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. 1983 Sep;72(3):826–835. doi: 10.1172/JCI111053

Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss. In vitro studies in guinea pig calvarial bones.

M Chojkier, R Spanheimer, B Peterkofsky
PMCID: PMC1129247  PMID: 6309911

Abstract

The question whether ascorbate regulates collagen production solely through its direct role in proline hydroxylation was investigated. Proteins in calvarial bones from control and scorbutic weanling guinea pigs were labeled in short-term cultures with radioactive proline. Proteins were digested with purified bacterial collagenase to distinguish between effects on collagen polypeptide production and hydroxyproline formation. There was a preferential decrease in the absolute rate of collagen biosynthesis beginning after 2 wk of ascorbate deficiency, and this effect was temporally dissociated from decreased proline hydroxylation. There were no significant changes in the absolute rates of collagen degradation or noncollagen protein production. In vitro inhibition of proline hydroxylation in normal bone with alpha, alpha'-dipyridyl did not affect the relative rate of collagen synthesis, further dissociating these functions. Ascorbate added to scorbutic bone cultures reversed defective proline hydroxylation but not defective collagen synthesis, suggesting that the latter was an indirect effect of scurvy. There was a linear correlation between the extent of body weight lost during the 3rd and 4th wk of scurvy and the rate of collagen synthesis in scorbutic bone. This correlation also applied to control animals receiving ascorbate, but with weight loss induced by food restriction. These studies establish for the first time that ascorbate deficiency in guinea pigs leads to a specific decrease in collagen polypeptide synthesis and suggest that this decrease results from the reduced food intake and/or weight-loss characteristic of scurvy.

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Selected References

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  1. Alfano M. C. Effect of acute ascorbic acid deficiency on the DNA content and permeability of guinea-pig oral mucosal epithelium. Arch Oral Biol. 1978;23(10):929–932. doi: 10.1016/0003-9969(78)90300-x. [DOI] [PubMed] [Google Scholar]
  2. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnes M. J., Constable B. J., Morton L. F., Kodicek E. 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. Biochem J. 1970 Sep;119(3):575–585. doi: 10.1042/bj1190575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barnes M. J., Kodicek E. Biological hydroxylations and ascorbic acid with special regard to collagen metabolism. Vitam Horm. 1972;30:1–43. doi: 10.1016/s0083-6729(08)60793-1. [DOI] [PubMed] [Google Scholar]
  5. Bates C. J., Prynne C. J., Levene C. I. The synthesis of underhydroxylated collagen by 3 T6 mouse fibroblasts in culture. Biochim Biophys Acta. 1972 Apr 15;263(2):397–405. doi: 10.1016/0005-2795(72)90091-8. [DOI] [PubMed] [Google Scholar]
  6. Bates C. J. Vitamin C deficiency in guinea pigs: variable sensitivity of collagen at different sites. Int J Vitam Nutr Res. 1979;49(1):77–86. [PubMed] [Google Scholar]
  7. Berg R. A., Prockop D. J. The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem Biophys Res Commun. 1973 May 1;52(1):115–120. doi: 10.1016/0006-291x(73)90961-3. [DOI] [PubMed] [Google Scholar]
  8. Berg R. A., Schwartz M. L., Crystal R. G. Regulation of the production of secretory proteins: intracellular degradation of newly synthesized "defective" collagen. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4746–4750. doi: 10.1073/pnas.77.8.4746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blanck T. J., Peterkofsky B. The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts. Arch Biochem Biophys. 1975 Nov;171(1):259–267. doi: 10.1016/0003-9861(75)90031-4. [DOI] [PubMed] [Google Scholar]
  10. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  11. Chen T. L., Raisz L. G. The effects of ascorbic acid deficiency on calcium and collagen metabolism in cultured fetal rat bones. Calcif Tissue Res. 1975;17(2):113–127. doi: 10.1007/BF02547284. [DOI] [PubMed] [Google Scholar]
  12. Chojkier M., Bateman J., Phang J. M., Peterkofsky B. Formation of proline metabolites in chick embryo bone: interference with the measurement of free hydroxyproline by ion-exchange chromatography. Anal Biochem. 1982 Mar 1;120(2):330–338. doi: 10.1016/0003-2697(82)90354-2. [DOI] [PubMed] [Google Scholar]
  13. Chojkier M., Peterkofsky B., Bateman J. New method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests. Anal Biochem. 1980 Nov 1;108(2):385–393. doi: 10.1016/0003-2697(80)90603-x. [DOI] [PubMed] [Google Scholar]
  14. Diegelmann R. F., Peterkofsky B. Collagen biosynthesis during connective tissue development in chick embryo. Dev Biol. 1972 Jul;28(3):443–453. doi: 10.1016/0012-1606(72)90028-0. [DOI] [PubMed] [Google Scholar]
  15. Dziewiatkowski D. D., Hascall V. C., Riolo R. L. Epimerization of trans-4-hydroxy-L-proline to cis-4-hydroxy-D-proline during acid hydrolysis of collagen. Anal Biochem. 1972 Oct;49(2):550–558. doi: 10.1016/0003-2697(72)90461-7. [DOI] [PubMed] [Google Scholar]
  16. Fabro S. P., Rinaldini L. M. Loss of ascorbic acid synthesis in embryonic development. Dev Biol. 1965 Jun;11(3):468–488. doi: 10.1016/0012-1606(65)90051-5. [DOI] [PubMed] [Google Scholar]
  17. Fern E. B., Garlick P. J. The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo. Biochem J. 1974 Aug;142(2):413–419. doi: 10.1042/bj1420413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. GOULD B. S. Biosynthesis of collagen. III. The direct action of ascorbic acid on hydroxyproline and collagen formation in subcutaneous polyvinyl sponge implants in guinea pigs. J Biol Chem. 1958 Jun;232(2):637–649. [PubMed] [Google Scholar]
  19. GROSS J. Studies on the formation of collagen. II. The influence of growth rate on neutral salt extracts of guinea pig dermis. J Exp Med. 1958 Feb 1;107(2):265–277. doi: 10.1084/jem.107.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. GROSS J. Studies on the formation of collagen. IV. Effect of vitamin C deficiency on the neutral salt-extractible collagen of skin. J Exp Med. 1959 Jun 1;109(6):557–569. doi: 10.1084/jem.109.6.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jimenez S., Harsch M., Rosenbloom J. Hydroxyproline stabilizes the triple helix of chick tendon collagen. Biochem Biophys Res Commun. 1973 May 1;52(1):106–114. doi: 10.1016/0006-291x(73)90960-1. [DOI] [PubMed] [Google Scholar]
  22. Kao W. W., Flaks J. G., Prockop D. J. Primary and secondary effects of ascorbate on procollagen synthesis and protein synthesis by primary cultures of tendon fibroblasts. Arch Biochem Biophys. 1976 Apr;173(2):638–648. doi: 10.1016/0003-9861(76)90301-5. [DOI] [PubMed] [Google Scholar]
  23. Kivirikko K. I., Prockop D. J. Enzymatic hydroxylation of proline and lysine in protocollagen. Proc Natl Acad Sci U S A. 1967 Mar;57(3):782–789. doi: 10.1073/pnas.57.3.782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuttan R., Parrott D. P., Kaplan S. R., Fuller G. C. Effect of ascorbic acid on prolyl hydroxylase activity, collagen hydroxylation and collagen synthesis in human synovial cells in culture. Res Commun Chem Pathol Pharmacol. 1979 Nov;26(2):337–345. [PubMed] [Google Scholar]
  25. LOFTFIELD R. B., HARRIS A. Participation of free amino acids in protein synthesis. J Biol Chem. 1956 Mar;219(1):151–159. [PubMed] [Google Scholar]
  26. Mata J. M., Assad R., Peterkofsky B. An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells. Arch Biochem Biophys. 1981 Jan;206(1):93–104. doi: 10.1016/0003-9861(81)90070-9. [DOI] [PubMed] [Google Scholar]
  27. Morris N. P., Fessler L. I., Weinstock A., Fessler J. H. Procollagen assembly and secretion in embryonic chick bone. J Biol Chem. 1975 Jul 25;250(14):5719–5726. [PubMed] [Google Scholar]
  28. Murad S., Grove D., Lindberg K. A., Reynolds G., Sivarajah A., Pinnell S. R. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci U S A. 1981 May;78(5):2879–2882. doi: 10.1073/pnas.78.5.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mussini E., Hutton J. J., Jr, Udenfriend S. Collagen proline hydroxylase in wound healing, granuloma formation, scurvy, and growth. Science. 1967 Aug 25;157(3791):927–929. doi: 10.1126/science.157.3791.927. [DOI] [PubMed] [Google Scholar]
  30. PETERKOFSKY B., UDENFRIEND S. ENZYMATIC HYDROXYLATION OF PROLINE IN MICROSOMAL POLYPEPTIDE LEADING TO FORMATION OF COLLAGEN. Proc Natl Acad Sci U S A. 1965 Feb;53:335–342. doi: 10.1073/pnas.53.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peterkofsky B., Diegelmann R. Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry. 1971 Mar 16;10(6):988–994. doi: 10.1021/bi00782a009. [DOI] [PubMed] [Google Scholar]
  32. Peterkofsky B. Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo fibroblasts. Biochem Biophys Res Commun. 1972 Dec 4;49(5):1343–1350. doi: 10.1016/0006-291x(72)90614-6. [DOI] [PubMed] [Google Scholar]
  33. Peterkofsky B. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch Biochem Biophys. 1972 Sep;152(1):318–328. doi: 10.1016/0003-9861(72)90221-4. [DOI] [PubMed] [Google Scholar]
  34. Richmond V., Stokstad E. L. Effect of ascorbic acid on guinea pig skin collagen synthesis. I. Total collagen. J Dent Res. 1969 Sep-Oct;48(5):863–871. doi: 10.1177/00220345690480054201. [DOI] [PubMed] [Google Scholar]
  35. Rokosova B., Chvapil M. Relationship between the dose of ascorbic acid and its structural analogs and proline hydroxylation in various biological systems. Connect Tissue Res. 1974;2(3):215–221. doi: 10.3109/03008207409152246. [DOI] [PubMed] [Google Scholar]
  36. Ross R. The fibroblast and wound repair. Biol Rev Camb Philos Soc. 1968 Feb;43(1):51–96. doi: 10.1111/j.1469-185x.1968.tb01109.x. [DOI] [PubMed] [Google Scholar]
  37. STONE N., MEISTER A. Function of ascorbic acid in the conversion of proline to collagen hydroxyproline. Nature. 1962 May 12;194:555–557. doi: 10.1038/194555a0. [DOI] [PubMed] [Google Scholar]
  38. Sato P., Udenfriend S. Studies on ascorbic acid related to the genetic basis of scurvy. Vitam Horm. 1978;36:33–52. doi: 10.1016/s0083-6729(08)60981-4. [DOI] [PubMed] [Google Scholar]
  39. Schneible P. A., Airhart J., Low R. B. Differential compartmentation of leucine for oxidation and for protein synthesis in cultured skeletal muscle. J Biol Chem. 1981 May 25;256(10):4888–4894. [PubMed] [Google Scholar]
  40. Schwarz R. I., Mandell R. B., Bissell M. J. Ascorbate induction of collagen synthesis as a means for elucidating a mechanism of quantitative control of tissue-specific function. Mol Cell Biol. 1981 Sep;1(9):843–853. doi: 10.1128/mcb.1.9.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stassen F. L., Cardinale G. J., Udenfriend S. Activation of prolyl hydroxylase in L-929 fibroblasts by ascorbic acid. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1090–1093. doi: 10.1073/pnas.70.4.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. VAN ROBERTSON W. B., HIWETT J., HERMAN C. The relation of ascorbic acid to the conversion of proline to hydroxyproline in the synthesis of collagen in the carrageenan granuloma. J Biol Chem. 1959 Jan;234(1):105–108. [PubMed] [Google Scholar]
  43. Van Venrooij W. J., Moonen H., Van Loon-Klaassen L. Source of amino acids used for protein synthesis in HeLa cells. Eur J Biochem. 1974 Dec 16;50(1):297–304. doi: 10.1111/j.1432-1033.1974.tb03898.x. [DOI] [PubMed] [Google Scholar]
  44. Zannoni V., Lynch M., Goldstein S., Sato P. A rapid micromethod for the determination of ascorbic acid in plasma and tissues. Biochem Med. 1974 Sep;11(1):41–48. doi: 10.1016/0006-2944(74)90093-3. [DOI] [PubMed] [Google Scholar]

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