Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Mechanism for the decreased biosynthesis of cartilage proteoglycan in the scorbutic guinea pig

Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

Complement component C1q is sensitive to acute vitamin C defficiency in guinea pigs

In acutely scorbutic guinea pigs, where interstitial collagen synthesis is markedly impaired, there was no significant reduction in total complement component C1 activity measured by a functional assay, and no significant reduction in the ratio of protein-bound hydroxyproline to protein-bound proline or to total serum protein, in comparison with pair-fed controls. There was a moderate increase in non-protein-bound hydroxyproline in the serum of the deficient animals.These result suggest that component C1q is largely resistant to the effects of severe acute scurvy, adn that some hydroxyproline-containing proteins may respond differently others, during vitamin C deficiency.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

A specific decrease in collagen synthesis in acutely fasted, vitamin C-supplemented, guinea pigs

Weight loss often results from various experimental conditions including scurvy in guinea pigs, where we showed that decreased collagen synthesis was directly related to weight loss, rather than to defective proline hydroxylation (Chojkier, M., Spanheimer, R., and Peterkofsky, B. (1983) J. Clin. Invest. 72, 826-835). In the study described here, this effect was reproduced by acutely fasting normal guinea pigs receiving vitamin C, as determined by measuring collagen and non-collagen protein production after labeling tissues in vitro with [3H]proline. Collagen production (dpm/microgram of DNA) decreased soon after initiating fasting and by 96 h it had reached levels 8-12% of control values. Effects on non-collagen protein were much less severe, so that the percentage of collagen synthesis relative to total protein synthesis was 20-25% of control values after a 96-h fast. These effects were not due to changes in the specific radioactivity of free proline. Refeeding reversed the effects on non-collagen protein production within 24 h, but collagen production did not return to normal until 96 h. The effect of fasting on collagen production was independent of age, sex, ascorbate status, species of animal, and type of connective tissue and also was seen with in vivo labeling. Pulse-chase experiments and analysis of labeled and pre-existing proteins by gel electrophoresis showed no evidence of increased collagen degradation as a result of fasting. Procollagen mRNA was decreased in tissues of fasted animals as determined by cell-free translation and dot-blot hybridization with cDNA probes. In contrast, there was no decrease in translatable mRNAs for non-collagen proteins. These results suggest that loss of nutritional factors other than vitamin C lead to a rapid, specific decrease in collagen synthesis mainly through modulation of mRNA levels.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

Alterations in the activities of intestinal enzymes in vitamin-C-deficient guinea pigs

The effect of vitamin C deficiency on various enzymes of the intestinal epithelium has been studied in guinea pigs. Brush border sucrase and alkaline phosphatase activities were considerably enhanced (p less than 0.001), but leucine aminopeptidase levels were reduced in scorbutic animals compared to the control group. There was essentially no change in the activity of maltase under these conditions. Kinetic studies with sucrase and alkaline phosphatase in control and scorbutic animals revealed that augmentation of the enzyme activities in scurvy is due to enhanced enzyme contents. Lactate dehydrogenase, succinate dehydrogenase, glucose-6-phosphatase and Mg+2 ATPase also exhibited reduced activities in the intestine of vitamin-C-deficient animals. Observed alterations in the activities of intestinal enzymes in scurvy were restored to control levels upon feeding of vitamin C to scorbutic guinea pigs.     

Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst

Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

Disruption of healed scars in scurvy -- the result of a disequilibrium in collagen metabolism.

Old scars break open in scorbutic patients because (1) the rate of collagen degradation is greater in an old scar than it is in normal skin, and (2) the rate of collagen synthesis is diminished throughout the body in ascorbate deficiency.     

WOUND HEALING AND COLLAGEN FORMATION : IV. Distortion of Ribosomal Patterns of Fibroblasts in Scurvy

The changes in scorbutic wounds following the administration of ascorbic acid have been investigated using the techniques of electron microscopy, histochemistry, and autoradioggraphy. Particular attention has been paid to the changes seen in the endoplasmic reticulum of the fibroblasts and to the identity of the extracellular filamentous material characteristic of scorbutic wounds. Seven-day-old wounds in scorbutic guinea pigs were examined prior to and from one to 72 hours following the administration of vitamin C. Fibroblasts from wounds of normal animals demonstrate a characteristic configuration of the ribosomes of the endoplasmic reticulum which is suggested to be analogous to polyribosomes described in cells synthesizing protein such as the reticulocyte. Tangential views of the membranes of the ergastoplasm show the ribosomes to be grouped in paired rows which take both straight and curved paths. This configuration is lost in scurvy and can be seen to begin to reappear as early as 4 hours after giving ascorbic acid. With increasing time, the morphology of the ribosomal aggregates approximates that seen in normal cells, so that by 24 hours their reorientation is complete. It is suggested that one of the disturbances in scurvy may relate to an alteration either in messenger RNA, in the ability of the ribosomes to relate to the messenger, or in the membranes of the ergastoplasm. In addition, the lack of formation of hydroxyamino acids necessary for completing collagen synthesis may be related to the architecture of the ribosomal aggregates. Extracellular collagen fibrils appear concomitant with the restoration of ribosomal and ergastoplasmic morphology as early as 12 hours after administration of ascorbic acid, with complete disappearance of the scorbutic extracellular material within 24 hours. Observations of this scorbutic material do not support the concept that it is a collagen precursor.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Extracellular matrix synthesis by articular chondrocytes and synovial fibroblasts in long-term monolayer culture

Synthesis of collagen and proteoglycan by rabbit articular chondrocytes and synovial fibroblasts has been studied over a 12-week period in primary monolayer culture. Chondrocytes, but not fibroblasts, accumulate large quantities of proteoglycan over the culture period studied. Radiolabeling studies with [35S]sulfate have shown that the major proteoglycan synthesized by cultured chondrocytes is similar to the proteoglycan of cartilage matrix. Chondrocytes also synthesize a smaller dermatan sulfate proteoglycan, which is apparently the only proteoglycan species produced by synovial fibroblasts. Collagen synthesis was studied by radiolabeling with [3H]proline. Cultured chondrocytes produce mainly Type II collagen, with lesser amounts of Type I, whereas synovial fibroblasts produce Type I collagen and some low molecular weight collagenous species. Therefore, long-term monolayer culture permits the production of extensive chondroid matrix by chondrocytes, but not fibroblasts.     

Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine

The effects of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and kidney carnitine levels were not affected by the deficiency, but scorbutic animals had 50% less carnitine in heart and skeletal muscle than control animals. Labeled carnitine precursors, 6-N-tri-methyl-L-lysine and 4-N-trimethylaminobutyrate, both of which require ascorbate for their enzymatic hydroxylation, were injected into the vena cava of control, pair-fed and scorbutic animals. The distribution of isotope in compounds present in the liver and kidney after 1 h was determined. The uptake of trimethyllysine by the liver was less than 2% in 1 h, while the kidney took up approx. 20% of the 14C. Control and pair-fed animals converted trimethyllysine to kidney trimethylaminobutyrate 8--10 times as well as did scorbutic animals. Trimethylaminobutyrate hydroxylase, present in the liver but almost absent from the kidney, converted nearly all of substrate taken up by the liver to carnitine in both the scorbutic and control animals.     

Effect of Aqueous and Nonaqueous Fixatives on the Quantitative Estimation of Collagen-Proteoglycan Interaction in Tissue Sections

The present study was designed to assess the influence of aqueous and nonaqueous fixatives on the quantitative estimation of collagen-proteoglycan interaction in tissue sections. Tissues containing different collagen types and distinct sulfated proteoglycan classes were isolated from pig costal cartilage, human skin, and the inner muscular layer of dog small intestine and fixed using aqueous or nonaqueous methods. The results showed that the best fixation method was exposure to paraformaldehyde gas. When using aqueous fixatives, proteoglycans were lost to different degrees among the various tissues analyzed, reflecting differences in chemical properties of proteoglycan classes and/or in their interactions with other matrix components such as collagen.     

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-³H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line. © 1995 Wiley-Liss, Inc.     

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.