Summary and Conclusion A Summary

HA is an acidic GAG of D-GlcA and D-GlcNA that is found in mammalian connected tissue. It is a polydisperse, unbranched chain whose molecular weight varies from 300 to >103 kDa. HA occurs as a free chain and covalently bonded to protein as the SHAP-HA complex. It can be found inside and outside the cells. Its secondary and tertiary structures in solution display both stiff and flexible domains that suggest deviations of its primary structure (sugar-sequence) from the alternating sequence of the sugars indicated earlier. In this review, the secondary and tertiary structures of HA in solution are analyzed, in an attempt to explain their relevance to normal biologic functions and aging. Hydrophilic sequences of D-GlcN and D-GlcA are used to explain the stiff and flexible domains detected in HA solutions. The hydrophilic segments should be more densely hydrogen bonded with water than the regions of alternating D-GlcA and D-GlaNAc, as water would form cages around them.

Stiff segments can originate from hydrogen bond networks or from van der Waals associations of the methyl moieties of D-GlcNAc in a sequence of this sugar only or in the classical alternating sequence of D-GlcA and D-GlcNAc. Stiff segments originating from acidic (D-GlcA) and basic (D-GlcN) sugars are disrupted by base (e.g., OH"), as it becomes the hydrogen acceptor in the hydrogen bonds HA makes with water. If stiffness results from hydrophobic interactions, a basic reagent relaxes it, as it removes methyl protons from the acetamido moiety of D-GlcNAc, since these protons are acidic.

Viscous solutions of HA in water are the result of highly dense, HA-water, hydrogen bond networks. These solutions appear to be involved in a variety of biologic functions such as the efficient support of healthy joints by synovial fluid; the protection of corneal endothelium from mechanical damage; cell differentiation in fetal tissue; fluidity in the vitreous body of the eye and many others. The therapeutic and medical uses of highly viscous HA are presently diverse and are growing rapidly.

In aging, the hypothetic D-GlcN sequence was used in an attempt to explain the N-deacetylation of human skin HA at 75 years of age. Because the skin composition of HA decreases by 77% (w/w) also at 75 years, and the basic sugar-sequence makes HA more hydrophilic than the alternating sequence of D-GlcA and D-GlcNAc, it was concluded that nature compensates for the loss of HA concentration in aging by having an enzyme that removes the N-acetyl groups, making HA more hydrophilic. Highly hydrophilic HA is needed in aged human skin, if no other molecules are made to retain the water, since about 50% (w/w) of this reagent is bonded to this GAG.

The reasons for the loss of HA from the skin were attributed to depolymerization induced by both endogenous and exogenous free radicals. This hypothesis is supported by findings that indicate that free radical scavengers inhibit UV-light-mediated cleavage of HA in vitro. It was found that the UV-light irradiation of skin in vivo resulted in increased GAG biosynthesis in hairless mice and albino rats; but providing vitamin E in the rat's diet reversed the latter results. Because vitamin E is a natural free radical scavenger, the data support a free radical involvement. In the absence of UV-light irradiation, depolymerization may be caused by endogenous free radicals, which are natural products of metabolism, or by enzymatic degradation. Results on this area are conflicting; some workers reported HA depolymerization in rat skin, while others found no significant age-related changes in HA size.

Analysis of the literature on the age-related effect of HA in cell aging (cell passage) in vitro revealed that the biosynthesis of HA increased with the number of cell passages in an UVA-dose-dependent way, and that there was an increment in hydrolytic enzymes. In the absence of UV-irradiation, the concentration of HA decreased as the number of cell passages increased.

Literature pertaining to the effect of age on the concentration of HA in body fluids has been investigated. It revealed that the HA content increased with aging in human serum.

Data on the alterations of HA in accelerated aging diseases like progeria and Down's syndrome are inconclusive. Nevertheless, it was found that urinary HA was increased in progeria patients. In the serum of patients with Down's syndrome, HA content was only slightly higher than in the normal serum.

B. Conclusion

HA is an essential heteropolysaccharide of mammalian connective tissue. It is a free radical scavenger that protects the system from endogenous and exogenous free radicals. Its broad involvement in biologic functions, aging, medicine and therapeutic processes requires that its exact sugar-sequence be elucidated. Such knowledge would shed light on its mechanism of action and facilitate: the development of ways to retard human aging; therapies to prevent or cure premature aging-like disorders as progeria and Down's syndrome; and drugs for the treatment or prevention of other diseases of the aged in which HA appears to play a role.


Scholarly Research Release Award from Purdue University Calumet supported this work.


1. Meyer K. Chemical structure of hyaluronic acid. Fed Proc 1958; 17:1075-1077.

2. Longas MO, Meyer K. Sequential hydrolysis of hyaluronate by b-N-acetylhexo-saminidase and b-glucuronidase. Biochem J 1981; 197:275-282.

3. Scott JE. Chemical morphology of hyaluronan. In: Laurent TC, ed. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. London: Portland Press, 1998:7-15.

4. Scott JE, Heatley F. Hyaluronan forms specific stable tertiary structures in aqueous solution: a 13C NMR study. Proc Natl Acad Sci USA 1999; 96:4850-4855.

5. Laurent TC. Structure of hyaluronic acid. In: Balazs EA, ed. Chemistry and Molecular Biology of the Intercellular Matrix. New York: Academic Press, 1970; vol. 2:703-732.

6. Piepkorn M, Hovingh P, Linker A. Glycosaminoglycan free chains. External plasma membrane components distinct from the membrane proteoglycans. J Biol Chem 1989:8662-8669.

7. Fessler JH, Fessler LJ. Electron microscopic visualization of the polysaccharide hyaluronic acid. Proc Natl Acad Sci USA 1966; 56:141-147.

8. Varma R, Varma RS, Allen WS, Wardi AH. On the carbohydrate-protein linkage groups in vitreous humor hyaluronate. Biochim Biophys Acta 1974; 362: 584-588.

9. Yoneda M, Suzuki S, Kimata K. Hyaluronic acid associated with the surfaces of cultured fibroblasts is linked to a serum-derived 85-kDa protein. J Biol Chem 1990; 265:5247-5257.

10. Zhao M, Yoneda M, Ohashi Y, Kurono S, Iwata H, Ohnuki Y, Kimata K. Evidence for the covalent binding of SHAP, heavy chains of inter-alpha-trypsin inhibitor, to hyaluronan. J Biol Chem 1995; 270:26657-26663.

11. Chem L, Zhang H, Powers RW, Russell PT, Larsen WJ. Covalent linkage between proteins of the inter-alpha-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells. J Biol Chem 1996; 271:19409-19414.

12. Rosenberg L, Hellmann W, Kleinschmidt AK. Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J Biol Chem 1975; 250: 1877-1883.

13. Hascall VC, Heingard D. Aggregation of cartilage proteoglycans I. The role of hyaluronic acid. J Biol Chem 1974; 249:4232-4241.

14. Hascall VC, Heinegard D. Aggraegation of cartilage proteoglycans II. Oligo-saccharide competitors of the proteoglycan-hyaluronic acid interaction. J Biol Chem 1974; 249:4242-4249.

15. Hascall VC, Heinegard D. The structure of cartilage proteoglycans. In: Slavkin HC, Greulich R, eds. Extracellular Matrix Influences on Gene Expression. New York: Academic Press, 1975:423-434.

16. Ripellino JA, Bailo M, Margolis RU, Margolis RK. Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum. J Cell Biol 1988; 106:845-855.

17. Bertolami CN, Berg S, Messadi DV. Binding and internalization of hyaluronate by human cutaneous fibroblasts. Matrix 1992; 11:11-21.

18. Eggli PS, Graber W. Association of hyaluronan with rat vascular endothelial cells and smooth muscle cells. J Histochem Cytochem 1995; 43:689-697.

19. Evanko SP, Wight TN. Intracellular localization of hyaluronan in proliferating cells. J Histochem Cytochem 1999; 47:1331-1341.

20. Tammi R, Rilla K, Pienimaki J-P, MacCallum DK, Luukkonen M, Hascall VC, Tammi M. Hyaluronan enters keratinocytes by a novel endocytic route for catabolism. J Biol Chem 2001; 276:35111-35122.

21. Brecht M, Mayer U, Schlosser E, Prehm P. Increased hyaluronate synthesis required for fibroblast detachment and mitosis. Biochem J 1986; 239:445-450.

22. Tammi R, Tammi M. Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes. Exp Cell Res 1991; 195: 524-527.

23. Ueno N, Chakrabarti B, Garg HG. Hyaluronic acid of human skin and post-burn scar: heterogeneity in primary structure and molecular weight. Biochem Int 1992; 26:787-796.

24. Roden L. Structure and metabolism of connective tissue proteoglycans. In: Lennarz WJ, ed. The Biochemistry of Glycoproteins and Proteoglycans. New York: Plenum Press, 1980:267-373.

25. Garrett RH, Grisham CM. Protein synthesis and degradation, signal peptide sequence. In: Garrett RH, Grishman CM, eds. Biochemistry, 2nd ed. New York:

Saunders College Publishing/Harcourt Brace College Publishers, 1999: 1089-1127.

26. McGuire PG, Castellot JJ, Orkin RW. Size-dependent hyaluronate degradation by cultured cells. J Cell Physiol 1987; 133:267-276.

27. Hascall VC, Hascall GK. Proteoglycans. In: Hay ED, ed. Cell Biology of Extracellular Matrix. New York: Plenum Press, 1981:39-63.

28. Fessler JH. A structural function of mucopolysaccharides in connective tissue. Biochem J 1960; 76:124-132.

29. Laurent TC, Bjork I, Pietruszkiewicz A, Persson H. On the interaction between polysaccharides and macromolecules. II. The transport of globular proteins through hyaluronic acid solutions. Biochim Biophys Acta 1963; 78:351-359.

30. Toole BP. Morphogenic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In: Barondes SH, ed. Neuronal Recognition. New York: Plenum Press, 1976:275-329.

31. Chakrabarti B, Park JW. Glycosaminoglycas: structure and interaction. CRC Rev Biochem 1980; 8:225-313.

32. Burd DAR, Siebert JW, Ehrlich HP, Garg HG. Human skin and post-burn scar hyaluronan: demonstration of the association with collagen and other proteins. Matrix 1989; 9:322-327.

33. Hall CL, Wang C, Lange LA, Turley EA. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity. J Cell Biol 1994; 126:575-588.

34. Knudson W, Knudson CB. The hyaluronan receptor, CD44. In: Laurent TC, ed. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. London: Portland Press, 1998:169-179.

35. Kumar R, Choudhury NR, Salunke DM, Datta K. Evidence for clustered mannose as a new ligand for hyaluronan binding protein (HABP1) from human fibroblasts. J Biosci 2001; 26:325-332.

36. Zhuo L, Yoneda M, Zhao M, Yingsung W, Yoshida N, Kitagawa Y, Kawamura K, Suzuki T, Kimata K. Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice. J Biol Chem 2001; 276:7693-7696.

37. Hascall VC, Sajdera SW. Physical properties and polydispersity of proteoglycan from bovine nasal cartilage. J Biol Chem 1970; 245:4920-4930.

38. Laurent TC, Ryan M, Pietruszkiewicz A. Fractionation of hyaluronic acid. The polydispersity of hyaluronic acid from bovine vitreous body. Biochim Biophys Acta 1960; 42:476-485.

39. Laurent TC, Fraser RE. Hyaluronan. FASEB J 1992; 6:2397-2404.

40. Garrett RH, Grisham CM. Proteins: their biologic functions and primary structure. In: Garrett RH, Grishman CM, eds. Biochemistry, 2nd ed. New York: Saunders College Publishing/Harcourt Brace College Publishers, 1999:158-208.

41. Darke A, Finer EG, Moorhouse R, Rees DA. Studies of hyaluronate solutions by nuclear magnetic relaxation measurements. Detection of covalently defined, stiff segments within the flexible chains. J Mol Biol 1975; 99:477-486.

42. Lubomir L Jr, Lubomir L, Desmedt S, Demeester J, Chabrecek P. Hyaluronan: preparation, structure, properties, and applications. Chem Rev 1998; 98:2663-2684.

43. Solomons TW, Fryhle CB. van der Waals forces. In: Solomons TW, Fryhle CB, eds. Organic Chemistry. 7th ed. upgrade New York: Wiley, 2002:73-74 see also pp. 152-153.

44. Solomons TW, Fryhle CB. The acidity of the a hydrogens of carbonyl compounds: enolate anions. In: Solomons TW, Fryhle CB, eds. 7th ed. upgrade, Organic Chemistry. New York: Wiley, 2002:767-776.

45. Atkins EDT, Sheehan JK. Hyaluronates: relation between molecular conformations. Science 1973; 179:562-564.

46. Pasqualli-Ronchetti I, Quaglino D, Mori G, Bacchelli B. Hyaluronan-phospholipid interactions. J Struct Biol 1997; 120:1-10.

47. Lee GM, Johnstone B, Jacobson K, Caterson B. The dynamic structure of the pericellular matrix of living cells. J Cell Biol 1993; 123:1899-1907.

48. Sheehan J, Brass A, Almond A. The conformation of hyaluronan in aqueous solution: comparison of theory and experiment. Biochem Soc Trans 1999; 2:121-124.

49. Balazs EA, Watson D, Duff IF, Roseman S. Hyaluronic acid in synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritic human fluids. Arthritis Rheum 1967; 10:357-375.

50. Graue EL, Polack FM, Balazs EA. The protective effect of Na-hyaluronate to corneal endothelium. Exp Eye Res 1980; 31:119-127.

51. Tammi R, Ripellino JA, Margolis RU, Tammi M. Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Investig Dermatol 1988; 90:412-414.

52. Becker A, Sandson J. The source of the inter-alpha trypsin inhibitor in pathologic hyaluronate protein. Arthritis Rheum 1971; 14:764-766.

53. Fransson L-A, Malmstrom A. Structure of pigskin dermatan sulfate. I. Distribution of D-glucuronic acid residues. Eur J Biochem 1971; 18:422-430.

54. Longas MO, Russell CS, He X-Y. Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging. Carbohydr Res 1987; 159:127-136.

55. Longas MO, Russell CS, He X-Y. Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy. Biochim Biophys Acta 1986; 884:265-269.

56. Perlish JS, Longas MO, Fleischmajer R. The role of glycosaminoglycans in aging of the skin. In: Balin AK, Kligman AM, eds. Aging and the Skin. New York: Raven Press, 1989:153-165.

57. Meyer LJ, Stern R. Age-dependent changes of hyaluronan in human skin. J Investig Dermatol 1994; 102:385-389.

58. Koichi S. Biochemical analysis of collagen, glycosaminoglycans and elastin in ISh rat dermis. Jpn J Dermatol 1992; 102:425-431.

59. Cechowskapasko M, Palka J. Age-dependent changes in glycosaminoglycan content in the skin of fasted rats. A possible mechanism. Exp Toxicol Path 2000; 52:127-131.

60. Morgan WTJ, Elson LA. A colorimetric method for the determination of N-acetylglucosamine and N-acetylchondrosamine. Biochem J 1934; 28:988-995.

61. Rissing JL, Strominger JL, Leloir LF. A modified colorimetric method for the estimation of N-acetylaminosugars. J Biol Chem 1955; 217:959-966.

62. Gilchrest BA. Aging of the skin. In: Gilchrest BA, ed. Skin and Aging Processes. Boca Raton: CRC Press, 1984:5-36.

63. Deguine V, Menasche M, Ferrari P, Fraisse L, Pouliquen Y, Robert L. Free radical depolymerization of hyaluronan by maillard reaction products. Role in liquefaction of aging vitreous. Biol Macromol 1998; 22:17-22.

64. Sato H, Takahashi T, Ide H, Fukushima T, Tabata M, Sekine F, Kobayashi K, Negishi M, Niwa Y. Antioxidant activity of synovial fluid, hyaluronic acid, and two subcomponents of hyaluronic acid. Arthritis Rheum 1988; 31:63-71.

65. Foschi D. Hyaluronan, a radical scavenger. Int J Tissue React XII 1990; 6:333-339.

66. Deeble DJ, Bothe E, Schuchmann H-P, Parsons BJ, Phillips GO, von Sonntag C. The kinetics of hydoxyl-radical-indiced strand breakage of hyaluronic acid. A pulse radiolysis study using conductometry and laser-light-scattering. Z Naturforsch 1990; 45c:1031-1043.

67. Lapcik L, Schurz J. Photochemical degradation of hyaluronic acid by singlet oxygen. Colloid Polym Sci 1991; 269:633-635.

68. Rehakova M, Bakos D, Soldan M, Vizarova K. Depolymerization reactions of hyaluronic acid in solution. Int J Biol Macromol 1994; 16:121-124.

69. Chatterjee R, Benzinger MJ, Ritter JL, Bissett DL. Chronic ultraviolet B radiation-induced biochemical changes in the skin of hairless mice. Photochem Photobiol 1990; 51:991-997.

70. Longas MO, Bhuyan DK, Bhuyan KC, Gutsch CM, Breitweiser KO. Dietary vitamin E reverses the effect of ultraviolet light on rat skin glycosaminoglycans. Biochim Biophys Acta 1993; 1156:239-244.

71. Miyamoto I, Nagase S. Changes in the molecular weight of hyaluronic acid from rat skin. Exp Anim 1984; 33:481-485.

72. Verdugo ME, Ray J. Age-related changes in activity of specific lysosomal enzymes in the human retinal pigment epithelium. Exp Eye Res 1997; 65:231-240.

73. Fodil-Bourahla I, Drubaix I, Robert L. Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation of the elastin-laminin receptor. Mech Ageing Dev 1999; 106:241-260.

74. Schachtschabel DO, Beerbaum MK. UVA-irradiaton stimulates hyaluronan synthesis of human skin fibroblast in vitro. Med Welt 1996; 47:191-195.

75. Sluke G, Schachtschabel DO, Weres J. Age-related changes in distribution pattern of glycosaminoglycans synthesized by cultured human diploid fibroblasts (WI-38). Mech Ageing Dev 1981; 16:19-27.

76. Moczar M, Robert L. Stimulation of cell proliferation by hyaluronidase during in vitro aging of human skin fibroblasts. Exp Gerontol 1993; 28:59-68.

77. Longas MO, Burden JD, Lesniak J, Booth RM, McPencow JA, Park JI. Hyaluronic acid N-deacetylase assay in whole skin. Biomacromolecules 2003; 4:189-192.

78. Manery JF, Haege LF. The extent to which radioactive chloride penetrates tissues and its significance. Am J Physiol 1941; 134:83-93.

79. Ogston AG. On water binding. Fed Proc 1966; 25:986-989.

80. Jung JW, Cha SH, Lee SC, Chun IK, Kim YP. Age-related changes of water content in the rat skin. J Dermatol Sci 1997; 14:12-19.

81. Lindqvist U, Laurent TC. Serum hyaluronan and amino terminal propeptide of type III procollagen: variation with age. Scand J Clin Lab Investig 1992; 52: 613-621.

82. Goto M. Urinary hyaluronic acid as marker of aging. Kotsu Yobo Igaku Kenkyu Zaidan Kenkyu Hokokusho 1995; 1993:47-53.

83. Sweeney KJ, Weiss AS. Hyaluronic acid in progeria and the aged phenotype. Gerontology 1992; 38:139-152.

84. Brown WT. Progeria: a human-disease model of accelerated aging. Am J Clin Nutr 1992; 55:1222S-1224S.

85. Hutchin T, Martin L, Prasher V, Sinclair AJ. Serum hyaluronic acid in Down's syndrome. Mech Ageing Dev 1998; 106:155-160.

Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) © 2004 Elsevier Ltd. All rights reserved.



Thank you for deciding to learn more about the disorder, Osteoarthritis. Inside these pages, you will learn what it is, who is most at risk for developing it, what causes it, and some treatment plans to help those that do have it feel better. While there is no definitive “cure” for Osteoarthritis, there are ways in which individuals can improve their quality of life and change the discomfort level to one that can be tolerated on a daily basis.

Get My Free Ebook

Post a comment