Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

STUDY QUESTION: Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? SUMMARY ANSWER: Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. WHAT IS KNOWN ALREADY: Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. STUDY DESIGN, SIZE, DURATION: We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). LIMITATIONS, REASONS FOR CAUTION: Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

Comparative analysis of adenoviral transgene delivery via tail or portal vein into rat liver.

The potential indications for gene therapy are expanding continuously. Currently, hepatotropic adenoviruses are useful vector systems for targeting liver in experimental animal models. Although this gene delivery technique is widely distributed, there is no common sense about how these viruses should be applied. In general, the local delivery into portal vein and the systemic application via tail vein induces above all substantial transgene expression. We here comparatively analysed both methods and found that the systemic administration of an adeno-virus expressing the green fluorescent protein resulted in a stronger infiltration, a more homogenous distribution, and a higher inter-individual reproducibility of reporter gene expression in rat liver than organ-specific administration via the portal vein.

Regulation of interleukin-8 synthesis in human lower uterine segment fibroblasts by cytokines and growth factors.

OBJECTIVE: To investigate the influence of lipopolysaccharide, cytokines, growth factors, and progesterone on the synthesis of interleukin-8 by human lower uterine segment fibroblasts. METHODS: Fibroblasts derived from a lower uterine segment biopsy specimen obtained from a woman undergoing elective cesarean delivery at term were exposed to lipopolysaccharide, interleukin-1beta, transforming growth factor-beta(1), platelet-derived growth factor-AB, and combinations of these substances. All experiments were performed in the absence and presence of progesterone. The concentration of interleukin-8 in the culture medium was determined by enzyme immunoassay after 24 hours. RESULTS: Compared with controls (0.71 +/- 0.04 ng interleukin-8/10(6) cells), fibroblasts exposed to lipopolysaccharide, transforming growth factor-beta(1), or platelet-derived growth factor-AB exhibited no increase, or at most, only a minor but significant increase, in interleukin-8 secretion. Incubation with interleukin-1beta led to a moderate increase, whereas the combinations interleukin-1beta/transforming growth factor-beta(1) (105.0 +/- 7.5 ng interleukin-8/10(6) cells) and interleukin-1beta/platelet-derived growth factor-AB (387.3 +/- 25.6 ng interleukin-8/10(6) cells) increased interleukin-8 secretion dramatically. No further increase was observed with the combination interleukin-1beta/platelet-derived growth factor-AB/transforming growth factor-beta(1). When progesterone was added, interleukin-8 secretion decreased significantly by 16-34%, depending on the stimulator, or did not change. CONCLUSION: The findings indicate that interleukin-8 secretion by human lower uterine segment fibroblasts in vitro is upregulated by interleukin-1beta, transforming growth factor-beta(1), and platelet-derived growth factor-AB in a synergistic fashion. Because interleukin-8 mediates the invasion of neutrophils into the cervical stroma, this may be an important mechanism controlling cervical dilatation during parturition.

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts.

BACKGROUND: The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. RESULTS: With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. CONCLUSIONS: Our results indicate that FuGENE6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.

Induction of alpha1-antitrypsin synthesis in human articular chondrocytes by interleukin-6-type cytokines: evidence for a local acute-phase response in the joint.

OBJECTIVE: We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflammatory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein alpha1-antitrypsin in human articular chondrocytes. METHODS: Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of alpha1-antitrypsin protein were determined by enzyme immunoassay. RESULTS: Stimulation of chondrocytes with oncostatin M (OSM) and IL-6 led to a 5-10-fold increase in alpha1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. CONCLUSION: Our data strongly suggest the existence of a local acute-phase response in the joint. Synthesis of the acute-phase protein alpha1-antitrypsin, a major inhibitor of serine proteinases, may be an important protective mechanism of articular chondrocytes to prevent cartilage damage in inflammatory joint diseases.

Interleukin-1beta and interleukin-8 concentrations in the lower uterine segment during parturition at term.

OBJECTIVE: To assess the roles of interleukin-1beta, interleukin-8, and fibroblasts in the lower uterine segment during parturition. METHODS: Lower uterine segment biopsy specimens were obtained from 36 women undergoing cesarean delivery at various stages of cervical dilation (less than 2 cm, n = 8; 2 to less than 4 cm, n = 9; 4-6 cm, n = 10; more than 6 cm, n = 9). The concentrations of interleukin-1beta and interleukin-8 in protein extracts prepared from the tissue samples were measured by enzyme immunoassays. The effect of incubation with interleukin-1beta (30 U/mL) on interleukin-8 secretion by lower uterine segment fibroblasts in vitro also was determined. RESULTS: The median interleukin-1beta concentration in the specimens increased from 1.3 pg/mg of total protein at less than 2 cm of dilation to 22.2 pg/mg of total protein at 4-6 cm of dilation (P < .05). No further increase was detectable after 6 cm of dilation. The interleukin-8 concentration increased from 17.2 pg/mg of total protein at less than 2 cm of dilation to 2080.7 pg/mg of total protein at 4-6 cm of dilation (P < .05), thus paralleling the increase in interleukin-1beta concentration. Interleukin-1beta induced a significant increase in interleukin-8 secretion by fibroblasts in vitro, from 0.8 ng/10(6) cells to 35.6 ng/10(6) cells. CONCLUSION: The increase in interleukin-8 concentration in the lower uterine segment during parturition may be induced by interleukin-1beta and fibroblasts may be one of the sources of this interleukin-8.

Oncostatin M differentially regulates tissue inhibitors of metalloproteinases TIMP-1 and TIMP-3 gene expression in human synovial lining cells.

Tissue inhibitor of metalloproteinases (TIMP) 1, 2 and 3 are related proteins that can form complexes with all known matrix metalloproteinases (MMPs). They inhibit the action of MMPs on extracellular matrix components. The balance of MMPs and TIMPs is important for tissue remodeling and its disturbance is believed to play a crucial role in pathophysiological processes such as tumor metastasis, destruction of cartilage and fibrosis. Cytokines and growth factors were found to regulate TIMPs and MMPs in a complex manner. In order to better understand the role of TIMPs in inflammatory joint diseases we have studied in vitro the regulation of TIMP-1 and TIMP-3 by inflammatory cytokines in cultured human synovial lining cells. We found that transforming growth factor beta 1 as well as interleukin-1 beta induce gene expression of both TIMP-1 and TIMP-3. In contrast, oncostatin M, an interleukin-6-type cytokine produced by activated T-lymphocytes and monocytes, had a differential effect on TIMP mRNA levels. After oncostatin M treatment, TIMP-1 expression was up-regulated but basal, as well as interleukin-1 beta-induced, TIMP-3 expression was inhibited. Interleukin-6 itself had no effect on synovial lining cells but a complex of interleukin-6 and the soluble interleukin-6 receptor induced activation of signal transducer and activator of transcription (STAT) factors in these cells and regulated TIMP-1 and TIMP-3 expression in a similar fashion as oncostatin M. Since TIMP-3 is matrix-associated whereas TIMP-1 is found in many body fluids, the role of oncostatin M during inflammatory processes might be to promote ECM degradation in the local environment but to prevent it systemically.

Transforming growth factor beta 1, a major stimulator of hyaluronan synthesis in human synovial lining cells.

OBJECTIVE: To investigate the role of cytokines and growth factors in the regulation of hyaluronan synthesis in human synovial lining cells. METHODS: Synovial lining cells were obtained from human knee joints, isolated by the explant method, and characterized by immunocytochemistry using monoclonal antibodies against monocyte/macrophage markers as well as antibodies against hyaluronan synthase. After stimulation by cytokines and growth factors, hyaluronan was measured by radiometric assay. The molecular weight distribution of the hyaluronan synthesized was determined by high-performance gel-permeation liquid chromatography. To test the effect of oxygen-derived free radicals, the concentration and molecular weight distribution of hyaluronan were determined in the presence and absence of catalase and superoxide dismutase. RESULTS: Hyaluronan synthesis was stimulated in synovial lining cells by transforming growth factor beta 1 (TGF beta 1), interleukin-1 beta (IL-1 beta), and to a lesser extent by tumor necrosis factor alpha (TNF alpha). Analysis of the molecular weight distribution of hyaluronan after stimulation of synovial lining cells with TGF beta 1, IL-1 beta, and TNF alpha indicated that hyaluronan is synthesized in a high molecular weight form and might be degraded in the course of inflammatory processes by oxygen-derived free radicals. CONCLUSION: Our findings suggest that TGF beta 1 is a major stimulator of hyaluronan synthesis in human synovial lining cells and might be involved in the pathogenic mechanisms of joint swelling in inflammatory and degenerative joint diseases.

Transforming growth factor beta 1 regulates tissue inhibitor of metalloproteinases-1 expression in differentiated human articular chondrocytes.

OBJECTIVE: To investigate the role of interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF beta 1) in the regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) synthesis in human articular chondrocytes. METHODS: Articular cartilage was obtained from human knee joints 24 hours after death. Chondrocytes were isolated by collagenase digestion and embedded in low-gelling-temperature agarose. After stimulation by cytokines, total RNA was isolated and analyzed by Northern blotting. TIMP-1 protein levels were determined using a competitive enzyme-linked immunosorbent assay. RESULTS: Human chondrocytes in agarose culture expressed messenger RNA (mRNA) for the IL-6 receptor (gp80) and its signal-transducing subunit gp130. In contrast to the findings in a previous study, IL-6 did not stimulate TIMP-1 expression in these cells, whereas TGF beta 1 was an important inducer of TIMP-1 mRNA and protein synthesis. CONCLUSION: Our findings suggest that TGF beta 1 has a protective effect on the extracellular matrix of human articular chondrocytes.

The metabolism of 2-fluoro-2-deoxy-D-glucose in human chondrocytes and its incorporation into keratan sulfate proteoglycans.

The incorporation of 2-fluoro-2-deoxy-D-[14C]glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high-cell-density thin-layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion-exchange-chromatography (DEAE-Sepharose CL-6B). With enzymic decomposition experiments concerning the glycosaminoglycan side-chains it could be shown that 65-69% were digestible by keratanase, whereas 21-29% of the 14C-labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2-fluoro-2-deoxy-D-[14C]glucose-metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2-fluoro-2-deoxy-D-[14C]galactose. Therefore, 2-fluoro-2-deoxy-D-glucose was preferentially incorporated into keratan sulfate. We investigated the effect of non-radioactive 2-fluoro-2-deoxy-D-glucose on UDP-sugar and proteoglycan biosynthesis after incubation periods of 1-30 h. A high 2-fluoro-2-deoxy-D-glucose concentration in the culture medium did not influence the pool size of UDP-N-acetylhexosamines, but UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, UDP-2-fluoro-2-deoxy-D-glucose, UDP-2-fluoro-2-deoxy-D-galactose and UDP-2-fluoro-2-deoxy-D-glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP-sugars was proved. The half-lives (t1/2) for UDP-2-fluoro-2-deoxy-D-glucose and UDP-2-fluoro-2-deoxy-D-galactose were about 7.7 h and 13.3 h, respectively. UDP-2-fluoro-2-deoxy-D-glucuronic acid could be found with delay. The incubation with 2-fluoro-2-deoxy-D-glucose and [14C]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate.

A radiochemical high-performance liquid chromatographic method for the analysis of 2-fluoro-2-deoxy-D-glucose-derived metabolites in human chondrocytes.

A high-performance liquid chromatography method with on-line radioactivity monitoring was developed for the measurement of 2-fluoro-2-deoxy-D-[U-14C]-glucose-derived metabolites in a cell culture system of human chondrocytes embedded in soft agarose. To optimize the chromatographic procedure, glucose-analogous substrates derived from 2-fluoro-2-deoxy-D-glucose by enzymatic synthesis in vitro were used. The synthesized metabolites could be separated by anion-exchange chromatography on a Partisil 10 SAX cartridge with a LiChrosorb RP 18-5 guard column eluted with a 35-min ion-strength/pH gradient performed from 15 mM NH4H2PO4, pH 3.8, to 0.75 M NH4H2PO4, pH 4.8, at a flow rate of 2 ml/min. Only by using an on-line radioactivity monitor instead of an off-line counting procedure was the resolution obtained sufficient for the determination of these intermediates. This method was applied to studying the metabolic pathway of 2-fluoro-2-deoxy-D-glucose in human chondrocytes. Due to the resistance of the chondrocytes embedded in soft agarose, the usual cell-lysing methods could not be used; therefore, an extraction procedure for acid-stable glucose metabolites, which may also be applied to other resistant cell lines or critical cell culture systems, was developed. With the procedure presented here, the existence of metabolites of 2-fluoro-2-deoxy-D-glucose resulting from enzymatic reactions following the hexokinase reaction could be proven.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes.

Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis.

Effect of diacetylrhein on the phagocytosis of polymorphonuclear leucocytes and its influence on the biosynthesis of hyaluronate in synovial cells.

1. The influence of diacetylrhein on the luminol-induced chemiluminescence of zymosan-activated polymorphonuclear leucocytes (PMNL) was investigated. At a concentration of 4 x 10(-5) mol/l diacetylrhein an inhibition of about 40% was found. 2. A model for the degradation of hyaline cartilage by frustrated phagocytosis was developed, in which human polymorphonuclear leucocytes cause a release of glycosaminoglycan peptides from hyaline cartilage slices (bovine nasal septum). We observed a 20% inhibition of this release at a concentration of 10(-4) mol/l diacetylrhein. 3. Human synovial fibroblasts synthesize the glycosaminoglycan hyaluronate. As a parameter of the rate of hyaluronate synthesis we measured the incorporation of 14C-glucosamine into hyaluronate. At a concentration of 2 x 10(-4) mol/l diacetylrhein a 4-fold increase of 14C-glucosamine incorporation in the membrane fraction of the synovial cells (tryptic fraction) and a 1.6-fold elevation of glucosamine release into the medium was measured. The synovial fibroblasts show a higher (1.5-fold) glucose consumption and lactate production in the presence of diacetylrhein (2 x 10(-4) mol/l).

Pathobiochemical significance of granulocyte elastase complexed with proteinase inhibitors: effect on glycosaminoglycan metabolism in cultured synovial cells.

Interactions between elastase inhibitor complexes and synovial cells are of special interest, since, in chronic joint diseases, granulocytes release large amounts of elastase into the synovial fluid and connective tissue, where the proteinase is bound to alpha 1-proteinase inhibitor and alpha 2-macroglobulin. To study the effect of elastase-alpha 2-macroglobulin and elastase-alpha 1-proteinase inhibitor complexes on the glycosaminoglycan metabolism of cultured synovial cells, we determined the distribution of [3H]glucosamine-labelled hyaluronate, which represents the main synthesized glycosaminoglycan, and of 35SO4(2-)-labelled chondroitin sulphate into the intracellular, pericellular and extracellular compartments of the cell culture. Exposure of the synovial cells to elastase-alpha 2-macroglobulin complexes leads to an enhanced synthesis and secretion of hyaluronate, and chondroitin sulphate, and also induces a rise of the fibronectin concentration in the medium. Analogous but less pronounced effects are observed in the presence of elastase-alpha 1-proteinase inhibitor complexes. Native uncomplexed elastase, however, causes no significant changes in hyaluronate metabolism. An increase of prostaglandin E2 in the culture medium during incubation with elastase inhibitor complexes occurs in parallel to the stimulatory effect on glycosaminoglycan metabolism. Our results demonstrate that elastase, whose enzymic activity is inactivated by the formation of complexes with alpha 1-proteinase inhibitor or alpha 2-macroglobulin, nevertheless acts as an inflammatory mediator, which in vitro induces metabolic changes closely resembling the in vivo findings in inflammatory joint diseases.

Interaction of synthetic polynucleotides with small rat liver ribosomal subunits possessing low and highly phosphorylated protein S6.

The interaction of some synthetic mRNAs (polyuridylate, polyadenylate, polycytidylate) with small rat liver ribosomal subunits which have protein S6 in different states of phosphorylation was studied by BioGel column chromatography, affinity chromatography on poly(U)-Sepharose 4B, and continuous diafiltration at 4 degrees C. 40-S subunits with low phosphorylated protein S6 (isolated from normal liver) and small subunits with highly phosphorylated protein S6 (from galactosamine-, thioacetamide-, dimethylnitrosamine-, puromycin-, and cycloheximide-treated livers) bind initially equal amounts of poly(U) but the dissociation of the radioactive polyuridylate occurs much more rapidly and to a greater extent from the low than from the highly phosphorylated type of subunits. From control- and galactosamine-4-S subunits 62% and 22%, respectively, of originally bound [3H]poly(U) was removed. The release of initially bound poly(A) from 40-S subunits of galactosamine-treated liver ws retarded but reached finally the same level as with control liver ribosomal subunits (removal of 40% of bound [3H]poly(A)). No differences between low and highly phosphorylated subunits were observed with poly(C). If the dissociation reaction was performed at 22 degrees C instead of 4 degrees C the differences in the release of poly(U) described above disappeared.

Metabolic and structural studies on serum- and liver-glycosaminoglycans in normal and liver-injured rats.

The incorporation of [35S]sulfate into total and specific types of serum glycosaminoglycans was studied in rats with acute, subacute or chronic liver injury (liver cirrhosis), and compared with that of normal rats. The macromolecular (protein-bound) nature of serum glycosaminoglycans in normal and diseased animals was also analysed. The results show a strong increase in rate and extent of [35S]sulfate incorporation into total serum glycosaminoglycans for acutely but a decrease for subacutely and chronically liver damaged rats. The time-course of distribution of label between serum chondroitin sulfate and dermatan sulfate exhibits significant changes in liver-injured animals, in particular a relatively high proportion of dermatan [35S]sulfate in rats with cirrhotic livers. In comparison with serum glycosaminoglycans the labeling profile of glycosaminoglycans in the cirrhotic liver was quite different (heparan sulfate:dermatan sulfate:chondroitin sulfate = 1:0.34:0.09) and changed only insignificantly during a 1 h labeling period. The protein-bound moiety of serum glycosaminoglycans was not affected by liver disease; but the elution profile of chondroitin [35S]sulfate from Dowex 1 X 2 for treated rats was altered, thus indicating a structural modification of its carbohydrate chain.