Photochemical degradation of hyaluronic acid by singlet oxygen. L. Lap~ik, jr.1 ... magnesium salt) solutions after exposure to UV radiation indicate a vigorous.
Colloid & Polymer Science
Colloid Polym Sci 269:633-635 (1991)
Short Communications Photochemical degradation of hyaluronic acid by singlet oxygen L. Lap~ik, jr.1 ) and J. Schurz 2) 1) Faculty of Chemistry and Technology, Slovak Technical University, Bratislava, CSFR 2) Institute for Physical Chemistry, University Graz, Graz, Austria Abstract: Changes of the rheological properties of hyaluronic acid (sodium-
magnesium salt) solutions after exposure to UV radiation indicate a vigorous decrease in their viscosity, but its still strong shear rate dependence. Whereasthe presence of the singlet oxygen sensitizer (anthracene-l-sulphonic acid) brings about a loss of shear dependence; the studied solutions show Newtonian behavior. HA-hyaluronic_acid;A-SOsH-anthracene-l-sulphonic acid; ~-shear rate; ~/_sp-specificviscosity
Key words:
Introduction
Materials and methods
Hyaluronic acid (HA) is a polysaccharide. Its primary structure consists of repeating disaccharide units of D-glucuronic acid and N-acetyl D-glucosamine with alternating 13(1--.3) and fl (1 -~ 4) interglycosidic linkages. H A is an important constituent of the intercellular matrix and of the vitreous body. One of the characteristic properties of its solutions is their visco-elasticity, which plays an important role in biological functions of HA in organisms. The eye is a major photoreceptor organ of the body. In the presence of a photosensitizer such as riboflavin in the normal eye [1], the possibility of the photogeneration of singlet oxygen in the vitreous body cannot be ignored [2]. The reactive species of the oxygen ( ' 0 2 , IO2, "OH) may be responsible for the chain splitting process in the macromolecule of HA [2-4], as well as for the changes of the physico-chemical and optical properties of the vitreous body matrix [4, 5]. The main aim of our studies on the rheological behavior of HA solutions was to achieve a better characterization of the degradation process provoked by UV-radiation.
Samples of hyaluronic acid (magnesium-sodiumsalt) used in our studies were obtained from MOVIS Co. (CSFR) (extract from rooster combs). The samples were purified according to the Czechoslovakian patent [6]. 6 mg of HA were dissolved in 10 cm3 of 0.2 M NaC1 at pH 7.4. As the singlet oxygen sensitizing system anthracene-1sulphonic acid (A-SO3H) was used. It was prepared according to the method described by Schmidt [7]. Purified A-SO3H was characterized by IR spectroscopy [8]. Rheological properties of the HA solutions were measured by a rotation viscosimeter (HAAKE Rotovisco RV-100 system with the sensor system ME 30). A 500 W high-pressure mercury lamp was used for irradiation (VEB Narva, GDR).
K 907
Results and discussion It is k n o w n that irradiation of polycyclic aromatic hydrocarbons with UV light produces corresponding transannular peroxides (for review see [9]). These substances undergo reversible photooxidation, e.g., they liberate singlet oxygen ( 1 0 2). In Figs. 1 and 2 the variation in HA solution viscosity (pure and in the presence of sensitizer) with shear rate is shown for a range of irradiation
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Colloid and Polymer Science, Vol. 269 9 No. 6 (1991)
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Fig. 1. Shear rate (y) dependence of hyaluronic acid solution viscosity before (O), after 30 rain ([~) and 60 rain (A) irradiation in 0.2 M NaC1 at 37 ~ pH, 7.0: HA concentration 6 mg/ml
Fig. 2. Shear rate (i) dependence of hyaluronic acid solution viscosity in the presence of anthracene-l-sulphonic acid before (O), after 30rain (E2) and 60rain (A)irradiation in 0.2M NaC] at 37 ~ pH, 7.0; HA concentration 6 mg/m]; A-SO3H concentration 6.3 mg/ml
time intervals. The initial shear independent value is known as the zero shear or Newtonian viscosity (r/0). The value of /7o for concentrated polymer solutions reflects the influence of molecular mass, concentration, and the extent of intermolecular interaction. Subsequent decrease in viscosity with increasing shear may be attributed [10] to progressive breakdown of intermolecular network character [11]. The remarkable decrease in viscosity of the studied HA solutions caused by UV radiation (both for pure HA (Fig. 1) and in the presence of A-SO3H (Fig. 2)), reflects the strong degradation process of the HA main macromolecular chain. In the presence of A-SO3H the degradation is so effective (see Fig. 2) that the studied solutions behave like Newtonian fluids (they loose their elasticity). Further addition of A-SO3H to the HA solution proportionally increases the degree of HA degradation with a proportional decrease in solution viscosity. UV irradiation of aqueous solutions brings about the production of hydrated electrons e~q (quantum yield 9 = 0.04), atomic hydrogen H" (~ = 0.37) and reactive "OH radicals (~ = 0.37) [12], which bring about consequent degradation reactions with HA (see Fig. 1). It is possible to
conclude from our measurements that the abovementioned reactive species (e.g., e~, H', and "OH) are less effective degrading agents than is singlet oxygen (see Fig. 2), which appears to be more dangerous in initiating the pathological processes in the eye during the irradiation. We plan to study these problems in more details in future work.
References 1. Berman ER, Voaden M (1970) In: Graymore CN (ed) Biochemistry of the eye. Academic Press, New York, pp 373-471 2. Andtey UP, Chakrabarti B (1983) Biochem Biophys Res Comms 115:894-901 3. Lal M (1985) J Radioanal Nuclear Chem, Articles 92:105-112 4. Lap~ik [ Jr, Omelka L, Kub6na K, Galatik A, Kell6 V (1990) Gen Physiol & Biophys 9 (in press) 5. Ueno N, Sebag J, Hirokawa H, Chakrabarti B (1987) Exp Eye Res 44:863-870 6. Galatik A, Kub6na K, Bla~ej A (1989) Pharmacological preparation on the basis of hyaturonic acid. CS-Patent 264:719 7. Schmidt RE, Tust P (1904) B Deutsch Chem Ges 37a:66-72
Lap~ik, Jr. and Schurz, Photochemical degradation of hyaluronic acid by singlet oxygen 8. Rohatgi-Mukherjee KK, Singh BP (1977) J Indian Chem Soc 54:527-533 9. LapSik [ Jr, Lap~ik L, Bako~ D, Kell6 V (1990) Chemick~ listy 84:582-605 10. Graessley WW (1974) The Entanglement Concept in Polymer Rheology. Advances in Polymer Science 16, Springer Verlag 11. Morris ER, Rees DA, Welsh EJ (1980) J Mol Biol 138:383-400 12. Khan KA, Parsons BJ, Phillips GO, Davies AK (1981) Polym Photochem 33-41
635 Received January 17, 1991; accepted February 7, 1991
Authors' address: Prof. Dr. J. Schurz Universitfit Graz Institut ffir Physikalische Chemie HeinrichstraBe 28 8010 Graz, Osterreich
