Hyaluronic acid oxidized at hydroxymethyl group displays an increased in vitro enzymatic resistance to testicular hyaluronidase. Hyaluronic acid belongs to the ...
ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2007, Vol. 417, pp. 341–342. © Pleiades Publishing, Ltd., 2007. Original Russian Text © I.Yu. Ponedel’kina, V.N. Odinokov, E.A. Saitgalina, U.M. Dzhemilev, 2007, published in Doklady Akademii Nauk, 2007, Vol. 417, No. 4, pp. 557–559.
BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY
The in vitro Resistance of Oxidized Hyaluronic Acid to Testicular Hyaluronidase I. Yu. Ponedel’kina, V. N. Odinokov, E. A. Saitgalina, and Corresponding Member of the RAS U. M. Dzhemilev Received July 4, 2007
DOI: 10.1134/S1607672907060142
Hyaluronic acid oxidized at hydroxymethyl group displays an increased in vitro enzymatic resistance to testicular hyaluronidase. Hyaluronic acid belongs to the class of acid glycosaminoglycans, heteropolysaccharides with a linear structure. This is the key compound of the extracellular matrix that performs important functions in the organism. It is composed of repeated units represented by glucuronic acid and N-acetyl-D-glucosamine. The interest to the derivatives of hyaluronic acid displaying a decreased enzymatic biodegradability has increased recently due to their potential as controlled
release drugs [1–3]. We have found that the oxidation of hydroxymethyl groups into carboxyl groups renders hyaluronic acid resistant to testicular hyaluronidase in vitro. Hyaluronic acid (I) was obtained by alkaline extraction from the umbilical cord of newborns and purified by anion exchange chromatography on DEAE cellulose as described in [4]. Modified hyaluronic acid II was obtained by treating hyaluronic acid I with sodium hypochlorite in the presence of sodium bromide and a catalytic amount of 2,2,6,6-tetramethylpiperidine-1oxyl (TEMPO) in water [5]:
OH O HO
COOH HO O O OH
O
NaOCl, NaBr, H2O, pH 10.2
O HO
NHAc N
n
COOH HO O O OH
COOH O NHAc . n
O
(I)
(II)
The 13C NMR spectrum of acid II contains three signals from carbonyl groups at δ 177.5, 177.2, and 176.4 ppm, which indicate the formation of a new COOH– group in addition to the present carboxyl and acetamido groups due to oxidation of the hydroxymethyl group in the initial acid I. The absence of the signal at δ 63.1 ppm proves its complete oxidation. Thus, the obtained carboxy hyaluronic acid II is composed of D-glucuronic and N-acetylglucosaminuronic acids. Note that N-acetyl-D-glucosaminuronic acid is a structural element of the extracellular polysaccharide generated by the black yeast-like fungus Rhinocladiella mansonii strain NRRL Y-6272 [6, 7].
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, pr. Oktyabrya 141, Ufa, 450075 Russia
We have found that the contents of D-glucuronic acid units determined by the carbazole assay based on the Dische’s reaction [8] in acids I and II are almost equal and amount to 49.5 and 50.9 mol %, respectively. This demonstrates that the assay used is insensitive to the presence of N-acetylglucosaminuronic acid units. Consequently, we used spectrophotometric assay for determining the degree of enzymatic biodegradation of carboxy hyaluronic acid II relative to the native hyaluronic acid I. The essence of this assay consists in determination of the relative absorption of the chromophores (λ = 530 nm) formed in the Dische’s reaction from D-glucuronic acid contained in the products obtained by enzymatic cleavage of acids I and II. Acid II virtually does not degrade as compared with the intact hyaluronic acid I at an enzyme concentration of 3 arb. unit/ml, which is close to the concentration in human blood serum [9] (figure). The biodegradability of acid II gradually increases with an increase in the
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Relative biodegradability, % 30 25 20 15 10 5 0
3
30
60
120 AU/ml
Dependence of in vitro biodegradability of carboxy-hyaluronic acid II on the concentration of hyaluronidase.
enzyme concentration. However, the relative biodegradability of acid II did not exceed 26% even at the hyaluronidase concentration 120 arb. unit/ml (figure). Thus, it is demonstrated that the oxidation at primary hydroxyl groups leads to an essential increase in the resistance of hyaluronic acid to testicular hyaluronidase in vitro. EXPERIMENTAL 13C NMR spectra of D O solutions were recorded in 2 a Bruker AMX-300 spectrometer using the sodium salt of 3-(trimethyl)-1-propanesulfonic acid as an internal standard; UV spectra were recorded in a Specord M-40 spectrophotometer; and pH of solutions was monitored with a pH-340 pH-meter. Testicular hyaluronidase (EC 3.2.1.35, pharmacopoeial preparation Lidaze) was used without additional purification. Sodium hypochlorite (13% aqueous solution) was purchased from Aldrich.
Oxidation of hyaluronic acid I. Hyaluronic acid I (200 mg, 0.5 mmol), sodium bromide (10.3 mg, 0.1 mmol), and TEMPO (0.84 mg, 0.0054 mmol) were dissolved in 40 ml of water, chilled to 0 ± 2°ë, and supplemented with 0.8 ml of 13% sodium hypochlorite under intensive stirring. Immediately after that, the pH of the reaction mixture was adjusted to 10.2 with 1 N HCl, and this value was maintained with 0.2 N NaOH to the end of the reaction (1 h). Then, 120 ml of methanol was added. The precipitate was separated by centrifugation, washed successively with methanol (40 ml) and diethyl ether (40 ml), and kept for 2 h at a reduced pressure and a temperature not greater than 60°ë. Thus, 190 mg (87%) of acid II were produced. The 13C NMR spectrum was as follows (D2O, δ, ppm): 177.5, 177.2,
176.4, 105.3, 102.8, 83.7, 82.8, 78.7, 78.2, 75.9, 74.7, 69.9, 56.6, and 24.9. Enzymatic cleavage of oxidized hyaluronic acid II. Each of four centrifuge tubes (V = 10 ml) was filled with 4.37 mg (0.01 mmol) of acid II, 0.9 ml of citrate buffer (30 mM citric acid, 150 mM Na2HPO4, and 150 mM NaCl; pH 6.3), and hyaluronidase solution (3, 30, 60, or 120 AU) in 0.1 ml of citrate buffer; the reaction mixture was allowed to incubate at 37°C for 20 h. Then, the reaction mixture in each tube was supplemented with 4 ml of the mixture of MeOH and Et2O (3 : 1) and incubated at 0°C for 1 h; the precipitate was separated by centrifugation. The supernatant was placed into a glass and evaporated; the solid residue was dissolved in a precisely measured volume of water (5 to 10 ml) to obtain a solution of the low-molecularweight products of enzymatic degradation. Then, the content of D-glucuronic acid was determined in each solution using the Dische’s reaction. For this purpose, the tube with the aliquot (0.5 ml) of each solution was supplemented with 3 ml of concentrated ç2SO4, heated for 20 min in a boiling water bath, chilled to the room temperature, and supplemented with 0.1 ml of 0.1% carbazole solution in alcohol. The tube was vigorously shaken, heated for 1–2 min in a boiling water bath, and chilled to measure the absorption (λ = 530 nm) of each solution (colored pink–violet) relative to the reference solution (compare with [10]). The enzymatic treatment of hyaluronic acid I and the Dische’s reaction for degradation products were performed in a similar way. The biodegradability of acid II relative to acid I at each concentration of hyaluronidase was determined from the ratio (%) of the absorptions of solutions prepared using the Dische’s reaction. REFERENCES 1. Vercruysse, K.P., Marecak, D.M., Marecak, J.F., and Prestwich, G.D., Bioconj. Chem., 1997, vol. 8, no. 5, pp. 686–694. 2. Prestwich, G.D., Marecak, D.M., Marecak, J.F., et al., J. Controll. Rel., 1998, vol. 53, p. 99. 3. Hirano, K., Sakai, S., Ishikawa, T., et al., Carbohydr. Res., 2005, vol. 430, no. 14, pp. 2297–2304. 4. Ponedel’kina, I.Yu., Odinokov, V.N., Vakhrusheva, E.S., et al., Bioorg. Khim., 2005, vol. 31, no. 1, pp. 90–95. 5. Jiang, B., Drouet, E., Milas, M., and Rinaudo, M., Carbohydr. Res., 2000, vol. 327, pp. 455–461. 6. Sanford, P.A., Burton, K.A., Watson, P.R., et al., Appl. Microbiol., 1975, vol. 29, no. 26, pp. 769–775. 7. Burton, K.A., Cadmus, M.C., Lagoda, A.A., et al., Biotechnol. Bioeng., 1976, vol. 18, no. 12, pp. 1669–1677. 8. Dische, Z., J. Biol. Chem., 1947, vol. 167, pp. 189–198. 9. Delpech, B., Bertrand, P., and Chauzy, C., J. Immunol. Methods, 1987, vol. 104, pp. 223–229. 10. Methods in Carbohydrate Chemistry, Whistler, R.L. and Wolfrom, M.L., Eds., New York: Academic, 1962–1965.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 417
2007
