364s
Biochemical SocietyTransactions ( 1 992) 20
Heparin inhibits production of thiobarbituric acidreactive substancesin the the presence of linolenic acid and Fe2+ ions. MARION A. ROSS, WILLIAM F. LONG and FRANK B. WILLIAMSON Department of Molecular and Cell Biology, University of Aberdeen. Marischal College. ABERDEEN AB9 1AS. Scotland. Heparins and heparan sulphates possess a variety of anionic groups that allow these molecules to interact with a range of biologically relevant micro- and macrocations. Such interactions are complex and not ex licable in terms of simple electrostatic theory. It is probaile that they lead to changes in the biological potentials of both partners i n the interaction. Copper [1,2] and iron [3-51 ions are among those cations that bind strongly to heparins and similar glycosaminoglycans. Such ions have a putative role in hydroxyl radical generation through Fenton reactions, and therefore perhaps in firstchain initiation of lipid peroxidation. Transition metal cation complexes also accelerate decomposition of lipid peroxides to alkoxyl and peroxyl radicals; both of these are capable of abstracting methylene roup hydrogen atoms and stimulating further l i p i f peroxidation. Binding of transition metal ions by particular proteins limits their ability to participate in such reactions. Because heparins and heparan sulphates also bind such ions, these polyanions might therefore also modulate their availability or reactivity. In accordance with this possibility, i t has been shown t h a t heparin, in a concentration-dependent manner, inhibits an early stage in Fe2+-catalyzed linolenic acid peroxidation, the production of u.v.-absorbing conjugated dienes [6,7]. In this transaction, we demonstrate the effect of heparin on a later stage of lipid peroxidation, the production of thiobarbituric acid-reactive substances.
12r
Sources of the heparin and of most of the other materials used have been described before [6,71. Additional reagents were of Analar grade and obtained from B.D.H., Poole, U.K. Assay of thiobarbituric acidreactive substances (nominally malondialdehyde present i n the sample assayed, together with t h a t formed by peroxide degradation during the acid heating stage of the assay process) was essentially by the method of Gutteridge [81. Glycosaminoglycans were incubated at 37oC for 10 min before addition of y-linolenic acid (0.2 mmol.dm-3 in 0.15 mmol.dm-3 NaCl) in polyoxyethylene ether W-1(1% w/v). After a further 5 min incubation, fresh ferrous ammonium sulphate (0.2 mmol.dm-3; deoxygenated) was added to give a final reaction volume of 0.5 cm3 containing components at t h e noted concentrations. After incubation for a further 10 min, thiobarbituric acid (0.5 cm3 of a 1% w/v solution in 0.5 mol.dm-3 NaOH) was then added followed by 0.1 cm3 of 10 mol.dm-3 hydrochloric acid. All reagents were dissolved in de-ionized distilled water. Final mixtures were heated at lOOoC for 10 min and then cooled. The absorbance of the coloured complex was measured at 532 nm. The molar absorption coefficient used to calculate the amounts of thiobarbituric acid-reactive substances produced i n the assay was 1.56 x 105 mol-~.dm3.cm-~ [9]. The figure shows that heparin, in a concentrationdependent manner, inhibited t h e generation of thiobarbituric acid-reactive substances in a reaction mix containing y-linolenic acid and Fez+. These results accord with the notion t h a t heparin and related glycosaminoglycans may contribute to antioxidant mechanisms In U L U O , perhaps by binding and sequestering ions such as Fe2+. 1. Rej, R.N., Holme, K.R. & Perlin, A.S. (1990) Carbohydrate Res. 207,143-152 2. Grant, D., Long, W.F., Moffat, C.F. & Williamson, F.B. (1992) Biochem. J. 283,243-246 3. Garcia-Segura, L.M. (1977) Acta Histochem. 59, 79-84 4. Kojima, S., Hama, Y., Sasaki, T. & Kubodera, A. (1983) J. Nucl. Med. 8,52-59 5. Grant, D., Long, W.F., Moffat, C.F. & Williamson, F.B. (1992) Biochem. J. 282,601-604 6. Ross, M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20,6S 7. Ross, M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20,216s 8. Gutteridge, J.M.C. (1985) in Handbook of Methods for
Oxygen Radical Research (Greenwald, R. A., ed.), pp. 303-307, CRC Press, Boca Raton 9. Buege, J.A. & Aust, S.D. (1978) i n Methods in Enzymology, volume 52 (Fleischer, S. & Packer, L., eds.), pp. 302-310, Academic Press, New York, San Francisco and London
10
"
0
0.01
0.05
0.1
0.25
0.5
1
[Heparin] (mmol.dm-3)
Fig.Effect of heparin on generationof thiobarbituricacidreactivesubstances
Numbers on the histogram refer t o final concentrations (in terms of hydrated disaccharide repeat unit) of heparin present in the reaction mix.
Biochemical SocietyTransactions ( 1 992) 20
Heparin inhibits production of thiobarbituric acidreactive substancesin the the presence of linolenic acid and Fe2+ ions. MARION A. ROSS, WILLIAM F. LONG and FRANK B. WILLIAMSON Department of Molecular and Cell Biology, University of Aberdeen. Marischal College. ABERDEEN AB9 1AS. Scotland. Heparins and heparan sulphates possess a variety of anionic groups that allow these molecules to interact with a range of biologically relevant micro- and macrocations. Such interactions are complex and not ex licable in terms of simple electrostatic theory. It is probaile that they lead to changes in the biological potentials of both partners i n the interaction. Copper [1,2] and iron [3-51 ions are among those cations that bind strongly to heparins and similar glycosaminoglycans. Such ions have a putative role in hydroxyl radical generation through Fenton reactions, and therefore perhaps in firstchain initiation of lipid peroxidation. Transition metal cation complexes also accelerate decomposition of lipid peroxides to alkoxyl and peroxyl radicals; both of these are capable of abstracting methylene roup hydrogen atoms and stimulating further l i p i f peroxidation. Binding of transition metal ions by particular proteins limits their ability to participate in such reactions. Because heparins and heparan sulphates also bind such ions, these polyanions might therefore also modulate their availability or reactivity. In accordance with this possibility, i t has been shown t h a t heparin, in a concentration-dependent manner, inhibits an early stage in Fe2+-catalyzed linolenic acid peroxidation, the production of u.v.-absorbing conjugated dienes [6,7]. In this transaction, we demonstrate the effect of heparin on a later stage of lipid peroxidation, the production of thiobarbituric acid-reactive substances.
12r
Sources of the heparin and of most of the other materials used have been described before [6,71. Additional reagents were of Analar grade and obtained from B.D.H., Poole, U.K. Assay of thiobarbituric acidreactive substances (nominally malondialdehyde present i n the sample assayed, together with t h a t formed by peroxide degradation during the acid heating stage of the assay process) was essentially by the method of Gutteridge [81. Glycosaminoglycans were incubated at 37oC for 10 min before addition of y-linolenic acid (0.2 mmol.dm-3 in 0.15 mmol.dm-3 NaCl) in polyoxyethylene ether W-1(1% w/v). After a further 5 min incubation, fresh ferrous ammonium sulphate (0.2 mmol.dm-3; deoxygenated) was added to give a final reaction volume of 0.5 cm3 containing components at t h e noted concentrations. After incubation for a further 10 min, thiobarbituric acid (0.5 cm3 of a 1% w/v solution in 0.5 mol.dm-3 NaOH) was then added followed by 0.1 cm3 of 10 mol.dm-3 hydrochloric acid. All reagents were dissolved in de-ionized distilled water. Final mixtures were heated at lOOoC for 10 min and then cooled. The absorbance of the coloured complex was measured at 532 nm. The molar absorption coefficient used to calculate the amounts of thiobarbituric acid-reactive substances produced i n the assay was 1.56 x 105 mol-~.dm3.cm-~ [9]. The figure shows that heparin, in a concentrationdependent manner, inhibited t h e generation of thiobarbituric acid-reactive substances in a reaction mix containing y-linolenic acid and Fez+. These results accord with the notion t h a t heparin and related glycosaminoglycans may contribute to antioxidant mechanisms In U L U O , perhaps by binding and sequestering ions such as Fe2+. 1. Rej, R.N., Holme, K.R. & Perlin, A.S. (1990) Carbohydrate Res. 207,143-152 2. Grant, D., Long, W.F., Moffat, C.F. & Williamson, F.B. (1992) Biochem. J. 283,243-246 3. Garcia-Segura, L.M. (1977) Acta Histochem. 59, 79-84 4. Kojima, S., Hama, Y., Sasaki, T. & Kubodera, A. (1983) J. Nucl. Med. 8,52-59 5. Grant, D., Long, W.F., Moffat, C.F. & Williamson, F.B. (1992) Biochem. J. 282,601-604 6. Ross, M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20,6S 7. Ross, M.A., Long, W.F. & Williamson, F.B. (1992) Biochem. SOC. Trans. 20,216s 8. Gutteridge, J.M.C. (1985) in Handbook of Methods for
Oxygen Radical Research (Greenwald, R. A., ed.), pp. 303-307, CRC Press, Boca Raton 9. Buege, J.A. & Aust, S.D. (1978) i n Methods in Enzymology, volume 52 (Fleischer, S. & Packer, L., eds.), pp. 302-310, Academic Press, New York, San Francisco and London
10
"
0
0.01
0.05
0.1
0.25
0.5
1
[Heparin] (mmol.dm-3)
Fig.Effect of heparin on generationof thiobarbituricacidreactivesubstances
Numbers on the histogram refer t o final concentrations (in terms of hydrated disaccharide repeat unit) of heparin present in the reaction mix.
