METABOLIC BALANCES OF SULFUR IN PATIENTS WITH
METACHROMATIC LEUCODYSTROPHY
HORÁCIO M . CANELAS * FRANCISCO B . DE JORGE **
WALDIR A . TOGNOLA * * *
Metachromatic leukodystrophy (MLD) is a lipidosis characterized by the
storage of sulfatides (Austin
2, Svennerholm
l 0). Moser, Moser and
Mc-Khann
9, working with
3 5S , showed that this accumulation is the result of
a deficient degradation of the sulfatides. Austin
4evidenced a low sulfatase
(particularly type A) activity in the brain, liver and kidney. In urine, the
mean arylsulfatase A specific activity of control children was found to be
47 times that of MLD children (Austin
3).
In three cases of the late juvenile type of MLD studied by Canelas,
Escalante, Iriya and De Jorge
5the sulfur content of urine was determined.
The total sulfur concentrations fell within the normal range, but the esterified
sulfur was increased.
In this report the metabolic balances of sulfur in two of those patients
are analyzed.
M A T E R I A L A N D M E T H O D S
T w o c o u s i n s (O. A. and M. A. R.) w i t h t h e l a t e j u v e n i l e type of MLD w e r e studied. T h e d i a g n o s i s w a s based on the c l i n i c a l p i c t u r e p l u s t h e finding of m e t a c h r o m a t i c bodies in urine ( A u s t i n 1
t e s t ) a n d m e t a c h r o m a t i c m a t e r i a l in biopsies of peripheral n e r v e , l i v e r a n d kidney. T h e c l i n i c a l and l a b o r a t o r i a l d a t a on t h e s e c a s e s are reported in full e l s e w h e r e ( C a n e l a s e t a l .5
) . T h e c o n c e n t r a t i o n s of esterified sulfur in u r i n e were, r e s p e c t i v e l y , 207 and 275 m g / 2 4 hr. ( n o r m a l range, 60-120 m g / 2 4 h r . ) .
P a t i e n t O. A. w a s s u b m i t t e d to a m i x e d diet c o n t a i n i n g 850 m g of sulfur e a c h day; p a t i e n t M. A. R. w a s s u b m i t t e d b o t h to m i x e d a n d v e g e t a r i a n diets, t h e l a t t e r c o n t a i n i n g 5 0 0 m g of s u l f u r by day.
P a t i e n t s O. A. and M. A. R. w e r e k e p t in m e t a b o l i c b a l a n c e for 19 and 37 d a y s { i n c l u d i n g 17 days on m i x e d diet a n d 2 0 d a y s on v e g e t a r i a n d i e t ) , r e s p e c t i v e l y . E a c h b a l a n c e w a s divided in periods of from t h r e e to s i x d a y s .
A t the end of e a c h period f a e c e s and a l i m e n t a r y residues, w i c h w e r e c o l l e c t e d i n g l a s s c o n t a i n e r s k e p t in a refrigerator, w e r e w e i g h e d , m i x e d and h o m o g e n i z e d in a W h a r i n g Bendor. D u p l i c a t e s a m p l e s of e a c h m a t e r i a l w e r e then s u b m i t t e d t o t h e a n a l y t i c a l m e t h o d described below. T h e i n g e s t a w e r e t h e d i f f e r e n c e b e t w e e n t h e k n o w n s u l f u r c o n t e n t of e a c h t y p e of d i e t and t h e c o n c e n t r a t i o n in t h e a l i -m e n t a r y residues. Urine w a s c o l l e c t e d daily under t h e s a -m e c o n d i t i o n s a s f a e c e s , a n d t h e 2 4 - h o u r v o l u m e d e t e r m i n e d .
T o t a l sulfur w a s d e t e r m i n e d by t h e f o l l o w i n g t u r b i d i m e t r i c m e t h o d ( D e J o r g e , S i l v a and Cintra ' ) :
Reagents — (1) F i v e p e r c e n t b a r i u m chloride s o l u t i o n in 0.5% p o l y v i n y l i c
a l c o h o l : e x a c t l y 500 m g of p o l y v i n y l i c a l c o h o l g r a d e 52.22 are w e i g h e d , and d i s s o l v e d in bidistilled w a t e r a t 70«C until a h o m o g e n o u s s o l u t i o n is o b t a i n e d , all i n a 100 m l v o l u m e t r i c flask, c o m p l e t i n g t h e v o l u m e w i t h bidistilled w a t e r ; t h e s o l u t i o n m u s t be h o m o g e n i z e d by s h a k i n g ; e x a c t l y 5 g m . of d e h y d r a t e d b a r i u m c h l o r i d e a r e w e i g h e d and d i s s o l v e d in t h e 0.5% p o l y v i n y l i c a l c o h o l s o l u t i o n , and t h e n filtered t h r o u g h W h a t m a n paper N o . 42. (2) Ten per c e n t s o l u t i o n of t r i c h l o r o a c e t i c acid ( 1 0 % T C A ) : 100 g m . of t r i c h l o r o a c e t i c acid R.A. are dissolved i n bidistilled w a t e r , c o m p l e t i n g the v o l u m e to 1,000 ml, and h o m o g e n i z e d by s h a k i n g . (3) S t a n d a r d s o l u t i o n of a m m o n i u m s u l f a t e 0.01 M: e x a c t l y 1.3214 g m . of a n h y d r o u s a m m o n i u m s u l f a t e are w e i g h e d and dried d u r i n g 4 h o u r s a t 100«C and dissolved in bidistilled w a t e r , u n t i l a final v o l u m e of 1,000 ml is o b t a i n e d ; this s o l u t i o n c o n t a i n s 320 m g of s u l f u r per liter. ( 4 ) S t a n d a r d s o l u t i o n for u s e : in a 100 m l v o l u m e t r i c flask 10 m l of t h e 0.01 M a m m o n i u m s u l f a t e s o l u t i o n are d i l u t e d w i t h bidistilled w a t e r , s h a k i n g t h e s o l u t i o n ; t h i s s o l u t i o n c o n t a i n s 32 m g of s u l f u r per m i l l i l i t e r . (5) B e n e d i c t s o l u t i o n for s u l f u r : 200 m g . of c r y s t a l l i z e d copper n i t r a t e , 5 0 g m . of s o d i u m or p o t a s s i u m c h l o r a t e and bidistilled w a t e r to 1,000 ml.
Determination of total sulfur in urine — In a porcelain c a p s u l e of 50 m l
c a p a c i t y , 2.0 m l of u r i n e a r e pipetted, and in a n o t h e r c a p s u l e 2.0 ml of bidistilled w a t e r a r e pipetted. To e a c h c a p s u l e 5.0 m l of t h e B e n e d i c t s o l u t i o n for sulfur are added. T h e m a t e r i a l is w e l l m i x e d , dried in a B u n s e n burner or h o t p l a t e , p u t in a f u r n a c e r e g u l a t e d to 530«C a n d l e f t o v e r n i g h t . The f u r n a c e is turned off and t h e c a p s u l e a l l o w e d to r e a c h r o o m t e m p e r a t u r e . T h e m a t e r i a l of t h e c a p s u l e is d i s s o l v e d in 10.0 m l of 1:4 hydrochloric acid s o l u t i o n , and diluted t o a final v o l u m e of 50.0 m l w i t h bidistilled w a t e r . I t is w e l l h o m o g e n i z e d and t h e s o l u t i o n filtered t h r o u g h a W h a t m a n paper no. 42. O n e m l of t h e f i l t r a t e is pipetted, added 3.0 m l of 5% TCA, h o m o g e n i z e d a n d t h e n 1.0 m l of t h e b a r i u m chloride s o l u t i o n is added. T h e m a t e r i a l is w e l l shaked, let s t a n d for 10 m i n u t e s a n d t h e turbidity compared to a b l a n k prepared w i t h w a t e r i n s t e a d of urine, in a K l e t t - S u m m e r s o n c o l o r i m e t e r w i t h a N o . 42 filter, or in a s p e c t r o p h o t o m e t e r , in 360 m i c r o n w a v e l e n g t h , a d j u s t i n g t h e b l a n k in m a x i m a l t r a n s m i s s i o n . If t h e f i l t r a t e is n o t c o m p l e t e l y clear, t h e r e a d i n g a g a i n s t t h e b l a n k should be m a d e before a d d i n g t h e b a r i u m r e a g e n t and a n y r e a d i n g o b t a i n e d s h o u l d be s u b t r a c t e d of t h e r e a d i n g a f t e r t h e addition of t h e r e a g e n t .
Determination of total sulfur in faeces and foods — A n e x a c t a m o u n t from
200 to 500 m g of m a t e r i a l is w e i g h e d in a porcelain c a p s u l e and t h e n t h e d e t e r -m i n a t i o n proceeds a s described for t o t a l s u l f u r in urine.
R E S U L T S
C O M M E N T S
As it is seen in Tables 1 and 2, a positive sulfur balance was evidenced
in both patients, particularly in O. A., who showed an average daily sulfur
balance of + 160 mg. In patient M.A.R., a significant difference was not
found between the balances on vegetarian ( + 5 6 ) or mixed ( + 4 6 ) diets.
These figures are definitely higher than the average found by De Jorge
and Cintra
6in 8 normal adults submitted to mixed (mean metabolic balance
of sulfur + 23.2 mg ± 17.3) or vegetarian ( + 23.5 mg ± 29.3) diets. If
the results in the two cases of MLD under mixed diet (average 103.0 mg ±
80.6) are compared with the normal values, a statistically significant
diffe-rence is found (t = 3.069; P < 0.02).
In the blood serum, the contents of inorganic sulfur were always above
the upper normal limit.
The evidence of a positive sulfur balance in these two patients with
MLD is in accordance with the facts already known on the pathogenesis of
the disease, which is essentially characterized by a storage of sulfatides,
probably due to a deficiency of the sulfatase activity ( A u s t i n
3-
4,
Jatzke-witz
8) .
This finding, allied to the deficiency of an enzyme (sulfatase), leading
to a storage of sulfatides in several body tissues, owing to deficient
degra-dation, the increased urinary excretion of esterifield sulfur, and the high
values of inorganic sulfur (sulfate) in the blood serum, build up a picture
with a striking similarity with that of hepatolenticular degeneration. In
Wilson's disease, likewise, copper absorption is raised, a copper oxidase
(ce-rulplasmin) is lacking, copper accumulates in the body tissues, cupriuresis
is high, and the blood serum contains higher levels of direct reacting
(non-ceruloplasmin) copper than in control subjects.
S U M M A R Y
The metabolic balances of sulfur in two cases of the late juvenile form
of metachromatic leucodystrophy were studied. A positive balance of sulfur
was found in both patients, apparently not influenced by the type of diet
(either mixed or vegetarian).
R E S U M O
Balanços metabólicos do enxôfre em pacientes com
leucodistrofia metacromática
Foram estudados os balanços metabólicos do enxofre em dois pacientes
com a forma juvenil da leucodistrofia metacromática. Foi verificado, em
ambos os casos, um balanço positivo desse metaloide, aparentemente não
in-fluenciado pelo tipo de dieta (geral ou vegetal).
Este resultado está de acordo com a atual concepção patogênica dessa
moléstia, que consiste, essencialmente, em uma sulfatidose com diminuição
da atividade das sulfatases.
R E F E R E N C E S
1 . A U S T I N , J . H . — M e t a c h r o m a t i c f o r m of diffuse c e r e b r a l s c l e r o s i s . D i a g n o s i s
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2 . A U S T I N , J . H . — M e t a c h r o m a t i c f o r m of diffuse c e r e b r a l s c l e r o s i s . S i g n i f i c a n c e of s u l f a t i d e a n d o t h e r lipid a b n o r m a l i t i e s in w h i t e m a t t e r a n d k i d n e y . N e u r o -l o g y ( M i n n e a p o -l i s ) 1 0 : 4 7 0 - 4 8 3 , 1960.
3 . A U S T I N , J . H . — M e t a c h r o m a t i c f o r m of diffuse c e r e b r a l s c l e r o s i s . L o w
s u l f a t a s e a c t i v i t y in t h e u r i n e of n i n e l i v i n g p a t i e n t s w i t h m e t a c h r o m a t i c l e u k o d y s t r o p h y ( M L D ) . A r c h . N e u r o l . ( C h i c a g o ) 12:447-455, 1 9 6 5 .
4 . A U S T I N , J . H . — M e t a c h r o m a t i c f o r m of diffuse c e r e b r a l s c l e r o s i s . T h e n a t u r e a n d s i g n i f i c a n c e of l o w s u l f a t a s e a c t i v i t y : a c o n t r o l l e d s t u d y of b r a i n , l i v e r a n d k i d n e y i n f o u r p a t i e n t s w i t h m e t a c h r o m a t i c l e u k o d y s t r o p h y ( M L D ) . A r c h . N e u r o l . ( C h i c a g o ) 13:593-614, 1965.
5 . C A N E L A S , H . M.; E S C A L A N T E , O. D.; I R I Y A , K. & D e J O R G E , F . B . —
L e u c o d i s t r o f i a m e t a c r o m á t i c a : e s t u d o c l í n i c o - l a b o r a t o r i a l d e c i n c o c a s o s n a m e s m a f a m í l i a . R e v . p a u l . Med. 63:355-376, 1963.
6 . D e J O R D E , F . B. & C I N T R A , A. B . U. — M e t a b o l i s m o d o e n x ô f r e . B a l a n ç o s m e t a b ó l i c o s e m a d u l t o s n o r m a i s s o b d i e t a g e r a l e s o b d i e t a e x c l u s i v a m e n t e v e g e t a l . R e v . p a u l . M s d . 69:25-30, 1966.
7 . D e J O R G E , F . B . ; S I L V A , A. G. & C I N T R A , A B . U. — D e t e r m i n a ç ã o q u a n t i -t a -t i v a d o e n x ô f r e n o s m a -t e r i a i s b i o l ó g i c o s . R e v . b r a s . Med. 21:491-494, 1964.
8 . J A T Z K E W I T Z , H . — T h e r o l e of c e r e b r o s i d e s u l p h u r i c e s t e r s in l e u c o d y s t r o p h y a n d a n e w m e t h o d for t h e q u a n t i t a t i v e u l t r a m i c r o d e t e r m i n a t i o n of t h e b r a i n
9 . M O S E R , H . W.; M O S E R , A. B . & M c K H A N N , G. M. — T h e d y n a m i c s of a l i p i d o s i s : t u r n o v e r of s u l f a t i d e , s t e r o i d s u l f a t e , a n d p o l y s a c c h a r i d e s u l f a t e i n m e t a c h r o m a t i c l e u k o d y s t r o p h y . A r c h . N e u r o l . ( C h i c a g o ) 1 7 : 4 9 4 - 5 1 1 , 1967.
1 0 . S V E N N E R H O L M , L . — S o m e a s p e c t s of t h e b i o c h e m i c a l c h a n g e s in l e u c o ¬
d y s t r o p h y . In J . F o l c h - P i & H . J . B a u e r : B r a i n L i p i d s a n d L i p o p r o t e i n s , a n d t h e L e u c o d y s t r o p h i e s , p p . 104-119. E l s e v i e r , A m s t e r d a m , 1963.
