Mecovet Dose Reference

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140 Journal o f the Neurological Sciences', 122 (199 4) 140 143Z~ 1994 Elsev ier Scien ce B.V . All rights reserv ed 11022-510X/t)4/$(17.0(IJNS 4251

Ultra-high dose m ethylcobalamin prom otes nerve regenerat ionin experimen tal acrylamide neuropathyTetsuya Watanabe, Ryuji Kaji *, Nobuyuki Oka, Will iam Bara and Jun Kimura

Depa rtment of Neurology, Kyoto U niversity Hospital, Shogoin, Sakyo ku, Kyoto 606-01, Japan(Received 11 June, 1993)(Revised, received 19 October, 1993)(Accepted 28 October, 1993)

K e y w or ds : Methylcoba lamin; Acrylamide neuropa thy; Nerve reg enera t ion; Therapy; Com pound musc le ac t ion potent i a lSummary

Despi te intens ive searches for therapeut ic agents , few substances have been convincingly shown to enhance nerve regenera-t ion in pat ients wi th peripheral neuropathies . Recent biochemical evidence suggests that an ul t ra-high dose of methylcobalamin(methyl-B12) may up - regula te gene t ransc r ip t ion and the reby pro te in synthesi s. We examined the e f fec t s of u l t ra -h igh do se ofmethyl -B12 on the ra t e o f ne rve regenera t ion in ra ts wi th ac ry lamide neuropa thy , us ing the ampl i tudes of compou nd muscleac t ion potent i a l s (CMAPs) a f t e r t i b i a l ne rve s t imula t ion as an index of the number of regenera t ing motor f ibe rs . Af te rintoxicat ion with acrylamide, al l the rats showe d equal ly decr eased C MA P am pli tudes . The animals were the n divided into 3groups ; ra t s t rea ted wi th u l t ra -h igh (50 0/~ g/k g body we ight, i n t raper i tonea l ly) and low (50 /xg /kg ) doses of me thyl-B12, andsal ine-t reated co ntrol ra ts. Tho se t reate d with ul t ra-high dose showed s ignificant ly fas ter CM AP re covery than sal ine -t reatedcontrol ra ts , whereas the low-dose group showed no difference from the control . Morphometric analys is revealed a s imilardifference in f iber densi ty between these groups . Ul t ra-high doses of methyl-B12 may be of c l inical use for pat ients wi thper iphera l neuropa th ies .

In t roduc t i onI n s p i te o f th e c o m m o n b e l i e f t h a t p e r i p h e r a l n e r v e s

a r e c a p a b l e o f e x te n s i v e r e g e n e r a t i v e g r o w t h a f t e r i n -j u r y , c li n ic a l r e c o v e r y s e l d o m i s c o m p l e t e i n p a t i e n t sw i t h p e r i p h e r a l n e u r o p a t h y . V a r i o u s a g e n t s a r e k n o w nt o e n h a n c e p e r i p h e r a l n e r v e r e g e n e r a t i o n , b u t n o t m a n yh a v e p r o v e d h e l p f u l i n c l in i c a l s e t t in g s ( H o r o w i t z 1 9 8 9 ).

V i t a m i n B 1 2 h a s a n i m p o r t a n t r o l e i n m e t h y l t r a n s -f e r r e a c t i o n s t h r o u g h f o l a t e m e t a b o l i s m , a n d a d e f t -c i e n c y o f i t c a u s e s h e m a t o l o g i c a n d n e u r o l o g i c d i s o r -d e r s s u c h a s m e g a l o b l a s ti c a n e m i a a n d p e r i p h e r a l n e u -r o p a t h y . N e u r o l o g i c m a n i f e s ta t i o n s h o w e v e r a r e n o ta l w a y s a c c o m p a n i e d b y h e m a t o l o g i c a b n o r m a l i t i e s n o ra r e t h e y r e v e r si b l e b y fo l a t e s u p p l e m e n t ( S h o r v o n 1 98 0;L i n d e n b a u m 1 9 8 8) . V i t a m i n B 1 2 m a y h a v e a s p e c i fi ca c t i o n o n t h e n e r v o u s s y s t e m , i n a d d i t i o n t o i t s a c t i o no n t h e h e m a t o p o i e t i c s y s t e m .

R e c e n t b i o c h e m i c a l e v i d e n c e s u g g e s t s t h a t m e t h y l -c o b a l a m i n ( m e t h y l - B 1 2 ) a c t s d i r e c t l y a s a m e t h y l d o n o ri n D N A m e t a b o l i s m ( P f o h l - L e s z k o w i c z 19 91 ). M o r ei m p o r t a n tl y , u lt r a -h i g h c o n c e n t r a t i o n s ( > 1 / ~ M o r 1 .3 4/ z g / m l o f m e t h y l- B 1 2 ) u p - r e g u l a t e g e n e t r a n s c r i p ti o n ,

* Cor respon ding auth or. T el.: 075-751-3770; Fax : 075-761-9780.

w h i c h m a y i n t u r n i n c r e a s e p r o t e i n s y n t h e s i s f o r n e r v er e g e n e r a t i o n .W e h a v e u s e d p h y s i o l o g i c a l a n d m o r p h o l o g i c a l

m e t h o d s t o i n v e s t i g a t e t h e e f f e c t o f u lt r a - h i g h ( 5 0 0~ g / k g ) v e rs u s l ow ( 5 0 / z g / k g ) d o s es o f m e t h y l- B 1 2 onp e r i p h e r a l n e r v e r e g e n e r a t i o n i n e x p e r i m e n t a l a c r y l -a m i d e n e u r o p a t h y .

Me t h od sA n i m a l t r e a t m e n t

T w e n t y - s ix m a l e W i s ta r r a ts ( b o d y w e i g h t 2 5 0 - 3 0 0g ) w e r e t r e a t e d w i th a c r y l a m i d e ( 25 m g / d a y i .p . f o r 5d a y s a w e e k f o r 4 w e e k s ) . A l l a n i m a l s d e v e l o p e d c l i n i -c a l s ig n s o f n e u r o p a t h y s u c h a s h i n d l i m b w e a k n e s s a n da t a x i a b y t h e e n d o f a d m i n i s t r a t i o n ( d a y 0 ). T h e a n i -m a l s w e r e a l l o w e d t o r e c o v e r f o r 9 0 d a y s ( d a y 0 - 9 0 ) ,a n d d i v i d e d i n t o 3 g r o u p s : 1 2 r a t s t r e a t e d w i t h u l t r a -h i g h d o s e s o f m e th y l - B 1 2 ( E i sa i C o . L t d , T o k y o , Ja p a n )( h i g h - d o s e g ro u p ; 5 0 0 I z g / k g b o d y w e i g h t / d a y , i .p .) , 5t r e a t e d w i t h l o w d o s e s m e t h y l - B 1 2 ( lo w - d o s e g r o u p ; 5 0~ g / k g , i . p . ) a n d 9 t r e a t e d w i t h n o r m a l s a l i n e ( c o n t r o lg r o u p ) . M e t h y l - B 1 2 o r s a l i n e w a s i n j e c t e d 5 d a y s aw e e k f o r 1 2 - 1 3 w e e k s a f t e r d a y 0 .

S S D I 0 0 2 2 - 5 1 0 X ( 9 3 ) E 0 2 6 2 - 8


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( ; M A P m e a s u r e m e n tP h y s i o l o g i c a l e v a l u a t i o n s w e r e m a d e b e f o r e a n d i m -

m e d i a t e l y a f t e r a c r y l a m i d e a d m i n i s t r a t i o n a n d 3 t i m e sd u r i n g t h c r e c o v e r y p h a s e ; d u r i n g t h e i n t e rv a l s f r o md a y 2 5 - 3 5 ( i n d i c a t ed a s " d a y 3 0 " ) , d a y 5 5 - 6 5 ( " d a yM ) ") a n d d a y 8 5 - 9 5 ( " d a y 9 0 " ) .

R a t s w e r e a n e s t h e t i z e d w i t h a m i x t u r e o f k e t a m i n c( 1 0 0 m g / k g , i . p . ) , x y l a z i n e ( 1 0 m g / k g , i . p . ) a n d a t -r o p i n e ( 0 .0 5 m g / k g , i .p .) . A p a i r o f n e e d l e e l e c t r o d e s{ N i h o n K o h d e n N E 2 3 3 S ) w a s p l a c e d a t t h e a n k l e w it ht h e c a t h o d e o n t h e m e d i a l a s p e c t . A s u p r a m a x i m a ls t i m u l a t i n g c u r r e n t w a s a p p l i e d t o t h e f i n a l n e r v e a tt h e a n k l e w i t h a c o n s t a n t v o l t a g e s t i m u l a t o r ( M e d e l e cM S 6 ) . W e r e c o r d e d t h e c o m p o u n d m u s c l e a c t i o n p o -t e n t i a l s ( ( ' M A P s ) f r o m t h e s m a l l h i n d f o o t m u s c l e sw i t h a r c c o r d i n g n e e d l e e l e c t r o d e ( N i h o n K o h d e nN E 2 3 3 S ) o n t h e d o r s u m a n d a r e f e r e n c e a t t h e s e c o n dd i gi t. C M A P a m p l i t u d e s , w h i c h r e f le c t t h e n u m b e r o fi n n e r v a t e d m u s c l e f i b e r s , w e r e m e a s u r e d f r o m t h e i n i -t i a l n e g a t i v e t o p o s i t i v e p e a k s . R e c o r d i n g s u s u a l l y w e r em a d e o n o n e s id e , th e s a m e s i d e b e i n g u s e d t h r o u g h -o u t t h e s t u d y . T h e o t h e r s i d e , h o w e v e r , w a s u s e dw h e n e v e r t h e r e p r o d u c i b i l i t y o f t h e p o t e n t i a l b e c a m ei n s uf f ic i e n t b e c a u s c o f lo c al h e m a t o m a f o r m a t i o n .R e p e t i t i v e n e r v e s t im u l a t i o n w a s p e r f o r m e d a t 3 H z ,w h e n e v e r p o s s i b l e , i n o r d e r t o a s s e s s t h e n e u r o m u s c u -l a r t r a n s m i s s i o n ( K i m u r a 1 98 9) . T h e s ki n t e m p e r a t u r ea t t h e h i n d l im b , w h i c h w a s m o n i t o r e d t h r o u g h o u t t h er e c o r d i n g s e s s i o n , w a s k e p t b e t w e e n 3 0 a n d 3 2 C ., 1 / I o r pho l ne / o ,

T h e m o r p h o l o g i c a l s t u d y u s e d s p e c i m e n s o f t h e t ib -i al n e r v c a t t h e m i d c a l f t a k e n f r o m 3 a n i m a l s o n d a y6 0, e a c h s p e c i m e n b e i n g r e p r e s e n t a t i v e o f a s p e c i f i cg r o u p w i t h C M A P a m p l i t u d e v a l u e s w i th i n 1 S D o f th em e a n . T h e a n a l y si s m e t h o d u s e d h a s a lr e a d y b e e nd e s c r i b e d e l s e w h e r e ( K a j i 1 9 89 ).

S t a ti s ti c al a n a l ys i s w a s m a d e w i t h a m i c r o c o m p u t e r{ M a c i n t o s h I I ) w i t h S t a t V i e w .... s o f t w a r e ( A b a c u s C o n -c e p t s , I n c . , B e r k c l c y , C A , U S A ) .V i t a m i n B I 2 a s s a y

S e r u m a n d C S F c o n c e n t r a t i o n s o f v i t a m i n B 1 2 w e r em e a s u r e d in tw o a n i m a l s f r o m h i g h - d o s e a n d l o w - d o s eg r o u p s d u r i n g t h e d a y 9 0 p e r i o d b y c o m p e t i t i v e p r o t e i nh i d i n g a s s a y , w h i c h w a s p e r f o r m e d a t S M I B r i s t o lL a b o r a t o r i e s , T o k y o , J a p a n , o n a c o m m e r c i a l b a si s.N o r m a l s e r u m l e v e ls i n th e r a t a r e 6 8 3 + 6 3 p g / d l(naean + S D. n - 4 ) (S a ga w a e t a l. 1987).

R e s u l t s

C M A P a m p l i t u d e sT h e C M A P a m p l i t u d e s d e c r e a s e d s h ar p l y a f t e r

a c r y l a m i d e t r e a t m e n t ( B e f o r e a n d A f t e r i n Fi g . 1, T a b l e

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D a y s b e l o r e a n d a l l e r a c r y l a m i d e i n t o x b c a l io nF i g . 1 . C o m p a r i s o n o f C M A P c h a n g e s am J,m g th e g r o u p s . B a r s

i n d i c a t c s t a n d a r d d e v i a t i o n s . * ~ P < ( ) . O 0 1 . * P 0 . 00 1

1 ). T h e s e a m p l i t u d e s d i d n o t d i f f e r st a t i st i c a ll y a m o n gt h e g r o u p s ( P = 0 .8 6 1 f o r B e f o r e : P - ( }.4 16 f o r A f t e r :o n e - w a y A N O V A ) . I n t h e r ec o v e r y p h a s e , t h e h i g h - d o seg r o u p s h o w e d a s ig n i fi c an t ly h i g h e r a m p l i t u d e s t h a nt h e o t h e r g r o u p s o n d a y 3 0 ( t ' < ( ) . 0 0 1 : o n e - w a yA N O V A , F i s h e r ' s P L S D ) , d a y 6 0 ( P < 0 .0 0 1) , a n d d a y9 0 ( P = ( ).0 01 ). T h e l o w - d o s e a n d t h e c o n t r o l g r o u p sd i d n o t s h o w s i g n i f i c a n t d i f f e r e n c e ( P > 0 . 0 5 ) . R e p r e -s e n t a t i v e r e c o r d i n g s f r o m e a c h g r o u p a r c s h o w n i n F i g .2 . R e p e t i t i v e n e r v e s t i m u l a t i o n a t 3 H z p r o d u c e d n os i g n i f i c an t d e c r e m e n t ( < 2( )( :f ) i n C M A P a m p l i t u d e s ,s u g g e s t in g t h a t th e d e c r e a s e d a m p l i t u d e s a r c n o tc a u s e d b y i m p a i r e d n e u r o m u s c u l a r t r a n s m i s s i o n b u tr e p r e s e n t t h e r e d u c e d n u m b e r o f m o t o r f i b er s i n n e r -v a t i n g t h e m u s c l e .Body weim

T h e t h r e e g r o u p s s h o w e d b o d y w e i g h t c h a n g e s w h i c hd i d n o t s i g n if i c a n t ly d i f f e r d u r i n g t h e e x p e r i m e n t( A N O V A P > 0 .0 5 : Fi g . 3 ).

T A B L E 1C M A P C H A N G E S ( m e a n + S I ) . m V )

G r o u p s C o n t r o l t t ig h d o s c L o w - d o s eB c f l l r e 1 3 . 9 2 + 1 . 16 1 3 . 8 0 + { } . 8 4 1 3 . 7 2 + 0 . 7 7P - 0 % 6 * ( n 9 ) ( n - 1 2 ) ( n 5 )A f t e r 7 . 7 6 + 1 . 2 0 7 . 8 2 + ( } 3t 4 7 . 3 2 + ( } .8 1P = 0 . 4 2 ~ ( n 9 ) I n 1 2 ) I n = 5 )D a y 3 0 7 . 7 0 + I .I 1 ~ L 3 2 + 0 . ~3 7 . 9 9 + 0 . 8 0P < 0 . ( ) 0 1 " ( n 9 ) ( ~ = I 1 ) ( n 5 )D a y 6 0 q . 3 ( } + f ) . 9 8 1 1 . 6 7 + 0 . 9 t} 9 . 5 1 + ( ). ,g 9P < ( ). () 0 1 * ( 1 1 - 8 ) i n - 1 ( )) ( ,1 5 )D a y 9 0 1 1 . 4 1 + _ 0 . 7 9 I 2 .S -+ - ( ) . 0 8 I 1 . 0 3 + 0 . 4 6P - 0 J ) 0 1 ,~ ( n 5 ) ( r ' S ) ( n - : 2 )* P - v a l u e o f t h e s t a t i st i c a l s i g n i fi c a n c e in t h e d i f f e r e n c e a m o n g 3

g r o u p s ( o n e w a y A N O V A ) . n : n u m b e r o f s am p l e s . S o m e a n i m a l sw e r e l o st d u e t o a n e s t h e t i c a c c k t e n t s d u r i n g t h e o b s e 1~ , a ti o n p cr i o d .


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b e f o r ea c r y l a m i d et r e a t m e n ta f t e ra c r y l a m i d ei n t o x i c a t i o n( D a y O )

C O N T R O L 5 0 0 ~ g / k g 5 0 ~ g / k g ~ 1 C , ~ , V ; ~

142

Fig. 2. Representative serial recordings of CMAPs in the sameanimals.

MorphometryResults of the morphometry performed on eachanimal whose CMAP is shown in Fig. 2 are given inFig. 4. Fiber densities were 212 5/mm 2 in the control,3290 /mm 2 in the low-dose, and 5360 /mm 2 in thehigh-dose groups. The histograms showed increasednumbers of small and medium-sized myelinated fiberswith two prominent peaks at 4 and 9 /zm in thehigh-dose group, as compared to the other groups. Themaximum fiber diameter was 15 /zm in the high-dosegroup, the largest among the groups.Vitamin B12 levelsSerum levels of vitamin B12 were 2500/~g/ml in arat from the high-dose group, and 180 /zg/ml in onefrom the low-dose group. CSF levels were 15.2/~g/mlin the former, and 1.2/zg/ml in the latter.

I q ~ " ~ 4

D i s c u s s i o n

We have shown here that animals treated with anultra-high dose of methyl-B12 promptly recoveredCMAP amplitude after acrylamide intoxication, as

50 0

J ~40 0

300 /k g'~ ~ Control

B12 50 ug/kg

20 0 ! ! I " ! !B e f o r e A f t e r 3 0 d 6 0 d 9 0 dD a y s b e fo r e a n d a f t e r a c r y la m i d e i n to x ic a t i o n

Fig. 3. Body weight change in three groups during th e experiment .

Fig. 4. Morphometric study in the groups in day 60. Representativemicrographs used for analysis are shown on the left.

compared to the controls and those treated with alow-dose of methyl-B12. Because the CMAP amplituderepresents the number of innervated muscle fibers, itsprompt recovery indicates accelerated reinnervation ofmuscles by axonal regeneration in the ultra-high dosegroup. In fact, nerve specimens from representativeanimals in each group provided morphometric evi-dence of increased numbers o f large-diameter fibers inthat group. Although the animals lost body weight afteracrylamide intoxication, the high dose of methyl-B12did not act through the improvement of the generalnutritional conditions, because the body weight recov-ery curve did not differ among the three groups.The response of a neuron to axonal damage is bestexemplified by wallerian degeneration. Soon after ax-onotomy, the soma undergoes a process called centralchromatolysis, which probably represents a metabolicshift from supporting transmitter production andsynaptic function to production of materials needed fornerve regenerat ion (Seckel 1990)~ This change shouldinvolve a surge in transcription of an entirely differentset of genes.

Acrylamide neuropathy is regarded as a prototypeof distal axonal neuropathy (Spencer and Schaumberg1974). It pathogenic mechanism has been intensivelystudied (Pleasure et al. 1969; Prineas 1969; Spencerand Schaumberg 1974; Spencer and Schaumberg 1977).A demise of the metabolism in the soma results in alength-dependent dying back of the distal axon (Prineas


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1 9 6 9 ) . I m p a i r e d a x o p l a s m i c f l o w m a y r e s u l t i n p a t h o -l o g ic c h a n g e s m o s t p r o n o u n c e d i n t h e d i s t a l p a r t o f t h ea x o n ( P l e a s u r e e t a l. 1 9 69 ). A c r y l a m i d e m a y d i r e c t l yd a m a g e t he ax , m a l m e m b r a n e ( S p e n c e r a n d S c h a u m -b e r g 1 97 7). V v ' h a te v c r t h e m c c h a n i s m , t h e n e u r o ns h o u l d s u s t a i n a m e t a b o l i c s h i f t s i m i l a r t o t h a t a f t e rw a l l c ri a n d e g c n c r a t i o n a f t c r a c r y la m i d e i n t ox i c a ti o n .

T h e r o l c o f v i t a m i n B 1 2 i n n e u r o n a l m e t a b o l i s m is am a t t e r o f c o n t ro v e r s y . T h e d i s s o ci a t io n o f n e u r o l o g i c a la n d h e m a t o l o g i c a l i m p r o v e m e n t s a f t e r f o l a t e s u p p l e -m e n t i n v i t a m i n B I 2 d e f i c i e n c y ( S h o r v o n 1 98 0; L i n d e n -b a u m 1 9 8 8 ) i s u n e x p l a i n e d b e c a u s e m o s t o f t h e p re v i -o u s l y k n o w n m e t a b o l i c p a t h w a y s r e q u i r i n g B 1 2 a r et h r o u g h t r a n s m e t h y l a t i o n u t il iz i ng t e t r a h y d r o f o l a t e a n d5 ; - a d c n o s y l m c t h i o n i n e . A r e c e n t b i o c h e m i c a l s t u d y h a sd c m o n s t r a t c d t h a t m e t h y l - B l 2 a c t s d ir e c tl y a s a m e t h y ld o n o r i n D N A m e t h y l a t i o n w h i ch n o r m a l l y d o w n - r e g u -l a t e s g c n c e x p r e s s i o n ( C e d a r 1 98 8: P f o h l - L e s z k o w i c z1 90 1). I n t c r c s t i n g l y , a t h i g h c o n c e n t r a t i o n ( > 1 m M o r1 .3 4 # g / r o l L m c t h y l - B 1 2 m a y u p - r e g u l a t e g e n e t r a n -s c r i p t i o n p o s s i b l y b y c o m p e t i t i v e i n h i b i t i o n o f D N Am e t h y l a t i o n w i t h S - a d e n o s y l m e t h i o n i n e ( P f o h l - k e s z k o -w i c z 1 9 t) l t. T h c C S F l e v e l s o f v i t a m i n B 1 2 i n l o w a n dh i g h d o s c g r o u p s i n o u r s t u d y f e l l e x a c t l y i n t o t h er a n g c s t h a t d o w n - o r u p - r e g u l a t e g e n e e x p r e s s i o n , r c -s p e c t i v e l x .

A l t h o u g h t h e p r e ci s e b i o c h c m i c a l m e c h a n i s m o f a c -t io n o f u l t r a -h i g h d o s e s o f m e t h y l - B l 2 is u n k n o w n , w ei n t e r p r e t t h e p r e s e n t r e s u l t s a s r e f l e c t i n g it s i m p o r t a n tp h y s io l o g ic a c ti o n o n m e t a b o l i s m . B e c a u s e a c r y l a m i d en e u r o t o x i c i t y is n o t l i k e ly to b e t h r o u g h t h e k n o w nm e t a b o l i c p a t h w a y s o f v i ta m i n B 1 2, w e s p e c u l a t e t h a tt h e e f f e c t o f u l t ra - h i g h d o s e m e t h y l - B l 2 i s n o t l i m i t e dt o a c r y l a m i d e n c u r o p a t h y b u t i s s e e n i n o t h e r a x o n a ln e t t r o p a t h i c s .

" l' hc h i g h b u t n o t t h e l o w d o s e g r o u p s h o w e d a c c e l -c r a t c d n e r v e r e g e n e r a t i o n . T h e b i o l o g i c a c t iv i t y o fm e t h y I - B l 2 m a y n o t b e l i m i t e d t o t h a t o f a v i t a m i nw h i c h c o r re c t s m c t a b o l i c d e r a n g e m e n t s o n l y w h e n t h e r ei s a d e f i c i e n c y o f i t . M e t h y l - B 1 2 a l s o m a y h a v e ap r o f o u n d e f f e ct o n m e t a b o l i s m w h e n p r e s e n t in su f fi -c i e n t a m o u n t s . R e s u l t s o f r e c e n t c l in i c a l t r i al s ( O k a w a1 99 0) t h a t u s c d h i g h d o s e s o f v i t a m i n B 1 2 t o t r e a ts l e e p - w a k e r h y t h m d i s o r d e r h a v e i n d i c at e d i t h a s b i o -l o g ic a ct i o n c v c n in t h e n o n - d e f i c i e n t c o n d i t i o n .

14 3

W e c o n c l u d e t h a i u l t r a - h i g h d o s e s of m c t h v I - B I 2m a y p r o v i d e a t h e r a p y t h a t e n h a n c e s p e r i p h e r a l n e r v er e g e n e r a t i o n i n v a r i o u s h u m a n n c u r o p a t h i c s .Acknowledgments T h i s w o r k w a s s u p p o r t e d b y S c ie n t i li c R e s e a r c hg r a n t s ( A - (1 1 4 4 0 9 6 , C - 0 4 6 7 0 4 8 7 , A - I) 44 0 41 1 43 1 f r o m t h e , l a p a n c s c M i n -i s tr y o f E d u c a t i o n , S c i e n c e a n d C u h u r c a n d G r a n t ~ , q n - A i d f o r a m y -o t r o p h i c l a t e r al s c l er o s i s a n d p e r i p h e r a l n c u r ~ p a t h y l m m t h eJ a p a n e s e M i n i s t w o f I fe a l t h a n d W e l f a r e . W e a r c i n d e h t c d t o l )r .M . K a m e y a m a l o r h e l p f u l a d x i c e .

Re f e r e n c e sC e d a r . H . (1 9 8 8 ) D N A m e t h y l a t i o n a n d g c n c a c ti vi t_ ~ . C e l l , 5 3: 3 - 4 .t t o r o w i t z , S . tt . ( 1 0 8 9 ) T h e r a p e u t i c s l r a t e g i c s m p r o m o t i n g p e r i p h e r a l

n e r v e r e g e n e r a t i o n . M u s c l e N e p . c , 1 2: 3 1 4 - 3 2 2 .K a j i , R . , L i u , Y . , D uc kc t t . S . a nd S umne r . A . J , ( 1989) S l ow i - e c ) vc l - y

o f c e n t ra l a x o n s i n a c r y la m i d c n c u r o p a t h 3 . M u s c l e N e l w c , 1 2:S 1~ 82~.

K i m u r a , J . (1 9 S 9 ) E l c c t ro d i a g n o s i s m D i s e a s e s ~ ,f N c r ~ c a n d M a s t i c ,F . A . D a v i s , P h i l a d e l p h i a , P A .

L i n d e n b a u m , J ., H e a l t o n , E . B . , S a v a g e , I ) . G . , B r u s t , J . ( ' . M . . G a r r e t t ,T . J . , P ode l l . E . R . , M a r c e l l , P . D . , S t a b l e r . S . P . a nd A l l e n . t ) , . | 1 .( 1t )8 8 ) N e u r o p s y c h i a t r i c d i s o r d e r s c a u s e d c o b a l a m i n d e f i c ie n c y i nt h e a b s e n c e o f a n e m i a o r m a c r o c y t o s i s N e w [ ~n g l. ,1 . M c d . , 3 1 8 :1720 1728.

O k a w a , M . , M i s h i m a , f . , N a n a m i , T . , S h i n l i z u . S .. l i ji m a , S .,t l i s h i k a v , a , Y . a n d T a k a h a s h i . K . ( 10 9 () ) V i t a m i n B I 2 t r e a t m e n to f s l e e p - w a k e r h y t h m d i s o r d e r s . S le e p . 1 3 : 1 5 2 3 .

P f o h l L c s z k o w i c z , A . , K e i t h , G . a n d D i r h e i m e r . G . ( l g q l ) E f f e c t o fc o h a l a m i n d e r i v a t i v e s o n i n v i t r o e n z y m a t i c D N A m c l h y l a l i o n :m e t h y l c o b a l a m i n c a n a c t a s a m e t h y l d o n o r . B i o c h c n f is t r x . 3 0:81)45 8/)51.

P l e a s u r e , D . , M i s c h l c r . K . c t a l . (1 9 {~ 9 ) A x t m a l t r a n s p o r t t~ l p r o t e i n si n e x p e r i m c n l a l n e u r o p a l h i c s . S c i cn c c , 1 ~ : 5 2 4 5 2 5 .

P r i n c a s , J . ( 1 % 9 ) T h e p a t h o g c n c s i s o f d y i n g - h a c k p o l y n c u r o p a l h i c s .I I. A n u l t r a s t r u c t u r a l s t u d y o f e x p e r i m e n t a l a c w l a r n i d c i n t~ x i c a-t i on i n t he c a t. J . N e ur o pa t ho l . E xp . N e u r o l . , 2S : 598 ~ '~21 .

S a g a w a , N . . N a k a m u r a , K . e t a l, ( 19871 E f f c c l o l v i t a m i n 1 3 1 2d e f i c i e n l d i e t o n l u n g a n d h e p a t i c p h o s p h o l i p i d s i n t h c r a t Ic t u s .M c l h y l B I 2 . K y o t o S y m p o s i u m . K y o w a K i k a k u . T o k y o , p . 7 4 .

S e c k e l. B . R . ( 1 9 9 0) E n h a n c e m e n t c~ l p e r i p h e r a l nc l - , c r c g c n e r a t i o n .M u s c l e N e r v e , 1 3 : 7 8 5 8 0 1L

S h o r v o n , S . D . , ( ' a r n c y , M . W . P . , ( ' h a n a r m , h a n d R c y n o l d s , E . ] t .(1 98 ,1 1) T h e n e u r o p s y c h i a t w o f m c g a l o b l a s t i c a n e r n ia t . B r , M e d . , 1.,281(1(I) : 1036 1038.

S p e n c e r . I '. a n d 1 t . S c h a u m b c r g ( 1 t) 7 4 ) , ,\ r ev i e w o f a c r y l a m i d cn e u r o t o x i c i t y . I 1 . E x p e r i m e n t a l a n i m a l n c u r o t o x i c i l } a n d p a t h o -l o g i c m e c h a n i s m s . C a n . J . N e u r o l . S c i ., 1 : 1 5 1 l ~t~ .

S p e n c e r , P . a n d t t . S c h a u m b e r g ( 1 97 7 ) U l l r a s t r u c t u r a l s t u d i e s o f th ed y i n g - h a c k p r o c e s s . I V . D i f f e r e n t i a l v u l n c r a b i l i t ~ o l P N S a n d( ' N S f i b e rs in e x p e r i m e n t a l c c n t r a l - p e r i p h c r a l d i s t al ax -o n o p a t h i c s . , h N e u r o p a t h o l . E x p . N c u r o l . , 3 , : 31111 3 2 {I .

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