6 The Administration of Radioactive
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L-Cystathionine to a Human Cystinuric VINCENT
du
VIGNEAUD,
JULIAN
R.
RACHELE,
JOHN
E.
WILSON,
FRED PLUM , AND LESTER J. REED Department of Biochemistry, and Department of Medicine, Cornell University Medical College, and New York Hospital, New York 21, Ν. Y.
The present communication is concerned with experimental work car ried out some years ago, in which cystathionine labeled with radioac tive sulfur was administered to a human cystinuric (4). In 1936, Brand, Block, Kassell, and Cahill (3) advanced the hypothesis that carboxyaminopropyl-S-cysteine [later called cystathionine (2)] might be an in termediate in the formation of cysteine from methionine in the body. The synthesis of L-cystathionine (5) made it possible to subject this in teresting hypothesis to experimental test. It was first demonstrated that cystathionine may be used in lieu of dietary cystine for the growth of rats (5) and subsequently that slices and extracts of rat liver are capable of cleaving cystathionine to cysteine (5). Tracer experiments then demonstrated that the sulfur of cystathionine may be utilized for cystine synthesis by the rat (10). In the present report direct evidence is offered that L-cystathionine serves as a source of sulfur for cystine synthesis in a human cystinuric. Procedure 6
Radioactive L-cystathionine (10) (765 mg.) containing 6.85 x 10 counts per minute of S was fed to the human cystinuric patient who had served previously as the subject in an experiment demonstrating the formation of cystine from sulfur-labeled methionine (11). The same precautions were followed with regard to human experimentation in volving radioactive material as in the latter experiment. After the feeding of the cystathionine, 24-hour urine specimens were collected for 3 days and sulfur distributions were determined by the titrimetric method of Fiske (6). Cystine determinations were carried out by the procedure of Sullivan, Hess, and Howard (12). Cystine was recovered from aliquots of the urine samples, follow ing the addition of carrier cystine by the procedure described pre viously (11), and after recrystallization the isolated cystine samples gave satisfactory sulfur analysis. 35
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6.
du VIGNEAUD ET AL L-Cystathionine in a Human Cystinuric Table I. Radioactivity Partition of Sulfur in the Urine of a Human Cystinuric 6
83 0
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765 mg. of L-cystathionine containing 6.85 χ 10 c.p.m. of S administered at beginning of day 1 Percentage of Urinary Total Administered Radioactivity, Sulfur Radioactivity C.P.M. per Mg. S Sulfur Content, Mg. Fraction Day 1 Day 2 Day 3 Total sulfur Total sulfate Inorg. sulfate Neutral sulfur Cystine sulfur a
327 163 149 164 35
468 309 259 159 92
490 277 234 213 64
Day 1 Day 2 Day 3 1522 1535 1527 1511 331
4022 5460 6196 1227 95
41 23 23 64 2
Day 1 Day 2 Day 3 7.3 3.7 3.3 3.6 0.17
27.5 24.7 23.4 2.9 0.13
0.29 0.09 0.08 0.20 0.002
Radioactivity of sulfur of administered cystathionine and of urinary sulfur fractions determined in benzidine sulfate derived therefrom with use of a thin mica end window Geiger-Mliller counter.
R a d i o a c t i v i t y m e a s u r e m e n t s w e r e made upon b e n z i d i n e sulfate r e c o v e r e d f r o m the sulfur d i s t r i b u t i o n d e t e r m i n a t i o n s , a s w e l l a s on b e n z i d i n e sulfate f r o m the sulfur a n a l y s i s of the c y s t i n e s a m p l e s . T h e r e s u l t s of these m e a s u r e m e n t s a r e given i n T a b l e I . Radioactive
Sulfur
in Urine
D u r i n g the two days f o l l o w i n g the i n g e s t i o n of the l a b e l e d c y s t a t h i onine, a p p r o x i m a t e l y 35% of the a d m i n i s t e r e d r a d i o a c t i v e s u l f u r a p p e a r e d i n the u r i n e , c o m p a r e d to 16% d u r i n g the two-day p e r i o d f o l l o w i n g the i n g e s t i o n of methionine by the s a m e patient (11). O n the other hand, 0.3% of the cystathionine s u l f u r appeared i n u r i n a r y c y s t i n e , i n c o n t r a s t to 1.4% of the methionine s u l f u r . T h e c o n v e r s i o n to u r i n a r y n e u t r a l sulfur compounds i n the e a r l i e r , e x p e r i m e n t appeared to be m u c h m o r e r a p i d i n i t s c o u r s e than i n the p r e s e n t e x p e r i m e n t . W h i l e the o x i d a t i o n of methionine s u l f u r to t o t a l sulfate had p r o g r e s s e d at a f a i r l y l e v e l r a t e , i n the p r e s e n t study d u r i n g the second d a y a v e r y r a p i d r i s e i n the oxidation of cystathionine sulfur to t o t a l sulfate o c c u r r e d . A p preciable radioactivity was present also i n cystine isolated from a h y d r o l y z a t e of a s a m p l e of h a i r , c l i p p e d on the 18th day of t h i s e x p e r i ment. O u r findings i n t h i s study a r e i n h a r m o n y w i t h the concept that L cystathionine i s a n i n t e r m e d i a t e i n the f o r m a t i o n of c y s t i n e f r o m m e thionine i n m a n . D i r e c t evidence f o r the e x i s t e n c e of cystathionine i n m a n w a s p r o v i d e d by the d e m o n s t r a t i o n b y T a l l a n , M o o r e , and S t e i n (13) of the o c c u r r e n c e of L - c y s t a t h i o n i n e i n e x t r a c t s of human b r a i n . M o r e o v e r , c a s e s of human c y s t a t h i o n i n u r i a have been r e p o r t e d b y H a r r i s , P e n r o s e , and T h o m a s (9) and b y F r i m p t e r , H a y m o v i t z , and H o r w i t h (8). The l a t t e r authors have a l s o stated that a n i n c r e a s e d r e n a l c l e a r a n c e of cystathionine i s not o b s e r v e d i n c y s t i n u r i a . It i s of c o n s i d e r a b l e i n t e r e s t , however, that the m i x e d d i s u l f i d e of L - c y s t e i n e and L - h o m o -
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ADVANCES IN CHEMISTRY SERIES
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c y s t e i n e w a s found by F r i m p t e r (7) i n the u r i n e s of a l l human c y s t i n u r i c s he had e x a m i n e d s p e c i f i c a l l y f o r t h i s u n u s u a l a m i n o a c i d . I n o u r own study, had the m i x e d d i s u l f i d e e x i s t e d i n the u r i n e of o u r patient, i t w o u l d have been c l e a v e d d u r i n g the c u p r o u s m e r c a p t i d e i s o l a t i o n , i t s c y s t e i n e m o i e t y b e c o m i n g p a r t of the f i n a l l y i s o l a t e d c y s t i n e .
Literature
Cited
(1) Binkley, F., Anslow, W. P., Jr., du Vigneaud, V., J . Biol. Chem. 143, 559 (1942). (2) Binkley, F . , du Vigneaud, V., Ibid., 144, 507 (1942). (3) Brand, E . , Block, R. J., Kassell, B., Cahill, G. F . , Proc. Soc. Exptl. Biol. Med. 35, 501 (1936). (4) du Vigneaud, V., "A Trail of Research in Sulfur Chemistry and Metabolism,'' p. 53, Cornell University Press, Ithaca, Ν. Y., 1952. (5) du Vigneaud, V., Brown, G. B., Chandler, J . P., J. Biol. Chem. 143, 59 (1942). (6) Fiske, C. H., Ibid., 47, 59 (1921). (7) Frimpter, G. W., Ibid., 236, PC51 (1961). (8) Frimpter, G. W., Haymovitz, A. Horwith, M., New Eng. J. Med. 268, 333 (1963). (9) Harris, H., Penrose, L . S., Thomas, D. H. H., Ann. Human Genet. 23, 442 (1959). (10) Rachele, J. R., Reed, L . J., Kidwai, A. R., Ferger, M. F., du Vigneaud, V., J . Biol. Chem. 185, 817 (1950). (11) Reed, L . J., Cavallini, D., Plum. F., Rachele, J . R., du Vigneaud, V., Ibid., 180, 783 (1949). (12) Sullivan, M. X., Hess, W. C., Howard, H. W., Ibid., 145, 621 (1942). (13) Tallan, H. H., Moore, S., Stein, W. H., Ibid., 230, 707 (1958). Received October 7, 1963
