Biochimica et Biophysica Acta, 381 (1975) 1--8
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27571 CYSTEINE METABOLISM IN VIVO OF VITAMIN B6-DEFICIENT RATS
KENJI YAMAGUCHI,SHOICHISHIGEHISA, SHIGEKISAKAKIBARA,YU HOSOKAWA and IWAOUEDA Department of Medical Chemistry, Osaka Medical College, 2--7, Daigakucho, Takatsuki, Osaka (Japan)
(Received July 8th, 1974).
Summary
The expirations of '4CO: from DL-[1-'4C] -, DL-[3-'4C] - and L-[U,4 C] cysteine used as isotopic tracers were estimated in order to determine the in vivo metabolic distribution of L-cysteine in pyridoxine deficient rats. The expired ' 4CO2 from L-[U-' 4C] cysteine was increased by pyridoxine deficiency. The loading of non-physiological dose of L-cysteine resulted in remarkable increase in the expiration of ' 4CO2 from each tracer in deficient rats as well as in controls. The in vivo metabolic distributions of L-cysteine were calculated from the expired '4CO2 from these isotopic tracers. The in vivo metabolic distribution of L-cysteine calculated showed that the remarkable lesion in taurine pathway occurred in pyridoxine deficient rats, and when non-physiological dose of L-cysteine was loaded the catabolism of L-cysteine of contro]s was markedly increased in either pyruvate or taurine pathway, whereas the Lcysteine catabolism in deficient rats was increased only in pyruvate but not in taurine pathway. The urinary excretions of 3SS.labeled metabolites such as sulfate or taurine were also examined in deficient and control rats.
Introduction As shown in Scheme I, the catabolism of cysteine in rats can occur by t w o main pathways; pyruvate (or sulfate) pa t hw ay and taurine pathway. Furthermore, two different pathways m ay be involved in the f o r m a t i o n of pyruvate f r o m cysteine. The first is a direct conversion of cysteine to pyruvate, NH3 and SH2, catalyzed by the e n z y m e cysteine desulfhydrase, and the second is an indirect f o r m a t i o n of pyr uva t e via cysteine sulfinate produced f r o m cysteine by the e n z y m e cysteine oxidase. In our l a b o r a t o r y it has been f o u n d that the cysteine oxidase of rat liver was inducible by h y d r o c o r t i s o n e , cysteine and nicotinamide [ 1 ] , and also d e m o n s t r a t e d t hat the metabolic distribution of
SerineMet hionine-~'~
Proteine•
~
Glutotnione
/ Cystgeine COA~ ~/ ~'3~)
(11 J 02
= ~Hp(~2)
• ~/1~ T. . Cysteamme CO 2 ~\~Cysteme sulfincrte T ~'p/ "" (5) I Hypotourine ~ . I Cu2 • NADI (g) -~__Cysteic
Cyst ine
NH3. SH2
"
~
Pyruvate
(8) ~ , ' ~ ~ h ~ Sulfate "w~/=J~-Sulf ini I J or ~l' pyruvatel etneral acid L ~ sulfate
Tauritne ~pA~
(6) " ~ ' ~ Tourocnolic acid
S c h e m e 1 . ( 1 ) Cysteine oxidase; (2) cysteine desulfhydrase; (3) cysteine d e c a r b o x y l a s e or p a n t o t h e n a t e cycle~ (4) cysteine sulfinate d e c a r b o x y l a s e ; (5) cysteine sulfinate d e h y d r o g c n a s e ; (6) cysteic acid decarb o x y l a s c (sazne e n z y m e to c y s t e i n e sulfinate d e c a r b o x y l a s e : (7) e y s t e a m i n e o x y g e n a s e ; (8) cysteine sulfinate desulfinicase or transaminase; (9) hypotauGdne d e h y d r o g e n a s e .
H
H-{~]- SH i H- [~-NH 2 rC']H~SO2H ^
Hypotaurine ~,, / ~ n I-~H2SO3 H
F~FI2NH2 Tau rine
Cysteme " ~
~ S~" j ~L~J- u ~ /
H - ~ - NH 2
['~=O L ~
Pyruvate
2
O-S-CoA yl-CoA [ t 2 ~]O2
/~OOH Cysteine sulfinate
Scheme 2.
L-cysteine in rats could be determined by the measurement in vivo based on the 14 COs expired from 14 C-labeled cysteine as tracers. This suggests that 31% of the degradation of L-cysteine occurred via the pyruvate pathway and 69% via the taurine pathway. The in vivo catabolism of L-cysteine was markedly stimulated by L-cysteine loading [2]. Furthermore, Sakakibara et al. presented evidence that the enzymic activity of purified cysteine oxidase from rat liver was remarkably activated by anaerobic preincubation with the substrate Lcysteine [3]. These findings strongly suggest that the enzyme cysteine oxidase may be participate in the metabolic regulation of L-cysteine catabolism through the substrate activation. On the other hand, it must be noted that cysteine is metabolized by many pyridoxal phosphate-depending enzymes, such as cysteine desulfhydrase, cysteine sulfinate decarboxylase, cysteine sulfinate aminotransferase and cysteine sulfinate desulfinicase. Some findings concerning the enzymic lesions of cysteine desulfhydrase [4] and cysteine sulfinate decarboxylase [5,6] in tissues of vitamin B6-deficient rats and the effects of vitamin B6 deficiency on the urinary or biliary metabolites from L-cysteine [5--8] and cystathionine [9,10] have been demonstrated. From these findings, it appears likely that the taurine pathway of L-cysteine catabolism in rats should be markedly inhibited by vitamin B6 deficiency. Therefore it is of interest that the abnormal metabolic situation caused by vitamin B0 deficiency will reflect on the in vivo metabolic distribution of L-cysteine measured by the in vivo measurement reported previously [2]. This study was carried out to clarify whether the in vivo measurements for L-cysteine metabolic distribution in rats will be
available to detect the in vivo metabolic alteration of L-cysteine caused by vitamin B6 deficiency and the in vivo cysteine oxidase activity in vitamin B6 -deficient rats will be activated by L-cysteine loading as well as in the control rats as previously reported [2]. Materials and Methods Pyridoxine-free diet and complete diet were obtained from Oriental Yeast Kogyo Co. The components of these diets include corn starch 38 g, vitamin free casein 25 g, wheat a-starch 10 g, filter paper powder 8 g, salad oil 6 g, cane sugar 5 g, vitamins A 1000 I.U., D3 200 I.U., B~ 2.4 rag, B2 8.0 mg, B6 1.6 mg, C 60.0 mg, E 10.0 mg biotin 0.04 mg, folic acid 0.4 mg, calcium pantophenate 10.0 mg, p-aminobenzoic acid 10.0 mg, niacin 12.0 mg, inositol 12.0 mg, choline--HC1 400 mg and minerals 6 g in 100 g of diet. Vitamin B6 was omitted in the pyridoxine-free diet. Wistar strain male albino rats weighing 130--150 g at the beginning of experiments were used. Animals were maintained with water and complete diet ad libitum for one week and then vitamin B0-deficient rats were given pyridoxine-free diet instead of complete diet. The diet ingestion of control rats was regulated by paired feeding with deficient rats. The animals were routinely used for the in vivo experiments at 5--7 weeks after feeding with pyridoxine-free diet. The injections were routinely given between 10.00 and 10.30 am by the intraperitoneal route. The urinary metabolites from cysteine were analyzed by means of column chromatography, using Dowex 50 × 8 and D o w e x 1 × 2 and the urinary content of taurine, cysteine sulfinate and cysteic acid were analyzed with HITACHI-HLB-3B amino acid analyzer as previously reported [2]. The measurement of L-cysteine catabolism in vivo was carried out as previous report using DL-[1 -~4C], DL-[3 -~4C]- and L-[U -~4C]cysteine as tracers [2]. The measurement is based on the following hypotheses: (1) the expired ~4CO2 is derived from cysteine through either the pyruvate or the taurine pathway, (2) ~4CO2 expired from DL-[1-14C]- and DL-[3 -14C]cysteine reflect the total catabolism and pyruvate pathway of DL-cysteine metabolism respectively, as shown in Scheme II, (3) taurine is produced from Lcysteine only, not from D-cysteine as demonstrated by previous reports [11,12], (4) ~4CO2 from L-[U -~ 4C] cysteine accounts for the whole portion and one third of L-cysteine metabolized via pyruvate and taurine pathways respectively, and (5) [~4C]taurine formed is not further metabolized to produce significantly detectable ~4 CO2. Based on these hypotheses, the following equations are used to calculate the metabolic distribution of L-cysteine and D-cysteine: 4 CO2 from L-cysteine via the taurine pathway ( e t a ) = 2 (Cm ax-1 - - C . . . . eta
14 CO2 from L-cysteine via the pyruvate pathway (Cpa) = Cm ax-u
3
Total metabolized L-cysteine ( C t o t a l ) = e t a -I- C p a ~4CO2 from D-isomer of DL-[1-~ 4C[ cysteine (Cd) = Cm ~x_l
Ctotal
2
3)
where C m a x - 1 is Cm ,x for DL-[1-14C] cysteine, C m a x - 3 is C m a x for DL-[314 C] cysteine, and C m ax-u is Cm ax for L-[U -14C] cysteine. The values of Cm ax and half-time of Cm ax were obtained as previously reported [2]. The metabolic turnover rate of L-cysteine (k value) was expressed as the value of Cmax.ult,/~ of C m a x . u • Results and Discussion
Measurement of Cm ~x and t,j~ for various labeled cysteine The expirations of 14CO2 from various labeled cysteine in control and vitamin B6-deficient rats were examined. As shown in Table I, when a trace a m o u n t of labeled c o m p o u n d (0.5pCi, 20--40 Ci/mole) was injected the Cm ax value for expiration from DL-[1 -I 4C] cysteine in deficient rats was 84% of that in controls but the Cm ax value from DL-[3 -I 4C]cysteine was not altered by vitamin B6 deficiency. These facts suggest that the total cysteine catabolism reflected by 14CO~ from [1-14C]cysteine was decreased but the catabolism through pyruvate pathway reflecting by 14CO2 from [3-14C] cysteine was not altered by vitamin B6 deficiency. Furthermore the C max for t4CO2 expired from L-[U-~4C]cysteine in deficient rats was increased to 130% of that in controls. This increase may be due to two possibilities; one is the increase of total catabolism of L-cysteine and the other is the shift of metabolic distribution from the taurine pathway to the pyruvate pathway, since 14 CO2 expired from L-[U-l 4 C] cysteine reflects one third of metabolized cysteine through the taurine pathway, whereas ~4 CO2 from the pyruvate pathway reflects an equivalent portion of metabolized cysteine. However it appears unlikely that the total catabolism of L-cysteine will be increased in deficient rats since the urinary excretions of 3 s S metabolites such as taurine or sulfate in deficient rats TABLE
I
THE EFFECT EXCRETION
OF L-CYSTEINE L O A D I N G O N T H E 14CO 2 E X P I R A T I O N AND ON THE URINARY OF 14C-METABOLITES FROM VARIOUS LABELED CYSTEINE IN INTACT RATS
The maximum 1 4 C O 2 e x p i r a t i o n ( C m a x ) a n d t h e u r i n a r y 14C e x c r e t i o n w e r e e x p r e s s e d as p e r c e n t o f t h e a d m i n i s t e r e d d o s e o f r a d i o a c t i v i t y and t h e C m a x h a l f - t i m e s (t 1/2) w e r e e x p r e s s e d in h o u r s . N u m b e r s o f a n i m a l s a r e given in t h e p a r e n t h e s e s . Labeled compound
Dose of loading of non-labeled L-cysteine (mg/100 g body wt) 0
DL-[1-14C]Cysteine
Cma x (%)
tl/2 DL-[3-14C] Cysteine
U r i n a r y 14C (%) Cma x (5)
L-[U-14C] Cysteine
U r i n a r y 14C C m a x (%)
tl/2 tl/2 U r i n a r y 14C ( % )
k (Cmax/tl/2)*
41.8 1.4 4.9 31.4 1.8 8.7 16.3 2.1 5.3 7.8
100 + 1.38 -+ 0 . 0 3 + 0.18 +- 0 . 8 1 -+ 0 . 0 7 + 0.63 -+ 0 . 9 7 -+ 0 . 0 5 +- 0 . 7 3
* The k values represent the in vivo turnover rates of L-cysteine.
(4) (4) (3) (5) (5) (3) (4) (4) (4)
61.0 1.5 6.0 35.9 2.0 11.4 40.9 1.5 8.8 27.3
+ 1.47 -+ 0 . 0 5 + 0.35 +- 1 . 1 7 +- 0 . 0 0 -+ 1 . 4 1 + 1,27 +- 0 . 0 1 +- 0 . 5 4
(3) (3) (2) (2) (2) (2) (4) (4) (3)
5 T A B L E II T H E E F F E C T O F L - C Y S T E I N E L O A D I N G ON T H E 14CO 2 E X P I R A T I O N A N D T H E U R I N A R Y EXC R E T I O N O F 1 4 C - M E T A B O L I T E S F R O M V A R I O U S L A B E L E D C Y S T E I N E IN V I T A M I N B6-DEFICIENT RATS T h e n u m b e r o f a n i m a l s is given in t h e p a r e n t h e s e s . Labeled compound
Dose of loading of non-labeled L-cysteine (rag/100 g body wt)
0 DL-[1-14C]Cysteine
C m a x (%)
tl/2 DEr[3-14C] C y s t e i n e
U r i n a r y 14CO2 (%) C m a x (%)
L-[U-14C] C y s t e i n e
U r i n a r y 14CO2 (%) C m a x (%)
tl/2 tl/2 U r i n a r y 14CO2 (%)
k (Cmax/tl/2)*
35.1 1.5 3.8 31.3 2.2 7.0 21.3 2.3 4.6 9.3
100 + ± ± ± + ± ± + ±
3.82 0.04 0.72 0.88 0.18 0.95 0.70 0.04 0.07
(2) (2) (2) (2) (2) (2) (2) (2) (2)
49.7 1.8 5.8 46.5 2.2 10.2 51.3 2.2 7.3 23.3
± + ± + ± + + ± ±
0.70 0.17 0.09 1.94 0.14 0.80 1.81 0.83 0.47
(3) (3) (3) (3) (3) (3) (3) (3) (3)
* T h e k values r e p r e s e n t t h e in vivo t u r n o v e r rate o f L - c y s t e i n e .
were almost equal to those of controls as s h o w n in Table IV. Therefore the latter possibility appears more likely to account for the increase of Cm ax for L-[U -14C] cysteine. As s h o w n in Tables I and II, when non-physiological doses of L-cysteine were loaded, the Cm ax for DL-[1 -l 4C] cysteine was increased by 15% over the unloading level in either deficient or control rats, whereas the Cm a x for D L - [ 3 - l a C]-cysteine was much higher increased in deficient rats by 15.2% over the unloading level than did in controls by 4.5% over. The Cm ax for L - [ U - 1 4 C ] c y s t e i n e in deficient rats was increased by 30% over the basal level by L-cysteine loading while the increase in control rats was 24%. On the other hand, the metabolic turn-over rate of L-cysteine, expressed as the k value, was also increased by L-cysteine loading, to 3.5- and 2.5-fold of unloading level in control and deficient rats respectively. According to the equations as mentioned above, the metabolic distribuT A B L E III M E T A B O L I C D I S T R I B U T I O N O F 14CO2 F R O M L A B E L E D L - C Y S T E I N E I N V I V O O F I N T A C T A N D VITAMIN B6-DEFICIENT RATS T h e 14 CO 2 e v o l u t i o n s w e r e e x p r e s s e d as p e r c e n t o f r a d i o a c t i v i t y o f L-[ 14 C ] c y s t e i n e a d m i n i s t e r e d . M e t a b o l i c d i s t r i b u t i o n s w e r e c a l c u l a t e d w a s d e s c r i b e d in the t e x t . Animals
Control V i t a m i n B6-de f i c i e n t
Loading dose of L-cysteine (mg/100 g body wt)
Metabolic pathway Total (Ctotal)
Pyruvate pathway (Cpa)
Taurine pathway (Cta)
none 100 none 100
30.2 74.4 26.4 55.8
9.4 24.2 18.8 49.2
20.8 50.2 7.6 6.4
14CO2 f r o m D-cysteine (Cd)
26.7 23.8 21.9 21.8
tions of L-cysteine and D-cysteine in deficient rats were calculated. As shown in Table III, when L-cysteine was not loaded, the ratio of taurine to pyruvate pathways in deficient rats remarkably decreased from 2.3 to 0.4 in control rats. It must be n o t e w o r t h y that when non-physiological doses of L-cysteine were loaded the pyruvate pathway of L-cysteine was increased aroung 2.6-fold either in controls or in deficient rats. This enhancement of pyruvate pathway of L-cysteine must be due to the stimulation of cysteine oxidase activity, but not of cysteine desulfhydrase activity, since the hepatic desulfhydrase activity is markedly suppressed in vitamin B6-deficient rats, and the enzyme activity was not induced by in vivo administration of L-cysteine, while the activity of cysteine oxidase in vitamin B6 -deficient rats was markedly induced as well as in controls as previously reported {data not shown) [1]. These facts also support the hypothesis, presented in a previous report [2], that the major catabolic route of L-cysteine in rats is through cysteine sulfinate as an obligate intermediate but the conversion of L-cysteine to pyruvate via the direct desulfhydration seems to be less significant. Therefore, it appears likely that the catabolic pathway of L-cysteine through cysteine sulfinate formation catalyzed by the enzyme cysteine oxidase plays a major role in cysteine catabolism in vitamin B6-deficient rats as well as in controls. As shown in Table III, in the deficient rats a minor portion of taurine pathway, 25% of controls, was detected by the in vivo measurement of cysteine metabolic distribution, but this taurine pathway was not increased by the L-cysteine loading, in contrast to the controls. These facts strongly suggest that the taurine formation in vitamin Bs-deficient rats may be catalyzed by an u n k n o w n enzyme system other than by the pathway via cysteine sulfinate catalyzed by cysteine oxidase. In this regard, an alternative taurine formation from L-cysteine reported by Cavallini et al. [13] and Dupr~ et al. [14--16] is of interest. They have demonstrated a possibility of the conversion of L-cysteine to hypotaurine through a hypothetical pathway, the pantotheic acid cycle involving pantothenate-4-phosphate, pantothenoylcysteine-4-phosphate, pantotheine-4-phosphate, pantotheine and cysteamine as intermediates in the cycle, connecting with hypotaurine formation through the action of cysteamine oxygenase. The 14CO 2 expired from D-cysteine was calculated by the equation mentioned above. As shown in Table III, 14CO2 derived from D-cysteine of D L[ i 4 C] cysteine used as tracers was 26.7% and 21.9%, in controls and in vitamin B6-deficient rats respectively. These values were not altered by L-cysteine loading either in controls or deficient rats. Furthermore, the loading of non-physiological doses (50 mg per 100 g of body weight) of D-cysteine did not increase the C m a x value for L-[U-~4C]cysteine in either controls or deficient rats as shown in Table IV. These facts strongly suggest that, in either deficient or control rats, D-cysteine must be converted to pyruvate via separate pathway from that of L-cysteine.
Urinary excretion of labeled sulfate and taurine from L _[3 s S]_cysteine injected intraperitoneally The amounts of urinary taurine in deficient rats were reduced to 30% of controls whereas the incorporation of 3 s S into taurine in deficient rats was remarkably decreased to less than 10% of controls. These facts strongly suggest
T A B L E IV T H E E F F E C T O F D - C Y S T E I N E L O A D I N G ON T H E 14CO2 E X P I R A T I O N F R O M V A R I O U S L A B E L E D C Y S T E I N E IN V I T A M I N B 6 - D E F I C I E N T R A T S T h e e x p e r i m e n t s w e r e c a r r i e d o u t u n d e r t h e s a m e c o n d i t i o n s as in T a b l e s I a n d II e x c e p t t h a t t h e l o a d i n g was of D - c y s t e i n e ( 5 0 m g / 1 0 0 g b o d y w e i g h t ) in p l a c e of L - c y s t e i n e . The n u m b e r of a n i m a l s is given in p a r e n t h e s e s . Labeled compound
C m a x value (%)
t 1/2 (h)
DL-[ 1-14C] C y s t e i n e DL-[3-14C] Cysteine L-[U-14C] C y s t e i n e
3 2 . 8 (1) 2 2 . 4 (1) 2 2 . 0 (1)
1.8 2.9 2.2
that the de novo synthesis of taurine from L-cysteine must be highly inhibited by vitamin B6 deficiency and a part of urinary taurine excreted in the deficient rats may be derived from exogenous source such as diet or from the production by intestinal bacteria suggested by Bergeret and Chatagner [8]. The similar situations as the changes in the taurine pathway measured by in vivo metabolic distribution of L-cysteine were also observed in urinary excretion of taurine. Thus the increases of urinary taurine and of the incorporation of radioactivity into urinary taurine from L-[ 3 s S] cysteine were not mediated by the loading of non-physiological dose of L-cysteine in vitamin B6-deficient rats in contrast to the controls as shown in Table V. On the other hand, the amounts and incorporations of 3 s S of urinary sulfate from L-[ 3 ss] cysteine were highly increased by L-cysteine loading in the deficient rats as well as in controls. These facts also TABLE V T H E U R I N A R Y I N C O R P O R A T I O N O F 35S I N T O T A U R I N E A N D I N O R G A N I C S U L F A T E L-[3SS] C Y S T E I N E A N D T H E U R I N A R Y C O N T E N T S
FROM
U r i n e w a s c o l l e c t e d for 24 h a f t e r i n t r a p e r i t o n e a l i n j e c t i o n of 0 . 5 #Ci o f L-[ 3SS] c y s t e i n e ( 2 0 - - 5 0 C i / m o l e ) w i t h or w i t h o u t L - c y s t e i n e ( 1 0 0 m g p e r 100 g b o d y w e i g h t ) . T h e p H of t h e c o l l e c t e d u r i n e was a d j u s t e d to 8.5 a n d f r a c t i o n a t e d b y c o l u m n c h r o m a t o g r a p h y w i t h D o w e x 5 0 - X 8 (H +) a n d the e f f l u e n t s f r o m t h e c o l u m n w e r e t r e a t e d w i t h b a r i u m a c e t a t e t o p r e c i p i t a t e i n o r g a n i c sulfate. A n a l i q u o t of t h e e f f l u e n t s was used f o r t h e analysis o f t a u r i n e c o n t e n t s e s t i m a t e d w i t h an a u t o m a t i c a m i n o acid a n a l y z e r . T h e i n c o r p o r a tion of 35S w a s e x p r e s s e d as p e r c e n t of the a d m i n i s t e r e d d o s e of r a d i o a c t i v i t y . T h e n u m b e r o f a n i m a l s axe given in p a r e n t h e s e s . T h e r a d i o a c t i v i t y in s u p e r n a t a n t s of the e f f l u e n t s a f t e r b a r i u m a c e t a t e t r e a t m e n t r e p r e s e n t s the i n c o r p o r a t i o n of 35S i n t o u r i n a r y t a u r i n c . Treatment
Total pmoles/ day
C o n t r o l rats None L-Cysteine loading D e f i c i e n t rats None L-Cysteine loading
Taurine
Sulfate
%
#moles/ day
%
#moles/ day
431.8
19.9
1624.4
59.9
1 1 5 . 2 ± 6.6 (3) 385.0 ± 28.4 (4)
5.7 ± 0.6 (3) 9.2 ± 0.7 (3)
3 1 6 . 6 ± 5.4 (3) 1239.4 ± 67.6 (5)
14.2 + 0.9 (3) 50.3 ± 2.7 (2)
351.5
16.9
1481.4
62.5
6 1 . 5 ± 8.4 (4) 81.3 ± 10.2 (3)
0.3 ± 0.07 (2) 0.4 ± 0.14 (2)
2 9 0 . 0 ± 21.5 (5) 1400.1 ± 74.9 (4)
16.6 ± 2.9 (3) 62.1 ± 4.8 (2)
support the above hypothesis, in which sulfate is converted from L-cysteine via cysteine sulfinate as an obligate intermediate, in either control or vitamin B6-deficient rats, but the taurine formation observed in vitamin B6-deficient rats may be catalyzed by an other pathway than via cysteine sulfinate, since if taurine is formed from L-cysteine via cysteine sulfinate in vitamin B6 -deficient rats as well as in controls, the excretion and 3 s S.incorporatio n of urinary taurine should be increased by the induced activity of cysteine oxidase mediated by the L-cysteine loading as well as urinary sulfate. From findings presented here, it is concluded that the abnormality of in vivo metabolic distribution of L-cysteine can be detected by the in vivo measurement of L-cysteine metabolism, and that the metabolism of L-cysteine in vitamin B6-deficient rats is characterized by remarkable lesion in the taurine pathway, and an u n k n o w n alternative pathway of taurine formation from L-cysteine other than via cysteine sulfinate may occur. However, the taurine pathway observed in vitamin B6-deficient rats was not stimulated by the nonphysiological dose of L-cysteine, in contrast to the controls. The metabolic turnover rate of L-cysteine represented as the k value was remarkably stimulated by the non-physiological dose of L-cysteine loaded in either control rats or vitamin B6 -deficient rats. Acknowledgements We are grateful to Dr Y. Sakamoto, Institute of Cancer Research, Osaka University Medical School, for his useful advices and encouragement. The able technical assistance of Mrs K. Tagawa is also gratefully acknowledged. This study is supported in part by research grants from the Scientific Research Fund of the Ministry of Education of Japan (No. 757045), from Tanabe Amino Acids Research Fund and from Kanae Shinyaku Kenkyu-kai Fund. The material herein was taken in part from the dissertation submitted to Osaka Medical College by Shioichi Shigehisa in partial fulfillment of the requirements for the Doctor of Science in Medicine. References 1 Y a m a g u c h i , K., S a k a k i b a r a , S., K o g a , K . a n d U e d a , I. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 3 7 , 5 0 2 2 Y a m a g u c h i , K . , S a k a k i b a r a , S., A s a m i z u , J. a n d U e d a , I, ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 2 9 7 , 4 8 3 S a k a k i b a r a , S., Y a m a g u c h i , K . , U e d a , I. a n d S a k a m o t o , Y. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 52, 1093 4 T h o m p s o n , R . Q . a n d G u e r r a n t , N.B. ( 1 9 5 3 ) J. N u t r . 5 0 , 1 6 1 5 B l a s c h k o , H., D a t t a , S.P. a n d H a r r i s , H. ( 1 9 5 3 ) Br. J. N u t r . 7, 3 6 4 6 C h a t a g n e r , F., T a b e c h i a n , H. a n d B e r g e r e t , B. ( 1 9 5 4 ) B i o c h i m . B i o p h y s . A c t a 1 3 , 3 1 3 7 B e r g e r e t , B., C h a t a g n e r , F. a n d F r o m a g e o t , C. ( 1 9 5 5 ) B i o c h i m . B i o p h y s . A c t a 1 7 , 1 28 8 B e r g e r e t , B. a n d C h a t a g n e r , F. ( 1 9 5 6 ) B i o c h i m . B i o p h y s . A c t a 2 2 , 2 7 3 9 H o p e , D.B. ( 1 9 5 7 ) B i o c h e m . J. 6 6 , 4 8 6 1 0 B r o w n , F.C. a n d G o r d o n , P.H. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 3 0 , 4 3 4 11 E w e t z , L. a n d S 6 r b o , B. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 8 , 2 9 6 1 2 Cavallini, D., M a r c o , C. a n d M o n d o v i , B. ( 1 9 5 8 ) J. Biol. C h e m . 2 3 0 , 2 5 1 3 Cavallini, D., D u p r 6 , S., G r a z i a n i , M.T. a n d T i n t i , M.G. ( 1 9 6 8 ) F E B S L e t t . 1, 1 1 9 1 4 D u p r 4 , S., G r a z i a n i , M . T . , R o s e i , M . A . , F a b i , A. a n d Del G r o s s o , E. ( 1 9 7 0 ) E u r . J. B i o c h e m . 1 6 , 5 7 1 1 5 D u p r 6 , S., R o s e i , M . A . , BeUussi, L., B a r b o n i , E. a n d S c a n d u r r a , R. ( 1 9 7 3 ) P r o c . 9 t h Int. C o n g . Biochem. 98 1 6 D u p r ~ , S., R o s e i , M . A . , BeUussi, L., G r o s s o , E . D . a n d C a v a n i n i , D. ( 1 9 7 3 ) E u r . J. B i o c h e m . 4 0 , 1 0 3
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27571 CYSTEINE METABOLISM IN VIVO OF VITAMIN B6-DEFICIENT RATS
KENJI YAMAGUCHI,SHOICHISHIGEHISA, SHIGEKISAKAKIBARA,YU HOSOKAWA and IWAOUEDA Department of Medical Chemistry, Osaka Medical College, 2--7, Daigakucho, Takatsuki, Osaka (Japan)
(Received July 8th, 1974).
Summary
The expirations of '4CO: from DL-[1-'4C] -, DL-[3-'4C] - and L-[U,4 C] cysteine used as isotopic tracers were estimated in order to determine the in vivo metabolic distribution of L-cysteine in pyridoxine deficient rats. The expired ' 4CO2 from L-[U-' 4C] cysteine was increased by pyridoxine deficiency. The loading of non-physiological dose of L-cysteine resulted in remarkable increase in the expiration of ' 4CO2 from each tracer in deficient rats as well as in controls. The in vivo metabolic distributions of L-cysteine were calculated from the expired '4CO2 from these isotopic tracers. The in vivo metabolic distribution of L-cysteine calculated showed that the remarkable lesion in taurine pathway occurred in pyridoxine deficient rats, and when non-physiological dose of L-cysteine was loaded the catabolism of L-cysteine of contro]s was markedly increased in either pyruvate or taurine pathway, whereas the Lcysteine catabolism in deficient rats was increased only in pyruvate but not in taurine pathway. The urinary excretions of 3SS.labeled metabolites such as sulfate or taurine were also examined in deficient and control rats.
Introduction As shown in Scheme I, the catabolism of cysteine in rats can occur by t w o main pathways; pyruvate (or sulfate) pa t hw ay and taurine pathway. Furthermore, two different pathways m ay be involved in the f o r m a t i o n of pyruvate f r o m cysteine. The first is a direct conversion of cysteine to pyruvate, NH3 and SH2, catalyzed by the e n z y m e cysteine desulfhydrase, and the second is an indirect f o r m a t i o n of pyr uva t e via cysteine sulfinate produced f r o m cysteine by the e n z y m e cysteine oxidase. In our l a b o r a t o r y it has been f o u n d that the cysteine oxidase of rat liver was inducible by h y d r o c o r t i s o n e , cysteine and nicotinamide [ 1 ] , and also d e m o n s t r a t e d t hat the metabolic distribution of
SerineMet hionine-~'~
Proteine•
~
Glutotnione
/ Cystgeine COA~ ~/ ~'3~)
(11 J 02
= ~Hp(~2)
• ~/1~ T. . Cysteamme CO 2 ~\~Cysteme sulfincrte T ~'p/ "" (5) I Hypotourine ~ . I Cu2 • NADI (g) -~__Cysteic
Cyst ine
NH3. SH2
"
~
Pyruvate
(8) ~ , ' ~ ~ h ~ Sulfate "w~/=J~-Sulf ini I J or ~l' pyruvatel etneral acid L ~ sulfate
Tauritne ~pA~
(6) " ~ ' ~ Tourocnolic acid
S c h e m e 1 . ( 1 ) Cysteine oxidase; (2) cysteine desulfhydrase; (3) cysteine d e c a r b o x y l a s e or p a n t o t h e n a t e cycle~ (4) cysteine sulfinate d e c a r b o x y l a s e ; (5) cysteine sulfinate d e h y d r o g c n a s e ; (6) cysteic acid decarb o x y l a s c (sazne e n z y m e to c y s t e i n e sulfinate d e c a r b o x y l a s e : (7) e y s t e a m i n e o x y g e n a s e ; (8) cysteine sulfinate desulfinicase or transaminase; (9) hypotauGdne d e h y d r o g e n a s e .
H
H-{~]- SH i H- [~-NH 2 rC']H~SO2H ^
Hypotaurine ~,, / ~ n I-~H2SO3 H
F~FI2NH2 Tau rine
Cysteme " ~
~ S~" j ~L~J- u ~ /
H - ~ - NH 2
['~=O L ~
Pyruvate
2
O-S-CoA yl-CoA [ t 2 ~]O2
/~OOH Cysteine sulfinate
Scheme 2.
L-cysteine in rats could be determined by the measurement in vivo based on the 14 COs expired from 14 C-labeled cysteine as tracers. This suggests that 31% of the degradation of L-cysteine occurred via the pyruvate pathway and 69% via the taurine pathway. The in vivo catabolism of L-cysteine was markedly stimulated by L-cysteine loading [2]. Furthermore, Sakakibara et al. presented evidence that the enzymic activity of purified cysteine oxidase from rat liver was remarkably activated by anaerobic preincubation with the substrate Lcysteine [3]. These findings strongly suggest that the enzyme cysteine oxidase may be participate in the metabolic regulation of L-cysteine catabolism through the substrate activation. On the other hand, it must be noted that cysteine is metabolized by many pyridoxal phosphate-depending enzymes, such as cysteine desulfhydrase, cysteine sulfinate decarboxylase, cysteine sulfinate aminotransferase and cysteine sulfinate desulfinicase. Some findings concerning the enzymic lesions of cysteine desulfhydrase [4] and cysteine sulfinate decarboxylase [5,6] in tissues of vitamin B6-deficient rats and the effects of vitamin B6 deficiency on the urinary or biliary metabolites from L-cysteine [5--8] and cystathionine [9,10] have been demonstrated. From these findings, it appears likely that the taurine pathway of L-cysteine catabolism in rats should be markedly inhibited by vitamin B6 deficiency. Therefore it is of interest that the abnormal metabolic situation caused by vitamin B0 deficiency will reflect on the in vivo metabolic distribution of L-cysteine measured by the in vivo measurement reported previously [2]. This study was carried out to clarify whether the in vivo measurements for L-cysteine metabolic distribution in rats will be
available to detect the in vivo metabolic alteration of L-cysteine caused by vitamin B6 deficiency and the in vivo cysteine oxidase activity in vitamin B6 -deficient rats will be activated by L-cysteine loading as well as in the control rats as previously reported [2]. Materials and Methods Pyridoxine-free diet and complete diet were obtained from Oriental Yeast Kogyo Co. The components of these diets include corn starch 38 g, vitamin free casein 25 g, wheat a-starch 10 g, filter paper powder 8 g, salad oil 6 g, cane sugar 5 g, vitamins A 1000 I.U., D3 200 I.U., B~ 2.4 rag, B2 8.0 mg, B6 1.6 mg, C 60.0 mg, E 10.0 mg biotin 0.04 mg, folic acid 0.4 mg, calcium pantophenate 10.0 mg, p-aminobenzoic acid 10.0 mg, niacin 12.0 mg, inositol 12.0 mg, choline--HC1 400 mg and minerals 6 g in 100 g of diet. Vitamin B6 was omitted in the pyridoxine-free diet. Wistar strain male albino rats weighing 130--150 g at the beginning of experiments were used. Animals were maintained with water and complete diet ad libitum for one week and then vitamin B0-deficient rats were given pyridoxine-free diet instead of complete diet. The diet ingestion of control rats was regulated by paired feeding with deficient rats. The animals were routinely used for the in vivo experiments at 5--7 weeks after feeding with pyridoxine-free diet. The injections were routinely given between 10.00 and 10.30 am by the intraperitoneal route. The urinary metabolites from cysteine were analyzed by means of column chromatography, using Dowex 50 × 8 and D o w e x 1 × 2 and the urinary content of taurine, cysteine sulfinate and cysteic acid were analyzed with HITACHI-HLB-3B amino acid analyzer as previously reported [2]. The measurement of L-cysteine catabolism in vivo was carried out as previous report using DL-[1 -~4C], DL-[3 -~4C]- and L-[U -~4C]cysteine as tracers [2]. The measurement is based on the following hypotheses: (1) the expired ~4CO2 is derived from cysteine through either the pyruvate or the taurine pathway, (2) ~4CO2 expired from DL-[1-14C]- and DL-[3 -14C]cysteine reflect the total catabolism and pyruvate pathway of DL-cysteine metabolism respectively, as shown in Scheme II, (3) taurine is produced from Lcysteine only, not from D-cysteine as demonstrated by previous reports [11,12], (4) ~4CO2 from L-[U -~ 4C] cysteine accounts for the whole portion and one third of L-cysteine metabolized via pyruvate and taurine pathways respectively, and (5) [~4C]taurine formed is not further metabolized to produce significantly detectable ~4 CO2. Based on these hypotheses, the following equations are used to calculate the metabolic distribution of L-cysteine and D-cysteine: 4 CO2 from L-cysteine via the taurine pathway ( e t a ) = 2 (Cm ax-1 - - C . . . . eta
14 CO2 from L-cysteine via the pyruvate pathway (Cpa) = Cm ax-u
3
Total metabolized L-cysteine ( C t o t a l ) = e t a -I- C p a ~4CO2 from D-isomer of DL-[1-~ 4C[ cysteine (Cd) = Cm ~x_l
Ctotal
2
3)
where C m a x - 1 is Cm ,x for DL-[1-14C] cysteine, C m a x - 3 is C m a x for DL-[314 C] cysteine, and C m ax-u is Cm ax for L-[U -14C] cysteine. The values of Cm ax and half-time of Cm ax were obtained as previously reported [2]. The metabolic turnover rate of L-cysteine (k value) was expressed as the value of Cmax.ult,/~ of C m a x . u • Results and Discussion
Measurement of Cm ~x and t,j~ for various labeled cysteine The expirations of 14CO2 from various labeled cysteine in control and vitamin B6-deficient rats were examined. As shown in Table I, when a trace a m o u n t of labeled c o m p o u n d (0.5pCi, 20--40 Ci/mole) was injected the Cm ax value for expiration from DL-[1 -I 4C] cysteine in deficient rats was 84% of that in controls but the Cm ax value from DL-[3 -I 4C]cysteine was not altered by vitamin B6 deficiency. These facts suggest that the total cysteine catabolism reflected by 14CO~ from [1-14C]cysteine was decreased but the catabolism through pyruvate pathway reflecting by 14CO2 from [3-14C] cysteine was not altered by vitamin B6 deficiency. Furthermore the C max for t4CO2 expired from L-[U-~4C]cysteine in deficient rats was increased to 130% of that in controls. This increase may be due to two possibilities; one is the increase of total catabolism of L-cysteine and the other is the shift of metabolic distribution from the taurine pathway to the pyruvate pathway, since 14 CO2 expired from L-[U-l 4 C] cysteine reflects one third of metabolized cysteine through the taurine pathway, whereas ~4 CO2 from the pyruvate pathway reflects an equivalent portion of metabolized cysteine. However it appears unlikely that the total catabolism of L-cysteine will be increased in deficient rats since the urinary excretions of 3 s S metabolites such as taurine or sulfate in deficient rats TABLE
I
THE EFFECT EXCRETION
OF L-CYSTEINE L O A D I N G O N T H E 14CO 2 E X P I R A T I O N AND ON THE URINARY OF 14C-METABOLITES FROM VARIOUS LABELED CYSTEINE IN INTACT RATS
The maximum 1 4 C O 2 e x p i r a t i o n ( C m a x ) a n d t h e u r i n a r y 14C e x c r e t i o n w e r e e x p r e s s e d as p e r c e n t o f t h e a d m i n i s t e r e d d o s e o f r a d i o a c t i v i t y and t h e C m a x h a l f - t i m e s (t 1/2) w e r e e x p r e s s e d in h o u r s . N u m b e r s o f a n i m a l s a r e given in t h e p a r e n t h e s e s . Labeled compound
Dose of loading of non-labeled L-cysteine (mg/100 g body wt) 0
DL-[1-14C]Cysteine
Cma x (%)
tl/2 DL-[3-14C] Cysteine
U r i n a r y 14C (%) Cma x (5)
L-[U-14C] Cysteine
U r i n a r y 14C C m a x (%)
tl/2 tl/2 U r i n a r y 14C ( % )
k (Cmax/tl/2)*
41.8 1.4 4.9 31.4 1.8 8.7 16.3 2.1 5.3 7.8
100 + 1.38 -+ 0 . 0 3 + 0.18 +- 0 . 8 1 -+ 0 . 0 7 + 0.63 -+ 0 . 9 7 -+ 0 . 0 5 +- 0 . 7 3
* The k values represent the in vivo turnover rates of L-cysteine.
(4) (4) (3) (5) (5) (3) (4) (4) (4)
61.0 1.5 6.0 35.9 2.0 11.4 40.9 1.5 8.8 27.3
+ 1.47 -+ 0 . 0 5 + 0.35 +- 1 . 1 7 +- 0 . 0 0 -+ 1 . 4 1 + 1,27 +- 0 . 0 1 +- 0 . 5 4
(3) (3) (2) (2) (2) (2) (4) (4) (3)
5 T A B L E II T H E E F F E C T O F L - C Y S T E I N E L O A D I N G ON T H E 14CO 2 E X P I R A T I O N A N D T H E U R I N A R Y EXC R E T I O N O F 1 4 C - M E T A B O L I T E S F R O M V A R I O U S L A B E L E D C Y S T E I N E IN V I T A M I N B6-DEFICIENT RATS T h e n u m b e r o f a n i m a l s is given in t h e p a r e n t h e s e s . Labeled compound
Dose of loading of non-labeled L-cysteine (rag/100 g body wt)
0 DL-[1-14C]Cysteine
C m a x (%)
tl/2 DEr[3-14C] C y s t e i n e
U r i n a r y 14CO2 (%) C m a x (%)
L-[U-14C] C y s t e i n e
U r i n a r y 14CO2 (%) C m a x (%)
tl/2 tl/2 U r i n a r y 14CO2 (%)
k (Cmax/tl/2)*
35.1 1.5 3.8 31.3 2.2 7.0 21.3 2.3 4.6 9.3
100 + ± ± ± + ± ± + ±
3.82 0.04 0.72 0.88 0.18 0.95 0.70 0.04 0.07
(2) (2) (2) (2) (2) (2) (2) (2) (2)
49.7 1.8 5.8 46.5 2.2 10.2 51.3 2.2 7.3 23.3
± + ± + ± + + ± ±
0.70 0.17 0.09 1.94 0.14 0.80 1.81 0.83 0.47
(3) (3) (3) (3) (3) (3) (3) (3) (3)
* T h e k values r e p r e s e n t t h e in vivo t u r n o v e r rate o f L - c y s t e i n e .
were almost equal to those of controls as s h o w n in Table IV. Therefore the latter possibility appears more likely to account for the increase of Cm ax for L-[U -14C] cysteine. As s h o w n in Tables I and II, when non-physiological doses of L-cysteine were loaded, the Cm ax for DL-[1 -l 4C] cysteine was increased by 15% over the unloading level in either deficient or control rats, whereas the Cm a x for D L - [ 3 - l a C]-cysteine was much higher increased in deficient rats by 15.2% over the unloading level than did in controls by 4.5% over. The Cm ax for L - [ U - 1 4 C ] c y s t e i n e in deficient rats was increased by 30% over the basal level by L-cysteine loading while the increase in control rats was 24%. On the other hand, the metabolic turn-over rate of L-cysteine, expressed as the k value, was also increased by L-cysteine loading, to 3.5- and 2.5-fold of unloading level in control and deficient rats respectively. According to the equations as mentioned above, the metabolic distribuT A B L E III M E T A B O L I C D I S T R I B U T I O N O F 14CO2 F R O M L A B E L E D L - C Y S T E I N E I N V I V O O F I N T A C T A N D VITAMIN B6-DEFICIENT RATS T h e 14 CO 2 e v o l u t i o n s w e r e e x p r e s s e d as p e r c e n t o f r a d i o a c t i v i t y o f L-[ 14 C ] c y s t e i n e a d m i n i s t e r e d . M e t a b o l i c d i s t r i b u t i o n s w e r e c a l c u l a t e d w a s d e s c r i b e d in the t e x t . Animals
Control V i t a m i n B6-de f i c i e n t
Loading dose of L-cysteine (mg/100 g body wt)
Metabolic pathway Total (Ctotal)
Pyruvate pathway (Cpa)
Taurine pathway (Cta)
none 100 none 100
30.2 74.4 26.4 55.8
9.4 24.2 18.8 49.2
20.8 50.2 7.6 6.4
14CO2 f r o m D-cysteine (Cd)
26.7 23.8 21.9 21.8
tions of L-cysteine and D-cysteine in deficient rats were calculated. As shown in Table III, when L-cysteine was not loaded, the ratio of taurine to pyruvate pathways in deficient rats remarkably decreased from 2.3 to 0.4 in control rats. It must be n o t e w o r t h y that when non-physiological doses of L-cysteine were loaded the pyruvate pathway of L-cysteine was increased aroung 2.6-fold either in controls or in deficient rats. This enhancement of pyruvate pathway of L-cysteine must be due to the stimulation of cysteine oxidase activity, but not of cysteine desulfhydrase activity, since the hepatic desulfhydrase activity is markedly suppressed in vitamin B6-deficient rats, and the enzyme activity was not induced by in vivo administration of L-cysteine, while the activity of cysteine oxidase in vitamin B6 -deficient rats was markedly induced as well as in controls as previously reported {data not shown) [1]. These facts also support the hypothesis, presented in a previous report [2], that the major catabolic route of L-cysteine in rats is through cysteine sulfinate as an obligate intermediate but the conversion of L-cysteine to pyruvate via the direct desulfhydration seems to be less significant. Therefore, it appears likely that the catabolic pathway of L-cysteine through cysteine sulfinate formation catalyzed by the enzyme cysteine oxidase plays a major role in cysteine catabolism in vitamin B6-deficient rats as well as in controls. As shown in Table III, in the deficient rats a minor portion of taurine pathway, 25% of controls, was detected by the in vivo measurement of cysteine metabolic distribution, but this taurine pathway was not increased by the L-cysteine loading, in contrast to the controls. These facts strongly suggest that the taurine formation in vitamin Bs-deficient rats may be catalyzed by an u n k n o w n enzyme system other than by the pathway via cysteine sulfinate catalyzed by cysteine oxidase. In this regard, an alternative taurine formation from L-cysteine reported by Cavallini et al. [13] and Dupr~ et al. [14--16] is of interest. They have demonstrated a possibility of the conversion of L-cysteine to hypotaurine through a hypothetical pathway, the pantotheic acid cycle involving pantothenate-4-phosphate, pantothenoylcysteine-4-phosphate, pantotheine-4-phosphate, pantotheine and cysteamine as intermediates in the cycle, connecting with hypotaurine formation through the action of cysteamine oxygenase. The 14CO 2 expired from D-cysteine was calculated by the equation mentioned above. As shown in Table III, 14CO2 derived from D-cysteine of D L[ i 4 C] cysteine used as tracers was 26.7% and 21.9%, in controls and in vitamin B6-deficient rats respectively. These values were not altered by L-cysteine loading either in controls or deficient rats. Furthermore, the loading of non-physiological doses (50 mg per 100 g of body weight) of D-cysteine did not increase the C m a x value for L-[U-~4C]cysteine in either controls or deficient rats as shown in Table IV. These facts strongly suggest that, in either deficient or control rats, D-cysteine must be converted to pyruvate via separate pathway from that of L-cysteine.
Urinary excretion of labeled sulfate and taurine from L _[3 s S]_cysteine injected intraperitoneally The amounts of urinary taurine in deficient rats were reduced to 30% of controls whereas the incorporation of 3 s S into taurine in deficient rats was remarkably decreased to less than 10% of controls. These facts strongly suggest
T A B L E IV T H E E F F E C T O F D - C Y S T E I N E L O A D I N G ON T H E 14CO2 E X P I R A T I O N F R O M V A R I O U S L A B E L E D C Y S T E I N E IN V I T A M I N B 6 - D E F I C I E N T R A T S T h e e x p e r i m e n t s w e r e c a r r i e d o u t u n d e r t h e s a m e c o n d i t i o n s as in T a b l e s I a n d II e x c e p t t h a t t h e l o a d i n g was of D - c y s t e i n e ( 5 0 m g / 1 0 0 g b o d y w e i g h t ) in p l a c e of L - c y s t e i n e . The n u m b e r of a n i m a l s is given in p a r e n t h e s e s . Labeled compound
C m a x value (%)
t 1/2 (h)
DL-[ 1-14C] C y s t e i n e DL-[3-14C] Cysteine L-[U-14C] C y s t e i n e
3 2 . 8 (1) 2 2 . 4 (1) 2 2 . 0 (1)
1.8 2.9 2.2
that the de novo synthesis of taurine from L-cysteine must be highly inhibited by vitamin B6 deficiency and a part of urinary taurine excreted in the deficient rats may be derived from exogenous source such as diet or from the production by intestinal bacteria suggested by Bergeret and Chatagner [8]. The similar situations as the changes in the taurine pathway measured by in vivo metabolic distribution of L-cysteine were also observed in urinary excretion of taurine. Thus the increases of urinary taurine and of the incorporation of radioactivity into urinary taurine from L-[ 3 s S] cysteine were not mediated by the loading of non-physiological dose of L-cysteine in vitamin B6-deficient rats in contrast to the controls as shown in Table V. On the other hand, the amounts and incorporations of 3 s S of urinary sulfate from L-[ 3 ss] cysteine were highly increased by L-cysteine loading in the deficient rats as well as in controls. These facts also TABLE V T H E U R I N A R Y I N C O R P O R A T I O N O F 35S I N T O T A U R I N E A N D I N O R G A N I C S U L F A T E L-[3SS] C Y S T E I N E A N D T H E U R I N A R Y C O N T E N T S
FROM
U r i n e w a s c o l l e c t e d for 24 h a f t e r i n t r a p e r i t o n e a l i n j e c t i o n of 0 . 5 #Ci o f L-[ 3SS] c y s t e i n e ( 2 0 - - 5 0 C i / m o l e ) w i t h or w i t h o u t L - c y s t e i n e ( 1 0 0 m g p e r 100 g b o d y w e i g h t ) . T h e p H of t h e c o l l e c t e d u r i n e was a d j u s t e d to 8.5 a n d f r a c t i o n a t e d b y c o l u m n c h r o m a t o g r a p h y w i t h D o w e x 5 0 - X 8 (H +) a n d the e f f l u e n t s f r o m t h e c o l u m n w e r e t r e a t e d w i t h b a r i u m a c e t a t e t o p r e c i p i t a t e i n o r g a n i c sulfate. A n a l i q u o t of t h e e f f l u e n t s was used f o r t h e analysis o f t a u r i n e c o n t e n t s e s t i m a t e d w i t h an a u t o m a t i c a m i n o acid a n a l y z e r . T h e i n c o r p o r a tion of 35S w a s e x p r e s s e d as p e r c e n t of the a d m i n i s t e r e d d o s e of r a d i o a c t i v i t y . T h e n u m b e r o f a n i m a l s axe given in p a r e n t h e s e s . T h e r a d i o a c t i v i t y in s u p e r n a t a n t s of the e f f l u e n t s a f t e r b a r i u m a c e t a t e t r e a t m e n t r e p r e s e n t s the i n c o r p o r a t i o n of 35S i n t o u r i n a r y t a u r i n c . Treatment
Total pmoles/ day
C o n t r o l rats None L-Cysteine loading D e f i c i e n t rats None L-Cysteine loading
Taurine
Sulfate
%
#moles/ day
%
#moles/ day
431.8
19.9
1624.4
59.9
1 1 5 . 2 ± 6.6 (3) 385.0 ± 28.4 (4)
5.7 ± 0.6 (3) 9.2 ± 0.7 (3)
3 1 6 . 6 ± 5.4 (3) 1239.4 ± 67.6 (5)
14.2 + 0.9 (3) 50.3 ± 2.7 (2)
351.5
16.9
1481.4
62.5
6 1 . 5 ± 8.4 (4) 81.3 ± 10.2 (3)
0.3 ± 0.07 (2) 0.4 ± 0.14 (2)
2 9 0 . 0 ± 21.5 (5) 1400.1 ± 74.9 (4)
16.6 ± 2.9 (3) 62.1 ± 4.8 (2)
support the above hypothesis, in which sulfate is converted from L-cysteine via cysteine sulfinate as an obligate intermediate, in either control or vitamin B6-deficient rats, but the taurine formation observed in vitamin B6-deficient rats may be catalyzed by an other pathway than via cysteine sulfinate, since if taurine is formed from L-cysteine via cysteine sulfinate in vitamin B6 -deficient rats as well as in controls, the excretion and 3 s S.incorporatio n of urinary taurine should be increased by the induced activity of cysteine oxidase mediated by the L-cysteine loading as well as urinary sulfate. From findings presented here, it is concluded that the abnormality of in vivo metabolic distribution of L-cysteine can be detected by the in vivo measurement of L-cysteine metabolism, and that the metabolism of L-cysteine in vitamin B6-deficient rats is characterized by remarkable lesion in the taurine pathway, and an u n k n o w n alternative pathway of taurine formation from L-cysteine other than via cysteine sulfinate may occur. However, the taurine pathway observed in vitamin B6-deficient rats was not stimulated by the nonphysiological dose of L-cysteine, in contrast to the controls. The metabolic turnover rate of L-cysteine represented as the k value was remarkably stimulated by the non-physiological dose of L-cysteine loaded in either control rats or vitamin B6 -deficient rats. Acknowledgements We are grateful to Dr Y. Sakamoto, Institute of Cancer Research, Osaka University Medical School, for his useful advices and encouragement. The able technical assistance of Mrs K. Tagawa is also gratefully acknowledged. This study is supported in part by research grants from the Scientific Research Fund of the Ministry of Education of Japan (No. 757045), from Tanabe Amino Acids Research Fund and from Kanae Shinyaku Kenkyu-kai Fund. The material herein was taken in part from the dissertation submitted to Osaka Medical College by Shioichi Shigehisa in partial fulfillment of the requirements for the Doctor of Science in Medicine. References 1 Y a m a g u c h i , K., S a k a k i b a r a , S., K o g a , K . a n d U e d a , I. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 3 7 , 5 0 2 2 Y a m a g u c h i , K . , S a k a k i b a r a , S., A s a m i z u , J. a n d U e d a , I, ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 2 9 7 , 4 8 3 S a k a k i b a r a , S., Y a m a g u c h i , K . , U e d a , I. a n d S a k a m o t o , Y. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 52, 1093 4 T h o m p s o n , R . Q . a n d G u e r r a n t , N.B. ( 1 9 5 3 ) J. N u t r . 5 0 , 1 6 1 5 B l a s c h k o , H., D a t t a , S.P. a n d H a r r i s , H. ( 1 9 5 3 ) Br. J. N u t r . 7, 3 6 4 6 C h a t a g n e r , F., T a b e c h i a n , H. a n d B e r g e r e t , B. ( 1 9 5 4 ) B i o c h i m . B i o p h y s . A c t a 1 3 , 3 1 3 7 B e r g e r e t , B., C h a t a g n e r , F. a n d F r o m a g e o t , C. ( 1 9 5 5 ) B i o c h i m . B i o p h y s . A c t a 1 7 , 1 28 8 B e r g e r e t , B. a n d C h a t a g n e r , F. ( 1 9 5 6 ) B i o c h i m . B i o p h y s . A c t a 2 2 , 2 7 3 9 H o p e , D.B. ( 1 9 5 7 ) B i o c h e m . J. 6 6 , 4 8 6 1 0 B r o w n , F.C. a n d G o r d o n , P.H. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 3 0 , 4 3 4 11 E w e t z , L. a n d S 6 r b o , B. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 8 , 2 9 6 1 2 Cavallini, D., M a r c o , C. a n d M o n d o v i , B. ( 1 9 5 8 ) J. Biol. C h e m . 2 3 0 , 2 5 1 3 Cavallini, D., D u p r 6 , S., G r a z i a n i , M.T. a n d T i n t i , M.G. ( 1 9 6 8 ) F E B S L e t t . 1, 1 1 9 1 4 D u p r 4 , S., G r a z i a n i , M . T . , R o s e i , M . A . , F a b i , A. a n d Del G r o s s o , E. ( 1 9 7 0 ) E u r . J. B i o c h e m . 1 6 , 5 7 1 1 5 D u p r 6 , S., R o s e i , M . A . , BeUussi, L., B a r b o n i , E. a n d S c a n d u r r a , R. ( 1 9 7 3 ) P r o c . 9 t h Int. C o n g . Biochem. 98 1 6 D u p r ~ , S., R o s e i , M . A . , BeUussi, L., G r o s s o , E . D . a n d C a v a n i n i , D. ( 1 9 7 3 ) E u r . J. B i o c h e m . 4 0 , 1 0 3
