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Chondroitin sulfate isomers in normal human urine
BIOCHIMICA ET BIOPHYSICA ACTA
583
BBA 26509 C H O N D R O I T I N SULFATE ISOMERS IN NORMAL HUMAN U R I N E KATSUMI MURATA, TOSHIE HARADA, TAKESHI FUJIWARA AND TAMOTSU FURUHASHI Department of Physical Therapy and Medicine, University of Tokyo School of Medicine, Tokyo, and Seikagaku Kogyo Co., Kurihama (Japan)
(Received September 8tb, 197o)
SUMMARY X. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nonsulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractionated by NaC1 elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.
INTRODUCTION Numerous authors have reported that the majority of urinary glycosaminoglycans in normal subiects is chondroitin sulfate concomitant with the other glycosaminoglycans a-5. The constituents of chondroitin sulfate isomers, as well as other glycosaminoglycans in normal urine, however, varied according to different determination methods 2-n. There is no universal agreement regarding the amounts of the nonsulfated or undersulfated isomer, namely chondroitin ~-6. Moreover, the presence of oversulfated chondroitin sulfate in human urine remains to be elucidated. Our previous enzymic analysis of urinary chondroitin sulfate isomers at the disaccharide level indicated that the major chondroitin sulfate of normal urine is chondroitin sulfate C with somewhat lesser amounts of chondroitin sulfate A, whereas chondroitin sulfate B is present in very small quantities :. The purpose of the present investigation was to study in more detail 'the chondroitin sulfate isomers in normal urine at the unsaturated disaccharide unit level by degradation with chondroitinases and chondrosulfatases. Since not much data are available regarding the presence of nonsulfated and oversulfated chondroitin sulfate isomers in human urine, special attention was given to the qualitative and quantitaBiochim. Biophys. Aeta, "~3o (197I) 583-594
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K. MURATA et al.
tive determination of unsaturated nonsulfated and di-sulfated disaccharides as the subunits of urinary chondroitin sulfate isomers. MATERIALS AND METHODS
Preparation of urinam glycosaminoglycans 3 ° 1 of pooled urinary specimens were collected with toluene from healthy individuals. The urinary glycosaminoglycans were prepared by a method similar to that reported previouslyL which is as follows. 15 ml of 5 % cetylpyridinium chloride were added per 1 of urine and the mixture was kept overnight at 4 °. The resulting precipitates were collected by centrifugation, washed with 95 % methanol saturated with potassium acetate, and digested with pronase (7° ooo P. U. K.*/g, Kaken Kagaku Co., Tokyo) at a rate of 2o rag/1 of starting urine volume in 0.067 M phosphate buffer (pH 7.8) for 24 h at 5 o°. After the proteolytic digestion had been repeated 3 times, trichloroacetic acid was added to a final concentration of 5 % (w/v) followed by cooling overnight at 4 ° and centrifugation. The supernatants were neutralized and dialyzed in cellulose tubing (Visking Co.) against running tap-water for 72 h. Following dialysis, glycosaminoglycans were concentrated in a flash evaporator to approx. 3o ml, and were then precipitated by adding 3 vol. of methanol saturated with potassium acetate. The precipitates were collected by centrifugation and were dissolved in distilled water. After the washing procedure had been repeated 5 times, the glycosaminoglycans were dried in a vacuum over P~O~. The urinary glycosaminoglycans thus prepared were then applied to Dowex I-X2 columns (C1- form, 200-40o mesh) and eluted with 0.5 and 3.0 M NaC1. Chondroitin sulfate isomers with other glycosaminoglycans, which were eluted with 3.0 M NaC1, were separated from hyaluronic acid eluted with 0.5 M NaC1. (Because hyaluronic acid interferes with the analysis of chondroitin sulfate isomers 8, the separation was carried out in this manner.) The eluates were dialyzed against running tap-water for 36 h to remove NaC1, and concentrated for the following analytical procedures. Further stepwise fractionation on Dowex columns was carried out in the selected specimens as described below. After the fractionation, glucuronic acid was determined by the borate-carbazole reaction as in the method of BITTER AND MUIR°.
Enzyme preparation Chondroitinase-ABC* * and chondro- 4- and chondro-6-sulfatases were prepared from Proteus vulgaris and separated by DEAE-cellulose chromatography. Chondroitinase-AC was prepared from Flavobacterium heparinum, and purified on a phosphocellulose column 1°. Further purification was carried out by the same procedure as reported previously l°,n. The enzymic activities were tested against standard chondroitin sulfates prior to application to the following enzymic separation and determination, to ensure there was sufficient activity.
Enzymic separation and identification procedures The amounts of nonsulfated and oversulfated chondroitin sulfates in urinary glycosaminoglycans might be relatively smalH, so special consideration was paid to • P . U . K . , u n i t of p r o t e o l y t i c a c t i v i t y of p r o n a s e m e a s u r e d b y t h e K a k e n C h e m i c a l C o m p a n y . • * The n o m e n c l a t u r e of e n z y m e s used here is b a s e d on t h e s u g g e s t i o n s m a d e b y SAITO et al. 8 a n d YAMAGATA et al. TM.
Biochim. Biophys. Acta, 230 (1971) 583-594
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585
the identification of the separated chondroitin sulfates in terms of unsaturated disaccharide subunits by rechromatographic procedure. The enzymic procedures were performed by the following methods, which are modifications of those described in refs. 7 and 8. Approx. 35/,moles of glycosaminoglycans (as glucuronic acid) eluted with 3.0 M NaC1 were exhaustively digested to the unsaturated disaccharide state with either chondroitinase-ABC (2 units) or chondroitinase-AC (6 units) in Tris buffer for 12o rain at 37 ° as reported previously 7. The resulting unsaturated disaccharides (after treatment with chondroitinase-ABC) were applied in a band (5 cm wide) to a filter paper of Toyo Roshi No. 5IA (55 cm × 20 cm, corresponding to W h a t m a n No. I) with standard markers of 4-unsaturated disaccharides : nonsulfated*, 4-sulfated, 6-sulfated and di-sulfated disaccharides. After desalting by chromatography in n - b u t a n o l - e t h a n o l - w a t e r (52:32:16, by vol.) for 24 h, descending paper chromatography was performed in 1-butyric acid0.5 M ammonia (5:3, by vol.) for 72 h at room temperature. The separated bands of the four different unsaturated disaccharides were visualized with a Mineralight at 232 nm. The regions corresponding to each unsaturated disaccharide were cut out, eluted with distilled water and then concentrated for quantitative determination and separation. The same procedure was carried out for digestion of urinary chondroitin sulfates with chondroitinase-AC. Appropriate amounts (o.15-o.8/,mole) of the eluted unsaturated disaccharides were again applied individually in spots to the filter paper for rechromatographic identification. Paper chromatography was carried out as described above, and the localization of the spots of the separated unsaturated disaccharides was investigated under Mineralight exposure. The regions of spots of each disaccharide were then cut out for elution with distilled water and the eluates were applied to a filter paper of Toyo Roshi No. 5IA. Paper electrophoresis was carried out in 0.05 M sodium citrate-citric acid buffer (pH 5.0) at a potential gradient of 30 V/cm for 4 ° min a2. After the unsaturated nonsulfated and 4-sulfated disaccharides (approx. 7/,moles each) eluted with distilled water had been hydrolyzed in 2 M HC1 for 4 h at IOO° and dried in a desiccator, IO/,1 of the hydrolyzates were applied in spots to a filter paper (Whatman No. I). The descending paper chromatographic separation of galactosamine and glucosamine from the unsaturated disaccharides was carried out in n - b u t a n o l - p y r i d i n e - f o r m i c a c i d - w a t e r (5o:3o:2:18, by vol.) for 42 h at room temperature. The residual portions of the eluted unsaturated disaccharides, except the unsaturated nonsulfated disaccharide, were further digested with chondro-4- and/or chondro-6-sulfatases to produce unsaturated nonsulfated disaccharide alone as a final product. Aliquots of the fractions were then added to each reaction tube for quantitatire determination by the Morgan-Elson reaction at 585 nm, instead of measurement at 232 nm (ref. 8) so as to eliminate a nucleate reaction. The linear relationship between the amounts of the isomeric chondroitin sulfates as the unsaturated disaccharide subunits and the absorbance at 585 nm was obtained in each product, as has been * Two u n s a t u r a t e d n o n s u l f a t e d d i s a c c h a r i d e s were used as s t a n d a r d s : one d e r i v e d f r o m c h o n d r o i t i n s u l f a t e i s o m e r s of s h a r k c a r t i l a g e , a n o t h e r d e r i v e d from h y a l u r o n i c a c i d of h u m a n u m b i l i c a l cord.
Biochim. Biophys. Acta, 23 ° (1971) 583-594
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reported in the previous papersT, 8,11. The result made it possible to analyze quantitatively the separated unsaturated disaccharides. For further fractionation, approx. 25/,moles of glycosaminoglycans (as glucuronic acid) were selectively prepared from normal urine. They were applied to a Dowex I-X2 column and fractionated by stepwise elation in o.25 M NaC1 increments. After dialysis against distilled water and suitable concentration, aliquots of the various fractions were each applied to cellulose acetate strip (Separax) for electrophoresis in o.I M veronal-phosphate buffer (pH 8.6) at o.25 mA/cm for 9 ° min. The strips were then stained by immersion in o.o 4 % toluidine blue in o.I % acetic acid for 5 rain, as reported previously 7. The fractionated glycosaminoglycans were then individually degraded to the unsaturated disaccharides with chondroitinase-ABC. Paper chromatographic separation of the unsaturated disaccharides was followed by digestion with the chondrosulfatases and determination by the Morgan-Elson reaction as mentioned above. The regions of the origins were also cut out in small pieces, the undigested glycosaminoglyeans were eluted with a small volume of distilled water at 37 ° for 3o rain, and the eluates were decanted into test-tubes. The elution was repeated 3 times. To the eluates, 4 vol. of ethanol saturated with potassium acetate were added and the solutions were kept overnight at 4 ° . The resulting precipitates were collected by centrifugation and dried over P205 in a vacuum. The yields of glycosaminoglyeans were determined b y the carbazole reaction 9. Paper chromatographic separation of hexosamines by the method mentioned above was performed on the samples that were selectively eluted from the separated unsaturated disaccharides and from the glycosaminoglycans that were not digested with chondroitinase-ABC at the region of origin. Analyses for hexosamine and sulfate were carried out by the methods described by STROMINGER et al. la and DODGSON 14, respectively. The anthrone reaction was measured by the method of SCOTT AND MELVIN15. RESULTS
The rechromatographic separation, as illustrated in Fig. i, shows that the unsaturated nonsulfated and di-sulfated disaccharides were clearly separated from the unsaturated 4-sulfated and 6-sulfated disaccharides after digestion of the chondroitin sulfates of normal urine with chondroitinase-ABC. The unsaturated nonsulfated disaccharide from urinary chondroitin sulfates migrated at nearly the same rate as a standard nonsulfated disaccharide derived from chondroitin sulfate isomers but more slowly than that from hyaluronic acid (not shown in the figure). The urinary unsaturated di-sulfated disaccharide was demonstrated clearly as a spot that migrated more slowly than that of the unsaturated 6-sulfated disaccharide. Nearly the same results were obtained b y digestion of urinary chondroitin sulfates of normal subjects with chondroitinase-AC. The paper chromatographic evidence was subsequently substantiated by the following paper electrophoretic identification. Fig. 2 illustrates that the unsaturated di-sulfated disaccharide migrated much faster than the unsaturated mono-sulfated (4-sulfated and 6-sulfated) disaccharides, whereas the unsaturated nonsulfated disaccharide moved distinctly more slowly than the latter on paper electrophoresis. Biochim. Biophys. Acta, 230 (1971) 583-594
URINARY CHONDROITIN SULFATE ISOMERS
587
Fig. i. Paper rechromatographic separation of the urinary unsaturated nonsulfated and di-sulfated disaccharides from the unsaturated 4-sulfated and 6-sulfated disaccharides found after digesting chondroitin sulfate isomers from normal human urine with chondroitinase-ABC. Descending paper chromatography was performed in i-butyric acid-o. 5 M ammonia (5:3, by vol.) for 72 h at room temperature. The separated bands of the unsaturated disaecharides were cut out, eluted with water and concentrated. Each was applied as a spot to a filter paper for rechromatographie identification. The photograph was taken under Mineralight exposure.
Paper c h r o m a t o g r a p h y of an acid hydrolyzate of the u n s a t u r a t e d n o n s u l f a t e d disaccharide eluted in the 1.25 M NaC1 fraction following the o.25 M NaC1 elution indicated t h a t the m a i n c o n s t i t u e n t was the galactosamine m o i e t y (Fig. 3). Moreover, a n acid hydrolyzate of the u n s a t t t r a t e d 4-sulfated disaccharide eluted u n d e r the same condition m i g r a t e d like galactosamine. These paper chromatographic a n d electrophoretic findings of the occurrence of the u n s a t u r a t e d n o n s u l f a t e d a n d di-sulfated disaccharides in n o r m a l h u m a n urine indicate t h a t n o n s u l f a t e d or undersulfated c h o n d r o i t i n sulfate, as well as oversulfated c h o n d r o i t i n sulfate, exist in h u m a n urine either i n d e p e n d e n t l y or as h y b r i d cons t i t u e n t s in addition to the major a m o u n t s of c h o n d r o i t i n sulfate A a n d C isomers. The u n s a t u r a t e d di-sulfated disaccharide of the 3,o M NaC1 fraction following t h e o.5 M NaC1 elation showed on analysis a molar ratio of uronic acid to hexosamine to sulfate of i.oo :1.o 5:2.o 4. No sulfate was detected in the u n s a t u r a t e d n o n s u l f a t e d disaccharide of the 1.25 M NaC1 fraction after the o.5 M NaC1 elution with a uronie acid to hexosamine molar ratio of I.O. The results of f r a c t i o n a t i o n of u r i n a r y glycosaminoglycans of a n o r m a l female b y stepwise elution in 0.25 M NaC1 i n c r e m e n t s on Dowex I - X 2 columns are illustrated in Fig. 4 a n d Table I. The m a i n peaks of u r i n a r y glycosaminoglycans appeared between the o.75 a n d 1. 5 M NaC1 fractions. The glycosaminoglycans t h a t were n o t Biochim. Biophys. Acta, 230 (I97 I) 583-594
588
K. MURATA et al.
\
Fig. 2. P a p e r electrophoretic identification of t h e u r i n a r y u n s a t u r a t e d n o n s u l f a t e d a n d di-sulfated disaccharides. T h e u n s a t u r a t e d disaccharides identical with t h o s e in Fig. I were applied to a filter p a p e r for p a p e r electrophoretic p r o c e d u r e w h i c h was carried o u t as m e n t i o n e d in t h e t e x t . T h e p h o t o g r a p h was t a k e n u n d e r Mineralight exposure.
Fig. 3- P a p e r c h r o m a t o g r a p h i c identification of t h e g a l a c t o s a m i n e derived from t h e u n s a t u r a t e d n o n s u l f a t e d disaccharide e l u t e d b y 1.25 M NaC1 a f t e r elution w i t h 0.25 M NaCI on D o w e x I - X 2 c o l u m n s . T h e p r o c e d u r e is described in t h e t e x t . T h e r e s u l t i n g s p o t s were s t a i n e d b y t h e A g N O 3 r e a c t i o n followed b y fixation w i t h s o d i u m thiosulfate.
digested with chondroitinase-ABC were mostly represented between the 0.25 and I.OO M NaC1 fractions, determined as glucuronic acid 9. A spectrum of electrophoretic mobilities of urinary glycosaminoglycans on cellulose acetate that were fractionated by stepwise eluti0n on Dowex I-X2 columns in 0.25 M NaC1 increments is shown in Fig. 5. The fractions between the 1. 5 and 1.75 M Biochim. Biophys. Acta, 23 ° (I97~) 583-594
URINARY
CHONDROITIN
0.(BM 0.25M
0. SM
589
SULFATE ISOMERS
0.15M
1.0M
1. SM
1.25M
1
1.75M
2.0M
3.0M
I 11
6(10
Pg
~. 1~
,
200
300
400
500
600
700
900 ml
800
Effluent F i g . 4. E l u t i o n d i a g r a m o f f r a c t i ~ n a t e d u r i n a r y g l y c o s a m i n o g l y c a n s o n a D o w e x I - X 2 c o l u m n . The glycosaminoglycans (approx. 25/tmoles as glucuronic acid) were fractionated by stepwise elution with NaC1 concentrations at 0.25 M increments. The eluates were collected in aliquots of io ml and the glucuronic acid contents were determined by the carbazole reaction.
TABLE THE AND
I
FRACTIONATION OF URINARY GLYCOSAMINOGLYCANS DIGESTED PARTS WITH CHONDROITINAsE-ABC
AND
THE
PROPORTION
OF
UNDIGESTED
Approx. 5 ° #moles of urinary glycosaminoglycans as uronic acid from a 45-year-old female were fractionated by stepwise elution on a Dowex I-X2 column in o.25 M NaC1 increments.
NaCl (moles)
Recovery * (%)
Undigested glyoosaminoglycans after chondroitinase-
Chondroitin sulfate isomers (%)
ABC** (%) 0'25 0"5 0.75 I.O 1-25 1-5 1.75
3 4 II 13 37 23 6
54 39 14 22 2 I 2
2.0
2
--
3.0
I
--
Digested glycosaminoglycans after chondroitinase-ABC* * * Unsaturated disaccharides (%) Non-sulfated 4-Sulfated
I 2 9 IO 36 23 6 1-2
6-Sulfated
-IOO
---
__ --
77
23
--
68 7 ---
32 22 23 42
__ 71 77 58
.
.
.
.
* Values were based on uronic acid. ** U r o n i c a c i d c o n t e n t s a f t e r d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C w e r e d i v i d e d b y t h e c o r r e sponding uronic acid values in each fraction before digestion. *** A f t e r d i g e s t i o n w i t h t h e e n z y m e , f o l l o w e d b y p a p e r c h r o m a t o g r a p h i c s e p a r a t i o n , t h e d e termination was made by the Morgan-Elson reaction, and the values are expressed as percentages in each fraction. Only the spots of unsaturated disaccharides visualized under a Mineralight were m e a s u r e d a n d l i s t e d ill t h e t a b l e . T h e r e f o r e t r a c e a m o u n t s o f t h e o t h e r u n s a t u r a t e d d i s a c c h a r i d e s at unvisualized parts are not included.
Biochim. Biophys. Acta, 2 3 o (1971) 5 8 3 - 5 9 4
K. MURATA et al.
590
NaC1 showed a fast migration rate identical with that of a standard chondroitin sulfate A prepared from whale cartilage. The fractions eluted by lower NaC1 concentrations migrated more slowly than those of higher fractions. The slowest migrating fraction at lower NaC1 concentrations moved somewhat faster than a standard hyaluronic acid prepared from umbilical cord and at a rate nearly identical with that of a standard chondroitin (sulfur content o.4%, and nitrogen 3.28%) prepared from shark cartilage by the desulfation method of KANTOR AND SCHUBERT16. KS
O
CS-A
O
3.0 M
O
2.0 M
O
Q O
1.75M 1.5 M
O
I.z9~
O
1.0 M
0.791 0.SM CH HA
O O
Q O
Fig. 5- Electrophoretic c o m p a r i s o n of f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s b y stepwise elution on a D o w e x I - X 2 c o l u m n in o.25 M NaC1 i n c r e m e n t s . I n c r e a s e d electrophoretic mobilities are s h o w n in fractions e l u t e d w i t h g r e a t e r NaC1 c o n c e n t r a t i o n s b e t w e e n t h e o. 5 a n d 2.o M e x c e p t for t h a t of k e r a t a n sulfate. S t a n d a r d g l y c o s a m i n o g l y c a n s were p r e p a r e d : h y a l u r o n i c acid (HA) f r o m h u m a n umbilical cord, c h o n d r o i t i n (CH) b y d e s u l f a t i o n of c h o n d r o i t i n s u l f a t e from s h a r k cartilage, c h o n d r o i t i n sulfate A (CS-A) from whale cartilage a n d k e r a t a n sulfate (KS) from bovine cornea.
It appears that the electrophoretic mobility of various urinary fractions is associated principally with the sulfate content of glycosaminoglycans. As shown in Table I, chondroitin sulfate isomers comprise the maior constituents in these fractions based on the ratios of the digested and undigested glycosaminoglycans found after treatment with chondroitinase-ABC. But a contribution of a smaller quantity of the glycosaminoglycans not digested with chondroitinase-ABC cannot be ruled out: heparan sulfate in various sulfate contents would be present at NaC1 concentrations between the o.75 and 1.25 M, and hyaluronic acid at the 0.25 and 0. 5 M NaC1, as mentioned below. Measurement of the undigested glycosaminoglycans at the region of the origin, indicated that less than one-fourth of the glycosaminoglycans in the 0.75 and I.O M NaC1 fractions is not digested. An acid hydrolyzate of pooled glycosaminoglycans from the regions of origin between the 0.75 and 1.25 M NaC1 fractions migrated like glucosamine, indicating that the substance is heparan sulfate. The residues that are not digested by chondroitinase-ABC at the origins of the 0.25 and 0.5 M NaC1 fractions evidently contained hyaluronic acid, because paper chromatographic separation indicated that an acid hydrolyzate migrated like glucosamine. These substances gave a positive anthrone reaction, indicating the concomitant presence of glycoproteins. Since these undigested original residues from the enzymic reactions consisted mainly Biochim. Biophys. Acta, 23o (1971) 583-594
591
URINARY CHONDROITIN SULFATE ISOMERS
of glucosamine, a possible overlap of heparan sulfate and hyaluronic acid should be considered, as has been described by VARADI et al3. The relationship between the electrophoretic mobilities of the fractionated chondroitin sulfates and the sulfate contents was substantiated by the following paper chromatographic separation of the unsaturated disaccharides. The proportion of unsaturated nonsulfated disaccharide to unsaturated 4-sulfated disaccharide obtained from the fractionated glycosaminoglycans in 0.25 M NaC1 increments is clearly a function of the concentration of NaC1 (Table I and Fig. 6). No Ct Standard Fraction 0.SM
0.75M
1.0M
1.25M
1.5M
1,75M
unsaturated : t di-sulfated disaccharicle
unsotur~.ed 4-sulfat.ed
"~
dis~:hari~ U d
m ~
~' e
•
i
Fig. 6. P a p e r c h r o m a t o g r a p h i c s e p a r a t i o n of t h e u n s a t u r a t e d disaccharides o b t a i n e d b y digestion of u r i n a r y c h o n d r o i t i n s u l f a t e i s o m e r s w i t h c h o n d r o i t i n a s e - A B C a f t e r I r a c t i o n a t i o n b y stepwise elution on D o w e x I - X 2 resins in 0.25 M NaC1 i n c r e m e n t s . T h e s a m p l e is t h e s a m e as t h a t u s e d for Table I. T h e e x p e r i m e n t a l p r o c e d u r e is described in t h e t e x t .
In the o. 5 M NaC1 fraction, one spot corresponding to unsaturated nonsulfated disaccharide was visualized by exposure to a Mineralight. Two spots were detected at the o.75 and I.O M NaC1, one predominant spot corresponding to unsaturated nonsulfated disaccharide and another faint spot corresponding to unsaturated 4-sulfated disaccharide. The 1.25 M NaC1 fraction showed three spots similar to unsaturated nonsulfated, 4-sulfated and 6-sulfated disaccharides. The ultraviolet absorption examined under a Mineralight at 232 nm, as well as the measurement by the MorganElson reaction, indicated that the greatest amount was shown in the unsaturated 6-sulfated disaccharide followed b y a lesser amount of the unsaturated 4-sulfated disaccharide and a minor amount of the unsaturated nonsulfated disaccharide. The findings indicated that the unsaturated nonsulfated disaccharide is the major constituent between the 0.5 and I.O M NaCI fractions, whereas the proportions of the unsaturated 4-sulfated disaccharide to the unsaturated nonsulfated disaccharide increased with NaC1 concentrations up to 1.25 M. The sum of the unsaturated nonsulfated disaccharide in urinary chondroitin sulfate of a 45-year-old female was nearly one-fifth of the total glycosaminoglycans, though lesser amounts of the unsaturated nonsulfated disaccharide were found in young individuals. Biochim. Biophys. Acta, 23 ° (I971) 583-594
592
K. MURATAet al.
The spot of the unsaturated 6-sulfated disaccharide appeared at NaC1 concentrations higher than 1.25 M. At NaC1 concentrations greater than 1.5 M, no spot of unsaturated nonsulfated disaccharide was detected. The ratio of unsaturated 6-sulfated disaccharide to unsaturated 4-sulfated disaccharide remained the same in the 1.25 and 1. 5 M NaC1 fractions, but it decreased in the 1.75 M NaC1 fraction. Three spots corresponding to the unsaturated 4-sulfated, 6-sulfated and all-sulfated disaccharides appeared in the 2.0 M NaC1 fraction, after staining by the AgNO 3 reaction v which is more sensitive than detection under a Mineralight. The sums of the unsaturated 4-sulfated disaecharides and the unsaturated 6-sulfated disaccharides in these fractions comprised approx. 25 and 4 ° % of the total glycosaminoglycans, respectively. Thus, greater electrophoretic mobility was associated with higher sulfate contents of the fractionated glycosaminoglycans, mainly chondroitin sulfate isomers, which were eluted with NaC1 concentrations between 0.5 and 2.0 M, except for that of keratan sulfate. The electrophoretic migration in 2.0 M NaC1 showed two spots, the faster spot corresponding to a standard chondroitin sulfate A and the slower one corresponding to a standard keratan sulfate prepared from bovine cornea (gift from Dr. T. Osawa, University of Tokyo). One spot obtained in the 3.0 M NaC1 fraction migrated somewhat faster than that of the slower spot of the 2.0 M NaC1 fraction. Anthrone-positive substances were also measured in the 2.0 and 3.0 M NaC1 fractions, and a faint spot corresponding to galactose in these fractions was detected on the paper chromatogram by the method mentioned above. These observations indicate the presence of keratan sulfate with different sulfate contents in both fractions. DISCUSSION Numerous investigations have indicated that urinary glycosaminoglycans constitute diverse compounds with heterogeneous structures 1-~. The present data determined at the level of unsaturated disaccharide units indicate that nonsulfated or undersulfated chondroitin sulfate and oversulfated chondroitin sulfate are present in human urine in addition to the major constituents of chondroitin monosulfate isomers. Several authors have reported the presence of nonsulfated or undersulfated chondroitin sulfate in human urine in various amounts 3-e. The differences in the undersulfated chondroitin sulfate contents m a y be due not only to the different extraction and determination procedures of chondroitin sulfate isomers, but also to the different rates of the partially or totally nonsulfated compounds. The present finding of the unsaturated nonsulfated disaccharide in normal urine determined b y the MorganElson method after paper chromatographic separation is consistent with our previous studies using another enzymic assay, indicating the presence of nonsulfated chondroitin sulfate in urinary glycosaminoglycans in normal individuals 7. On the other hand, it has not been clear whether an oversulfated chondroitin sulfate occurs in urinary glycosaminoglycans in normal human urine. We have recently reported the presence of an oversulfated chondroitin sulfate as one of the urinary chondroitin sulfate isomers in certain cases 7. The present results with paper rechromatographic and electrophoretic procedures with chemical analysis have clearly demonstrated the existence of an unsaturated di-sulfated disaccharide, indicating that an oversulfated chondroitin sulfate is present in human urine, Because of the limited Biochim. Biophys. Acta, 23° (1971) 583-594
URINARY CHONDROITIN SULFATE ISOMERS
593
quantities of unsaturated di-sulfated disaccharide available, further identification could not be made. Detailed analysis of the proportion of urinary chondroitin sulfate isomers appears to be worth while, since it m a y reflect their metabolic turnover in the body connective tissue. The present results confirm our previous studies performed b y a different enzymic approach ~that chondroitin sulfate C as well as chondroitin sulfate A are major components in urinary glycosaminoglycans of normal individuals. Inadequate data are as yet available to assess the relationships between the minor constituents of the undersulfated or oversulfated chondroitin sulfate isomers and the major component of monosulfated chondroitin sulfates. Since urinary glycosaminoglycans are parts of the final degraded products of the body connective tissue, the presence of such diverse chondroitin sulfate isomers would be interpretable. The diversity of urinary chondroitin sulfate isomers resulting from the degradation and desulfation would be partly due to such enzymic actions as those of hyaluronidase in human urine 17 and chondro-4-sulfatase in bovine aortic tissue TM. Our recent findings that a novel oversulfated chondroitin sulfate exists in human arterial tissue TM indicate a certain relationship between the oversulfated chondroitin sulfate isomer in circulatory connective tissue system and that in urine. It is uncertain whether the nonsulfated and oversulfated chondroitin sulfate isomers in urine are present independently or as hybrid structures. The possible contribution of aging or inflammatory effects on connective tissue m a y reflect the qualitative and quantitative changes of the chondroitin sulfate isomers. The problem that the electrophoretic mobility of fractionated urinary glycosaminoglycans is related to sulfate content has been indicated by VARADI et al. 4. Recently, MATHEWS AND DECKER1° reported that the greater migration rates on electrophoresis were also associated with the sulfate content of the products of degradation of chondroitin sulfate preparations with hyaluronidase. The present finding that the fractions with higher sulfate contents, which are associated with those of the higher ratios of the sums of the unsaturated 4-sulfated and 6-sulfated disaccharides to the unsaturated nonsulfated disaccharide, show distinctly faster migration on electrophoresis is in accord with the reports by VARADI g~ al. 4 and MATHEWS AND DECKER 19.
The appearance of a single spot corresponding to the unsaturated nonsulfated disaccharide at 0.5 M NaC1 elution is of interest, since it m a y suggest the presence of totally nonsulfated chondroitin sulfate in the fraction. The observation of two spots in the 0.75 and I.O M NaCI fractions, which correspond to the unsaturated nonsulfated and 4-sulfated disaccharides but not to the unsaturated 6-sulfated disaccharide, m a y indicate a close relationship between these two compounds. The finding would suggest that nonsulfated chondroitin sulfate and chondroitin sulfate A exist independently and/or form possible hybrid structures such as undersulfated chondroitin sulfate A. The interaction of heparan sulfate or hyaluronic acid in these fractions is not clear in the present study. Tile findings reported here suggest heterogeneous structures of urinary glycosaminoglycans. Further extensive studies of urinary chondroitin sulfate isomers at disaccharide units would assist in the understanding of the diverse urinary chondroitin sulfate isomers in connection with the metabolic states and pathogenesis of connective tissue disorders. Biochim. Biophys. Acta, 230 (I97 I) 583-594
594
K. MURATAet al.
ACKNOWLEDGMENTS
The authors wish to thank Professor Y. Osbima for his interest and encouragement. We are indebted to Dr. T. Okuyama for providing chondroitinases and chondrosulfatases, and to Miss T. Aoyama and Miss H. Azuma for skillful technical assistance. This investigation was supported in part by a research grant from the Ministry of Education, Japan. REFERENCES I 2 3 4 5 6 7 8 9 io 1i 12 13 14 I5 16 17 18 I9
N. Dx F:ERRANTE, J. Lab. Clin. Med., 61 (1963) 633. A. LINKER AND K. D. TERRY, Pro~. Soc. Exptl. Biol. Med., I i 3 (1963) 743G. S. BERENSON AND E. R. DALFERS, JR., Biochim. Biophys. Acta, IOi (1965) 183. D. P. VARADI, J. A. CIFONELLI AND A. DORFMAN,Biochim. Biophys. Acta, 141 (1967) lO 3. T. ORII, Z. Physiol. Chem., 349 (1968) 816. T. ORXI, Biochim. Biophys. Acta, 17o (I968) 204. K. MURATA, T. ISHIKAWA AND Y. OSHIMA, Clin. Chim. Acta, 28 (197 o) 213. H. SAITO, T. YAMAGATA AND S. SUZUKI, J. Biol. Chem., 243 (1968) 1536. T. BITTER AND H. M. MUIR, Anal. Biochem., 4 (1962) 33 o. T. YAMAGATA, H. SAITO, O. HABUCHI AND S. SUZUKI, J. Biol. Chem., 243 (1968) 1923. K. MURATA,T. HARADA AND K. OKUBO,J. Atherosclerosis Res., 8 (I968) 951. T. HARADA, K. MURATA, T. FUJIWARA AND T. FURUHASHI, Biochim. Biophys. Acta, 177 (1969) 676. J. L. STROMINGER, J. T. PARK AND R. E. THOMPSON, J. Biol. Chem., 234 (I959) 3263. K. S. DODGSON, Biochem. J., 78 (1961) 312. T. A. SCOTT, JR AND E. H. MELVIN, Anal. Chem., 25 (1953) 1656T. G. KANTOR AND M. SCHUBERT, J. Am. Chem. Soc., 79 (1957) 152, D. ALLALOUF AND A. HER, Biochim. Biophys. Acta, 2Ol (197 o) 61. E. HELD AND E. BUDDECKE, Z. Physiol. Chem., 348 (i967) lO47. M. B. MATHEWS AND L. DECKER, Biochim. Biophyo. Acta, 156 (1968) 419 •
Bioehim. Biophys. Acta, 230 (I97I) 835-594
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BBA 26509 C H O N D R O I T I N SULFATE ISOMERS IN NORMAL HUMAN U R I N E KATSUMI MURATA, TOSHIE HARADA, TAKESHI FUJIWARA AND TAMOTSU FURUHASHI Department of Physical Therapy and Medicine, University of Tokyo School of Medicine, Tokyo, and Seikagaku Kogyo Co., Kurihama (Japan)
(Received September 8tb, 197o)
SUMMARY X. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nonsulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractionated by NaC1 elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.
INTRODUCTION Numerous authors have reported that the majority of urinary glycosaminoglycans in normal subiects is chondroitin sulfate concomitant with the other glycosaminoglycans a-5. The constituents of chondroitin sulfate isomers, as well as other glycosaminoglycans in normal urine, however, varied according to different determination methods 2-n. There is no universal agreement regarding the amounts of the nonsulfated or undersulfated isomer, namely chondroitin ~-6. Moreover, the presence of oversulfated chondroitin sulfate in human urine remains to be elucidated. Our previous enzymic analysis of urinary chondroitin sulfate isomers at the disaccharide level indicated that the major chondroitin sulfate of normal urine is chondroitin sulfate C with somewhat lesser amounts of chondroitin sulfate A, whereas chondroitin sulfate B is present in very small quantities :. The purpose of the present investigation was to study in more detail 'the chondroitin sulfate isomers in normal urine at the unsaturated disaccharide unit level by degradation with chondroitinases and chondrosulfatases. Since not much data are available regarding the presence of nonsulfated and oversulfated chondroitin sulfate isomers in human urine, special attention was given to the qualitative and quantitaBiochim. Biophys. Aeta, "~3o (197I) 583-594
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K. MURATA et al.
tive determination of unsaturated nonsulfated and di-sulfated disaccharides as the subunits of urinary chondroitin sulfate isomers. MATERIALS AND METHODS
Preparation of urinam glycosaminoglycans 3 ° 1 of pooled urinary specimens were collected with toluene from healthy individuals. The urinary glycosaminoglycans were prepared by a method similar to that reported previouslyL which is as follows. 15 ml of 5 % cetylpyridinium chloride were added per 1 of urine and the mixture was kept overnight at 4 °. The resulting precipitates were collected by centrifugation, washed with 95 % methanol saturated with potassium acetate, and digested with pronase (7° ooo P. U. K.*/g, Kaken Kagaku Co., Tokyo) at a rate of 2o rag/1 of starting urine volume in 0.067 M phosphate buffer (pH 7.8) for 24 h at 5 o°. After the proteolytic digestion had been repeated 3 times, trichloroacetic acid was added to a final concentration of 5 % (w/v) followed by cooling overnight at 4 ° and centrifugation. The supernatants were neutralized and dialyzed in cellulose tubing (Visking Co.) against running tap-water for 72 h. Following dialysis, glycosaminoglycans were concentrated in a flash evaporator to approx. 3o ml, and were then precipitated by adding 3 vol. of methanol saturated with potassium acetate. The precipitates were collected by centrifugation and were dissolved in distilled water. After the washing procedure had been repeated 5 times, the glycosaminoglycans were dried in a vacuum over P~O~. The urinary glycosaminoglycans thus prepared were then applied to Dowex I-X2 columns (C1- form, 200-40o mesh) and eluted with 0.5 and 3.0 M NaC1. Chondroitin sulfate isomers with other glycosaminoglycans, which were eluted with 3.0 M NaC1, were separated from hyaluronic acid eluted with 0.5 M NaC1. (Because hyaluronic acid interferes with the analysis of chondroitin sulfate isomers 8, the separation was carried out in this manner.) The eluates were dialyzed against running tap-water for 36 h to remove NaC1, and concentrated for the following analytical procedures. Further stepwise fractionation on Dowex columns was carried out in the selected specimens as described below. After the fractionation, glucuronic acid was determined by the borate-carbazole reaction as in the method of BITTER AND MUIR°.
Enzyme preparation Chondroitinase-ABC* * and chondro- 4- and chondro-6-sulfatases were prepared from Proteus vulgaris and separated by DEAE-cellulose chromatography. Chondroitinase-AC was prepared from Flavobacterium heparinum, and purified on a phosphocellulose column 1°. Further purification was carried out by the same procedure as reported previously l°,n. The enzymic activities were tested against standard chondroitin sulfates prior to application to the following enzymic separation and determination, to ensure there was sufficient activity.
Enzymic separation and identification procedures The amounts of nonsulfated and oversulfated chondroitin sulfates in urinary glycosaminoglycans might be relatively smalH, so special consideration was paid to • P . U . K . , u n i t of p r o t e o l y t i c a c t i v i t y of p r o n a s e m e a s u r e d b y t h e K a k e n C h e m i c a l C o m p a n y . • * The n o m e n c l a t u r e of e n z y m e s used here is b a s e d on t h e s u g g e s t i o n s m a d e b y SAITO et al. 8 a n d YAMAGATA et al. TM.
Biochim. Biophys. Acta, 230 (1971) 583-594
URINARY CHONDROITIN SULFATE ISOMERS
585
the identification of the separated chondroitin sulfates in terms of unsaturated disaccharide subunits by rechromatographic procedure. The enzymic procedures were performed by the following methods, which are modifications of those described in refs. 7 and 8. Approx. 35/,moles of glycosaminoglycans (as glucuronic acid) eluted with 3.0 M NaC1 were exhaustively digested to the unsaturated disaccharide state with either chondroitinase-ABC (2 units) or chondroitinase-AC (6 units) in Tris buffer for 12o rain at 37 ° as reported previously 7. The resulting unsaturated disaccharides (after treatment with chondroitinase-ABC) were applied in a band (5 cm wide) to a filter paper of Toyo Roshi No. 5IA (55 cm × 20 cm, corresponding to W h a t m a n No. I) with standard markers of 4-unsaturated disaccharides : nonsulfated*, 4-sulfated, 6-sulfated and di-sulfated disaccharides. After desalting by chromatography in n - b u t a n o l - e t h a n o l - w a t e r (52:32:16, by vol.) for 24 h, descending paper chromatography was performed in 1-butyric acid0.5 M ammonia (5:3, by vol.) for 72 h at room temperature. The separated bands of the four different unsaturated disaccharides were visualized with a Mineralight at 232 nm. The regions corresponding to each unsaturated disaccharide were cut out, eluted with distilled water and then concentrated for quantitative determination and separation. The same procedure was carried out for digestion of urinary chondroitin sulfates with chondroitinase-AC. Appropriate amounts (o.15-o.8/,mole) of the eluted unsaturated disaccharides were again applied individually in spots to the filter paper for rechromatographic identification. Paper chromatography was carried out as described above, and the localization of the spots of the separated unsaturated disaccharides was investigated under Mineralight exposure. The regions of spots of each disaccharide were then cut out for elution with distilled water and the eluates were applied to a filter paper of Toyo Roshi No. 5IA. Paper electrophoresis was carried out in 0.05 M sodium citrate-citric acid buffer (pH 5.0) at a potential gradient of 30 V/cm for 4 ° min a2. After the unsaturated nonsulfated and 4-sulfated disaccharides (approx. 7/,moles each) eluted with distilled water had been hydrolyzed in 2 M HC1 for 4 h at IOO° and dried in a desiccator, IO/,1 of the hydrolyzates were applied in spots to a filter paper (Whatman No. I). The descending paper chromatographic separation of galactosamine and glucosamine from the unsaturated disaccharides was carried out in n - b u t a n o l - p y r i d i n e - f o r m i c a c i d - w a t e r (5o:3o:2:18, by vol.) for 42 h at room temperature. The residual portions of the eluted unsaturated disaccharides, except the unsaturated nonsulfated disaccharide, were further digested with chondro-4- and/or chondro-6-sulfatases to produce unsaturated nonsulfated disaccharide alone as a final product. Aliquots of the fractions were then added to each reaction tube for quantitatire determination by the Morgan-Elson reaction at 585 nm, instead of measurement at 232 nm (ref. 8) so as to eliminate a nucleate reaction. The linear relationship between the amounts of the isomeric chondroitin sulfates as the unsaturated disaccharide subunits and the absorbance at 585 nm was obtained in each product, as has been * Two u n s a t u r a t e d n o n s u l f a t e d d i s a c c h a r i d e s were used as s t a n d a r d s : one d e r i v e d f r o m c h o n d r o i t i n s u l f a t e i s o m e r s of s h a r k c a r t i l a g e , a n o t h e r d e r i v e d from h y a l u r o n i c a c i d of h u m a n u m b i l i c a l cord.
Biochim. Biophys. Acta, 23 ° (1971) 583-594
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reported in the previous papersT, 8,11. The result made it possible to analyze quantitatively the separated unsaturated disaccharides. For further fractionation, approx. 25/,moles of glycosaminoglycans (as glucuronic acid) were selectively prepared from normal urine. They were applied to a Dowex I-X2 column and fractionated by stepwise elation in o.25 M NaC1 increments. After dialysis against distilled water and suitable concentration, aliquots of the various fractions were each applied to cellulose acetate strip (Separax) for electrophoresis in o.I M veronal-phosphate buffer (pH 8.6) at o.25 mA/cm for 9 ° min. The strips were then stained by immersion in o.o 4 % toluidine blue in o.I % acetic acid for 5 rain, as reported previously 7. The fractionated glycosaminoglycans were then individually degraded to the unsaturated disaccharides with chondroitinase-ABC. Paper chromatographic separation of the unsaturated disaccharides was followed by digestion with the chondrosulfatases and determination by the Morgan-Elson reaction as mentioned above. The regions of the origins were also cut out in small pieces, the undigested glycosaminoglyeans were eluted with a small volume of distilled water at 37 ° for 3o rain, and the eluates were decanted into test-tubes. The elution was repeated 3 times. To the eluates, 4 vol. of ethanol saturated with potassium acetate were added and the solutions were kept overnight at 4 ° . The resulting precipitates were collected by centrifugation and dried over P205 in a vacuum. The yields of glycosaminoglyeans were determined b y the carbazole reaction 9. Paper chromatographic separation of hexosamines by the method mentioned above was performed on the samples that were selectively eluted from the separated unsaturated disaccharides and from the glycosaminoglycans that were not digested with chondroitinase-ABC at the region of origin. Analyses for hexosamine and sulfate were carried out by the methods described by STROMINGER et al. la and DODGSON 14, respectively. The anthrone reaction was measured by the method of SCOTT AND MELVIN15. RESULTS
The rechromatographic separation, as illustrated in Fig. i, shows that the unsaturated nonsulfated and di-sulfated disaccharides were clearly separated from the unsaturated 4-sulfated and 6-sulfated disaccharides after digestion of the chondroitin sulfates of normal urine with chondroitinase-ABC. The unsaturated nonsulfated disaccharide from urinary chondroitin sulfates migrated at nearly the same rate as a standard nonsulfated disaccharide derived from chondroitin sulfate isomers but more slowly than that from hyaluronic acid (not shown in the figure). The urinary unsaturated di-sulfated disaccharide was demonstrated clearly as a spot that migrated more slowly than that of the unsaturated 6-sulfated disaccharide. Nearly the same results were obtained b y digestion of urinary chondroitin sulfates of normal subjects with chondroitinase-AC. The paper chromatographic evidence was subsequently substantiated by the following paper electrophoretic identification. Fig. 2 illustrates that the unsaturated di-sulfated disaccharide migrated much faster than the unsaturated mono-sulfated (4-sulfated and 6-sulfated) disaccharides, whereas the unsaturated nonsulfated disaccharide moved distinctly more slowly than the latter on paper electrophoresis. Biochim. Biophys. Acta, 230 (1971) 583-594
URINARY CHONDROITIN SULFATE ISOMERS
587
Fig. i. Paper rechromatographic separation of the urinary unsaturated nonsulfated and di-sulfated disaccharides from the unsaturated 4-sulfated and 6-sulfated disaccharides found after digesting chondroitin sulfate isomers from normal human urine with chondroitinase-ABC. Descending paper chromatography was performed in i-butyric acid-o. 5 M ammonia (5:3, by vol.) for 72 h at room temperature. The separated bands of the unsaturated disaecharides were cut out, eluted with water and concentrated. Each was applied as a spot to a filter paper for rechromatographie identification. The photograph was taken under Mineralight exposure.
Paper c h r o m a t o g r a p h y of an acid hydrolyzate of the u n s a t u r a t e d n o n s u l f a t e d disaccharide eluted in the 1.25 M NaC1 fraction following the o.25 M NaC1 elution indicated t h a t the m a i n c o n s t i t u e n t was the galactosamine m o i e t y (Fig. 3). Moreover, a n acid hydrolyzate of the u n s a t t t r a t e d 4-sulfated disaccharide eluted u n d e r the same condition m i g r a t e d like galactosamine. These paper chromatographic a n d electrophoretic findings of the occurrence of the u n s a t u r a t e d n o n s u l f a t e d a n d di-sulfated disaccharides in n o r m a l h u m a n urine indicate t h a t n o n s u l f a t e d or undersulfated c h o n d r o i t i n sulfate, as well as oversulfated c h o n d r o i t i n sulfate, exist in h u m a n urine either i n d e p e n d e n t l y or as h y b r i d cons t i t u e n t s in addition to the major a m o u n t s of c h o n d r o i t i n sulfate A a n d C isomers. The u n s a t u r a t e d di-sulfated disaccharide of the 3,o M NaC1 fraction following t h e o.5 M NaC1 elation showed on analysis a molar ratio of uronic acid to hexosamine to sulfate of i.oo :1.o 5:2.o 4. No sulfate was detected in the u n s a t u r a t e d n o n s u l f a t e d disaccharide of the 1.25 M NaC1 fraction after the o.5 M NaC1 elution with a uronie acid to hexosamine molar ratio of I.O. The results of f r a c t i o n a t i o n of u r i n a r y glycosaminoglycans of a n o r m a l female b y stepwise elution in 0.25 M NaC1 i n c r e m e n t s on Dowex I - X 2 columns are illustrated in Fig. 4 a n d Table I. The m a i n peaks of u r i n a r y glycosaminoglycans appeared between the o.75 a n d 1. 5 M NaC1 fractions. The glycosaminoglycans t h a t were n o t Biochim. Biophys. Acta, 230 (I97 I) 583-594
588
K. MURATA et al.
\
Fig. 2. P a p e r electrophoretic identification of t h e u r i n a r y u n s a t u r a t e d n o n s u l f a t e d a n d di-sulfated disaccharides. T h e u n s a t u r a t e d disaccharides identical with t h o s e in Fig. I were applied to a filter p a p e r for p a p e r electrophoretic p r o c e d u r e w h i c h was carried o u t as m e n t i o n e d in t h e t e x t . T h e p h o t o g r a p h was t a k e n u n d e r Mineralight exposure.
Fig. 3- P a p e r c h r o m a t o g r a p h i c identification of t h e g a l a c t o s a m i n e derived from t h e u n s a t u r a t e d n o n s u l f a t e d disaccharide e l u t e d b y 1.25 M NaC1 a f t e r elution w i t h 0.25 M NaCI on D o w e x I - X 2 c o l u m n s . T h e p r o c e d u r e is described in t h e t e x t . T h e r e s u l t i n g s p o t s were s t a i n e d b y t h e A g N O 3 r e a c t i o n followed b y fixation w i t h s o d i u m thiosulfate.
digested with chondroitinase-ABC were mostly represented between the 0.25 and I.OO M NaC1 fractions, determined as glucuronic acid 9. A spectrum of electrophoretic mobilities of urinary glycosaminoglycans on cellulose acetate that were fractionated by stepwise eluti0n on Dowex I-X2 columns in 0.25 M NaC1 increments is shown in Fig. 5. The fractions between the 1. 5 and 1.75 M Biochim. Biophys. Acta, 23 ° (I97~) 583-594
URINARY
CHONDROITIN
0.(BM 0.25M
0. SM
589
SULFATE ISOMERS
0.15M
1.0M
1. SM
1.25M
1
1.75M
2.0M
3.0M
I 11
6(10
Pg
~. 1~
,
200
300
400
500
600
700
900 ml
800
Effluent F i g . 4. E l u t i o n d i a g r a m o f f r a c t i ~ n a t e d u r i n a r y g l y c o s a m i n o g l y c a n s o n a D o w e x I - X 2 c o l u m n . The glycosaminoglycans (approx. 25/tmoles as glucuronic acid) were fractionated by stepwise elution with NaC1 concentrations at 0.25 M increments. The eluates were collected in aliquots of io ml and the glucuronic acid contents were determined by the carbazole reaction.
TABLE THE AND
I
FRACTIONATION OF URINARY GLYCOSAMINOGLYCANS DIGESTED PARTS WITH CHONDROITINAsE-ABC
AND
THE
PROPORTION
OF
UNDIGESTED
Approx. 5 ° #moles of urinary glycosaminoglycans as uronic acid from a 45-year-old female were fractionated by stepwise elution on a Dowex I-X2 column in o.25 M NaC1 increments.
NaCl (moles)
Recovery * (%)
Undigested glyoosaminoglycans after chondroitinase-
Chondroitin sulfate isomers (%)
ABC** (%) 0'25 0"5 0.75 I.O 1-25 1-5 1.75
3 4 II 13 37 23 6
54 39 14 22 2 I 2
2.0
2
--
3.0
I
--
Digested glycosaminoglycans after chondroitinase-ABC* * * Unsaturated disaccharides (%) Non-sulfated 4-Sulfated
I 2 9 IO 36 23 6 1-2
6-Sulfated
-IOO
---
__ --
77
23
--
68 7 ---
32 22 23 42
__ 71 77 58
.
.
.
.
* Values were based on uronic acid. ** U r o n i c a c i d c o n t e n t s a f t e r d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C w e r e d i v i d e d b y t h e c o r r e sponding uronic acid values in each fraction before digestion. *** A f t e r d i g e s t i o n w i t h t h e e n z y m e , f o l l o w e d b y p a p e r c h r o m a t o g r a p h i c s e p a r a t i o n , t h e d e termination was made by the Morgan-Elson reaction, and the values are expressed as percentages in each fraction. Only the spots of unsaturated disaccharides visualized under a Mineralight were m e a s u r e d a n d l i s t e d ill t h e t a b l e . T h e r e f o r e t r a c e a m o u n t s o f t h e o t h e r u n s a t u r a t e d d i s a c c h a r i d e s at unvisualized parts are not included.
Biochim. Biophys. Acta, 2 3 o (1971) 5 8 3 - 5 9 4
K. MURATA et al.
590
NaC1 showed a fast migration rate identical with that of a standard chondroitin sulfate A prepared from whale cartilage. The fractions eluted by lower NaC1 concentrations migrated more slowly than those of higher fractions. The slowest migrating fraction at lower NaC1 concentrations moved somewhat faster than a standard hyaluronic acid prepared from umbilical cord and at a rate nearly identical with that of a standard chondroitin (sulfur content o.4%, and nitrogen 3.28%) prepared from shark cartilage by the desulfation method of KANTOR AND SCHUBERT16. KS
O
CS-A
O
3.0 M
O
2.0 M
O
Q O
1.75M 1.5 M
O
I.z9~
O
1.0 M
0.791 0.SM CH HA
O O
Q O
Fig. 5- Electrophoretic c o m p a r i s o n of f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s b y stepwise elution on a D o w e x I - X 2 c o l u m n in o.25 M NaC1 i n c r e m e n t s . I n c r e a s e d electrophoretic mobilities are s h o w n in fractions e l u t e d w i t h g r e a t e r NaC1 c o n c e n t r a t i o n s b e t w e e n t h e o. 5 a n d 2.o M e x c e p t for t h a t of k e r a t a n sulfate. S t a n d a r d g l y c o s a m i n o g l y c a n s were p r e p a r e d : h y a l u r o n i c acid (HA) f r o m h u m a n umbilical cord, c h o n d r o i t i n (CH) b y d e s u l f a t i o n of c h o n d r o i t i n s u l f a t e from s h a r k cartilage, c h o n d r o i t i n sulfate A (CS-A) from whale cartilage a n d k e r a t a n sulfate (KS) from bovine cornea.
It appears that the electrophoretic mobility of various urinary fractions is associated principally with the sulfate content of glycosaminoglycans. As shown in Table I, chondroitin sulfate isomers comprise the maior constituents in these fractions based on the ratios of the digested and undigested glycosaminoglycans found after treatment with chondroitinase-ABC. But a contribution of a smaller quantity of the glycosaminoglycans not digested with chondroitinase-ABC cannot be ruled out: heparan sulfate in various sulfate contents would be present at NaC1 concentrations between the o.75 and 1.25 M, and hyaluronic acid at the 0.25 and 0. 5 M NaC1, as mentioned below. Measurement of the undigested glycosaminoglycans at the region of the origin, indicated that less than one-fourth of the glycosaminoglycans in the 0.75 and I.O M NaC1 fractions is not digested. An acid hydrolyzate of pooled glycosaminoglycans from the regions of origin between the 0.75 and 1.25 M NaC1 fractions migrated like glucosamine, indicating that the substance is heparan sulfate. The residues that are not digested by chondroitinase-ABC at the origins of the 0.25 and 0.5 M NaC1 fractions evidently contained hyaluronic acid, because paper chromatographic separation indicated that an acid hydrolyzate migrated like glucosamine. These substances gave a positive anthrone reaction, indicating the concomitant presence of glycoproteins. Since these undigested original residues from the enzymic reactions consisted mainly Biochim. Biophys. Acta, 23o (1971) 583-594
591
URINARY CHONDROITIN SULFATE ISOMERS
of glucosamine, a possible overlap of heparan sulfate and hyaluronic acid should be considered, as has been described by VARADI et al3. The relationship between the electrophoretic mobilities of the fractionated chondroitin sulfates and the sulfate contents was substantiated by the following paper chromatographic separation of the unsaturated disaccharides. The proportion of unsaturated nonsulfated disaccharide to unsaturated 4-sulfated disaccharide obtained from the fractionated glycosaminoglycans in 0.25 M NaC1 increments is clearly a function of the concentration of NaC1 (Table I and Fig. 6). No Ct Standard Fraction 0.SM
0.75M
1.0M
1.25M
1.5M
1,75M
unsaturated : t di-sulfated disaccharicle
unsotur~.ed 4-sulfat.ed
"~
dis~:hari~ U d
m ~
~' e
•
i
Fig. 6. P a p e r c h r o m a t o g r a p h i c s e p a r a t i o n of t h e u n s a t u r a t e d disaccharides o b t a i n e d b y digestion of u r i n a r y c h o n d r o i t i n s u l f a t e i s o m e r s w i t h c h o n d r o i t i n a s e - A B C a f t e r I r a c t i o n a t i o n b y stepwise elution on D o w e x I - X 2 resins in 0.25 M NaC1 i n c r e m e n t s . T h e s a m p l e is t h e s a m e as t h a t u s e d for Table I. T h e e x p e r i m e n t a l p r o c e d u r e is described in t h e t e x t .
In the o. 5 M NaC1 fraction, one spot corresponding to unsaturated nonsulfated disaccharide was visualized by exposure to a Mineralight. Two spots were detected at the o.75 and I.O M NaC1, one predominant spot corresponding to unsaturated nonsulfated disaccharide and another faint spot corresponding to unsaturated 4-sulfated disaccharide. The 1.25 M NaC1 fraction showed three spots similar to unsaturated nonsulfated, 4-sulfated and 6-sulfated disaccharides. The ultraviolet absorption examined under a Mineralight at 232 nm, as well as the measurement by the MorganElson reaction, indicated that the greatest amount was shown in the unsaturated 6-sulfated disaccharide followed b y a lesser amount of the unsaturated 4-sulfated disaccharide and a minor amount of the unsaturated nonsulfated disaccharide. The findings indicated that the unsaturated nonsulfated disaccharide is the major constituent between the 0.5 and I.O M NaCI fractions, whereas the proportions of the unsaturated 4-sulfated disaccharide to the unsaturated nonsulfated disaccharide increased with NaC1 concentrations up to 1.25 M. The sum of the unsaturated nonsulfated disaccharide in urinary chondroitin sulfate of a 45-year-old female was nearly one-fifth of the total glycosaminoglycans, though lesser amounts of the unsaturated nonsulfated disaccharide were found in young individuals. Biochim. Biophys. Acta, 23 ° (I971) 583-594
592
K. MURATAet al.
The spot of the unsaturated 6-sulfated disaccharide appeared at NaC1 concentrations higher than 1.25 M. At NaC1 concentrations greater than 1.5 M, no spot of unsaturated nonsulfated disaccharide was detected. The ratio of unsaturated 6-sulfated disaccharide to unsaturated 4-sulfated disaccharide remained the same in the 1.25 and 1. 5 M NaC1 fractions, but it decreased in the 1.75 M NaC1 fraction. Three spots corresponding to the unsaturated 4-sulfated, 6-sulfated and all-sulfated disaccharides appeared in the 2.0 M NaC1 fraction, after staining by the AgNO 3 reaction v which is more sensitive than detection under a Mineralight. The sums of the unsaturated 4-sulfated disaecharides and the unsaturated 6-sulfated disaccharides in these fractions comprised approx. 25 and 4 ° % of the total glycosaminoglycans, respectively. Thus, greater electrophoretic mobility was associated with higher sulfate contents of the fractionated glycosaminoglycans, mainly chondroitin sulfate isomers, which were eluted with NaC1 concentrations between 0.5 and 2.0 M, except for that of keratan sulfate. The electrophoretic migration in 2.0 M NaC1 showed two spots, the faster spot corresponding to a standard chondroitin sulfate A and the slower one corresponding to a standard keratan sulfate prepared from bovine cornea (gift from Dr. T. Osawa, University of Tokyo). One spot obtained in the 3.0 M NaC1 fraction migrated somewhat faster than that of the slower spot of the 2.0 M NaC1 fraction. Anthrone-positive substances were also measured in the 2.0 and 3.0 M NaC1 fractions, and a faint spot corresponding to galactose in these fractions was detected on the paper chromatogram by the method mentioned above. These observations indicate the presence of keratan sulfate with different sulfate contents in both fractions. DISCUSSION Numerous investigations have indicated that urinary glycosaminoglycans constitute diverse compounds with heterogeneous structures 1-~. The present data determined at the level of unsaturated disaccharide units indicate that nonsulfated or undersulfated chondroitin sulfate and oversulfated chondroitin sulfate are present in human urine in addition to the major constituents of chondroitin monosulfate isomers. Several authors have reported the presence of nonsulfated or undersulfated chondroitin sulfate in human urine in various amounts 3-e. The differences in the undersulfated chondroitin sulfate contents m a y be due not only to the different extraction and determination procedures of chondroitin sulfate isomers, but also to the different rates of the partially or totally nonsulfated compounds. The present finding of the unsaturated nonsulfated disaccharide in normal urine determined b y the MorganElson method after paper chromatographic separation is consistent with our previous studies using another enzymic assay, indicating the presence of nonsulfated chondroitin sulfate in urinary glycosaminoglycans in normal individuals 7. On the other hand, it has not been clear whether an oversulfated chondroitin sulfate occurs in urinary glycosaminoglycans in normal human urine. We have recently reported the presence of an oversulfated chondroitin sulfate as one of the urinary chondroitin sulfate isomers in certain cases 7. The present results with paper rechromatographic and electrophoretic procedures with chemical analysis have clearly demonstrated the existence of an unsaturated di-sulfated disaccharide, indicating that an oversulfated chondroitin sulfate is present in human urine, Because of the limited Biochim. Biophys. Acta, 23° (1971) 583-594
URINARY CHONDROITIN SULFATE ISOMERS
593
quantities of unsaturated di-sulfated disaccharide available, further identification could not be made. Detailed analysis of the proportion of urinary chondroitin sulfate isomers appears to be worth while, since it m a y reflect their metabolic turnover in the body connective tissue. The present results confirm our previous studies performed b y a different enzymic approach ~that chondroitin sulfate C as well as chondroitin sulfate A are major components in urinary glycosaminoglycans of normal individuals. Inadequate data are as yet available to assess the relationships between the minor constituents of the undersulfated or oversulfated chondroitin sulfate isomers and the major component of monosulfated chondroitin sulfates. Since urinary glycosaminoglycans are parts of the final degraded products of the body connective tissue, the presence of such diverse chondroitin sulfate isomers would be interpretable. The diversity of urinary chondroitin sulfate isomers resulting from the degradation and desulfation would be partly due to such enzymic actions as those of hyaluronidase in human urine 17 and chondro-4-sulfatase in bovine aortic tissue TM. Our recent findings that a novel oversulfated chondroitin sulfate exists in human arterial tissue TM indicate a certain relationship between the oversulfated chondroitin sulfate isomer in circulatory connective tissue system and that in urine. It is uncertain whether the nonsulfated and oversulfated chondroitin sulfate isomers in urine are present independently or as hybrid structures. The possible contribution of aging or inflammatory effects on connective tissue m a y reflect the qualitative and quantitative changes of the chondroitin sulfate isomers. The problem that the electrophoretic mobility of fractionated urinary glycosaminoglycans is related to sulfate content has been indicated by VARADI et al. 4. Recently, MATHEWS AND DECKER1° reported that the greater migration rates on electrophoresis were also associated with the sulfate content of the products of degradation of chondroitin sulfate preparations with hyaluronidase. The present finding that the fractions with higher sulfate contents, which are associated with those of the higher ratios of the sums of the unsaturated 4-sulfated and 6-sulfated disaccharides to the unsaturated nonsulfated disaccharide, show distinctly faster migration on electrophoresis is in accord with the reports by VARADI g~ al. 4 and MATHEWS AND DECKER 19.
The appearance of a single spot corresponding to the unsaturated nonsulfated disaccharide at 0.5 M NaC1 elution is of interest, since it m a y suggest the presence of totally nonsulfated chondroitin sulfate in the fraction. The observation of two spots in the 0.75 and I.O M NaCI fractions, which correspond to the unsaturated nonsulfated and 4-sulfated disaccharides but not to the unsaturated 6-sulfated disaccharide, m a y indicate a close relationship between these two compounds. The finding would suggest that nonsulfated chondroitin sulfate and chondroitin sulfate A exist independently and/or form possible hybrid structures such as undersulfated chondroitin sulfate A. The interaction of heparan sulfate or hyaluronic acid in these fractions is not clear in the present study. Tile findings reported here suggest heterogeneous structures of urinary glycosaminoglycans. Further extensive studies of urinary chondroitin sulfate isomers at disaccharide units would assist in the understanding of the diverse urinary chondroitin sulfate isomers in connection with the metabolic states and pathogenesis of connective tissue disorders. Biochim. Biophys. Acta, 230 (I97 I) 583-594
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K. MURATAet al.
ACKNOWLEDGMENTS
The authors wish to thank Professor Y. Osbima for his interest and encouragement. We are indebted to Dr. T. Okuyama for providing chondroitinases and chondrosulfatases, and to Miss T. Aoyama and Miss H. Azuma for skillful technical assistance. This investigation was supported in part by a research grant from the Ministry of Education, Japan. REFERENCES I 2 3 4 5 6 7 8 9 io 1i 12 13 14 I5 16 17 18 I9
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