Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

Oestradiol inhibits collagen breakdown in the involuting rat uterus

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

In diatoms, the siliceous cell walls are enveloped by an organic component which includes 4-hydroxyproline and 3,4-dihydroxy-L-proline. The formation of these two amino acids were studied in Nitzschia angularis in Si-starvation synchrony. Both appear to arise from peptidyl proline. Its conversion to peptidyl hydroxyproline was shown in cell-free extracts and in kinetic studies using [¹⁴C]proline. Two lines of evidence indicate that dihydroxyproline does not arise from the further hydroxylation of peptidyl hydroxyproline: First, there was a lag of several minutes between the incorporation of [¹⁴C]proline into protein and the appearance therein of [¹⁴C]hydroxyproline but no such lag for the appearance of dihydroxyproline. Second, ,-dipyridyl blocked the formation of hydroxyproline, but not of dihydroxypyroline, from peptidyl proline. Cell walls made in the presence of dipyridyl differed little in overall chemical composition from walls made in its absence and were morphologically identical. [¹⁴C]dehydroproline was rapidly metabolized in the cells, with [¹⁴C]dihydroxyproline a prominent product. Studies of the conversion of [¹⁴C]proline to [¹⁴C]hydroxyproline at various stages of wall formation showed an increased synthesis of [¹⁴C]dihydroxyproline at the end of cell separation.     

A study of the biosynthesis of collagenous and non-collagenous proteins and of the proline/hydroxyproline ratios of collagens isolated from embryonic tissues

1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.     

Hydroxyproline in Chiorella and its Metabolism in Tobacco Leaves

Hydroxyproline and other compounds were labeled with C¹⁴ by Chlorella pyrenoidosa supplied with C¹⁴O₂ in the light. The hydroxyproline recovered from a hydrolysate of the algae was administered through the cut bases of tobacco leaves. The leaves formed little proline from the hydroxyproline, but the C¹⁴ label was transferred to a variety of other amino acids. Although hydroxyproline is not abundant in plants, it appears to be an active metabolite.     

Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [¹⁴C]proline and determining the percentage of total [¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [¹⁴C]proline is often contaminated with [¹⁴C]hydroxyproline and must be purified before use. (2) Salt and [¹⁴C]proline interfere with the determination of [¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [¹⁴C]hydroxyproline during processing varies; [³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.     

Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Metabolites of -[¹⁴C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 HCL for elution or on DC-6A using 0.1 sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [¹⁴C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.     

Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

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.     

Inhibition of collagen breakdown by diphenylhydantoin

Gingival tissue obtained from diphenylhydantoin-treated patients was cultured in the presence of [¹⁴C]proline for 24 h. The radioactive medium was removed and the tissue cultured for three days more. DNA, protein, hydroxyproline, proline and radioactivity determinations in the tissue indicated increased cellular proliferation, increased collagen contents and decreased breakdown of collagen in the affected tissues. The media were assayed for dialyzable and non-dialyzable hydroxyproline contents. It was found that the media in which diphenylhydantoin tissues were cultured contained more than twice as much non-dialyzable hydroxyproline than the controls. It was concluded that diphenylhydantoin brought about a reduction in collagen breakdown thus explaining the accumulation of hydroxylated collagen in the tissue.     

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [¹⁴C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [¹⁴C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [¹⁴C]lysine, [¹⁴C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

Effect of colchicine on atherosclerosis. II. Biochemical studies on skin biopsies from patients treated perorally with colchicine

The biosynthesis of proteins and glycosaminoglycans (GAGs) was determined in skin biopsies from atherosclerotic patients treated perorally for 3 months with 1 mg/day colchicine. The biopsies were incubated with 3H-glucosamine and 14C-proline for 5 h and subsequently digested with pronase. In an aliquot of the pronase digest, the specific radioactivity of 14C-proline and 14C-hydroxyproline were determined. The 3H-glucosamine-labeled GAGs were identified by specific enzymic assay and quantified after electrophoretic separation. 3 months treatment with colchicine did not modify the total amounts of proline and hydroxyproline in skin proteins, but diminished the amount of the GAGs as expressed by uronic acid content. Colchicine treatment decreased also the specific radioactivity of proline and hydroxyproline, which reflects a decrease of total protein and collagen synthesis. The incorporation of 3H-glucosamine in the 3H-GAGs was also decreased, mainly in hyaluronic acid. These results suggest that peroral administration of colchicine modifies the synthesis of extracellular matrix proteins and polysaccharides by skin fibroblasts.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

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.     

16,16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16,16 dimethyl prostaglandin E2 (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more 14C-proline and produced more 14C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10(-10) M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG: while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.     

Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.