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Isolation and preliminary characterization of glycosaminoglycans in human plasma
319
Clinica
Chimica
@ Elsevier
Acta,
Scientific
50 (1974) 319-328 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 5979
ISOLATION AND PRELIMINARY CHARACTERIZATION GLYCOSAMINOGLYCANS IN HUMAN PLASMA
NOBORU
TANIGUCHI,
Department
(Received
of Pediatrics,
NAOKI
MORIYA
Kanazawa
and ICHIRO
University
School
OF
NANBA of Medicine,
Kanazawa
(Japan)
June 24,1973)
Summary Plasma glycosaminoglycans were isolated, after pronase digestion by precipitation with cetylpyridinium chloride. The glycosaminoglycans in plasma were found exclusively in the protein fraction precipitated by trichloroacetic acid. The concentration of glycosaminoglycans in normal plasma was 168 to 272 pg per 100 ml. Electrophoresis of plasma glycosaminoglycans in barium acetate buffer on cellulose acetate revealed three fractions, all resistant to streptomyces hyaluronidase but susceptible to testicular hyaluronidase. Electrophoretic mobility of the major fraction was similar to dermatan sulfate and hyaluronic acid. Enzymatic assay with chondroitinases, however, indicated that the major constituent of plasma glycosaminoglycans was undersulfated chondroitin sulfate composed of equimolar amounts of unsaturated non-sulfated disaccharides and unsaturated 4-sulfated disaccharides at the disaccharide subunit levels. The other two fractions were identical with chondroitin sulfate A and C. The quantity of chondroitin sulfate C was least. Hyaluronic acid and over-sulfated chondroitin sulfate were not demonstrated in plasma.
Introduction The presence of glycosaminoglycans (GAG) in human plasma has been reported by several investigators [l-7]. Badin et al. [l] demonstrated that a uranic acid-containing polysaccharide was present in the “euglobulin” fraction of human plasma. With their procedure, the average value from normal individuals was 206 pg per 100 ml of plasma. However, investigations concerning the GAG in human plasma have been relatively few, probably owing to their low concentration. Bassiouni [ 21 isolated, from normal human plasma, trace amounts of acid mucopolysaccharides which could be separated electrophoretically into a component migrating at the same rate as cartilage chondroitin sulfate and another
with lower mobility. Bollct et. al. 13 J olm~~rd that the GAG in human plasma were separated into two components on paper chromatography; I)oth were labile to testicular hyaluronidase. Chromatographic characteristics of one’ (‘omponcnt resembled those of cartilage chondroitin sulfate. Schiller [ 4 ] rf>l)orted positive evidence for chondroitin sulfate> A Ijeing the major GAG in human plasma. The nature of the other electrophorctic fraction rramains to IW cblucidated. In the present work, the GAG in human plasma were isolated, after digestion of plasma proteins with pronase, by precipitation with crtyll)yridinium chloride. Electrophoretic characteristics and enzymatic susceptibility of this material were examined. Also, the disaccharide subunits of chondroitin sulfate isomers in plasma GAG were estimated chromatographically by using the enzymatic assay method with chondroitinases. Materials
and Methods
Isolation ofplasma GAG Blood specimens (approximately 50 to 100 ml) were obtained from healthy adults with the addition of 1.3 mg of EDTA- 2Na per ml. After mixing, plasma was separated by ccntrifugation. Plasma proteins were precipitated with 10 volumes of 10% trichloroacetic acid. After standing for 2 h at O”, the precipitates were obtained by centrifugation and the supernatant solutions were also collected. The precipitated mat,erial was washed twice with 10%’ trichloroacetic acid, twice with ether to be free from trichloroacetic acid and dried at 70”. The dried material was suspended in 0.1% calcium acetate, pH 8.0, and digested with 50 mg of pronase (Kaken Kagaku Co., Tokyo, Japan) per g of protein for 48 h at 37”. The pH of the solution was adjusted several times h3 adding sodium hydroxide. Then, trichloroacetic acid was added to a final con centration of 10%. The precipitates which formed after standing for 2 h at O”, were centrifuged down. The clear supernatant was dialyzed against running tap water overnight and concentrated to approximately 3 to 5 ml by evaporation in air. The trichloroacetic acid-treated supernatant of original plasma was also dialyzed and concentrated as above. Crude GAG were obtained by adding 4 volumes of ethanol saturated with sodium acetate. After standing for 4 h at 0”. the resultant precipitates were collected, washed with ethanol and dried with ether. The crude materials were dissolved in 0.1 M NaCl and the GAG were precipitated with 5% cetylpyridinium chloride added dropwise until no further precipitation occurred. The precipitates which formed after stallding for several and dissolved in a small volume of 2 M NaCl. hours at 37”, were collected Insoluble materials were centrifuged down and the GAG were reprecipitated by adding 4 volumes ethanol for 4 h at 0”. The resultant precipitates were dissolved in a small volume of water and the GAG were precipitated by adding 4 volumes of ethanol saturated with sodium acetate. The last procedure was repeated and the material obtained was washed with ethanol and ether, and dried in air. Further experiments were carried out on this material. Recovery of chondroitin sulfate added to plasma Various amounts of commercial chondroitin
sulfate
(A + C) were added
to
321
each 5.0 ml of plasma. The isolation and purification of GAG were performed as described above. Uranic acid was determined by the carbazole method of Dische [ 141. Effects of enzymatic digestion Streptomyces hyaluronidase (AMANO) from Streptomyces hyalurolyticus nou. was purchased from Seikagaku Kogyo Co., Tokyo, Japan. Only hyaluronic acid is degraded by this enzyme; chondroitin sulfates A, B, C, and D, chondroitin, keratosulfate, heparin and chitin are not attacked. Enzymatic digestion with streptomyces hyaluronidase was carried out in 0.02 M acetate - 0.15 M NaCl buffer, pH 5.0, at 60” for 60 min by the method of Ohya and Kaneko [9]. Digestion with testicular hyaluronidase (Fluka Co.) was performed in 0.1 M acetate-O.15 M NaCl buffer, pH 5.0, at 37” for 24 h. The specimens were also treated with chondroitinase ABC (Seikagaku Kogyo Co.) by the method of these specimens were Saito et al. [8] for 60 min at 37“. After incubation, deproteinized by adding trichloroacetic acid to a final concentration of 10%. After centrifugation, the supernatant solution was dialyzed against distilled water and the undigested GAG were recovered with the addition of 4 volumes of ethanol saturated with sodium acetate after standing for 4 h at 0”. Electrophoresis on cellulose acetate membrane Electrophoresis on Separax membrane (Joko Sangyo Co., Tokyo, Japan) was carried out by Wessler’s method [lo] in 0.05 M barium acetate for 2 to 3 h with a potential gradient of 15 V/cm and in 0.1 M HCl for 1 h with a potential gradient of 5 V/cm. The strips were then transfered without drying to 1% alcian blue in 2% acetic acid for 30 min and washed with running tap water. The reference standards of dermatan sulfate and heparan sulfate were kindly supplied by Dr M.B. Mathews (Chicago University). Chondroitin sulfate A and C were products of Seikagaku Kogyo Co. Hyaluronic acid was prepared by a stepwise elution technique of Schmidt [ll] on a DEAE Sephadex A-25 column (Cl-) from a commercial product of chondroitin sulfate which was contaminated with trace amounts of hyaluronic acid. Isolation of GAG from electrophoretically separated fractions Electrophoresis of plasma GAG was performed in 0.05 M barium acetate as mentioned above. Alcian blue-positive fractions were located by staining narrow strips cut from the edges of the cellulose acetate membrane and the areas of the unstained strips corresponding to each fraction were cut out. The GAG of each fraction were eluted with distilled water at 37” for several hours. The eluates of the same fraction were combined from several strips, dialyzed against distilled water and then concentrated. The GAG of each fraction were precipitated with the addition of ethanol saturated with sodium acetate. Enzymatic assay in plasma GAG The plasma method of Saito specimens were
of disaccharide
constituents
of isomeric chondroitin
sulfates
GAG were digested with chondroitinase ABC and AC by the et al. [8] at 37” for 60 min. Following complete digestion, the spotted onto Toyo Roshi No. 51 paper (45 cm long) with
standard markers of unsaturated disaccharides. The al)hreviations used foi unsaturated disaccharides are as follows: ADi-OS, 2-acetamido-2-dcox~-~~-~~-(~D -gluc~o-4-e~~epyranosyluronic acid)-I,-galac~tosc; ADi-OSFJ . 2-ac,rtamiclo-2-dc~oxy-3-0-(0-D -gluco-4-enepyranosyluronic acid)-D -glucose; A Di-4S, 2-acetamido-2-deoxy-3-o-(fl-D -gluc~o-l-enepyral~osyiuronic acid)-4-O-sulfa-D -galactosc,; ADi-GS, 2-acetamido-2-desox;y-3-0-(P_n-gluco-4-ent~l~~~ranosyluronic acid)-6-Osulfo-D -galactose. ADi-OS. ADi-4s and ADi-6S were purchased from Seikagaku was prepared from hyaluronic acid by t,he digestion with Kogyo Co. ADi-OS, chondroitinase AC. Descending paper chromatography was carried out in the following solvents; Solvent I, n-butanol - ethanol - water (52:32:16, by vol.); and Solvent II, n-hutanol - acetic acid - 1 iV:I ammonia (2:3:1, by vol.). After desalting chromatography for 23 h in Solvent I, descending paper chromatography was carried out in Solvent II for 15 h at room temperature. The unsaturated disaccharides were detected by viewing under a Minerallight. Model S-2537, and hy ultraviolet photography. In some cases, reducing sugars were detected on the chromatogram with a silver nitrat,e reagent. The regions containing unsaturated disaccharides and the corresponding areas of the blank were cut out and eluted with 0.01 M HCl at 50” for 10 min. After centrifugation, the absorbance of the supernatant solution was measured at 232 nm against the corresponding blank solution. The amount of unsaturated disaccharide products was calculated from the change in absorbance with the use of 5.7, 5.1 and 5.5 as millimolar ahsorption coefficients for ADi-OS ADi-4S and ADi-GS, respectively [ 12 ] .
Other analytical
methods
Uranic acid was estimated by carbazole method of Dische [14]. Hexosamine content of plasma GAG was determined after the hydrolysis in 4 M HCl at 100” for 15 h by a Hitachi amino acid autoanalyzer, KLA-3B. Hexosamine content in unsaturated disaccharides was estimated after hydrolysis in 4 M HCl for 3 h in boiling water bath, by the amino acid analyzer. Under these conditions, the maximal yield of hexosamine in these disaccharides was achieved. Results
with TABLE
Plasma GAG trichloroacetic
(MEASURED
Amount
protein fraction acid-containing
which was precipitated polysaccharides were
I
RECOVERY
added
were found in the acid. No uranic
OF AS
CHONDROITIN
of chondroitin sulfate
SULFATE
GLUCURONIC
(@g)
ADDED
TO
5.0-ml
SAMPLES
OF
NORMAL
PLASMA
ACID)
TCA-precipitated’
TCA-supernatant
Recovery
fraction
fraction
(3:
(ug)
(pg)
) _
0.0
6.8
0.0
17.5
6.2
14.0
80
35.0
7.2
27.0
77
70.0
6.6
58.0
83
* The
protein
fraction
of plasma
which
was precipitated
\*rith tnchloroacetic
acid.
323
TABLE
II
ANALYTICAL
DATA
OF
GLYCOSAMINOGLYCANS
IN HUMAN
Plasma
GAG
Molar
volume
isolated
of uranic
PLASMA
Molar
ratio
ratio
Molar
of
ratio
of
Gal-NH*-Glu-NH2
ADi-OS-ADi-4s
-IS:22
1.00:
1.20
1.00:0.92
83:17
1.00:
1.34
1.00:0.90
84:
1.00:
1.25
1.00:
1.02
acid:hexosamine
(ml)
50
91
25
(182 42
1.00:
!Jg*
0.96
fig/d11
(168 /.z/dl) 25
68
(272 /.oz/dl) * Measured
as glucuonic
16
Fraction
1 in Fig.1.
acid.
detectable in the trichloroacetic acid-treated supernatant of plasma. Recovery experiments indicated that various amounts of chondroitin sulfate added to plasma were recovered in the supernatant fraction with no significant amounts in the protein fraction. Recovery rates were approximately 80% (Table I). Plasma GAG levels in this experiment were found to be 182, 168, and 272 pg per 100 ml, respectively (Table II). Electrophoresis of plasma GAG in barium acetate buffer revealed three akian blue-positive spots clearly (Fig. 1). These fractions were numbered 1, 2 and 3 in order of increasing electrophoretic mobility. Fraction 1 corresponded
Fig.
1.
barium
Electrophoretic acetate
with
pattern a potential
of
plasma
gradient
glycosaminoglycans. of
15
V/cm
for
2 h.
Electrophoresis
was
carried
out
in 0.05
M
+ ~trept0aryce.s HYaluroniaase ( mAi
Plasma
+
)
GAG
Testicular iiyaluronidase
Plasma
GAG
+ chOnarOitinase AE
Fig. 2. Electrophoretic enrymes. Electrophoretic
pattern of plasma glycosamu~ug~y~~ns condition was the same as in Fig.
before
and after
the treatment
with
various
1.
in mobility to hyaluronic acid and dermatan sulfate. Fractions 2 and 3 were similar in electrophoretic mobility to chondroitin sulfate A and C, respectively. As shown in Fig. 2, these three fractions were all resistant to enzymatic digestion with streptomycrs hyaluronidase (AMANO). However, treatment with testicular hyaluronidase or chondroitinase ABC resulted in complete disappearance of these alcian blue-positive spots. In some cases, a faint spot which migrated more slowly than authentic samples of GAG, was observed.
I
I I I
I
I I 1 Fig. 3. Electrophoretic HCl with a potential
pattern of plasma glycosaminoglycans. gradient of 5 V/cm for 1 h.
Electrophoresis
was carried
out
in 0.1
M
325
STD
STV
L m-66
AVi-6S
A Dl-bS
ADI-~S
ADI-CJ
ADi-OS
Fig. 4. Paper chromatographic separation of unsaturated disaccharides obtained by digestion of plasma glycosaminoglycans with chondroitinase ABC and AC. Ultraviolet absorption print is represented.
Electrophoresis in 0.1 M HCl indicated that the electrophoretic mobility of the major fraction of plasma GAG was intermediate between those of hyaluronic acid and isomeric chondroitin sulfates. A faint spot migrating at the same rate as hyaluronic acid was also observed. However, this fraction was not degraded by streptomyces hyaluronidase (AMANO) (Fig. 3). The plasma GAG were found to have an average uranic acid:hexosamine molar ratio of 1.00:0.93. The hexosamine of plasma GAG was found to be approximately 80% galactosamine and 20% glucosamine (Table II). Paper chromatographic analyses of the unsaturated disaccharides which appeared after the digestion of plasma GAG with chondroitinase ABC or AC, revealed two spots corresponding to ADi-4S and ADi-OS under a Minerallight exposure (Fig. 4). No appreciable differences were observed between chondroitinase ABC and AC. The molar ratios of ADi-OS and ADi-4S were. on an
AM-68
ADl4s
AM-4S
AM48
ADI-@
ADi-CS Af-%
Fig. 5. Paper chromatographic separation of unsaturated disaccharides obtained by digestion glycosaminoglycans with chondroitinase. Tracing of ultraviolet absorption print is presented.
of plasma
Fig. 6. Paper chromatographic separation of unsaturated disaccharides obtained by digestion of minor fractions of plasma glycosaminoglycans with chondroitinase .4C. Stained with the silver nitrate reagent. Dotted circles indicate faint spots corresponding to ADi-GS and ADi-4S. respectively.
average, 1.00:1.30. The materials isolated from Fraction 1 in Fig. 1, contained approximately equimolar amounts of ADi-OS and ADi-4S (Table II). No detectof hyaluronic acid, were able amounts of ADi-OSn, which is the constituent found in the enzymatic digests of plasma GAG (Fig. 5). On paper chromatography, the enzymatic digests of the materials obtained from Fractions 2 and 3 in Fig. 1, showed faint spots corresponding to ADi-4S and ADi-GS, respectively, by staining with the silver nitrate reagent (Fig. 6). The quantity of ADi-6S was extremely small. Insufficient materials prevented further characterization of these fractions. The assay of uranic acid and hexosamine of the eluates from the areas corresponding to ADi-OS and ADi-4S, indicated that galactosamine was a sole aminosugar and the molar ratio of uranic acid to hexosamine was approximately 1:l in both eluates. Discussion Badin et al. [1] found the uranic acid content of the GAG isolated from plasma of seven normal adults to range between 153 and 281 pg per 100 ml of plasma. Bollet [ 31 reported that sera of 34 normal adults had an average level of GAG of 277 pg per 100 ml, measured as glucuronic acid. Inherent losses of GAG in the method employed here for isolation and purification were unavoidable, especially when a small amount of plasma was used. Although the actual concentration of plasma GAG could not be inferred from the present results alone, the values, 168 to 272 pg of uranic acid per 100 ml of plasma, agree well with previous reports. Calantroni et al. [6] reported that, on adsorption on an ECTEOLA column, plasma GAG were present in two forms. One was adsorbed readily
327
(free type), the other after treatment with proteolytic enzyme (bound type). Badin et al. [l] reported that the uranic acid-containing polysaccharide in fraction, since examination of the plasma was found entirely in “euglobulin” euglobulin-free plasma revealed no uranic acid. In the present work, plasma GAG were found exclusively in the protein fraction precipitated by trichloroacetic acid. Because of the different isolation techniques, further discussion on these “free” and “bound” types of plasma GAG seems to be meaningless. Previous reports indicated the major GAG in plasma to be chondroitin sulfate A [4,6,7]. But the nature of the remaining GAG was not adequately identified. Recently, Murata et al. [7] reported that, using the enzymatic assay method with chondroitinases, chondroitin sulfate isomers in plasma GAG contained various types of unsaturated disaccharides as constituents; that is, ADi-OS, ADi-4S, ADi-6S and oversulfated unsaturated disaccharide. They concluded that chondroitin sulfate A was the major constituent of plasma GAG and chondroitin sulfate C was also present in plasma in lesser quantity. The presence of ADi-OS and oversulfated unsaturated disaccharide at the subunit levels indicated the heterogeneous structures of plasma chondroitin sulfate isomers with regard to the degree of sulfation. In the present experiments, electrophoretic mobility of the major fraction of plasma GAG was not identical with chondroitin sulfate A in barium acetate buffer or 0.1 M HCl, but was similar to dermatan sulfate and hyaluronic acid in barium acetate buffer. In this system, dermatan sulfate and hyaluronic acid have nearly identical mobility. However, the major fraction of plasma GAG was not susceptible to streptomyces hyaluronidase (AMANO), but labile to testicular hyaluronidase completely. These results indicated that the major GAG in plasma was neither hyaluronic acid nor dermatan sulfate. Electrophoresis of plasma GAG in 0.1 M HCl showed the major GAG having an intermediate mobility between those of hyaluronic acid and isomeric chondroitin sulfates. Wessler [lo] reported that in 0.1 M HCl, the carboxyl groups of GAG are not dissociated so that the migration rate depends exclusively on sulfate content. These results strongly suggest the major fraction of plasma GAG being undersulfated when compared with authentic samples of isomeric chondroitin sulfates. Enzymatic assay with chondroitinases clearly demonstrated the major GAG in plasma to contain equimolar amounts of ADi-4S and ADi-OS as unsaturated disaccharide subunits suggesting it might be composed of hybrid structures of ADi-4S and ADi-OS. Probably, predominance of ADi-4S in plasma GAG disaccharide subunits might have been erroneously interpretated by previous workers as indicating the major GAG in plasma to be chondroitin sulfate A. The other two minor fractions of plasma GAG were similar in electrophoretic mobility in barium acetate buffer with those of chondroitin sulfate A and C. Enzymatic assay of these fractions at the disaccharide subunit levels showed that one was identical with chondroitin sulfate A and the other probably identical with chondroitin sulfate C; but insufficient materials prevented further characterization of these fractions. Deutsch [13] isolated hyaluronic acid from the sera of two patients, one with reticulum cell sarcoma and the other with neuroblastoma, but was unable to demonstrate the presence of
hyaluronic acid in normal serum. In the present work, t~nz~~matic dtgests o I’ plasma GAG with c*hondroitinase AC contained no detc~ctabltl amounts 01 ADi-OSII , which is the constituent of hyaluronic* acid. El~~c,trol)hor~~sis of l)lasma GAG in 0.1 M HCl rrvealcd a faint sl’ot (*orresponding in mol)ility with hyaluronic acid. hut this fraction was not degraded by streptotnyccs hyaluronidase (AMRNO). Although the exact nature of this fraction remains to IW elucidated, il seems likely to 1~ acidic, glycoprotcins. Contrary to the> data tx~~)orted by Murata et al. 17 1. the prcs~lnc*r of oversulfatcd unsaturate(l tlisac~llarid~~ was nol demonstrated in the disaccharide subunits of plasma GAG. The source of GAG in plasma is not known. The oc’currenccl of undersulfated chondroibin sulfate, which is composed of both A Di4S and A Di-OS, is of interest in particular with regard to the process of sulfation. li’urther work is required to elucidate whether these undersulfated chondroitin sulfates in plasma might he waiting for intracellular sulfation process or not. Acknowledgment This Education
work was supported in Japan.
in part
by a research
grant
from
the Ministry
of
References 1 2 3 4 5 6 7 8 9 10
.I. Badin, M. Schubert and M. Vouras, J. Clin. Invest., 34 (1955) 1317. M. Bassiouni. Ann. Rheum. Dis.. 14 (1955) 288. A.J. Ballet, M.W. Seravdarian and W.F. Simpson, J. Clin. Invest.. 36 (1957) 1328. S. Srhitler. Biochim. Biophys. Acta, 28 (1958) 413. C. Friman and R. Brunish. Proc. Sot. Exutl. Blol. Med.. 122 (1966) 599. A. Catantroni. P.V. Doneliy and N. DIFe&antr, J. Clin. Invest., 48 (1969) 332 K. Muratd, T. Ogura and T. Okuvama. Blochem. Med.. 6 (1972) 223. II. Saito. T. Yamagata and S. Suruki. ,I. Biol. Chem.. 243 (1968) 1536. ‘I’. Ohya and Y. Kaneko, Biochim. Biophys. Acta. 198 (1970) 607. F:. \vesstrr. in E.A. Balazs (Ed.). Chemistry and Molecular Biology of the Intercrxllulu Academic Press. London and New York. Vol. 2 1970. I). 895. 11 M. Schmidt, Biochim. Biophvs. Acta, 63 (1962) 346. 12 T. Yamagata. H. Saitn. 0. Habuchi and S. Suzuki. .J. Biol. Chcm.. 243 (1968) 152.3. 13 H.F. Deutsch, J. Biot. Chem., 224 (1957) 767 14 Z. Dischr. .I. Biol. Chem.. 167 (1947) 189.
Matnx.
Clinica
Chimica
@ Elsevier
Acta,
Scientific
50 (1974) 319-328 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 5979
ISOLATION AND PRELIMINARY CHARACTERIZATION GLYCOSAMINOGLYCANS IN HUMAN PLASMA
NOBORU
TANIGUCHI,
Department
(Received
of Pediatrics,
NAOKI
MORIYA
Kanazawa
and ICHIRO
University
School
OF
NANBA of Medicine,
Kanazawa
(Japan)
June 24,1973)
Summary Plasma glycosaminoglycans were isolated, after pronase digestion by precipitation with cetylpyridinium chloride. The glycosaminoglycans in plasma were found exclusively in the protein fraction precipitated by trichloroacetic acid. The concentration of glycosaminoglycans in normal plasma was 168 to 272 pg per 100 ml. Electrophoresis of plasma glycosaminoglycans in barium acetate buffer on cellulose acetate revealed three fractions, all resistant to streptomyces hyaluronidase but susceptible to testicular hyaluronidase. Electrophoretic mobility of the major fraction was similar to dermatan sulfate and hyaluronic acid. Enzymatic assay with chondroitinases, however, indicated that the major constituent of plasma glycosaminoglycans was undersulfated chondroitin sulfate composed of equimolar amounts of unsaturated non-sulfated disaccharides and unsaturated 4-sulfated disaccharides at the disaccharide subunit levels. The other two fractions were identical with chondroitin sulfate A and C. The quantity of chondroitin sulfate C was least. Hyaluronic acid and over-sulfated chondroitin sulfate were not demonstrated in plasma.
Introduction The presence of glycosaminoglycans (GAG) in human plasma has been reported by several investigators [l-7]. Badin et al. [l] demonstrated that a uranic acid-containing polysaccharide was present in the “euglobulin” fraction of human plasma. With their procedure, the average value from normal individuals was 206 pg per 100 ml of plasma. However, investigations concerning the GAG in human plasma have been relatively few, probably owing to their low concentration. Bassiouni [ 21 isolated, from normal human plasma, trace amounts of acid mucopolysaccharides which could be separated electrophoretically into a component migrating at the same rate as cartilage chondroitin sulfate and another
with lower mobility. Bollct et. al. 13 J olm~~rd that the GAG in human plasma were separated into two components on paper chromatography; I)oth were labile to testicular hyaluronidase. Chromatographic characteristics of one’ (‘omponcnt resembled those of cartilage chondroitin sulfate. Schiller [ 4 ] rf>l)orted positive evidence for chondroitin sulfate> A Ijeing the major GAG in human plasma. The nature of the other electrophorctic fraction rramains to IW cblucidated. In the present work, the GAG in human plasma were isolated, after digestion of plasma proteins with pronase, by precipitation with crtyll)yridinium chloride. Electrophoretic characteristics and enzymatic susceptibility of this material were examined. Also, the disaccharide subunits of chondroitin sulfate isomers in plasma GAG were estimated chromatographically by using the enzymatic assay method with chondroitinases. Materials
and Methods
Isolation ofplasma GAG Blood specimens (approximately 50 to 100 ml) were obtained from healthy adults with the addition of 1.3 mg of EDTA- 2Na per ml. After mixing, plasma was separated by ccntrifugation. Plasma proteins were precipitated with 10 volumes of 10% trichloroacetic acid. After standing for 2 h at O”, the precipitates were obtained by centrifugation and the supernatant solutions were also collected. The precipitated mat,erial was washed twice with 10%’ trichloroacetic acid, twice with ether to be free from trichloroacetic acid and dried at 70”. The dried material was suspended in 0.1% calcium acetate, pH 8.0, and digested with 50 mg of pronase (Kaken Kagaku Co., Tokyo, Japan) per g of protein for 48 h at 37”. The pH of the solution was adjusted several times h3 adding sodium hydroxide. Then, trichloroacetic acid was added to a final con centration of 10%. The precipitates which formed after standing for 2 h at O”, were centrifuged down. The clear supernatant was dialyzed against running tap water overnight and concentrated to approximately 3 to 5 ml by evaporation in air. The trichloroacetic acid-treated supernatant of original plasma was also dialyzed and concentrated as above. Crude GAG were obtained by adding 4 volumes of ethanol saturated with sodium acetate. After standing for 4 h at 0”. the resultant precipitates were collected, washed with ethanol and dried with ether. The crude materials were dissolved in 0.1 M NaCl and the GAG were precipitated with 5% cetylpyridinium chloride added dropwise until no further precipitation occurred. The precipitates which formed after stallding for several and dissolved in a small volume of 2 M NaCl. hours at 37”, were collected Insoluble materials were centrifuged down and the GAG were reprecipitated by adding 4 volumes ethanol for 4 h at 0”. The resultant precipitates were dissolved in a small volume of water and the GAG were precipitated by adding 4 volumes of ethanol saturated with sodium acetate. The last procedure was repeated and the material obtained was washed with ethanol and ether, and dried in air. Further experiments were carried out on this material. Recovery of chondroitin sulfate added to plasma Various amounts of commercial chondroitin
sulfate
(A + C) were added
to
321
each 5.0 ml of plasma. The isolation and purification of GAG were performed as described above. Uranic acid was determined by the carbazole method of Dische [ 141. Effects of enzymatic digestion Streptomyces hyaluronidase (AMANO) from Streptomyces hyalurolyticus nou. was purchased from Seikagaku Kogyo Co., Tokyo, Japan. Only hyaluronic acid is degraded by this enzyme; chondroitin sulfates A, B, C, and D, chondroitin, keratosulfate, heparin and chitin are not attacked. Enzymatic digestion with streptomyces hyaluronidase was carried out in 0.02 M acetate - 0.15 M NaCl buffer, pH 5.0, at 60” for 60 min by the method of Ohya and Kaneko [9]. Digestion with testicular hyaluronidase (Fluka Co.) was performed in 0.1 M acetate-O.15 M NaCl buffer, pH 5.0, at 37” for 24 h. The specimens were also treated with chondroitinase ABC (Seikagaku Kogyo Co.) by the method of these specimens were Saito et al. [8] for 60 min at 37“. After incubation, deproteinized by adding trichloroacetic acid to a final concentration of 10%. After centrifugation, the supernatant solution was dialyzed against distilled water and the undigested GAG were recovered with the addition of 4 volumes of ethanol saturated with sodium acetate after standing for 4 h at 0”. Electrophoresis on cellulose acetate membrane Electrophoresis on Separax membrane (Joko Sangyo Co., Tokyo, Japan) was carried out by Wessler’s method [lo] in 0.05 M barium acetate for 2 to 3 h with a potential gradient of 15 V/cm and in 0.1 M HCl for 1 h with a potential gradient of 5 V/cm. The strips were then transfered without drying to 1% alcian blue in 2% acetic acid for 30 min and washed with running tap water. The reference standards of dermatan sulfate and heparan sulfate were kindly supplied by Dr M.B. Mathews (Chicago University). Chondroitin sulfate A and C were products of Seikagaku Kogyo Co. Hyaluronic acid was prepared by a stepwise elution technique of Schmidt [ll] on a DEAE Sephadex A-25 column (Cl-) from a commercial product of chondroitin sulfate which was contaminated with trace amounts of hyaluronic acid. Isolation of GAG from electrophoretically separated fractions Electrophoresis of plasma GAG was performed in 0.05 M barium acetate as mentioned above. Alcian blue-positive fractions were located by staining narrow strips cut from the edges of the cellulose acetate membrane and the areas of the unstained strips corresponding to each fraction were cut out. The GAG of each fraction were eluted with distilled water at 37” for several hours. The eluates of the same fraction were combined from several strips, dialyzed against distilled water and then concentrated. The GAG of each fraction were precipitated with the addition of ethanol saturated with sodium acetate. Enzymatic assay in plasma GAG The plasma method of Saito specimens were
of disaccharide
constituents
of isomeric chondroitin
sulfates
GAG were digested with chondroitinase ABC and AC by the et al. [8] at 37” for 60 min. Following complete digestion, the spotted onto Toyo Roshi No. 51 paper (45 cm long) with
standard markers of unsaturated disaccharides. The al)hreviations used foi unsaturated disaccharides are as follows: ADi-OS, 2-acetamido-2-dcox~-~~-~~-(~D -gluc~o-4-e~~epyranosyluronic acid)-I,-galac~tosc; ADi-OSFJ . 2-ac,rtamiclo-2-dc~oxy-3-0-(0-D -gluco-4-enepyranosyluronic acid)-D -glucose; A Di-4S, 2-acetamido-2-deoxy-3-o-(fl-D -gluc~o-l-enepyral~osyiuronic acid)-4-O-sulfa-D -galactosc,; ADi-GS, 2-acetamido-2-desox;y-3-0-(P_n-gluco-4-ent~l~~~ranosyluronic acid)-6-Osulfo-D -galactose. ADi-OS. ADi-4s and ADi-6S were purchased from Seikagaku was prepared from hyaluronic acid by t,he digestion with Kogyo Co. ADi-OS, chondroitinase AC. Descending paper chromatography was carried out in the following solvents; Solvent I, n-butanol - ethanol - water (52:32:16, by vol.); and Solvent II, n-hutanol - acetic acid - 1 iV:I ammonia (2:3:1, by vol.). After desalting chromatography for 23 h in Solvent I, descending paper chromatography was carried out in Solvent II for 15 h at room temperature. The unsaturated disaccharides were detected by viewing under a Minerallight. Model S-2537, and hy ultraviolet photography. In some cases, reducing sugars were detected on the chromatogram with a silver nitrat,e reagent. The regions containing unsaturated disaccharides and the corresponding areas of the blank were cut out and eluted with 0.01 M HCl at 50” for 10 min. After centrifugation, the absorbance of the supernatant solution was measured at 232 nm against the corresponding blank solution. The amount of unsaturated disaccharide products was calculated from the change in absorbance with the use of 5.7, 5.1 and 5.5 as millimolar ahsorption coefficients for ADi-OS ADi-4S and ADi-GS, respectively [ 12 ] .
Other analytical
methods
Uranic acid was estimated by carbazole method of Dische [14]. Hexosamine content of plasma GAG was determined after the hydrolysis in 4 M HCl at 100” for 15 h by a Hitachi amino acid autoanalyzer, KLA-3B. Hexosamine content in unsaturated disaccharides was estimated after hydrolysis in 4 M HCl for 3 h in boiling water bath, by the amino acid analyzer. Under these conditions, the maximal yield of hexosamine in these disaccharides was achieved. Results
with TABLE
Plasma GAG trichloroacetic
(MEASURED
Amount
protein fraction acid-containing
which was precipitated polysaccharides were
I
RECOVERY
added
were found in the acid. No uranic
OF AS
CHONDROITIN
of chondroitin sulfate
SULFATE
GLUCURONIC
(@g)
ADDED
TO
5.0-ml
SAMPLES
OF
NORMAL
PLASMA
ACID)
TCA-precipitated’
TCA-supernatant
Recovery
fraction
fraction
(3:
(ug)
(pg)
) _
0.0
6.8
0.0
17.5
6.2
14.0
80
35.0
7.2
27.0
77
70.0
6.6
58.0
83
* The
protein
fraction
of plasma
which
was precipitated
\*rith tnchloroacetic
acid.
323
TABLE
II
ANALYTICAL
DATA
OF
GLYCOSAMINOGLYCANS
IN HUMAN
Plasma
GAG
Molar
volume
isolated
of uranic
PLASMA
Molar
ratio
ratio
Molar
of
ratio
of
Gal-NH*-Glu-NH2
ADi-OS-ADi-4s
-IS:22
1.00:
1.20
1.00:0.92
83:17
1.00:
1.34
1.00:0.90
84:
1.00:
1.25
1.00:
1.02
acid:hexosamine
(ml)
50
91
25
(182 42
1.00:
!Jg*
0.96
fig/d11
(168 /.z/dl) 25
68
(272 /.oz/dl) * Measured
as glucuonic
16
Fraction
1 in Fig.1.
acid.
detectable in the trichloroacetic acid-treated supernatant of plasma. Recovery experiments indicated that various amounts of chondroitin sulfate added to plasma were recovered in the supernatant fraction with no significant amounts in the protein fraction. Recovery rates were approximately 80% (Table I). Plasma GAG levels in this experiment were found to be 182, 168, and 272 pg per 100 ml, respectively (Table II). Electrophoresis of plasma GAG in barium acetate buffer revealed three akian blue-positive spots clearly (Fig. 1). These fractions were numbered 1, 2 and 3 in order of increasing electrophoretic mobility. Fraction 1 corresponded
Fig.
1.
barium
Electrophoretic acetate
with
pattern a potential
of
plasma
gradient
glycosaminoglycans. of
15
V/cm
for
2 h.
Electrophoresis
was
carried
out
in 0.05
M
+ ~trept0aryce.s HYaluroniaase ( mAi
Plasma
+
)
GAG
Testicular iiyaluronidase
Plasma
GAG
+ chOnarOitinase AE
Fig. 2. Electrophoretic enrymes. Electrophoretic
pattern of plasma glycosamu~ug~y~~ns condition was the same as in Fig.
before
and after
the treatment
with
various
1.
in mobility to hyaluronic acid and dermatan sulfate. Fractions 2 and 3 were similar in electrophoretic mobility to chondroitin sulfate A and C, respectively. As shown in Fig. 2, these three fractions were all resistant to enzymatic digestion with streptomycrs hyaluronidase (AMANO). However, treatment with testicular hyaluronidase or chondroitinase ABC resulted in complete disappearance of these alcian blue-positive spots. In some cases, a faint spot which migrated more slowly than authentic samples of GAG, was observed.
I
I I I
I
I I 1 Fig. 3. Electrophoretic HCl with a potential
pattern of plasma glycosaminoglycans. gradient of 5 V/cm for 1 h.
Electrophoresis
was carried
out
in 0.1
M
325
STD
STV
L m-66
AVi-6S
A Dl-bS
ADI-~S
ADI-CJ
ADi-OS
Fig. 4. Paper chromatographic separation of unsaturated disaccharides obtained by digestion of plasma glycosaminoglycans with chondroitinase ABC and AC. Ultraviolet absorption print is represented.
Electrophoresis in 0.1 M HCl indicated that the electrophoretic mobility of the major fraction of plasma GAG was intermediate between those of hyaluronic acid and isomeric chondroitin sulfates. A faint spot migrating at the same rate as hyaluronic acid was also observed. However, this fraction was not degraded by streptomyces hyaluronidase (AMANO) (Fig. 3). The plasma GAG were found to have an average uranic acid:hexosamine molar ratio of 1.00:0.93. The hexosamine of plasma GAG was found to be approximately 80% galactosamine and 20% glucosamine (Table II). Paper chromatographic analyses of the unsaturated disaccharides which appeared after the digestion of plasma GAG with chondroitinase ABC or AC, revealed two spots corresponding to ADi-4S and ADi-OS under a Minerallight exposure (Fig. 4). No appreciable differences were observed between chondroitinase ABC and AC. The molar ratios of ADi-OS and ADi-4S were. on an
AM-68
ADl4s
AM-4S
AM48
ADI-@
ADi-CS Af-%
Fig. 5. Paper chromatographic separation of unsaturated disaccharides obtained by digestion glycosaminoglycans with chondroitinase. Tracing of ultraviolet absorption print is presented.
of plasma
Fig. 6. Paper chromatographic separation of unsaturated disaccharides obtained by digestion of minor fractions of plasma glycosaminoglycans with chondroitinase .4C. Stained with the silver nitrate reagent. Dotted circles indicate faint spots corresponding to ADi-GS and ADi-4S. respectively.
average, 1.00:1.30. The materials isolated from Fraction 1 in Fig. 1, contained approximately equimolar amounts of ADi-OS and ADi-4S (Table II). No detectof hyaluronic acid, were able amounts of ADi-OSn, which is the constituent found in the enzymatic digests of plasma GAG (Fig. 5). On paper chromatography, the enzymatic digests of the materials obtained from Fractions 2 and 3 in Fig. 1, showed faint spots corresponding to ADi-4S and ADi-GS, respectively, by staining with the silver nitrate reagent (Fig. 6). The quantity of ADi-6S was extremely small. Insufficient materials prevented further characterization of these fractions. The assay of uranic acid and hexosamine of the eluates from the areas corresponding to ADi-OS and ADi-4S, indicated that galactosamine was a sole aminosugar and the molar ratio of uranic acid to hexosamine was approximately 1:l in both eluates. Discussion Badin et al. [1] found the uranic acid content of the GAG isolated from plasma of seven normal adults to range between 153 and 281 pg per 100 ml of plasma. Bollet [ 31 reported that sera of 34 normal adults had an average level of GAG of 277 pg per 100 ml, measured as glucuronic acid. Inherent losses of GAG in the method employed here for isolation and purification were unavoidable, especially when a small amount of plasma was used. Although the actual concentration of plasma GAG could not be inferred from the present results alone, the values, 168 to 272 pg of uranic acid per 100 ml of plasma, agree well with previous reports. Calantroni et al. [6] reported that, on adsorption on an ECTEOLA column, plasma GAG were present in two forms. One was adsorbed readily
327
(free type), the other after treatment with proteolytic enzyme (bound type). Badin et al. [l] reported that the uranic acid-containing polysaccharide in fraction, since examination of the plasma was found entirely in “euglobulin” euglobulin-free plasma revealed no uranic acid. In the present work, plasma GAG were found exclusively in the protein fraction precipitated by trichloroacetic acid. Because of the different isolation techniques, further discussion on these “free” and “bound” types of plasma GAG seems to be meaningless. Previous reports indicated the major GAG in plasma to be chondroitin sulfate A [4,6,7]. But the nature of the remaining GAG was not adequately identified. Recently, Murata et al. [7] reported that, using the enzymatic assay method with chondroitinases, chondroitin sulfate isomers in plasma GAG contained various types of unsaturated disaccharides as constituents; that is, ADi-OS, ADi-4S, ADi-6S and oversulfated unsaturated disaccharide. They concluded that chondroitin sulfate A was the major constituent of plasma GAG and chondroitin sulfate C was also present in plasma in lesser quantity. The presence of ADi-OS and oversulfated unsaturated disaccharide at the subunit levels indicated the heterogeneous structures of plasma chondroitin sulfate isomers with regard to the degree of sulfation. In the present experiments, electrophoretic mobility of the major fraction of plasma GAG was not identical with chondroitin sulfate A in barium acetate buffer or 0.1 M HCl, but was similar to dermatan sulfate and hyaluronic acid in barium acetate buffer. In this system, dermatan sulfate and hyaluronic acid have nearly identical mobility. However, the major fraction of plasma GAG was not susceptible to streptomyces hyaluronidase (AMANO), but labile to testicular hyaluronidase completely. These results indicated that the major GAG in plasma was neither hyaluronic acid nor dermatan sulfate. Electrophoresis of plasma GAG in 0.1 M HCl showed the major GAG having an intermediate mobility between those of hyaluronic acid and isomeric chondroitin sulfates. Wessler [lo] reported that in 0.1 M HCl, the carboxyl groups of GAG are not dissociated so that the migration rate depends exclusively on sulfate content. These results strongly suggest the major fraction of plasma GAG being undersulfated when compared with authentic samples of isomeric chondroitin sulfates. Enzymatic assay with chondroitinases clearly demonstrated the major GAG in plasma to contain equimolar amounts of ADi-4S and ADi-OS as unsaturated disaccharide subunits suggesting it might be composed of hybrid structures of ADi-4S and ADi-OS. Probably, predominance of ADi-4S in plasma GAG disaccharide subunits might have been erroneously interpretated by previous workers as indicating the major GAG in plasma to be chondroitin sulfate A. The other two minor fractions of plasma GAG were similar in electrophoretic mobility in barium acetate buffer with those of chondroitin sulfate A and C. Enzymatic assay of these fractions at the disaccharide subunit levels showed that one was identical with chondroitin sulfate A and the other probably identical with chondroitin sulfate C; but insufficient materials prevented further characterization of these fractions. Deutsch [13] isolated hyaluronic acid from the sera of two patients, one with reticulum cell sarcoma and the other with neuroblastoma, but was unable to demonstrate the presence of
hyaluronic acid in normal serum. In the present work, t~nz~~matic dtgests o I’ plasma GAG with c*hondroitinase AC contained no detc~ctabltl amounts 01 ADi-OSII , which is the constituent of hyaluronic* acid. El~~c,trol)hor~~sis of l)lasma GAG in 0.1 M HCl rrvealcd a faint sl’ot (*orresponding in mol)ility with hyaluronic acid. hut this fraction was not degraded by streptotnyccs hyaluronidase (AMRNO). Although the exact nature of this fraction remains to IW elucidated, il seems likely to 1~ acidic, glycoprotcins. Contrary to the> data tx~~)orted by Murata et al. 17 1. the prcs~lnc*r of oversulfatcd unsaturate(l tlisac~llarid~~ was nol demonstrated in the disaccharide subunits of plasma GAG. The source of GAG in plasma is not known. The oc’currenccl of undersulfated chondroibin sulfate, which is composed of both A Di4S and A Di-OS, is of interest in particular with regard to the process of sulfation. li’urther work is required to elucidate whether these undersulfated chondroitin sulfates in plasma might he waiting for intracellular sulfation process or not. Acknowledgment This Education
work was supported in Japan.
in part
by a research
grant
from
the Ministry
of
References 1 2 3 4 5 6 7 8 9 10
.I. Badin, M. Schubert and M. Vouras, J. Clin. Invest., 34 (1955) 1317. M. Bassiouni. Ann. Rheum. Dis.. 14 (1955) 288. A.J. Ballet, M.W. Seravdarian and W.F. Simpson, J. Clin. Invest.. 36 (1957) 1328. S. Srhitler. Biochim. Biophys. Acta, 28 (1958) 413. C. Friman and R. Brunish. Proc. Sot. Exutl. Blol. Med.. 122 (1966) 599. A. Catantroni. P.V. Doneliy and N. DIFe&antr, J. Clin. Invest., 48 (1969) 332 K. Muratd, T. Ogura and T. Okuvama. Blochem. Med.. 6 (1972) 223. II. Saito. T. Yamagata and S. Suruki. ,I. Biol. Chem.. 243 (1968) 1536. ‘I’. Ohya and Y. Kaneko, Biochim. Biophys. Acta. 198 (1970) 607. F:. \vesstrr. in E.A. Balazs (Ed.). Chemistry and Molecular Biology of the Intercrxllulu Academic Press. London and New York. Vol. 2 1970. I). 895. 11 M. Schmidt, Biochim. Biophvs. Acta, 63 (1962) 346. 12 T. Yamagata. H. Saitn. 0. Habuchi and S. Suzuki. .J. Biol. Chcm.. 243 (1968) 152.3. 13 H.F. Deutsch, J. Biot. Chem., 224 (1957) 767 14 Z. Dischr. .I. Biol. Chem.. 167 (1947) 189.
Matnx.