Austin 4 evidenced a low sulfatase. (particularly type A) activity in the brain, liver and kidney. ... metachromatic bodies in urine (Austin 1 .... Msd. 69:25-30, 1966.
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 , Svennerholm ). Moser, Moser and McKhann , working with S , showed that this accumulation is the result of a deficient degradation of the sulfatides. Austin evidenced 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 ). 2
9
l0
35
4
3
In three cases of the late juvenile type of MLD studied by Canelas, Escalante, Iriya and De Jorge the sulfur content of urine was determined. The total sulfur concentrations fell within the normal range, but the esterified sulfur was increased. 5
In this report the metabolic balances of sulfur in two of those patients are analyzed.
MATERIAL A N D METHODS T w o cousins (O. A. and M. A. R.) w i t h the late j u v e n i l e type of MLD w e r e studied. The diagnosis w a s based on the clinical picture plus the finding of m e t a c h r o m a t i c bodies in urine ( A u s t i n t e s t ) and 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 nerve, liver and kidney. The clinical and laboratorial d a t a on these cases are reported in full e l s e w h e r e (Canelas e t a l . ) . The c o n c e n t r a t i o n s of esterified sulfur in urine were, respectively, 207 and 275 m g / 2 4 hr. (normal range, 60-120 m g / 2 4 h r . ) . 1
5
P a t i e n t O. A. w a s submitted 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 both to m i x e d and v e g e t a r i a n diets, the l a t t e r c o n t a i n i n g 500 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. were 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 days { i n c l u d i n g 17 days on m i x e d diet and 20 d a y s on v e g e t a r i a n d i e t ) , respectively. E a c h b a l a n c e w a s divided in periods of from three to s i x days.
F r o m t h e D e p a r t m e n t s of N e u r o l o g y (* A s s o c i a t e P r o f e s s o r , *** I n s t r u c t o r ) a n d M e d i c i n e (** Chief of L a b o r a t o r y ) , U n i v e r s i t y of S ã o P a u l o S c h o o l of Medicine, São Paulo, Brazil
At the end of e a c h period faeces and a l i m e n t a r y residues, w i c h were collected in g l a s s c o n t a i n e r s kept in a refrigerator, w e r e weighed, mixed 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 material w e r e then submitted 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 the difference b e t w e e n t h e k n o w n sulfur c o n t e n t of each type of d i e t and the c o n c e n t r a t i o n in the ali m e n t a r y residues. Urine w a s collected daily under the s a m e conditions as f a e c e s , a n d t h e 24-hour v o l u m e determined. Total sulfur w a s determined by t h e f o l l o w i n g turbidimetric method S i l v a and Cintra ' ) :
(De
Jorge,
Reagents — (1) F i v e percent b a r i u m chloride solution in 0.5% polyvinylic a l c o h o l : e x a c t l y 500 m g of polyvinylic alcohol grade 52.22 are weighed, and dissolved in bidistilled w a t e r a t 70«C until a h o m o g e n o u s solution is obtained, all in a 100 ml v o l u m e t r i c flask, c o m p l e t i n g the 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 dehydrated b a r i u m chloride are w e i g h e d and dissolved in t h e 0.5% polyvinylic alcohol solution, 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 cent solution of trichloroacetic acid (10% T C A ) : 100 g m . of trichloroacetic acid R.A. are dissolved in 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 shaking. (3) Standard solution 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 gm. of anhydrous 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 during 4 hours a t 100«C and dissolved in bidistilled water, until a final v o l u m e of 1,000 ml is obtained; this s o l u t i o n c o n t a i n s 320 m g of sulfur per liter. (4) Standard solution for use: in a 100 ml v o l u m e t r i c flask 10 ml of t h e 0.01 M a m m o n i u m s u l f a t e solution are diluted w i t h bidistilled water, s h a k i n g t h e s o l u t i o n ; this solution c o n t a i n s 32 m g of sulfur per milliliter. (5) B e n e d i c t solution for sulfur: 200 m g . of crystallized copper nitrate, 5 0 gm. of s o d i u m or p o t a s s i u m chlorate and bidistilled w a t e r to 1,000 ml. Determination of total sulfur in urine — In a porcelain capsule of 50 ml capacity, 2.0 ml of urine a r e pipetted, and in a n o t h e r capsule 2.0 ml of bidistilled w a t e r are pipetted. To e a c h capsule 5.0 m l of t h e Benedict solution for sulfur are added. The m a t e r i a l is w e l l mixed, dried in a B u n s e n burner or h o t plate, p u t in a f u r n a c e regulated to 530«C and left 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 capsule is dissolved in 10.0 ml of 1:4 hydrochloric acid solution, 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 the solution filtered t h r o u g h a W h a t m a n paper no. 42. One m l of t h e filtrate is pipetted, added 3.0 ml of 5% TCA, h o m o g e n i z e d and t h e n 1.0 m l of t h e b a r i u m chloride solution is added. T h e m a t e r i a l is w e l l shaked, let stand for 10 m i n u t e s a n d the turbidity compared to a blank prepared w i t h w a t e r instead of urine, in a K l e t t - S u m m e r s o n colorimeter w i t h a N o . 42 filter, or in a spectrophotometer, in 360 micron w a v e l e n g t h , a d j u s t i n g the blank in m a x i m a l transmission. If the filtrate 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 blank should be m a d e before a d d i n g the barium r e a g e n t and a n y reading obtained should be subtracted of the reading after the addition of the reagent. Determination of total sulfur in faeces and foods — An 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 capsule and then the deter m i n a t i o n proceeds as described for t o t a l sulfur in urine.
RESULTS The results are s u m m a r i z e d in Tables 1 and 2.
COMMENTS
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 in 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). 6
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 (Austin - , Jatzkewitz ) . 3
4
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 (cerulplasmin) is lacking, copper accumulates in the body tissues, cupriuresis is high, and the blood serum contains higher levels of direct reacting (nonceruloplasmin) copper than in control subjects.
SUMMARY
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). This finding is in accordance with the current views on the pathogenesis of the disease, namely a sulfatidosis with low sulfatase activity.
RESUMO
Balanços metabólicos do enxôfre em pacientes leucodistrofia metacromática
com
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 influenciado 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. REFERENCES 1.
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