disability status scales (DSS) experienced by each patient during the study. The 16 patients range from DSS 1 to 7. Some show great DSS change, e.g. patients 4, 10, and 16, while others do not. Concentrations of no amino acid, other than glutamic acid, correlated with clinical status and values for these other amino acids are therefore not included in the tables. In individual patients the greatest glutamate values occur during a period of relapse. The increase in serum glutamate shown by all the patients does not occur sharply during the
* Arginine, lysine, histidine, aspartic acid, serine, threonine, glutamine, asparagine, proline, valine, cystine, isoleucine, methionine, leucine, and alanine.
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359 TABLE 3B GLUTAMIC ACID VALUES FROM 2 MS PATIENTS OF RELAPSING-PROGRESSIVE TYPES Relapse onset
Sample day
Sample value
Patient 15 (5, 6) a Relapsing-progressive
12/ 3/73
Sample day
Sample value
Patient 16 (3-7) a
Progressive after 7/4/74
29/ 7/74
0.0916 --0.0854
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0.0660 0.0674 0.0729 0.0562 0.0892 0.1644 0.1688 0.0505 0.0488 0.0597
26/ 3/74 17/ 6/74
Relapse onset
15/ 1/73
0.1408
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0.0929 0.1346 0.0692
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0.1331 0.1263 0.1688 0.1703 0.1331 0.1456 0.1346
26/ 1/73
11/ 9/74 10/12/73 28/12/73 7/ 4/74
a Disability status.
onset of the relapse but gradually rises within a month or two prior to the onset of the relapse, to reach a peak during the relapse followed by slow decline*. There are only 3 instances where a relapse value is preceded by a higher value - 29/5/75 sample of patient 5 and samples 13/2/75 and 8/9/75 from patient 13. In no instance is the value following the relapse higher than the relapse value. In 7 instances a rise in glutamate value was not associated with a clinical relapse. This could represent subclinical disease activity or may indicate that the glutamate value may fluctuate unrelated to disease activity. As mentioned above (Table 3B) patient 16 possesses a relapsing and progressive type of MS. The disease in patient 16 became progressive only after mid 1974. With this change in status the patient's glutamic acid values increased markedly. Table 4 compares the averaged glutamic acid values obtained during the relapses for each patient with those collected during stationary phase MS. The average relapse values are a composite of all those values obtained beginning one month prior to the onset of the clinical relapse and extending to the next stationary p h a s e . P a t i e n t 16 is e x c l u d e d f r o m t h e t a b l e s i n c e h e d o e s n o t h a v e s t r i c t l y r e l a p s i n g
* In 8/11 instances in which a sample was obtained before, during and after a relapse the value during the relapse was higher than the value preceding or following the relapse.
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362 TABLE 4 COMPARISON OF NORMALIZED GLUTAMIC ACID VALUES OBTAINED DURING RELAPSE AND STATIONARY PHASES
Average normalized glutamic acid valuc¢' Patient
relapsc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.1675 0.1562 0.2153 0.1484 0.1686 0.1636 0.1579 0.1346 0.1099 0.1337 0.1548 0.1541 0.1339 0.1801 0.0810
stationary (3) h (3) (3) (6) (3) (3) (2) (2) ( 1) (2) (2) (3) (6) (3) (3)
0.1038 0.0994 0.1792 0.1094 0.1333 0.1514 0.0897 0.1289 0.0843 0.1173 0.1062 0.1106 0.1316 (I.1315 0.0861
(101 h (6) (9) (7) (5) (3) (14) (2) (4) (3) (5) (11 (9) (4) (9)
"o difference~ 61 57 20 36 26 8 76 4 30 14 46 39 2
37 -9 Av. 33
" Average values are composite of all those values obtained beginning one month prior to onset of
relapse and lasting until next stationary phase. Number of samples in ( ). ~" Average relapse/average stationary - 1 × 100 = ",, difference. h
MS. Otherwise, for every patient except No. 15 the averaged relapse value is higher than the corresponding stationary phase value. The difference ranges from 2'~ to 76~i with an average of 33 °,~,. DISCUSSION
There are several possible sources of the high serum glutamic acid levels found. The normal ratio of concentrations of glutamic acid in brain compared to blood plasma is approximately 150 to 1 (McIlwain 1966). It is maintained by an active transport system. Therefore, by disruption of the blood brain barrier the higher glutamic acid values could be due to a change in this normally observed glutamic acid gradient. However, this is unlikely. We have not found any of the other amino acids which are at higher concentrations in the brain than in blood (such as aspartate) to be elevated either in serum or urine (Robinson and Westall 1974; Robinson et al. 1974). Therefore, unless the glutamic acid transport system is selectively disrupted, the high concentrations of glutamic acid in sera are not due to disruption of the blood-brain barrier.
363 It is also highly unlikely that the changes are due to alterations in the life-style of the patients during relapses. The glutamate values increase a substantial time prior to the patient's awareness of a clinical exacerbation. We have attempted to determine which metabolic pathways involving glutamic acid are affected during disease. In the serum samples tested, none of the values for the amino acids other than the ones listed in Table 2 were either statistically elevated or depressed. Therefore, the pathways involving production of glutamine (via L-glutaminase), transamination (via L-glutamate transaminase) or histidine degradation (via histidine-deaminase) apparently are not specifically affected by the glutamate abnormality. Cerkez et al. (1962) have shown that ~-ketoglutaric acid levels are normal in MS patients. However, during glucose tolerance tests ~-ketoglutarate concentrations were higher than expected in their patients' sera. This is not surprising since ~-ketoglutaric acid is utilized to form glutamic acid by both glutamate dehydrogenase and glutamate transaminase. Glutamic acid-7-decarboxylase converts glutamic acid to gamma-aminobutyric acid (GABA). Since the GABA concentration is normally extremely low in serum, we have not attempted to measure it. However, recently GABA concentrations have been reported in MS cerebrospinal fluid (Achar et al. 1976). Detectable GABA appeared related to spinal cord lesions in multiple sclerosis. Therefore, the glutamate-GABA pathway may be selectively affected in MS. However, because of the extent of the abnormality it is unlikely that it is entirely due to disturbance of this one pathway. Conceivably, hormonal aberrations due to the disease process might affect the serum glutamate. Whether this abnormality is part of the process which initiates clinical MS or is just a secondary effect of a relapse is not known. It is known that in MS plaque margins there is an increase in oxidative enzymes, possibly involving oligodendroglia (Ibrahim and Adams 1963). One would expect this change to begin before the onset of observable clinical changes. Our data support this assumption. Currently there are no objective methods for following disease activity in patients with MS. Clinical measurements are the most commonly used but it is well recognized that these are inadequate because of subclinical disease activity (Rose 1974). Dowling and Cook (1976) have suggested that measurement of the serum acute phase reactants, C-reactive protein, C~, proactivator and orosomucoid, may be of value in assessing disease activity but longitudinal studies have not yet been reported. Following the level of myelin basic protein in the spinal fluid with a radioimmunoassay may provide a means of measuring disease activity (Cohen et al. 1976; Whitaker 1977) but because of the difficulty of performing routine serial lumbar punctures this does not appear to be practical except for special research protocols. Serial determination of serum glutamic acid levels offers a more practical measurement. Certainly it warrants further study to determine its potential to predict relapses.
364 ACKNOWLEDGEMENTS W e w o u l d like t o t h a n k R a c h e l C o r o n a d o
and Cheryl Look for their valuable
a s s i s t a n c e . M a r i e H o e v e t a s s i s t e d in s a m p l e c o l l e c t i o n , s t o r a g e , a n d in d a t a p r o c e s sing, w h i l e A l i c e V i g i l a s s i s t e d in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t . REFERENCES Achar, V. S., K. M. A. Welch, E. Chabi, K. Bartosh and J.S. Meyer (1976) Cerebrospinal fluid gammaaminobutyric acid in neurologic disease, Neurology (Minneap.), 26:777 780. Cerkez, C., J. M. Chandler and D. Chandler (1962) Glucose metabolism in multiple sclerosis, Dis. Nerv. Syst., 23:377 382. Claveria, L. E., G. Lurzon, M. S. G. Harrison, et al. (1974) Amine metabolites in the cerebrospinal fluid of patients with disseminated sclerosis, J. Neurol. Neurosurg. Psychiat., 37 : 715 718. Cohen, S. R., R. M. Herndon and G. M. McKhann (1976) Radioimmunoassay of myelin basic protein in spinal fluid, New Engl. J. Med., 295 (26): 1455 1459. Dowling, P. C. and S. D. Cook (1976) Disease markers in acute multiple sclerosis, Arch. Neurol. (Chic.), 33 (10): 668 670. Gottesfeld, Z., D. Teitelbaum, C. Webb and R. Arnon (1976) Changes in the GABA system in experimental allergic encephalomyelitis-induced paralysis, J. Neurochem., 27 (3) : 695-699. Ibrahim, M.Z. and C. W. M. Adams (1963) The relationship between enzyme activity and neurologia in early plaques of multiple sclerosis, J. Neurol. Neurosurg. Psychiat., 26:101 -110. Johansson, B. and B.F. Roos (1976) 5-HIAA and homovanillic acid in cerebrospinal fluid of patients with neurological diseases, Europ. Neurol., 11 : 3745. Kurtzke, J.F. (1961) On the evaluation of disability in multiple sclerosis, Neurology (Minneap.), 11: 686694. McIlwain, H., Biochemist O' and the Central Nervous System, Longman, New York, NY, 1966, p. 78. Moore, S. and N. M. Stein (1951) Chromatography of amino acids on sulfonated polystyrene resins, J. biol. Chem., 192:663 681. Robinson, A.B. and F.W. Westall (1974) The use of urinary amine measurements for orthomolecular diagnosis of multiple sclerosis, J. Orthomol. Psych., 3:1 10. Robinson, A. B., F. C. Westall and G. Ellison (1974) Multiple sclerosis Urinary amine measurements for orthomolecular diagnosis, L(/b Sci., 14:1747 1753. Rose, A. S. (1974) Multiple sclerosis A clinical and theoretical review, J. Neurosurg., 41 : 279 284. Schumacher, G.A., G. Beebe, R.F. Kibler, et al. (1965) Problems of experimental trails of therapy in multiple sclerosis, N. Y. Aead. Sci., 122: 552-568. Siegel, S. (1956) Non-Parametric Statistics, McGraw-Hill, New York, NY, pp. 117 126. Sonninen, V., P. Riekkinen and U. K. Rinne (1973) Acid monoamine metabolities in cerebrospinal fluid in multiple sclerosis, Neurology (Minneap.), 23: 760-763. Whitaker, J. N. (1977) Myelin encephalitogenic protein fragments in cerehralspinal fluid in persons with multiple sclerosis, Neurology (Minneap.), 27 (10): 911-920.