Journal o/'the Neurological Sciences, 1976, 29: 351-359
3 51
cC) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
H Y P E R P H E N Y L A L A N I N A E M I A AND E X P E R I M E N T A L A L L E R G I C ENCEPHA LOM YEL1TIS
JORGEN MERTIN and RUTH HUNT Clinical Research Centre, Wa([brd Road, Harrow, Middlesex HAl 3UJ (Great Britain)
!Received 23 February, 1976)
SUMMARY Previous suggestions to the effect that a reduced proportion of long chain and unsaturated fatty acids in the CNS as induced by experimental hyperphenylalaninaemia in young rats may alter the biochemical reactivity and stability of the myelin have been examined using experimental allergic encephalomyelitis (EAE). Lewis rats treated chronically with phenylalanine during development showed a higher susceptibility to EAE and a more severe course of the disease than their mediumtreated litter mates. The possible implications of this observation for EAE as an experimental model of multiple sclerosis (MS) is discussed briefly in the light of the decreased levels of unsaturated fatty acids found in the CNS of MS patients.
INTRODUCTION Hypomyelination and breakdown of myelin occurring in phenylketonuria (PKU) are looked upon as a result of abnormal lipid content of the white matter of the brain caused by hyperphenylalaninaemia (Menkes 1967). Phenylalanine (PHE) and, to an even greater extent, its metabolites phenylpyruvic and phenyllactic acid have been shown to inhibit [14U-C] glucose incorporation into the lipids of the brain in vitro (Shah, Peterson and McKean 1970). It has been suggested that by inhibition of pyruvate decarboxylation, phenylpyruvate would diminish the availability of acetyl-CoA for fatty acid (FA) and cholesterol synthesis. This would bring about a decrease in myelin formation (Bowden and McArthur 1972). Phenyllactate accumulation in early life has been observed in the brains of rats treated with PHE (Edwards and Blau 1972). Johnson and Shah (1973) reported that rats treated chronically with PHE during development showed a reduced proportion of long chain and unsaturated FA in the total lipids of the whole brain and in the individual lipids of purified myelin. The authors suggest that these alterations may affect the biochemical reactivity and decrease the stability of the myelin in hyperphenylalaninaemia. Hyperphenyl-
352 alaninaemia may also cause an altered incorporation of various amino acids into cerebral proteins (Swaiman, Hosfield and Lemieux 1968) but it is, to our knowledge, not yet known to what degree the proteins of the myelin might be affected by that. Experimental allergic encephalomyelitis (EAE), as an incomplete but as yet the only experimental model for MS (Paterson 1968) has been used to investigate the effects of polyunsaturated fatty acids (PUFA) on CNS autoimmune disease. Supplementation of the diet with PUFA has proved to exert a markedly protective effect in rats with EAE (Selivonchick and Johnston 1975). Clausen and Moiler (1969) and others (Selinvonchick and Johnston 1975) have shown that rats bred and raised on a PUFA-deficient diet will show increased susceptibility to EAE and a more severe course of this disease. This observation supports the hypothesis that a decrease in certain FA may alter the reactivity and stability of myelin (Johnson and Shah 1973). However, dietary deficiency not only lowers PUFA levels in the CNS but will have a more general effect involving other systems as well. An essential FA deficiency syndrome in young animals is well known (Aaes-Jorgensen 1961) and its characteristics include developmental disturbances, the most obvious symptom being growth retardation. One must therefore be cautious in interpreting the results of an increased EAE susceptibility found in animals with a dietary PUFA deficiency. This might not only be the result of myelin alterations but also, for example, of an enhanced cellmediated immune (CM1) response brought about by decreased PUFA serum levels (Mertin 1976; Mertin and Hunt 1976). Thus, a less general PUFA deficiency affecting preponderantly the CNS would help to ascertain the degree to which FA alterations in the CNS may increase the vulnerability to CMI attack. We have thereiore studied susceptibility to EAE and its clinical course in rats with FA disturbances induced by experimental hyperphenylalaninaemia. MATERIALS AND METHODS Pregnant Lewis rats were obtained from Charles River Laboratories. Between days 3-5 after birth the litters were randomly divided into 2 groups. One group (experimental) was treated with 45 #moles/g body weight of L-phenylalanine IPHE) (Sigma Chem.) as follows: PHE was dissolved in balanced salt solution (BSS) and injected intraperitoneally twice a day. The other group (control) were injected according to the same schedule but with an equal volume of BSS only, Usually, the treatments were continued until after the animals had passed the climax of EAE in the second part of the experiment. However, in one of the experiments PHE and BSS injections were discontinued at the time of puberty. At an age of about 40-50 days, control and experimental animals received an injection of fresh guinea pig CNS tissue in Freund's complete adjuvant (FCA) or (in experiment B) FCA alone. The antigenic emulsion was prepared as follows: Fresh guinea pig (strain Duncan-Hartley) brain stem and spinal cord were mixed with an equal amount (w/v) of BSS and this mixture was emulsified together with an equal amount (v/v) of Freund's complete adjuvant (Difco). 0.2 ml of this emulsion was injected into all 4 foot pads. According to our experience in other experiments,
353 this regimen normally induces EAE of moderate clinical severity in Lewis rats of this age and weight range (130-180 g), with most of the affected animals recovering from the disease. Body weights were checked daily starting some days before sensitisation with CNS tissue. The clinical scores were evaluated twice daily (morning and evening) the criteria for the evaluation being - - (grading in brackets) : !. No disease (0). 11. Atonia of the abdominal muscles, "limp tail" sign (1). Ill. As Ii plus ataxia or slight weakness of the hind (or front) legs (2). IV. Distinct weakness of the hind (or front) legs (3). V. As 1V plus neurogenic "overflow bladder" (4). VI. Total paralysis of the hind legs (5). VII. As VI plus "overflow bladder" (6). VIII. As VI plus marked weakness of the front legs (7). IX. Death (8). Because of our main interest in the time course of the disease and the occurrence of relapses, histopathologic criteria for EAE were not evaluated in this study. Three experiments (experiments A, B and C) were carried out (Table I). Agematched animals without the pretreatment described, but raised on a PUFA-deficient diet (Mertin and Hunt 1976) were included in experiment B. Scoring in experiment B was done blind up to day 30 after sensitisation. RESULTS There was no recognisable difference in the development and behaviour of the animals of control and experimental groups. An example for their similar growth as reflected in their body weights is shown in Fig. 1. In contrast, age-matched animals
TABLE I EXPERIMENTAL DESIGN Number of animals per group, pretreatment and EAE induction in 3 experiments. Pretreatment
Experiment A (CNS tissue
BSS continuous BSS until puberty PHE continuous PHE until puberty PUFA-deficient diet Continuous
Experiment B
Experiment C (CNS tissue -~ CFA)
+ CFA)
(CFA alone)
(CNS tissue + CFA)
17 (6/11)~ n.d. b 18 (5/13) n.d.
8 (4/4) n.d. 13 (7/6) n.d.
24 (12/12) n.d. 24 (I 2/12) n.d.
n.d. 15 (6/9) n.d. 16 (7/9)
n.d.
n.d.
14 (7/7)
n.d.
'~ Actual numbers of male and female rats per group (if/?). b n.d. : not done. Abbreviations: BSS = balanced salt solution, PHE unsaturated fatty acids, CFA -- complete Freund's adjuvant.
L-phenylalanine,PUFA = poly-
354 180 -
-.I~ Females [~
140-
~ates
r
._~ m
o
i00
-
PHE
DD
Fig. 1. Mean body weight (:k t SE) of groups of age-matched mate and female Lewis rats on the day of sensitisation with fresh CNS tissue (experiment B); C = control animals; PHE = phenylataninetreated animals; DD -- age-matched animals on PUFA-deficient diet. Rats of the two former groups were fed with normal rodent diet (Spratts Laboratory Diet 1, Spillers Ltd.) and water ad lib. Fatty acid levels of the PUFA-deficient diet have been stated elsewhere (Mertin and Hunt 1976). which were raised simultaneously on a PUFA-deficient diet showed a significant growth retardation (Fig. 1). As shown in Fig. 2 the onset of the disease occurred earlier in the PHE-treated group. In experiment A only 10 control animals developed clinical EAE as compared with 14 in the PHE group. In both groups the disease was relatively severe but comparison of the highest overall clinical scores showed a higher score in the PHE group, the difference lying just outside the 5 ~ limit of statistical significance. In experiment B both groups showed a generally less severe but more protracted clinical course. However, 2 animals died in the control and 3 in the PHE treated group. Clinical EAE occurred in 19 control and 20 experimental animals but affected the latter group to a greater degree as shown by the statistical difference between the maximal scores reached by each group. N o marked difference was observed in disease onset and clinical course between the control group and the animals raised on a PUFA-deficient diet. N o n e of the animals which received F C A alone developed clinical disease nor did they show weight loss as usually observed during the period o f disease onset. In experiment A no second episode was observed, whereas in experiment B, 5 relapses occurred in the controls (BSS) and 9 in the PHE-treated animals. Similar results were observed in experiment C, these animals having been PHE treated only up to the time of puberty (Table 2). DISCUSSION
Experimental hyperphenylalaninaemia is able to bring about an increased susceptibility to EAE and a more severe course of the disease in young rats. This effect might be due to the hyperphenylalaninaemia-induced decrease in t h e central nervous system (CNS) of long-chain and unsaturated F A described by ~ o ~ s o n and Shah (1973) and to the subsequent alteration in biochemical reactivity a n d stability of the myelin as postulated by these authors.
355 100
aa
5O
O"t, o..,.....o.1.~4~_
"
•
\
°~"
O 20
10
~
//' 0 JD'4P'O 4b-04 0-41,~4
/'j
I
lO
20
I
S/ -
3O lO
I
[
20
30
Days after sensitisation Fig. 2. T i m e course of the daily clinical score per g r o u p a n d the incidence of clinical E A E * in Lewis rats of 2 experiments A a n d B. O - - - O = control animals; O - - - - O ~ phenylalaninine-treated animals.
The relatively moderate dose of PHE given in our experiments did not cause gross general metabolic disturbances as indicated by the fact that the animals did not show growth retardation. The serum and tissue levels of essential PUFA are regulated by dietary intake (Aaes-Jorgensen 1961) and hormonal modulation (Raben 1965) -the main PUFA here being linoleic, linolenic and arachidonic acids. An interference with this regulation by hyperphenylalaninaemia is not known. In the myelin,
* Statisticalevaluation
The clinical scores are arbitrary figures and have no quantitative m e a n i n g except to indicate a rank order of disease intensity. We have therefore applied a n o n - p a r a m e t r i c test (Wilcoxon test). First a p p e a r a n c e o f clinical disease was significantly earlier in the PHE-treated groups o f both experiments A a n d B t h a n in the control groups (exp. A: P ~-. 0.01 ; exp. B: 0.02 ~ P ~> 0.01). C o m p a r i s o n o f the highest clinical scores reached in control a n d PHE-treated animals at any time during the e x p e r i m e n t s showed greater m a x i m u m severity of the disease in the P H E groups, this difference being statistically significant in experiment B: (Significance exp. A: 0.05 < P < 0.10; exp. B: P ~ 0.01).
356 TABLE 2 E A E IN P H E - T R E A T E D LEWIS RATS E A E incidence and clinical scores in the animals (Lewis rats) of experiment C on day 16 after sensitisation with fresh guinea pig C N S tissue plus complete Freund's adjuvant (at climax o f the clinical disease). Animals .................. No.
Sex
1
M
2 3 4 5 6
M M M M M
BSS-treated controls (c) and PilE-treated experimentals (e)
1
] r i
c
~!
EAE No disease ( - - ) and disease (-+)
Clinical score (see text and footnote to Fig. 2) (0-8)
---
0
i -• ....
2 0 1 t 0
1
M
i
i
1
2 3 4 5 6 7
M M M M M M
j [
} ~ ~ + i ~
4 I 7 3 5 1
}
e
~ ]
1
F
+
I
2
F
j
-I-
2
3 4 5 6 7 8 9
F F F F F F F
I
-~ ~
-
0 5 5 0 0 I 0
+
1
+ 4.-~ , ; q ~ ~-
3 1 l 2 2 4 1 6
1
F
2 3 4 5 6 7 8 9
F F F F F F F F
¢
c
~ 1
-
-
-
-
q
j I e [ 1
however, the PUFA quoted above are merely precursors for PUFA of a longer carbon chain, docosahexanenoic acid for example (Bernsohn and Steplmnides 1967), chain elongation taking place in the CNS. It is for this elongation process that various enzymes are needed and where hyperphenylalaninaemia may interfere (Johnson and Shah 1973). Our results indicate that it is possible to modify the expression of an experiment-
357 al immune disease by an active direct interference with metabolic pathways of the target organ resulting in alterations of its composition. Such interference with immune mechanisms is different from and appears more specific than those brought about, for example, by corticosteroid treatment (Stavy, Cohen and Feldman 1973), by dietary modifications in protein (Jose and Good 1973) or FA levels (Mertin 1976; Mertin and Hunt 1976). The fact that in our investigation rats raised on a PUFAdeficient diet did not develop more severe EAE than the animals fed with a normal diet, does not contradict the results of Clausen and Moller (1969) who observed differences in EAE susceptibility only when the rats were bred and raised on the deficient diet. There are indications of some involvement of FA metabolism in the aetiology of multiple sclerosis (MS). Biochemical investigations have supplied evidence for decreased FUFA levels in the lipids of the CNS in MS (Gerstl, Kahnke, Smith, Tavaststjerna and Hayman 1961 ; Thompson 1966; Gerstl, Eng, Tavaststjerna, Smith and Kruse 1970; Thompson 1973). Epidemiologically, an association between MS prevalence and low dietary PUFA intake has been described (reviewed by Alter, Yamoor and Harshe 1974). On the basis of these observations, Thompson (1966, 1973) has postulated an inborn error of FA metabolism in MS, probably accentuated by dietary factors, that would allow an environmental factor, such as a virus infection, to precipitate the pathological changes of the disease. In EAE similarities between the pathological events and those occurring in acute MS attacks have suggested that similar immune mechanisms may be responsible for the pathological changes in both diseases (Paterson 1968). An MS-like relapsing course in EAE was observed after provision of supportive care to the animals during the first acute episode (McFarlin, Blanck and Kibler 1974). This finding has been considered either as a result of a second immune response (McFarlin et al. 1974) or simply as a secondary phenomenon involving increased corticosteroid secretion in the animals caused by non-specific stress (kevine and Sowinski 1975). Whatever the outcome of this discussion may be, we feel that, in order to gain greater insight into M S aetiology by the use of experimental models, one should concentrate on the causal mechanisms rather than on factors able to perpetuate the experimental disease once it has been established. If, as has been postulated (Thompson 1966, 1973) alteration of CNS FA metabolism is really one of the preconditions for the development of MS, animals with experimentally induced FA disturbances of the CNS may provide a useful tool for the search for a better experimental MS model than the existing one. For example, a deficit in certain lipid components such as PUFA may influence packaging of paramyxovirus at the site of the oligodendroglial cell membrane. This could bring about an atypical mode of infection such as is assumed to exist in MS (Eylar 1972). Alteration of CNS FA levels may bring about an altered susceptibility to virus infection of the CNS in experimental animals. We are aware of the fact that no direct conclusions can be drawn from our findings with respect to the aetiology of MS. However, increased blood levels of pyruvate (Jones, Jones and Bunch 1950) and altered pyruvate tolerance has been described in MS patients (Jeanes and Cumings 1958; McArdle, Mackenzie and Web-
358 ster 1960) and these o b s e r v a t i o n s c o u l d indicate a d i s t u r b a n c e in the interlinked P H E - g l u c o s e m e t a b o l i s m (Shah et al. t970) o f the C N S in this disease. There is, on the o t h e r h a n d , no evidence for the co-existence o f P K U a n d MS. H o w e v e r , we feet t h a t this fact does not exclude the possibility o f a m e t a b o l i c lesion limited 1o the C N S causing local a l t e r a t i o n s only. In P K U , for example, h y p e r p h e n y l a l a n i n a e m i a is n o r m a l l y b a s e d on a d i m i n i s h e d P H E - h y d r o x y l a s e level in the liver. Nevertheless, it has recently been p o i n t e d out t h a t despite n o r m a l P H E - h y d r o x y l a s e liver levels, d i s t u r b a n c e s can occur due to the decrease o f an essential c o m p o n e n t o f the P H E h y d r o x y l a s e system, d i h y d r o p t e r i d i n e reductase, in v a r i o u s tissues including the brain ( K a u f m a n , M i l s t i e n a n d B a r t h o l o m 6 1975). A decrease or lack o f such a c o m p o n e n t o f the P H E - h y d r o x y l a s e system l i m i t e d to the C N S could b r i n g a b o u t a d i s t u r b a n c e in the P H E - P U F A m e t a b o l i s m in MS. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Sir Peter M e d a w a r for helpful discussion a n d Dr. C. J. M e a d e for s u p p l y i n g the statistical analysis.
REFERENCES Aaes-Jorgensen, E. (1961) Essential fatty acids, Physiol. Rev., 41 : 1-51. Alter, M., M. Yamoor and M. Harshe (I 974) Multiple sclerosis and nutrition, Arch. Neurol. (Chic.), 31 : 267-272. Bernsohn, J. and L. M. Stephanides (1967) Aetiology of multiple sclerosis, Nature (Lond.), 215: 821-823. Bowden, J. A. and C. L. McArthur (1972) Possible biochemical model for phenylketonuria. Nature (Lond.), 235: 230. Clausen, J. and J. Moiler (1969) Allergic encephalomyelitis induced by brain antigen after deficiency in polyunsaturated fatty acids during myelination, Int. Arch. Allergy, 36: 224-233, Edwards, D. J. and K. Blau (1972) Aromatic acids derived from phenyalanine in the tissues of rats with experimentally induced phenylketonuria-like characteristics, Biochem. J.. 130: 495-503. Eylar, E. H. (1972) Experimental allergic encephalomyelitis and multiple sclerosis. In: F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews (Eds.), Multiple Sclerosis - - hnmunology. Virology and Ultrastructure (Ucla Forum. No. 16), Academic Press. New York, London, p. 449. Gerstl, B., L. Eng, M. G. Tavaststjerna, J. K. Smith and S. Kruse (1970) Lipids and proteins in multiple sclerosis white matter. J. Neurochem., 17: 677-689. Gerstl, B., M. J. Kahnke, J. K. Smith, M. G. Tavaststjerna and R. B. Hayman 11961 ) Brain lipids in multiple sclerosis and other diseases. Brain, 84:310-319. Jeanes, A. L. and J. N. Cumings (1958) Some laboratory investigations in multiple sclerosis. Confin. neurol. (Basel), 18 : 397404. Johnson, R. C. and S. N. Shah (1973) Effect of hyperphenylalaninemia on fatty acid composition of lipids of rat brain myelin, J. Neurochem., 21: 1225-1240. Jones, H. H., H. H. Jones, Jr. and L. D. Bunch (1950) Biochemical studies in multiple sclerosis. Ann. intern. Med., 33: 831-840. Jose, D. G. and R. A. Good (1973) Quantitative effects of nutritional essential amino acid deficiency upon immune responses to tumours in mice, J. exp. Med., 137: I-9. Kaufrnan, S., S. Milstien and K. Bartholom~ (1975) New forms of phenylketonuria, Lancet. 2: 708. Levine, S. and R. Sowinski (1975) The role of the adrenal in relapses of experimental allergic encephalomyelitis, Proc. Soc. exp. Biol. /N. Y.), 149: 1032-1035. McArdle, B., I. C. K. Mackenzie and G. R. Webster (1960) Studies on intermediate carbohydrate metabolism in multiple sclerosis, J. NeuroL Neurosurg. Psychiat., 23: 127-132.
359 McFarlin, D. E., S. E. Blank and R. F. Kibler (1974) Recurrent experimental allergic encephalomyelitis in the Lewis rat, J. lmmunol., 113: 712-715. Menkes, J. H. (1967) The pathogenesis of mental retardation in phenylketonuria and other inborn errors of amino acid metabolism, Pediatrics, 39:297 308. Mertin, J. (1976) Effect of polyunsaturated fatty acids (PUFA) on skin allograft survival and primary and secondary cytotoxic response in mice, Tramplantation, 21 : 1-4. Mertin, J. and R. Hunt (1976) Influence of polyunsaturated fatty acids on survival of skin allografts and tumour incidence in mice, Proc. nat. Aead. Sei. (Wash.), 73: 928-931. Paterson, P. Y. (1968) Experimental autoimmune (allergic) encephalomyelitis. In: P. A. Miescher and H. J. Mfiller-Eberhard (Eds.), Textbook of hnmunology, Grune and Stratton, New York, N.Y., p. 132. Raben, M. S. (1965) Regulation of fatty acid release with particular reference to pituitary factors. In: A. E. Renold and G. 1. Cabil, Jr. (Eds.), Handbook of Physiology, Section 5 (Adipose Tissue), Williams and Wilkins, Baltimore, Md., p. 331. Selinvonchick, D. P. and P. V. Johnston (1975) Fat deficiency in rats during development of the central nervous system and susceptibility to experimental allergic encephalomyelitis, J. Nutr., 105: 288-300. Shah, S. N., N. A. Peterson and C. M. McKean (1970) Cerebral lipid metabolism in experimental hyperphenylalaninemia - - Incorporation of 14C-labelled glucose into total lipids, J. Neuroehem., 17 : 279-284. Stavy, L., I. R. Cohen and M, Feldman (1973) The effect of hydrocortisone on lymphocyte mediated cytolysis, Cellular hnmunol., 7:302-312. Swaiman, K. F., W. B. Hosfield and B. Lemieux (1968) Elevated plasma phenylalanine concentration and lysine incorporation into ribosomal protein of developing brain, J. Neurochem., 15:687 690. Thompson, R. H. S. (1966) A biochemical approach to the problem of multiple sclerosis, Proc. roy. Soc. Med., 59:269 276. Thompson, R. H. S. (1973) Fatty acid metabolism in multiple sclerosis, Biochem. Sac. Sym., 35 103 111.
3 51
cC) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
H Y P E R P H E N Y L A L A N I N A E M I A AND E X P E R I M E N T A L A L L E R G I C ENCEPHA LOM YEL1TIS
JORGEN MERTIN and RUTH HUNT Clinical Research Centre, Wa([brd Road, Harrow, Middlesex HAl 3UJ (Great Britain)
!Received 23 February, 1976)
SUMMARY Previous suggestions to the effect that a reduced proportion of long chain and unsaturated fatty acids in the CNS as induced by experimental hyperphenylalaninaemia in young rats may alter the biochemical reactivity and stability of the myelin have been examined using experimental allergic encephalomyelitis (EAE). Lewis rats treated chronically with phenylalanine during development showed a higher susceptibility to EAE and a more severe course of the disease than their mediumtreated litter mates. The possible implications of this observation for EAE as an experimental model of multiple sclerosis (MS) is discussed briefly in the light of the decreased levels of unsaturated fatty acids found in the CNS of MS patients.
INTRODUCTION Hypomyelination and breakdown of myelin occurring in phenylketonuria (PKU) are looked upon as a result of abnormal lipid content of the white matter of the brain caused by hyperphenylalaninaemia (Menkes 1967). Phenylalanine (PHE) and, to an even greater extent, its metabolites phenylpyruvic and phenyllactic acid have been shown to inhibit [14U-C] glucose incorporation into the lipids of the brain in vitro (Shah, Peterson and McKean 1970). It has been suggested that by inhibition of pyruvate decarboxylation, phenylpyruvate would diminish the availability of acetyl-CoA for fatty acid (FA) and cholesterol synthesis. This would bring about a decrease in myelin formation (Bowden and McArthur 1972). Phenyllactate accumulation in early life has been observed in the brains of rats treated with PHE (Edwards and Blau 1972). Johnson and Shah (1973) reported that rats treated chronically with PHE during development showed a reduced proportion of long chain and unsaturated FA in the total lipids of the whole brain and in the individual lipids of purified myelin. The authors suggest that these alterations may affect the biochemical reactivity and decrease the stability of the myelin in hyperphenylalaninaemia. Hyperphenyl-
352 alaninaemia may also cause an altered incorporation of various amino acids into cerebral proteins (Swaiman, Hosfield and Lemieux 1968) but it is, to our knowledge, not yet known to what degree the proteins of the myelin might be affected by that. Experimental allergic encephalomyelitis (EAE), as an incomplete but as yet the only experimental model for MS (Paterson 1968) has been used to investigate the effects of polyunsaturated fatty acids (PUFA) on CNS autoimmune disease. Supplementation of the diet with PUFA has proved to exert a markedly protective effect in rats with EAE (Selivonchick and Johnston 1975). Clausen and Moiler (1969) and others (Selinvonchick and Johnston 1975) have shown that rats bred and raised on a PUFA-deficient diet will show increased susceptibility to EAE and a more severe course of this disease. This observation supports the hypothesis that a decrease in certain FA may alter the reactivity and stability of myelin (Johnson and Shah 1973). However, dietary deficiency not only lowers PUFA levels in the CNS but will have a more general effect involving other systems as well. An essential FA deficiency syndrome in young animals is well known (Aaes-Jorgensen 1961) and its characteristics include developmental disturbances, the most obvious symptom being growth retardation. One must therefore be cautious in interpreting the results of an increased EAE susceptibility found in animals with a dietary PUFA deficiency. This might not only be the result of myelin alterations but also, for example, of an enhanced cellmediated immune (CM1) response brought about by decreased PUFA serum levels (Mertin 1976; Mertin and Hunt 1976). Thus, a less general PUFA deficiency affecting preponderantly the CNS would help to ascertain the degree to which FA alterations in the CNS may increase the vulnerability to CMI attack. We have thereiore studied susceptibility to EAE and its clinical course in rats with FA disturbances induced by experimental hyperphenylalaninaemia. MATERIALS AND METHODS Pregnant Lewis rats were obtained from Charles River Laboratories. Between days 3-5 after birth the litters were randomly divided into 2 groups. One group (experimental) was treated with 45 #moles/g body weight of L-phenylalanine IPHE) (Sigma Chem.) as follows: PHE was dissolved in balanced salt solution (BSS) and injected intraperitoneally twice a day. The other group (control) were injected according to the same schedule but with an equal volume of BSS only, Usually, the treatments were continued until after the animals had passed the climax of EAE in the second part of the experiment. However, in one of the experiments PHE and BSS injections were discontinued at the time of puberty. At an age of about 40-50 days, control and experimental animals received an injection of fresh guinea pig CNS tissue in Freund's complete adjuvant (FCA) or (in experiment B) FCA alone. The antigenic emulsion was prepared as follows: Fresh guinea pig (strain Duncan-Hartley) brain stem and spinal cord were mixed with an equal amount (w/v) of BSS and this mixture was emulsified together with an equal amount (v/v) of Freund's complete adjuvant (Difco). 0.2 ml of this emulsion was injected into all 4 foot pads. According to our experience in other experiments,
353 this regimen normally induces EAE of moderate clinical severity in Lewis rats of this age and weight range (130-180 g), with most of the affected animals recovering from the disease. Body weights were checked daily starting some days before sensitisation with CNS tissue. The clinical scores were evaluated twice daily (morning and evening) the criteria for the evaluation being - - (grading in brackets) : !. No disease (0). 11. Atonia of the abdominal muscles, "limp tail" sign (1). Ill. As Ii plus ataxia or slight weakness of the hind (or front) legs (2). IV. Distinct weakness of the hind (or front) legs (3). V. As 1V plus neurogenic "overflow bladder" (4). VI. Total paralysis of the hind legs (5). VII. As VI plus "overflow bladder" (6). VIII. As VI plus marked weakness of the front legs (7). IX. Death (8). Because of our main interest in the time course of the disease and the occurrence of relapses, histopathologic criteria for EAE were not evaluated in this study. Three experiments (experiments A, B and C) were carried out (Table I). Agematched animals without the pretreatment described, but raised on a PUFA-deficient diet (Mertin and Hunt 1976) were included in experiment B. Scoring in experiment B was done blind up to day 30 after sensitisation. RESULTS There was no recognisable difference in the development and behaviour of the animals of control and experimental groups. An example for their similar growth as reflected in their body weights is shown in Fig. 1. In contrast, age-matched animals
TABLE I EXPERIMENTAL DESIGN Number of animals per group, pretreatment and EAE induction in 3 experiments. Pretreatment
Experiment A (CNS tissue
BSS continuous BSS until puberty PHE continuous PHE until puberty PUFA-deficient diet Continuous
Experiment B
Experiment C (CNS tissue -~ CFA)
+ CFA)
(CFA alone)
(CNS tissue + CFA)
17 (6/11)~ n.d. b 18 (5/13) n.d.
8 (4/4) n.d. 13 (7/6) n.d.
24 (12/12) n.d. 24 (I 2/12) n.d.
n.d. 15 (6/9) n.d. 16 (7/9)
n.d.
n.d.
14 (7/7)
n.d.
'~ Actual numbers of male and female rats per group (if/?). b n.d. : not done. Abbreviations: BSS = balanced salt solution, PHE unsaturated fatty acids, CFA -- complete Freund's adjuvant.
L-phenylalanine,PUFA = poly-
354 180 -
-.I~ Females [~
140-
~ates
r
._~ m
o
i00
-
PHE
DD
Fig. 1. Mean body weight (:k t SE) of groups of age-matched mate and female Lewis rats on the day of sensitisation with fresh CNS tissue (experiment B); C = control animals; PHE = phenylataninetreated animals; DD -- age-matched animals on PUFA-deficient diet. Rats of the two former groups were fed with normal rodent diet (Spratts Laboratory Diet 1, Spillers Ltd.) and water ad lib. Fatty acid levels of the PUFA-deficient diet have been stated elsewhere (Mertin and Hunt 1976). which were raised simultaneously on a PUFA-deficient diet showed a significant growth retardation (Fig. 1). As shown in Fig. 2 the onset of the disease occurred earlier in the PHE-treated group. In experiment A only 10 control animals developed clinical EAE as compared with 14 in the PHE group. In both groups the disease was relatively severe but comparison of the highest overall clinical scores showed a higher score in the PHE group, the difference lying just outside the 5 ~ limit of statistical significance. In experiment B both groups showed a generally less severe but more protracted clinical course. However, 2 animals died in the control and 3 in the PHE treated group. Clinical EAE occurred in 19 control and 20 experimental animals but affected the latter group to a greater degree as shown by the statistical difference between the maximal scores reached by each group. N o marked difference was observed in disease onset and clinical course between the control group and the animals raised on a PUFA-deficient diet. N o n e of the animals which received F C A alone developed clinical disease nor did they show weight loss as usually observed during the period o f disease onset. In experiment A no second episode was observed, whereas in experiment B, 5 relapses occurred in the controls (BSS) and 9 in the PHE-treated animals. Similar results were observed in experiment C, these animals having been PHE treated only up to the time of puberty (Table 2). DISCUSSION
Experimental hyperphenylalaninaemia is able to bring about an increased susceptibility to EAE and a more severe course of the disease in young rats. This effect might be due to the hyperphenylalaninaemia-induced decrease in t h e central nervous system (CNS) of long-chain and unsaturated F A described by ~ o ~ s o n and Shah (1973) and to the subsequent alteration in biochemical reactivity a n d stability of the myelin as postulated by these authors.
355 100
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Days after sensitisation Fig. 2. T i m e course of the daily clinical score per g r o u p a n d the incidence of clinical E A E * in Lewis rats of 2 experiments A a n d B. O - - - O = control animals; O - - - - O ~ phenylalaninine-treated animals.
The relatively moderate dose of PHE given in our experiments did not cause gross general metabolic disturbances as indicated by the fact that the animals did not show growth retardation. The serum and tissue levels of essential PUFA are regulated by dietary intake (Aaes-Jorgensen 1961) and hormonal modulation (Raben 1965) -the main PUFA here being linoleic, linolenic and arachidonic acids. An interference with this regulation by hyperphenylalaninaemia is not known. In the myelin,
* Statisticalevaluation
The clinical scores are arbitrary figures and have no quantitative m e a n i n g except to indicate a rank order of disease intensity. We have therefore applied a n o n - p a r a m e t r i c test (Wilcoxon test). First a p p e a r a n c e o f clinical disease was significantly earlier in the PHE-treated groups o f both experiments A a n d B t h a n in the control groups (exp. A: P ~-. 0.01 ; exp. B: 0.02 ~ P ~> 0.01). C o m p a r i s o n o f the highest clinical scores reached in control a n d PHE-treated animals at any time during the e x p e r i m e n t s showed greater m a x i m u m severity of the disease in the P H E groups, this difference being statistically significant in experiment B: (Significance exp. A: 0.05 < P < 0.10; exp. B: P ~ 0.01).
356 TABLE 2 E A E IN P H E - T R E A T E D LEWIS RATS E A E incidence and clinical scores in the animals (Lewis rats) of experiment C on day 16 after sensitisation with fresh guinea pig C N S tissue plus complete Freund's adjuvant (at climax o f the clinical disease). Animals .................. No.
Sex
1
M
2 3 4 5 6
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BSS-treated controls (c) and PilE-treated experimentals (e)
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Clinical score (see text and footnote to Fig. 2) (0-8)
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however, the PUFA quoted above are merely precursors for PUFA of a longer carbon chain, docosahexanenoic acid for example (Bernsohn and Steplmnides 1967), chain elongation taking place in the CNS. It is for this elongation process that various enzymes are needed and where hyperphenylalaninaemia may interfere (Johnson and Shah 1973). Our results indicate that it is possible to modify the expression of an experiment-
357 al immune disease by an active direct interference with metabolic pathways of the target organ resulting in alterations of its composition. Such interference with immune mechanisms is different from and appears more specific than those brought about, for example, by corticosteroid treatment (Stavy, Cohen and Feldman 1973), by dietary modifications in protein (Jose and Good 1973) or FA levels (Mertin 1976; Mertin and Hunt 1976). The fact that in our investigation rats raised on a PUFAdeficient diet did not develop more severe EAE than the animals fed with a normal diet, does not contradict the results of Clausen and Moller (1969) who observed differences in EAE susceptibility only when the rats were bred and raised on the deficient diet. There are indications of some involvement of FA metabolism in the aetiology of multiple sclerosis (MS). Biochemical investigations have supplied evidence for decreased FUFA levels in the lipids of the CNS in MS (Gerstl, Kahnke, Smith, Tavaststjerna and Hayman 1961 ; Thompson 1966; Gerstl, Eng, Tavaststjerna, Smith and Kruse 1970; Thompson 1973). Epidemiologically, an association between MS prevalence and low dietary PUFA intake has been described (reviewed by Alter, Yamoor and Harshe 1974). On the basis of these observations, Thompson (1966, 1973) has postulated an inborn error of FA metabolism in MS, probably accentuated by dietary factors, that would allow an environmental factor, such as a virus infection, to precipitate the pathological changes of the disease. In EAE similarities between the pathological events and those occurring in acute MS attacks have suggested that similar immune mechanisms may be responsible for the pathological changes in both diseases (Paterson 1968). An MS-like relapsing course in EAE was observed after provision of supportive care to the animals during the first acute episode (McFarlin, Blanck and Kibler 1974). This finding has been considered either as a result of a second immune response (McFarlin et al. 1974) or simply as a secondary phenomenon involving increased corticosteroid secretion in the animals caused by non-specific stress (kevine and Sowinski 1975). Whatever the outcome of this discussion may be, we feel that, in order to gain greater insight into M S aetiology by the use of experimental models, one should concentrate on the causal mechanisms rather than on factors able to perpetuate the experimental disease once it has been established. If, as has been postulated (Thompson 1966, 1973) alteration of CNS FA metabolism is really one of the preconditions for the development of MS, animals with experimentally induced FA disturbances of the CNS may provide a useful tool for the search for a better experimental MS model than the existing one. For example, a deficit in certain lipid components such as PUFA may influence packaging of paramyxovirus at the site of the oligodendroglial cell membrane. This could bring about an atypical mode of infection such as is assumed to exist in MS (Eylar 1972). Alteration of CNS FA levels may bring about an altered susceptibility to virus infection of the CNS in experimental animals. We are aware of the fact that no direct conclusions can be drawn from our findings with respect to the aetiology of MS. However, increased blood levels of pyruvate (Jones, Jones and Bunch 1950) and altered pyruvate tolerance has been described in MS patients (Jeanes and Cumings 1958; McArdle, Mackenzie and Web-
358 ster 1960) and these o b s e r v a t i o n s c o u l d indicate a d i s t u r b a n c e in the interlinked P H E - g l u c o s e m e t a b o l i s m (Shah et al. t970) o f the C N S in this disease. There is, on the o t h e r h a n d , no evidence for the co-existence o f P K U a n d MS. H o w e v e r , we feet t h a t this fact does not exclude the possibility o f a m e t a b o l i c lesion limited 1o the C N S causing local a l t e r a t i o n s only. In P K U , for example, h y p e r p h e n y l a l a n i n a e m i a is n o r m a l l y b a s e d on a d i m i n i s h e d P H E - h y d r o x y l a s e level in the liver. Nevertheless, it has recently been p o i n t e d out t h a t despite n o r m a l P H E - h y d r o x y l a s e liver levels, d i s t u r b a n c e s can occur due to the decrease o f an essential c o m p o n e n t o f the P H E h y d r o x y l a s e system, d i h y d r o p t e r i d i n e reductase, in v a r i o u s tissues including the brain ( K a u f m a n , M i l s t i e n a n d B a r t h o l o m 6 1975). A decrease or lack o f such a c o m p o n e n t o f the P H E - h y d r o x y l a s e system l i m i t e d to the C N S could b r i n g a b o u t a d i s t u r b a n c e in the P H E - P U F A m e t a b o l i s m in MS. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Sir Peter M e d a w a r for helpful discussion a n d Dr. C. J. M e a d e for s u p p l y i n g the statistical analysis.
REFERENCES Aaes-Jorgensen, E. (1961) Essential fatty acids, Physiol. Rev., 41 : 1-51. Alter, M., M. Yamoor and M. Harshe (I 974) Multiple sclerosis and nutrition, Arch. Neurol. (Chic.), 31 : 267-272. Bernsohn, J. and L. M. Stephanides (1967) Aetiology of multiple sclerosis, Nature (Lond.), 215: 821-823. Bowden, J. A. and C. L. McArthur (1972) Possible biochemical model for phenylketonuria. Nature (Lond.), 235: 230. Clausen, J. and J. Moiler (1969) Allergic encephalomyelitis induced by brain antigen after deficiency in polyunsaturated fatty acids during myelination, Int. Arch. Allergy, 36: 224-233, Edwards, D. J. and K. Blau (1972) Aromatic acids derived from phenyalanine in the tissues of rats with experimentally induced phenylketonuria-like characteristics, Biochem. J.. 130: 495-503. Eylar, E. H. (1972) Experimental allergic encephalomyelitis and multiple sclerosis. In: F. Wolfgram, G. W. Ellison, J. G. Stevens and J. M. Andrews (Eds.), Multiple Sclerosis - - hnmunology. Virology and Ultrastructure (Ucla Forum. No. 16), Academic Press. New York, London, p. 449. Gerstl, B., L. Eng, M. G. Tavaststjerna, J. K. Smith and S. Kruse (1970) Lipids and proteins in multiple sclerosis white matter. J. Neurochem., 17: 677-689. Gerstl, B., M. J. Kahnke, J. K. Smith, M. G. Tavaststjerna and R. B. Hayman 11961 ) Brain lipids in multiple sclerosis and other diseases. Brain, 84:310-319. Jeanes, A. L. and J. N. Cumings (1958) Some laboratory investigations in multiple sclerosis. Confin. neurol. (Basel), 18 : 397404. Johnson, R. C. and S. N. Shah (1973) Effect of hyperphenylalaninemia on fatty acid composition of lipids of rat brain myelin, J. Neurochem., 21: 1225-1240. Jones, H. H., H. H. Jones, Jr. and L. D. Bunch (1950) Biochemical studies in multiple sclerosis. Ann. intern. Med., 33: 831-840. Jose, D. G. and R. A. Good (1973) Quantitative effects of nutritional essential amino acid deficiency upon immune responses to tumours in mice, J. exp. Med., 137: I-9. Kaufrnan, S., S. Milstien and K. Bartholom~ (1975) New forms of phenylketonuria, Lancet. 2: 708. Levine, S. and R. Sowinski (1975) The role of the adrenal in relapses of experimental allergic encephalomyelitis, Proc. Soc. exp. Biol. /N. Y.), 149: 1032-1035. McArdle, B., I. C. K. Mackenzie and G. R. Webster (1960) Studies on intermediate carbohydrate metabolism in multiple sclerosis, J. NeuroL Neurosurg. Psychiat., 23: 127-132.
359 McFarlin, D. E., S. E. Blank and R. F. Kibler (1974) Recurrent experimental allergic encephalomyelitis in the Lewis rat, J. lmmunol., 113: 712-715. Menkes, J. H. (1967) The pathogenesis of mental retardation in phenylketonuria and other inborn errors of amino acid metabolism, Pediatrics, 39:297 308. Mertin, J. (1976) Effect of polyunsaturated fatty acids (PUFA) on skin allograft survival and primary and secondary cytotoxic response in mice, Tramplantation, 21 : 1-4. Mertin, J. and R. Hunt (1976) Influence of polyunsaturated fatty acids on survival of skin allografts and tumour incidence in mice, Proc. nat. Aead. Sei. (Wash.), 73: 928-931. Paterson, P. Y. (1968) Experimental autoimmune (allergic) encephalomyelitis. In: P. A. Miescher and H. J. Mfiller-Eberhard (Eds.), Textbook of hnmunology, Grune and Stratton, New York, N.Y., p. 132. Raben, M. S. (1965) Regulation of fatty acid release with particular reference to pituitary factors. In: A. E. Renold and G. 1. Cabil, Jr. (Eds.), Handbook of Physiology, Section 5 (Adipose Tissue), Williams and Wilkins, Baltimore, Md., p. 331. Selinvonchick, D. P. and P. V. Johnston (1975) Fat deficiency in rats during development of the central nervous system and susceptibility to experimental allergic encephalomyelitis, J. Nutr., 105: 288-300. Shah, S. N., N. A. Peterson and C. M. McKean (1970) Cerebral lipid metabolism in experimental hyperphenylalaninemia - - Incorporation of 14C-labelled glucose into total lipids, J. Neuroehem., 17 : 279-284. Stavy, L., I. R. Cohen and M, Feldman (1973) The effect of hydrocortisone on lymphocyte mediated cytolysis, Cellular hnmunol., 7:302-312. Swaiman, K. F., W. B. Hosfield and B. Lemieux (1968) Elevated plasma phenylalanine concentration and lysine incorporation into ribosomal protein of developing brain, J. Neurochem., 15:687 690. Thompson, R. H. S. (1966) A biochemical approach to the problem of multiple sclerosis, Proc. roy. Soc. Med., 59:269 276. Thompson, R. H. S. (1973) Fatty acid metabolism in multiple sclerosis, Biochem. Sac. Sym., 35 103 111.
