Hydroxyproline Synthesis Research Articles

Free and small peptide-bound [ 14c]hydroxyproline synthesis in vitro in ethanol-induced hepatic injury in the rat liver

To clarify the significance of free and small peptide-bound hydroxyproline synthesis in ethanol-induced liver injury, we measured the in vitro synthesis of [ 14C]hydroxyproline in the 67% ethanol-soluble fraction in rat liver slices, together with hepatic protein-bound [ 14C]hydroxyproline synthesis. The synthesis of free and small peptide-bound [ 14C]hydroxyproline was 11.1 ± 2.0 dpm × 10 −4/ g liver/3 hr and the synthesis of protein-bound [ 14C]hydroxyproline was 10.1 ± 3.3 dpm × 10 −4/g liver/ 3 hr in control rat liver. In the ethanol-fed rat liver, the synthesis of free and small peptide-bound [ 14C]hydroxyproline significantly increased 1.5-fold and the synthesis of protein-bound [ 14C]hydroxy-proline significantly increased 1.6-fold, while the hepatic collagen content did not change. There was a significant correlation between free and small peptide-bound [ 14C]hydroxyproline synthesis and protein-bound [ 14C]hydroxyproline synthesis. These results suggest that free and small peptide-bound hydroxy-proline synthesis plays an important role in regulating the content of hepatic collagens.

Effects of dietary NaCl supplementation on bone synthesis of hydroxyproline, urinary hydroxyproline excretion and bone 45Ca uptake in the rat.

High sodium chloride intakes are regarded as a risk factor for osteoporosis because they increase the obligatory urinary calcium loss and stimulate parathyroid activity. Sodium chloride loads induce osteopenia in the rat. The effect could be due to a decrease in bone formation or a rise in bone resorption. Two experiments were undertaken to study the effects of dietary NaCl supplementation on 3H-hydroxyproline synthesis and 45Ca uptake in femoral bone. Salt-treated rats excreted 1.7 times more total urinary hydroxyproline (P less than 0.001) and 2.1 times more recently labelled 3H-hydroxyproline than controls (P less than 0.02) but they did not accumulate less 3H-hydroxyproline or less 45Ca in their bones than controls. These results indicate that salt-mediated osteopenia is due to an increase in bone resorption, rather than to a decrease in bone formation.

Effects of minoxidil on human skin fibroblasts in culture

Collagens are a family of extracellular proteins that have evolved to serve many specialized biologic functions. Fifteen collagen types with at least 18 genetically distinct polypeptide chains have been identified.1 In skin, eight of these collagens make up a major portion of the extracellular matrix including specialized structures such as basement membrane (Type IV collagen) and anchoring fibrils (Type VII collagen). All collagens are synthesized as procollagen precursor molecules. In addition to synthesis and precise association and alignment of the three polypeptide chains, biosynthesis involves many posttranslational modifications.2,3 These include synthesis of hydroxyproline catalyzed by prolyl hydroxylase and synthesis of hydroxylysine catalyzed by lysyl hydroxylase. Hydroxyproline is essential for maximum helical stability, and hydroxylysine participates in collagen cross-link formation as well as synthesis of specific glycosides catalyzed by collagen glycosyl transferases. Other enzymes incorporate into the procollagen molecule complex carbohydrate residues typical of glycoproteins. Following secretion from the cell, amino and carboxyterminal propeptides of the procollagen molecule are cleaved by separate enzymes. Certain lysyl and hydroxylysyl residues are then modified by lysyl oxidase to corresponding aldehydes, which interact with certain hydroxylysine residues to form intermodular cross-links.

Modulation of collagen metabolism by glucocorticoids: Receptor-mediated effects of dexamethasone on collagen biosynthesis in chick embryo fibroblasts and chondrocytes

The steroid modulation of collagen metabolism was studied by injecting chick embryos with dexamethasone in vivo, and collagen synthesis was subsequently assayed by pulse-labeling the tissue with [ 14C]proline in vitro. The synthesis of [ 14]Chydroxyproline in tendons and sterna from chick embryos treated with dexamethasone was markedly reduced as compared with untreated controls. The inhibition of [ 3H]hydroxyproline synthesis was accompanied by a similar reduction in type I and II procollagen mRNA levels, as detected by Northern blot and dot blot hybridizations with chick proα1(I), proα2(I) and proα1(II) sequence specific cDNAs. The reduction in type II procollagen mRNA level was shown to be dose dependent. Control experiments indicated that the post-translational hydroxylation of prolyl residues was only slightly decreased in dexamethasone treated animals, and that the specific activity of the intracellular free proline pool and the intracellular degradation of collagen were unchanged. To address the mechanisms of the inhibition of collagen biosynthesis, specific binding of dexamethasone to glucocorticoid receptors in chick embryo tendon and cartilage cells was studied in a whole cell assay using [ 3H]dexamethasone as the ligand. Matrix-free tendon and cartilage cells had approximately 19,000 and 15,000 receptor sites per cell, respectively, and the binding affinities ( K d) for dexamethasone in tendon and cartilage cells were 2.9 x 10 −9 and 2.3 x 10 −9 M. Comparable values were obtained using a cytosol binding assay. The nuclear binding of dexamethasone in tendon and cartilage cells were similar. The results suggest that the dexamethasone-induced inhibition of collagen production is primarily due to decreased levels of funtional procollagen mRNA, possibly resulting from receptor-mediated inhibition of the gene expression on the transcriptional level.

Free and small peptide-bound [ 14C]hydroxyproline synthesis in rat liver in vitro in CCl 4-induced hepatic fibrosis

To clarify the process of free and small peptide-bound hydroxyproline synthesis in hepatic fibrogenesis, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. In control rat liver, the amount of free and small peptide-bound [14C]hydroxyproline synthesized was 13.1 +/- 2.6 10(-4) x dpm/g liver/3 hr. In the CCl4-treated rat liver, where the hepatic hydroxyproline content was increased 4.6-fold, the protein-bound [14C]hydroxyproline synthesis was significantly increased 1.5-fold, but free and small peptide-bound [14C]hydroxyproline synthesis was decreased into 70%. There was a significant inverse correlation between free and small peptide-bound [14C]hydroxyproline synthesis, and hepatic hydroxyproline content. These results suggest that the combination of an increase in collagen synthesis and a decrease in free and small peptide-bound [14C]hydroxyproline synthesis contributes to rapid accumulation of collagen in hepatic fibrosis.

Doxorubicin-induced inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures. Relevance to imparied wound healing.

Previous clinical and experimental observations have indicated that wound healing is impaired as a result of treatment with doxorubicin, a chemotherapeutic agent. In this study, the effects of doxorubicin were examined in human skin fibroblast cultures with respect to collagen production and fibroblast proliferation. The results indicated that the synthesis of hydroxyproline as a marker of collagen production was markedly reduced, with an approximate concentration of inhibitor yielding 50% inhibition of 1 microM. This inhibition could be explained, in part, by generalized inhibition of total protein synthesis, but in addition, there was a significant inhibition of prolyl hydroxylation during collagen biosynthesis, as indicated by a reduction in the ratio of [3H]hydroxyproline/([3H]hydroxyproline + [3H]proline). The latter effect was shown to result from inhibition of prolyl hydroxylase by doxorubicin. As a consequence of reduced prolyl hydroxylation, the stability of newly synthesized procollagen triple helix was shown to be compromised. At the same time, doxorubicin significantly reduced fibroblast proliferation in vitro, as determined by [3H]thymidine incorporation. Thus, reduced collagen production and inhibition of fibroblast proliferation may explain the reduced wound healing in patients undergoing treatment with doxorubicin.

Argon laser—welded arteriovenous anastomoses

This study compared the healing of laser-welded and sutured canine femoral arteriovenous anastomoses. Arteriovenous fistulas 2 cm in length were created bilaterally in the femoral vessels of 10 dogs and were studied at 1 (n = 2), 2 (n = 2), 4 (n = 3), and 8 (n = 3) weeks. In each animal, one anastomosis (control) was closed with running 6-0 polypropylene sutures, and the contralateral anastomosis (experimental) was sealed with an argon laser (0.5 watt, 4 minutes of exposure, 1830 J/cm2/1 cm length of anastomosis). At removal all experimental anastomoses were patent without hematomas, aneurysms, or luminal narrowing. Histologic examination at 4 weeks revealed that laser-welded anastomoses had less inflammatory response and almost normal collagen and elastin reorientation. At 8 weeks sutured anastomoses had significant intimal hyperplasia whereas laser repairs had normal luminal architecture. Tensile strength and collagen production, measured by the synthesis of hydroxyproline and the steady-state levels of type I and type III procollagen messenger ribonucleic acids, at the anastomoses and in adjacent vein and artery specimens were similar in sutured and laser-welded repairs at 2, 4, and 8 weeks. We conclude that argon laser welding of anastomoses is an acceptable alternative to suture techniques, with the advantage of improved healing without foreign body response and possible diminished intimal hyperplasia at the anastomotic line.

Preventive effect of malotilate on carbon tetrachloride-induced liver damage and collagen accumulation in the rat.

Malotilate is a new drug suggested for use in chronic liver diseases. It is shown here to prevent liver damage caused by CCl4. The concomitant administration of malotilate with CCl4 significantly decreased hydroxyproline accumulation in the liver, liver prolyl 4-hydroxylase and liver and serum galactosylhydroxylysyl glucosyltransferase activities. However, it had no effect on the daily urinary hydroxyproline excretion or the hydroxyproline content of the skin, liver or lungs in normal young growing rats. It also had no specific inhibitory effect on hydroxyproline synthesis or secretion in fibroblast cultures, and did not affect the amount of procollagen-alpha 1(I)-specific mRNAs in these cultures. Thus it seems to have no direct inhibitory effect on collagen metabolism. In addition to inhibition of liver collagen accumulation, malotilate was also able to prevent the development of morphological changes in the liver such as focal necrosis, fatty infiltration and inflammatory changes. It also normalized almost completely the standard liver-function tests. It is possible that malotilate may prevent excessive collagen deposition by inhibiting the inflammation caused by CCl4-induced liver damage.

Lipoid proteinosis: Ultrastructural and biochemical studies

Lipoid proteinosis, a rare autosomal recessive disease, is histologically characterized by deposition of hyalinlike material in the dermis. In this study the pathologic processes of lipoid proteinosis were evaluated by ultrastructural and biochemical analysis of skin and cultured fibroblasts from a patient with classic features of the disease. Transmission electron microscopy revealed the presence of hyalinlike material with a granular appearance interspersed between collagen fibers. Immediately surrounding the blood vessel walls, there was reduplication of basal laminae in an "onionskin" arrangement. The fibroblastic cells in the affected dermis contained peculiar cytoplasmic inclusions. Biochemical studies with the cultured fibroblasts showed that the total synthesis of extracellular matrix components, as detected by the synthesis of radioactive hydroxyproline or the incorporation of 35SO4(2-) and [3H]glucosamine into macromolecules, was not altered in lipoid proteinosis. However, the relative expression of type I and type III procollagen genes, as detected by molecular hybridizations with pro-alpha 1(I) and pro-alpha 1(III) procollagen complementary deoxyribonucleic acid probes, was markedly altered in cultured fibroblasts. Specifically, the type I procollagen messenger ribonucleic acid (mRNA) levels were significantly reduced, resulting in a decreased type I/III procollagen mRNA ratio. Furthermore, the replicative capacity of lipoid proteinosis fibroblasts, as detected by the incorporation of radioactive thymidine, was reduced. Thus the skin fibroblasts from lipoid proteinosis demonstrate ultrastructural changes, as well as alterations in their phenotypic characteristics, and these changes may have relevance to the pathologic processes of this systemic disease affecting the skin and other organs.

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.

A possible mechanism of cadmium-copper interaction in embryonic chick bone in tissue culture

Femurs from 9-day-old embryo were cultured for 4 days by the roller-tube method in the presence of Cd and/or Cu. The combination of both Cd and Cu caused a significantly interactive decrease in hydroxyproline (Hyp) synthesis as well as bone growth compared with that in the presence of Cd (1.1 or 3.3 μ m) or Cu (1.1 or 2.2 μ m) alone. The presence of both 2.2 μ m Cd and 3.3 μ m Cu also showed a significantly interactive decrease in the incorporation of [ 3H]proline (Pro) into collagenase-digestible protein (CDP), but it showed no interactive inhibition of the hydroxylation of [ 3H]Pro in CDP. The two metals were also interactive with respect to the inhibition of the synthesis of protein, RNA and DNA. Culture of epiphysis in the presence of both Cd and Cu resulted in higher content of Cd and Cu compared to those cultured in Cd or Cu alone. Subcellular fractions from epiphysis cultured in Cd plus Cu contained more Cd than those cultured in Cd alone. Cu was increased in two fractions, nuclei and cytosol, following co-incubation. The cytosol from epiphysis cultured in the presence of both Cd and Cu contained more Cd in both a metallothionein (MT)-like protein and a high-molecular-weight (HM) protein than cytosol from Cd-treated bones. The amount (nmol) of Cu in the MT fraction was nearly equal to the sum of the increased amounts (nmol) of Cd in HM fraction of cytosol and particulate fractions. This indicates that some Cd in MT-like protein induced by Cd is replaced with Cu and the released Cd redistribution to HM fraction and particulate fractions. Most of the Cd in the solubilized particulate fractions was detected in the HM fraction. A marker enzyme or component in each fraction was interactively inhibited by both Cd and Cu. Therefore, the interactive inhibition of bone metabolism by both Cd and Cu in the cultured bone is at least partly due to the increase in Cd content of HM fraction of cytosol and particulate fractions.

The in vitro effect of gold complexes on bone resorption.

Mouse calvaria were maintained in organ culture without serum additives. The effects of three gold complexes--aurothioglucose, aurothiomalate, and auranofin--on active bone resorption (45Ca release) and hydroxyproline synthesis were determined. The influence of these compounds on DNA and protein synthesis and lysosomal enzyme release from calvaria was also assessed. All gold complexes reduced bone resorption to some extent, with auranofin being the most potent within a narrow concentration range (10(-6) M). This concentration of auranofin also significantly inhibited collagen synthesis, although DNA and protein synthesis were unaffected. None of the compounds tested appeared to mediate their action via significant inhibition of lysosomal enzyme release.

Collagen synthesis in the midgut connective sheath of the mealwormTenebrio molitorL. (Insecta, Coleoptera) during metamorphosis

Summary The biosynthesis of the collagen in the mesenteric sheath of the insect mealworm Tenebrio molitor L. has been investigated during the last larval, pupal and adult stages. The synthesis of collagenous protein was estimated by the quantitation of proline incorporation into hydroxy[3H]proline in the mesenteron. The maximum conversion of [3H]proline into hydroxy[3H]proline occurs in the 5 day old pupa, which appears correlated with the pupal ecdysteroid peak. Relationships between the synthesis of hydroxyproline, histological events and hormonal events are discussed.

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.

Phenytoin Modulates Connective Tissue Metabolism and Cell Proliferation in Human Skin Fibroblast Cultures

Phenytoin has been proposed for the treatment of certain dermatologic conditions involving connective tissue abnormalities. To understand the biochemical basis of connective tissue changes, we incubated human skin fibroblasts in culture with varying concentrations of phenytoin. The results indicated that fibroblast proliferation, detected by tritiated thymidine incorporation into cells, was slightly stimulated when short incubation periods and low concentrations of phenytoin were employed. However, with longer incubation times and higher phenytoin concentrations, a significant reduction in fibroblast proliferation was observed. Further studies demonstrated that incubation of cells with phenytoin did not affect the production of procollagen, measured as synthesis of radioactive hydroxyproline in the cultures. However, assay of prolyl hydroxylase, an enzyme participating in the post-translational synthesis of hydroxyproline during collagen biosynthesis, was significantly reduced in the fibroblast cultures. The activity of collagenase, an enzyme participating in degradation of collagen, was markedly decreased in cultures treated with phenytoin. Thus, phenytoin may modulate collagen metabolism primarily by affecting the degradation of collagen. The results support previous suggestions that phenytoin may be useful for treatment of patients with increased levels of collagenase, such as in recessive dystrophic epidermolysis bullosa.

Photochemie von Aminoketonen. VII. Eine diastereoselektive Synthese von 3‐Hydroxy‐prolinen

Photochemistry of Aminoketones. VIII. Diastereoselective Synthesis of 3‐Hydroxy‐prolinesGlycine esters react in two steps (aminoalkylation and N‐acylation) to N‐acyl‐N‐(β‐benzoylethyl)‐glycine ester 3. In less polar solvents these ketonic compounds are n,π*‐excited by 300 nm irradiation. In ether 3* yield mixtures of aminocyclopropanoles 4, hydroxyprolines 5 and products of additions and cleavages. In benzene and cyclohexane/benzene, respectively, the regioselective photocyclization to 5 is favoured. Furthermore this hydroxyproline synthesis has the advantages of high diastereoselectivity yielding the (2,3) E–5 and of high overall‐yields in connection with the one pot method. The influence of solvents and substituents on the regio‐ and diastereoselectivity are explained using a model with stabilization of special conformations of intermediates by dipol‐dipol interactions.

Increased pulmonary collagen synthesis in mice treated with cyclophosphamide.

Cyclophosphamide is a widely used anticancer drug which damages lung tissue and elicits a progressive fibrotic lesion in mice. The early time course of collagen accumulation and the changes in collagen synthesis which may accompany this lung damage and fibrosis have not been reported. The total lung concentration of hydroxyproline, an index of collagen content and the extent of pulmonary fibrosis, was not significantly increased in mice until 12 days after treatment with 200 mg/kg cyclophosphamide. The change in total lung hydroxyproline content was preceded, on Day 6, by an increase in the rate of acid-insoluble hydroxyproline synthesis. This rate remained elevated to Day 21, but was no longer significantly increased by Day 28. The percentage of total protein synthesis devoted to the production of collagen was significantly increased 9 and 12 days after cyclophosphamide, compared to saline-treated controls. The rate of pulmonary non-collagen protein synthesis was significantly decreased 3 days after cyclophosphamide, and increased 6 and 15 days after this treatment. These data indicate that cyclophosphamide-induced increases in pulmonary collagen synthesis precede the accumulation of this protein. Increased collagen synthesis may occur in the absence of changes in overall protein synthesis although cyclophosphamide also alters non-collagen protein synthesis.

Auxin-induced cell expansion of potato tuber and chicory root: Role of hydroxyproline synthesis

We have reinvestigated the role of protein-bound hydroxyproline (extensin) in auxininduced cell enlargement using discs excised from tubers of Solanum tuberosum L. cv. Pentland Crown and from roots of Cichorium intybus L. cv. Magdeburg. Extensin increases markedly in potato tuber discs treated with water and auxin, and the hydroxyproline is primarily in the cell wall. 2,2′-Dipyridyl totally inhibits both hydroxyproline synthesis and auxin-induced cell expansion in potato with the inhibitions being reversed in parallel by Fe2+. Free hydroxyproline also totally prevents induced cell enlargement. Pretreatment with gibberellic acid totally inhibits subsequent auxin-induced cell expansion but does not inhibit hydroxyproline synthesis. Therefore, the level of hydroxyproline does not control auxin-induced cell enlargement in potato tuber discs. Other interpretations are discussed but we conclude that extensin biosynthesis is necessary for auxin-dependent cell expansion as inhibition of the synthesis prevents the induced expansion. Dipyridyl and free hydroxyproline partially inhibit auxin-induced cell enlargement in chicory root discs. Thus a component of the auxin-dependent cell enlargement in chicory is also dependent on extensin synthesis.

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by 45Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10 −7 M). Prostaglandin F and prostaglandin E 2 production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10 −6M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10 −7M) both PGE 2 and PGF 2α stimulated resorption in a dose-dependent manner, with PGE 2 being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE 2 had a marked inhibitory action at a higher concentration (10 −6M). In combination, the effects of PGE 2 and PGF 2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE 2 production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.

Lysyl Oxidase and Collagenase in Experimental Acute and Chronic Liver Injury

Lysyl oxidase and collagenase activities were measured in experimental acute and chronic liver injury in mice and rats, and correlated with collagen synthesis and accumulation. Acute liver injury was induced in mice and rats by a single dose of carbon tetrachloride given by gavage, and also in mice by a single injection of murine hepatitis virus. Chronic liver injury was induced in rats by repeated injections of carbon tetrachloride. Elevated plasma glutamic oxaloacetic transaminase levels, increased hepatic prolyl hydroxylase activity, and increased synthesis of collagen-bound hepatic hydroxyproline occurred in animals with acute as well as with chronic liver injury. However, only chronic liver injury appeared to be associated with fibrosis, increased collagen-bound hydroxyproline content, increased hepatic lysyl oxidase and collagenase activities, as well as with increased serum lysyl oxidase activity. These data suggest that lysyl oxidase and collagenase may play an important role in the collagen accumulation associated with hepatic fibrosis.