Articular cartilage, blood serum glycosaminoglycans and glycoproteins in osteoarthritis deformans

9

Clinica Chimica Acta, 94 (1979) 9-21 0 Elsevier/North-Holland Biomedical Press

CCA 10016

ARTICULAR CARTILAGE, BLOOD SERUM GLYCOS~INOGLYCANS AND GLYCOPROTEINS IN OSTEOARTHRITIS DEFORMANS

N.A. YUSIF’OVA and AS. KRIUK Minsk Medical Institute,

Lenin Prospect

6, Minsk (U.S.S.R.)

(Received September 8th, 1978)

Summary

Glycosaminoglycans in articular cartilage from human femoral heads have been isolated and fractionated by means of a cellulose microscale technique. The glucosaminoglycans have been identified by different procedures as keratan sulphate, hy~uroni~ acid, heparitin sulphate, ~hondroitin 4-sulpha~, chondroitin 6-sulphate and dermatan sulphate. Osteoarthritic cartilage showed a significant reduction of dermatan sulphate, chondroitin 6-sulphate, keratan sulphate and heparitin sulphate. The content of glycopro~in carbohydrate components in the cartilage (neur~ini~ acid derivatives, hexoses, 6-deoxyhexoses) also appeared to be reduced. It has been shown that chondroitin 4-sulphate, keratan sulphate and dermatan sulphate content was considerably increased in osteoarthritic serum. Among serum glycoprotein carbohydrate constituents only the content of 6-deoxyhexoses was slightly increased.

Introduction

The composition, structure and degree of polymerization of glycosaminoglycans (GAG) determine the main properties of articular cartilage, i.e., its resilience, strength, elasticity [l--3 1. The disturbance of the metabolism of these polymers plays an important part in the development of osteoarthritis deformans [Z--4]. According to some authors, collagen is involved in the pathological process secondarily owing to changes in the composition and properties of proteoglycans taking part in processes of its synthesis and fibrillation, in processes of cartilage tissue ossification and calcification, in water and salt metabolism regulation and in many other physiological phenomena {4-101. Nevertheless, the most metabolic~ly active and fun~tion~ly the most important structural components of cartilage (GAG) have not been sufficiently studied under pathological or even under normal conditions. Information con-

10

cerning the composition of cartilage glycoproteins is also extremely scanty. The present report covers the results of fractionations, identification and quantitative analysis of individual GAG from normal articular cartilage and from osteoarthritic cartilage. The fractional composition of blood serum GAG has also been studied with special reference to the importance of haemostatic changes. The content of some glycoprotein carbohydrate compounds was investigated in the same tissues. Materials and methods Articular cartilage was obtained from one femoral head of healthy subjects who had died as a result of trauma. Similar tissue was obtained from patients suffering from osteoarthritis deformans in stages II--III during resection of the femoral head. All subjects were between the ages of 30 and 60. All articular cartilage from femoral heads was excised with a scalpel. The entire thickness of the cartilage was saved but the bone was removed. Articular cartilage was thoroughly minced, ground to powder in liquid nitrogen, treated with acetone, dried and screened. GAG were isolated and fractionated by means of a cellulose microcolumn technique as described by Svejcar and Robertson [ll]. Unmodified cellulose was used as a filler (Whatman, Great Britain). The original method was modified in the following way. For proteolysis of the tissue papain (EC 3.4.4.10) (Chemische Fabrik, F.R.G.) was used. The incubation mixture consisted of 40 mg acetone powder, 0.1 ml 1.2% papain solution in acetate buffer, pH 6.5; 1 ml acetate buffer. Incubation conditions: for 24 h at 65°C. As a sorbent for GAG in a column, a 1% water solution of cetyltrimethylammonium bromide (CTAB) was used. Completeness of GAG sedimentation in the column was checked by uranic acid determinations in a l-ml sample of distilled water used for rinsing the column, after filling with the GAG mixture. In the same sample the content of the following glycoprotein components was estimated: neuraminic acid derivatives [ 121, hexoses [13], 6-deoxyhexoses [14]. GAG was eluated with solutions recommended by some workers [ 111 with the exception that the eluent CTAB concentration for fraction 4 was reduced from 0.5 to 0.05%. GAG contents in all the fractions were estimated from uranic acid [15] or hexosamine [16] levels. GAG for identification were obtained by analogous macroscale fractionation, they were precipitated with ethanol and dried by lyophilization. Hexosamines in lyophilized samples (after hydrolysis for 4 h in 0.25 M H,SO,) were identified by paper chromatography on Whatman No. 1 paper. The solvent mixture consisted of pyridine/butyl alcohol/O.1 M HCl (3 : 5 : 2, v/v), and spots were detected with an alkaline solution of AgNO+ Infrared spectrophotometry was done using a UR-10 spectrophotometer (Karl Zeiss, Jena, G.D.R.) and with the KBr pellet technique [17]. Infrared spectra of the lyophilized GAG fractions and of commercial hyaluronic acid preparations (potassium salt; Reanal, Hungary), chondroitin sulphate A (Olaine, Latvian S.S.R.); heparin (Minsk, Byelorussian S.S.R.) were obtained. Sensitivity to testicular hyaluronidase was tested by the turbidimetric method [18]. Anticoagulant activity was determined according to Lee and Whit

11

[ 191 by adding 1 mg of lyophilized GAG preparation, dissolved in 0.3 ml 0.85% NaCl, to 0.7 ml of fresh rat blood. Qualitative reactions for galactase [ 201, heparin, iduronic and glucuronic acids were carried out according to Dische [ 211. Blood serum from healthy and diseased subjects was diluted with ethyl alcohol (1 : 7, v/v); lipids were extracted twice with a ten-fold volume of acetone/ ether mixture (1 : 1, v/v), for 24 h each time; the precipitate was washed with the same mixture, dried from distilled acetone in a vacuum desiccator under CaCl,; for GAG isolation and separation, acetone powder (0.4 g) was used by the above-described microcolumn method. The content of serum glycoprotein carbohydrate components (neuraminic acid derivatives, hexoses, 6-deoxyhexoses) was estimated by the methods described above. Statistical significance of differences between the means has been evaluated by Student’s method. Results We characterized every isolated GAG fraction by means available to us. The data obtained are given in Table I and in Figures l-4. The presence of galactose, the absence of uranic acids in the lyophilized sample, its low sensitivity to testicular hyaluronidase (Fig. 2), a substance with glucosamine-like chromatographic mobility found in the preparation hydrolysate (Fig. l), all these findings allow us to conclude that fraction 1 consists mainly of keratan sulphate *. This conclusion is also supported by infra-red spectroscopic data, in that the “finger print” region of a sample from fraction 1 was similar to that of chondroitin 6-sulphate (Fig. 4, No. 1 and No. 2). This is in agreement with published data on keratan sulphate [ 121. Furthermore, the infra-red spectrum revealed a high degree of sulphation in the sample. Fraction 2 consisted mainly of hyaluronic acid. This fact is evidenced by localization of the marker (a commercial hyaluronic acid preparation) (Fig. 3), by the high sensitivity of the lyophilized sample to testicular hyaluronidase (Fig. 2), by the presence of glucosamine and the absence of galactosamine in its hydrolysate (Fig. l), by the identify of the infra-red spectra of a sample from fraction 2 and of the marker (commercial hyaluronic acid preparation) (Fig. 4, No. 3 and No. 4, respectively). In the infra-red spectrum of the sample attention is drawn to the absence of a marked absorption band in the 1260-1240 -I region, which reflects the absence of significant contamination with sulErated polysaccharides, and the presence of acetamide bands I and II (1660 cm-l and 1550 cm-l, respectively). The fact that there is no absorption band in the 1735 cm-l zone shows that the carboxyl group in the polymer is ionized, i.e., the fraction consists of hyaluronate. Fraction 3 in the reports by Svejcar and Robertson [ 111 is defined as heparitin sulphate. According to our data this fraction should be considered as a mixture of heparitin sulphate and of glycosaminoglycan which we failed to * The traditional use of the singular their heterogeneity.

to designate

individual

GAG does not mean

that

the authors

deny

High

LOW

Glucuronic acid * Galactosamine

Iduronic acid *

* Found from qualitative reactions. * * Qualitative reaction on heparin was positive

only

High

Glueuranic acid * Galactosamine

Glucuronic acid

very low

Glucuronic acid * Glucosamine Galactosamine

3

High

Giucuronic acid * Glucosamine

2

LOW

2 min

min

Does not coagulate for 24 h

Does not coagulate for 24 h

6-l

6-9

5-l 0 min

-

Heparin

Dermatan sulphate Heparin

-

Chondroitin 6-sulphate

Chondroitin 4sulphate

Heparitin sulphate

Hyaluronic acid

Ketatan sulphate

Polysaccharide pm dominating in fraction

1000; 820; 770

1000; 830: 780

-

930: 856-640; 730-740

1250: 850; 1660: 1550

1660: 1550

1000: 630; 770: 1260-1240

Chondroitin Psulphate

-

Hyaluronic acid

-

-

Typical bands of infrared absorption (cm-l) (Fig. 4)

FROM HEALTHY SUB3ECTS

Polysaccharide eluted into fraction of mixture GAG markers (Fig. 3)

CARTILAGE

Clotting time of white rat blood after adding fraction

with a sample from this fraction.

Sensitivitiy to testicular hyaluronidase (Fig. 2)

Galactose * Glucosamine

Carbohydrate in polymer composition (Fig, 1)

1

NO.

FUhiOtl

~D~~T~FICATION DATA ON GAG FRACTIONS ISOLATED FROM ARTICULAR

TABLE I

13

Fig. 1. Chromatograms of hydrolysates of samples from fractions l-5. For chromatograms we used a sample containing 500 /a of substance in 0.05 ml of hydrolysate. (a) Glucosamine. (b) Galactosamine.

identify. The fact that there is an admixture is evidenced by certain quantities of galactosamine in the preparation hydrolysate, in addition to glucosamine (Fig. 1). It is hardly possible to suppose that Fraction 3 was simply contaminated with adjacent hyaluronic acid or chondroitin sulphates during elution, A530 k

0.60. 0.50. 0.40 0.30 0.201

Fig. 2. Sensitivity of lyophilized GAG fraction samples to testicular hyaluronidase. polysaccharide in a sample after incubation with hyaluronidase; in fractions 1-S. Fig. 3. Elution profiles tin sulphate A); . - . -

of the markers.

., heparin.

-,

hyaluronic

acid; -

-

-.

chondroitin

Percent

4-sulphate

of unsplit

(chondmi-

N3

N4 I

,

,

1

I

,

1

I

,

Fig. 4. Infra-red spectrum of fraction samples. No. 1, fraction 1 (keratan eulphate); No. 2, fraction 5 (chondroitin 6-sulphate); No. 3, fraction 2 (hyaluronic acid, salt form); No. 4. a marker (potassium hyaluronate); No. 5, fraction 3 (heparitin sulphate); No. 6, a marker (heparin); No. 7. fraction 4 (chondroitin 4sulphate); No. 8, a marker (chondroitin lsulphate (chondroitin sulphate A)); No. 9, fraction 6 (dermatan sulphate).

for the following reasons. A sample from this fraction was practically unaffected by testicular hyaluronidase, in contrast to highly sensitive hyaluronic acid, chondroitin 4- and chondroitin 6-sulphates (Fig. 2), i.e., the fraction demonstrated the properties of heparitin sulphate [ 8,231. The preparation differed from dermatan sulphate (neither of these are split by testicular hy~uronid~e~ in that the former did not contain iduronic acid (during the reaction with thioglycolic and sulphuric acids a typical pink coloration develops after adding mannose), and demonstrated no anticoagulant properties (Table I). The infrared spectrum of the fraction (Fig. 4, No. 5) showed a high degree of sulphation of the polymer and the presence of acetamide groups. The latter distinguishes this fraction from heparin (Fig. 4, No. 6), as does the absence of ~ticoa~ant activity which is peculiar to heparitin sulphate (Table I). Having taken into account all the considerations mentioned above, we have for the time being defined fraction 3 as heparitin sulphate, although it contained a certain amount of other GAG or “hybrid form” of heparitin sulphate. According to our data fraction 4 comprised mainly chondroitin 4-sulphate.

15

This was shown by marker (commercial preparation) localization of the course of microcolumn fractionation (Fig. 3), by the similarity of the “finger print” zone in the infra-red spectra of the marker and of the fraction sample (Fig. 4, No. 7 and No. 8, respectively). Like chondroitin 4-sulphate, according to data in the literature [23] fraction 4 showed no anticoagulant activity. But it appeared to be very sensitive to testicular hyaluronidase (Fig. 2), which is also characteristic of chondroitin 4-sulphate [ 8,231. It should be noted that the prerequisite of this polysaccharide elution into fraction 4 was a decrease in the CTAB concentration in the eluent solvent to one-tenth. Use of the sorbent concentration (0.5 g per 100 ml of eluent) recommended by a number of workers [11,23] led to chondroitin 4-sulphate elution mainly in fraction 5 (together with chondroitin 6-sulphate). Apparently this amount of CTAB in the eluent solvent prevented complete dissolution of the CTAB-chondroitin 4-sulphate complex in organic solvents. Fraction 5 exhibited galactosamine after hydrolysis (Fig. l), it was split by testicular hyaluronidase (Fig. 2) and demonstrated no anticoagulant ability (Table I). The two last-mentioned properties as well as the absence of iduronic acid distinguished it from dermatan sulphate. Fraction 5 differed from chondroitin 4-sulphate in that it was practically insoluble in organic solvents, as well as in regard to infra-red spectroscopy date. The presence of marked absorption bands in the 1000 cm-i and 830 cm-l regions and of a band in the 780 cm-l region on the sample spectrum indicated equatorial disposition of the sulphate group, i.e., its linkage to C6 (Fig. 4, No. 2). All these findings suggest that fraction 5 consisted mainly of chondroitin 6-sulphate. Fraction 6 might be identified as dermatan sulphate due to the presence of iduronic acid and the absence of glucuronic acid. Actually, in contrast to chondroitin 4-sulphate and chondroitin 6-sulphate, a sample from this fraction was not split by testicular hyaluronidase (Fig. 2) and exhibited anticoagulant activity (Table I), i.e., demonstrated the properties of dermatan sulphate. It is suggested that these biological peculiarities of the polymer are associated with iduronic acid in its composition [ 8,231. The infra-red spectrum of the lyophilized sample (Fig. 4) resembled that of chondroitin 6-sulphate (absorption bands in the 1000 cm-l, 820 cm-l, and 770 cm-’ regions), and this is in good agreement with published data concerning dermatan sulphate [ 221. Fraction 7 comprised mainly heparin, as demonstrated by the marker elution profile (Fig. 3). In anticoagulant properties the fraction was also similar to heparin (Table I). And finally, the qualitative reaction on heparin was positive only with this sample. Thus, using the above-mentioned methods it was possible to separate a mixture of cartilage GAG into individual fractions. The results of identification (Table I) according to the prevalence of a certain polysaccharide, allow us to define fraction 1 as keratan sulphate, fraction 2 as hyaluronic acid (hyaluronate), fraction 3 as presumably heparitin sulphate, fraction 4 as chondroitin 4-sulphate, fraction 5 as chondroitin 6-sulphate, fraction 6 as dermatan sulphate, fraction 7 as heparin. In normal cartilage the amount of proteoglycans was 12% of dry fat extracted tissue weight (120 + 10.0 pg per 1 mg of the powder). In GAG from femoral head articular cartilage of healthy subjects we found significant

16 TABLE II DATA

ON FRACTIONATION

OF GAG ISOLATED

FROM ARTICULAR

CARTILAGE

Uranic acids, mg, per 1 g of dry tissue, M + S,

Fraction No. Normal cartitilage (n = 10) Arthritic cartilage (n = 11) P

1 *

2

3

4

5

6*

7

3.09 f 0.6 0.94 f 0.20 (0.001

0.52 + 0.1 0.64 t 0.1 >0.05

4.86 i 0.18 2.43 + 0.3
3.72 f 0.7 3.44 f 0.4 >0.2

3.00 mt0.7 0.86 * 0.2 <0.05

3.45 + 0.8 0.58 + 0.1
0.40 r 0.13 0.33 f 0.1 >0.05

* Contents of fractions 1 and 6 were estimated by the amount of hexosamines (mg per 1 g of dry tissue).

amounts of keratan sulphate (fraction I), heparitin sulphate (fraction 3), chondroitin 4-sulphate {fraction 4), chondroitin 6-sulphate (fraction 5), dermatan sulphate (fraction 6) and some hyaluronic acid (fraction 2) and heparin (fraction 7), see Table II and Fig. 5. In osteoarthritis deformans the total content of articular cartilage proteoglycans decreased to 7% (70 ? 8.0 pg per 1 mg of the powder). A quantitative assay of pathological cartilage GAG revealed a reduction in the contents of all sulphated polysaccharides in comparison with normal cartilage (Table II, Fig. 5). However, the decrease in the amounts of chondroitin 4-sulphate and heparin was not marked. There was a significant reduction in the amount of heparitin sulphate to half, keratan sulphate to 30.3%, chondroitin 6-sulphate to 29%, dermatan sulphate to 16.6%. The content of glycopro~in c~bohydrate components also appeared to be reduced (Table III): 6-deoxyhexoses were estimated at 40% neuraminic acid derivatives at 43%, hexoses at 66% of the corresponding values in normal cartilage.

5.0

4.0

3.0 .

2.0

.

1.0 -

4

5

6

7

Fig. 5. Diagram of elution profiles of GAG fractions from normal and pathological cartilage. 1-7, numbers. - - -. hexosamines (mg/g of dry tissue): 1 uronie acids (mg/g of dry tissue); =, mal cartilage’,-.- - pathological cartilage.

fraction nor-

17 TABLE III CONTENT OF GLYCOPROTEIN

CARBOHYDRATE

CONSTITUENTS

IN ARTICULAR

CARTILAGE

Results presented as mg per 1 g dry tissue, M k s. Tested materiat

Carbohydrate constituent

NormaI cartilage (n = 10) Arthritic cartilage (n = 11) P

Neuraminic acid derivatives

6-Deoxyhexoses

Hexoses

2.1 t 6.4

4.5 + 1.3

15.0 t 2.3

0.9 + 0.1

1.8 ?: 0.3

9.1 t 1.6




TABLE IV FRACTIONAL

COMPOSITION OF GAG ISOLATED FROM BLOOD SERUM

Uranic acids, pg. ner 1 g of dry tissue, M t s. Tested material Fraction No. serum (n = 20) Arthritic serum (n = 22) P Normal

Uranic acids 1 *

2

3

4

5

6*

7

11.2 + 1.2 29.9 r 4.4
4.9 f 0.8 5.4 f 1.5 >0.5

2.6 zt0.7 4.2 i 0.9 >0.2

1.2 ?: 0.4 15.1 t 2.1
2.8 + 0.6 3.4 t 1.0 >0.5

7.5 t 1.3 18.5 i 2.5
4.8 k 0.8 7.4 f 1.9 >O.l

* Content of Fractions 1 and 6 were estimated by the amount of hexosamines (pg per 1 g of dry tissue).

A 30.

20.

Fig. 6. Diagram of elution profiles of blood serum GAG fractions. 1-7, fraction numbers. - - -, hexosamines @g/g of dry tissue); -, uranic acids C&g/gof dry tissue); ---_, healthy serum; =z,z_ serum from diseased subjects.

18 TABLE

V

CONTENT

OF

HEALTHY

AND

Results

GLYCOPROTEIN

CARBOHYDRATE

OSTEOARTHRITIC

are presented

as mg%.

M

Glycoprotein material

Donor (n

Neuraminic

acid

derivatives

6-Deoxyhexoses HCXOSeS Total

protein

serum

= 32)

BLOOD

SERUM

FROM

carbohydrate Patient’s

serum

P

(n = 65)

70.3

? 2.3

66.5

* 5.5

>0.2

9.5

t 0.2

13.7

? 0.6


108.7 (g%)

IN

t s.

Constituents Tested

CONSTITUENTS

SUBJECTS

6.8

f 1.8 f 0.3

102.5 9.3

t 6.0

>0.2

+ 0.3


Data concerning the fractional composition of blood serum GAG in healthy and osteoarthritic subjects are given in Table IV and Fig. 6. As in the case of cartilage, the changes here concerned sulphated polysaccharides, while the amount of hyaluronic acid (fraction 2) remained practically unchanged. But the trend of the changes in blood serum was different, i.e. the content of all the sulphated GAG tended to increase. The increase in the amount of heparitin sulphate (fraction 3), chondroitin 6-sulphate (fraction 5) and heparin (fraction 7) was statistically insignificant. The chondroitin 4-sulphate content (fraction 4) exceeded the normal value by more than 10 times, and the keratan sulphate content (fraction 1) and dermatan sulphate content (fraction 6) by more than two times. From Table V it can be seen that among the serum glycoprotein carbohydrate constituents studied in osteoarthritis only 6-deoxyhexoses exceeded the norm, by 34%. The amount of neuraminic acid and hexose did not change. Total protein content was on the average 36% higher. Discussion Our data concerning the total amount of normal articular cartilage proteoglycans (12% of tissue dry weight) are in good agreement with the results reported by other workers [2,9,25]. The presence of chondrotin 4-sulphate, chondrotiin 6-sulphate and keratan sulphate in integumentary cartilage was reported earlier [l-3]. In addition to these polymers, we found in the cartilage under investigation considerable amounts of heparitin sulphate, dermatan sulphate, as well as some hyaluronic acid and heparin. The chondroitin 4-sulphate and chondroitin 6-sulphate content in GAG were nearly the same (ratio 1 : 0.8) respectively). Some discrepancies [ 2,3,25] in the estimation of chondroitin 4-sulphate content in normal cartilage are probably, at least to some extent, caused by peculiarities in the methods used for GAG separation by different investigators. In patients suffering from osteoarthritis the proteoglycan content was 42% lower in affected than in normal cartilage. The results obtained from quantitative analyses of GAG fractions indicate a sharp drop in the sulphated polysaccharide content of this tissue. A great many workers [24-261 have related

19

the decrease in their total content to the reduced content of chondroitin sulphates. According to our investigations the changes concerned mainly dermatane sulphate, chondroitin 4-sulphate, keratan sulphate and heparitin sulphate. The ratio of chondroitin 4-sulphate to chondroitin 6-sulphate was 1 : 0.25. It should be noted that cartilage GAG populations were investigated, both in the present and in the above-mentioned works, only in diseased subjects in the IIIII stages of the disease. Mankin [2] found analogous tissue in III-IV stage of the disease to have a surplus of chondroitin 4-sulphate and a deficiency of keratan sulphate. Such a distribution is characteristic of GAG in young, immature articular cartilage. These findings formed the basis for a hypothesis suggesting that as the pathological process develops chondroblasts synthesize proteoglycans peculiar to immature tissue. This mechanism may act in the final, most severe stage of the disease (III-IV). The decrease in sulphated GAG of affected tissue could be noticed, according to our data, even earlier. It was accomplished by a drop in the glycoprotein content estimated from the decrease in neuraminic acid derivatives, 6-deoxyhexoses and hexoses. Similar results have been obtained by Bollet and Nance [25] in studies on the neutral protein-bound polysaccharides in articular cartilage from such patients. Phenomena like these may be associated with the intensification of processes of polysaccharide-protein polymer degradation in an affected articular cartilage. This suggestion is supported by data on GAG structural breakdown, intensification of sulphated polysaccharide synthesis in induced osteoarthritis [ 27,281 and by the results of enzymological examination of patients suffering from osteoarthritis which attest to the fact of activization of some lysosomal hydrolases in affected cartilage sites [ 291. Intensification of the synthesis of carbohydrate polymers under these conditions is apparently of a compensatory nature. Bosman [,30] and FittonJackson [31] in experiments on chick embryo bone rudiments found that following special ferment treatment chondrocytes responded to a decrease in the amount of GAG in the matrix which elevated chondroitin sulphate synthesis and restored it to the initial amount in five days. Cell reaction was dose-dependent, which suggests strict regulation of GAG synthesis in chondrocytes by quantitative composition of carbohydrate polymers in the matrix. It is also probable that the intensification of carbohydrate-protein polymer synthesis is associated with a rise in the mitotic activity of chondrocytes [2,3]. In particular, chondroitin sulphate synthesis is attended by DNA synthesis [32]. At the same time it was shown experimentally that chondrocytes affected by lysosomal ferments do not lose the ability for synthesis, but produce qualitatively different proteoglycans. For example, a molecule of chondroitin sulphate-protein had less than the normal amount of polysaccharide chains for one protein chain. Such molecules can be split easier by hydrolases [ 3,31,33]. This fact is probably the reason for the predominance of hydrolytic processes in osteoarthritic cartilage, in spite of active mitosis and the intensification of synthetic reactions in chondrocytes. Disturbances in DNA metabolism of a similar nature were found in articular cartilage in patients suffering from osteoarthritis, i.e., the intensification of polymer synthesis was accompanied by a decrease in its quantity [ 2,3]. Now proteoglycan molecules of connective tissue in a native state are believed to exist in the form of multi-chain macromolecule aggregates, as has

20

been shown for heparin [34]. It is possible that aggregate formation may prevent manifestation of specific biological activity of the molecule. Horner [35] reports that the lipoprotein-lipase activity of heparin in rodent mast cells is manifested only after partial depolymerization of polymer macromolecules. The enzymes able to perform such hydrolysis was found in lysosomes [ 361. In this case intensification of hydrolytic processes in cartilage may lead not only to structural breakdown but also to the formation of molecules unusual for native unaffected tissue and possessing certain biological activity. It should be noted that the pattern of quantitative changes in blood serum carbohydrate polymers from affected subjects distinctly differs from that in cartilage. Among serum glycoprotein carbohydrate constituents only the content of 6-deoxyhexoses was slightly increased, which may reflect some intensification of processes of antibody formation [ 371. Disturbances in the quantitative composition of GAG concerned sulphated polysaccharides and became apparent through a considerable increase in chondroitin 4-sulphate, keratan sulphate and dermatan sulphate content. According to literature data, blood serum is an important extracellular agent regulating the activities in cells that synthesize sulphated GAG [ 33,381. It also exerts a stimulating influence upon cell mitosis [ 331. The activating effect of chondroitin sulphates upon factor 12 (Hageman factor) in the kinin system is known. It was shown that factor 12 was a normal constituent of articular fluid [ 391. Chondroitin sulphate accumulation in the blood serum of patients suffering from osteoarthritis may lead to a local increase in kinin production in an affected articulation (probably due to changes in composition and in the properties of synovial tissue). This in its turn, according to Whitehouse [40] results in the intensification of the mitotic activity of chondrocytes. In the light of our data, the changes in the permanent composition of polysaccharide-protein polymers in the blood serum presented here may be considered as pathogenetic factors possibly closing the vicious circle of biochemical disturbances in osteoarthritis. Acknowledgements We wish to thank Professor E.L. Rosenfeld for her constant interest in our work and her valuable advice in the discussion of the results; we are also grateful to A.N. Balesnaya and W.A. Mamontova for their technical assistance. References 1 2 3 4 5 6 7

Caygill, J.C. (1971) Ann. Biol. CIin. 29, 85 Mankin, H.J. (1971) J. CIin. Invest. 50, 1712 McDevitt, C.A. and Pond. M.J. (1973) Biochem. Sot. Trans. 1,287 Howell, D.S. (1975) Arthritis Rheum. 18, 167 Rosenfeld. E.L. (1962) Usp. Biol. Khim. 4, 218 Bichkof, S.M. (1968) USP. Sovr. Biol. 65, 328 Slutskiy, L.I. (1968) in Biokhimiya normalnoi i patologicheski p. 89. Medicina, Leningrad 8 Onodera. K. (1972) in Retrospect and Prospect in Carbohydrate moirs of the College of Agriculture. Kyoto, Vol. 102, p. 50. Kyoto 9 Hardingham. T.E. and Muir, H. (1973) Biochem. Sot. Trans. 1,282

izmenionnoi Chemistry University,

soyedinitelnoi and Biochemistry. Kyoto

tkani, Me-

21 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Franzblau. C., Schmid. K.. Faris, B., Beldekas, J.. Garvin, P., Kagan. H.M. and Baum. B.J. (1976) Biochim. Biophys. Acta 427, 302 Svejcar, J. and Robertson, W.B. (1967) Anal. Biochem. 18,333 Warren, L. (1959) J. Biol. Chem. 234,197l Winzler, R.I. (1955) in Methods of Biochemical Analysis (GIick. D.. ed.). Vol. 2, P. 290 Dische, Z. (1955) in Methods of Biochemical Analysis (Glick. D., ed.), Vol. 2, P. 338 Bitter, F. and Muir, H. (1962) Anal. Biochem. 4. 330 Antonopoulos, C.A.. Garde& S.. Szirmai, J.A. and de Tyssonsk, E.R. (1964) Biochim. Biophys. Acta 83.1 Schiedt, M. and Reinwein. H. (1952) Z. Naturforsch. 7, 270 Dorfman, A. and Ott, M. (1948) J. Biol. Chem. 172, 367 Lee, T. and Whit. R. (1968) in Spravochnik PCI klinicheskim laborat, metodam isslyedouniya, P. 124, Medicina. Moscow Dische. Z. (1967) in Met,odi khimii uglevodov. P. 30, Mir, Moscow Dische. Z. (1967) in Metodi khimii uglevodov. P. 40, Mir. Moscow Mathews, M.B. (1958) Nature 181. 42 Stacy. M. and Barker, S. (1965) in Uglevodi zhivikh tkanei, Mir. Moscow Ichida, T. and Kalant, N. (1968) Can. J. Biochem. 46,249 Bollet, Al. and Nance, J.L. (1966) J. CIin. Invest. 45, 1170 Franson. L.A. and Anseth, A. (1967) Expt. Eye Res. 6.107 Collins. O.H. and McElIigot, T.F. (1960) Ann. Rheum. Dis. 19. 318 Lust. L. and Pronsky. W. (1972) Clin. Chim. Acta 39.281 AIi, S.I. and Evans, L. (1973) Fed. Proc. 32. 1494 Bosman. H.B. (1968) Proc. R. Sot. Land. Ser. B 169,399 FittonJackson, S. (1970) Proc. R. Sot. Lond. Ser. B 175, 405 Nemeroff, M. and Holtzer, H. (1967) Dev. Biol. 16, 250 Schwartz. E.R., Kirkpatrick, R. and Thompson, R.C. (1974) J. Clin. Invest. 54.1056 Jansson, L., Ogren, S. and Lindahl. U. (1975) Biochem. J. 145, 53 Homer, A.A. (1972) Proc. Natl. Acad. Sci. U.S.A. 69.3469 &en. S. and Lindahl, U. (1976) Biochem. J. 154,605 Rosenfeld, E.L. and Kostyukovskaya, O.M. (1961) Vopr. Med. Khim. 6.620 Kurtz, M.J. and Stidworthy, G.H. (1975) Biochim. Biophys. Acta 399,90 Eisen. V. (1969) in Scientific Basis of Medicine, P. 146, Atblone Press, University of London Whitehouse. M.W. (1967) J. Pharm. Pharmacol. 19, 590