Reactive Carbonyls and Polyunsaturated Fatty Acids Produce a Hydroxyl Radical-like Species

The pattern of oxidized amino acids in aortic proteins of nonhuman primates suggests that a species resembling hydroxyl radical damages proteins when blood glucose levels are high. However, recent studies argue strongly against a generalized increase in diabetic oxidative stress, which might instead be confined to the vascular wall. Here, we describe a pathway for glucose-stimulated protein oxidation and provide evidence of its complicity in diabetic microvascular disease. Low density lipoprotein incubated with pathophysiological concentrations of glucose became selectively enriched in ortho-tyrosine and meta-tyrosine, implicating a hydroxyl radical-like species in protein damage. Model system studies demonstrated that the reaction pathway requires both a reactive carbonyl group and a polyunsaturated fatty acid, involves lipid peroxidation, and is blocked by the carbonyl scavenger aminoguanidine. To explore the physiological relevance of the pathway, we used mass spectrometry and high pressure liquid chromatography to quantify oxidation products in control and hyperglycemic rats. Hyperglycemia raised levels of ortho-tyrosine, meta-tyrosine, and oxygenated lipids in the retina, a tissue rich in polyunsaturated fatty acids. Rats that received aminoguanidine did not show this increase in protein and lipid oxidation. In contrast, rats with diet-induced hyperlipidemia in the absence of hyperglycemia failed to exhibit increased protein and lipid oxidation products in the retina. Our observations suggest that generation of a hydroxyl radical-like species by a carbonyl/polyunsaturated fatty acid pathway might promote localized oxidative stress in tissues vulnerable to diabetic damage. This raises the possibility that antioxidant therapies that specifically inhibit the pathway might delay the vascular complications of diabetes. The pattern of oxidized amino acids in aortic proteins of nonhuman primates suggests that a species resembling hydroxyl radical damages proteins when blood glucose levels are high. However, recent studies argue strongly against a generalized increase in diabetic oxidative stress, which might instead be confined to the vascular wall. Here, we describe a pathway for glucose-stimulated protein oxidation and provide evidence of its complicity in diabetic microvascular disease. Low density lipoprotein incubated with pathophysiological concentrations of glucose became selectively enriched in ortho-tyrosine and meta-tyrosine, implicating a hydroxyl radical-like species in protein damage. Model system studies demonstrated that the reaction pathway requires both a reactive carbonyl group and a polyunsaturated fatty acid, involves lipid peroxidation, and is blocked by the carbonyl scavenger aminoguanidine. To explore the physiological relevance of the pathway, we used mass spectrometry and high pressure liquid chromatography to quantify oxidation products in control and hyperglycemic rats. Hyperglycemia raised levels of ortho-tyrosine, meta-tyrosine, and oxygenated lipids in the retina, a tissue rich in polyunsaturated fatty acids. Rats that received aminoguanidine did not show this increase in protein and lipid oxidation. In contrast, rats with diet-induced hyperlipidemia in the absence of hyperglycemia failed to exhibit increased protein and lipid oxidation products in the retina. Our observations suggest that generation of a hydroxyl radical-like species by a carbonyl/polyunsaturated fatty acid pathway might promote localized oxidative stress in tissues vulnerable to diabetic damage. This raises the possibility that antioxidant therapies that specifically inhibit the pathway might delay the vascular complications of diabetes. Diabetes mellitus markedly increases the risk of microvascular and macrovascular disease (1Skyler J. Endocrinol. Metab. Clin. North. Am. 2001; 30: 833-856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). An elevated level of glucose is the metabolic hallmark of diabetes, and the degree of glycemic control has long been known to be a major risk factor for diabetic complications (1Skyler J. Endocrinol. Metab. Clin. North. Am. 2001; 30: 833-856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 2Nathan D.M. Lancet. 1998; 352: 832-833Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). These observations have given rise to the glucose hypothesis, which proposes that toxic effects of glucose mediate many of the deleterious effects of this disorder (1Skyler J. Endocrinol. Metab. Clin. North. Am. 2001; 30: 833-856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 2Nathan D.M. Lancet. 1998; 352: 832-833Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). This hypothesis was strongly supported by the Diabetes Control and Complications Trial and the United Kingdom Prospective Diabetes Study, which demonstrated that intensive glucose-lowering therapy dramatically lowered the incidence of retinopathy and nephropathy (3Control Diabetes Group Complications Trial Research N. Engl. J. Med. 1993; 329: 977-986Crossref PubMed Scopus (22705) Google Scholar, 4UK Prospective Diabetes Study (UKPDS) Group Lancet. 1998; 352: 837-853Abstract Full Text Full Text PDF PubMed Scopus (18951) Google Scholar). Several mechanisms might explain the link between glucose and vascular disease, including mitochondrial dysfunction (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar), formation of advanced glycation end products (AGEs) 1The abbreviations used are: AGE, advanced glycation end-product; BHT, butylated hydroxytoluene; DTPA, diethylenetriaminepentaacetic acid; GC/MS, gas chromatography-mass spectrometry; HODE, hydroxyoctadecadienoic acid; HPLC, high pressure liquid chromatography; LDL, low density lipoprotein; PUFA, polyunsaturated fatty acid; RNase A, ribonuclease A; STZ, streptozotocin. 1The abbreviations used are: AGE, advanced glycation end-product; BHT, butylated hydroxytoluene; DTPA, diethylenetriaminepentaacetic acid; GC/MS, gas chromatography-mass spectrometry; HODE, hydroxyoctadecadienoic acid; HPLC, high pressure liquid chromatography; LDL, low density lipoprotein; PUFA, polyunsaturated fatty acid; RNase A, ribonuclease A; STZ, streptozotocin. (6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, 7Bucala R. Cerami A. Adv. 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Feener E.P. King G.L. Science. 1996; 272: 728-731Crossref PubMed Scopus (1081) Google Scholar). Another important factor might be oxidative stress, because in vitro oxidation of glucose produces reactive intermediates, carbonyl compounds, and AGEs, all of which have been linked to artery wall damage (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar, 6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, 7Bucala R. Cerami A. Adv. Pharmacol. 1992; 23: 1-34Crossref PubMed Scopus (478) Google Scholar, 8Brownlee M. Annu. Rev. Med. 1995; 46: 223-234Crossref PubMed Scopus (1146) Google Scholar, 15Hunt J.V. Dean R.T. Wolff S.P. Biochem. J. 1988; 256: 205-212Crossref PubMed Scopus (743) Google Scholar, 16Monnier V.M. Arch Biochem. Biophys. 2003; 419: 1-15Crossref PubMed Scopus (177) Google Scholar, 17Wells-Knecht K.J. Zyzak D.V. Litchfield J.E. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 3702-3709Crossref PubMed Scopus (558) Google Scholar, 18Wolff S.P. Dean R.T. Biochem. J. 1987; 245: 243-250Crossref PubMed Scopus (1146) Google Scholar). For example, collagen exposed to glycoxidation reactions in vitro (6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google the oxidized amino acids ortho-tyrosine and AGEs, and of and with the of diabetic complications A. P. J. V.M. Diabetes. 1992; PubMed Google Scholar). levels of and increased levels of of lipid oxidation products with (6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, 2003; 23: PubMed Scopus Google Scholar), that oxidative stress might a in diabetic vascular disease. observations the hypothesis that hyperglycemia increases oxidative stress, Thorpe S.R. Baynes J.W. J. Clin. 1997; PubMed Scopus Google used and to that levels of ortho-tyrosine and are in collagen of diabetic and studies have failed to levels of glycoxidation products in and blood of in (6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, Thorpe S.R. Baynes J.W. J. Clin. 1993; PubMed Scopus Google Scholar). observations argue strongly against increase in generalized oxidative stress in diabetes, in the of studies the possibility of a pathway that increases oxidative stress in tissues the and artery wall that are to diabetic damage. pathway involves reactive species by (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar). in (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google and the of aortic J. P. K. Am. J. PubMed Scopus Google Scholar), and of mitochondrial this In its products and promote formation (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar). evidence of reactive in the and of diabetic rats Med. 1998; PubMed Scopus Google Scholar, Diabetes Clin. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Another important might the of glucose to protein kinase C (14Ishii H. Jirousek M.R. Koya D. Takagi C. Xia P. Clermont A. Bursell S.E. Kern T.S. Ballas L.M. Heath W.F. Stramm L.E. Feener E.P. King G.L. Science. 1996; 272: 728-731Crossref PubMed Scopus (1081) Google Scholar, G.L. H. Koya D. 1997; Google Scholar), which to by a of mechanisms (13Semenkovich C.F. Heinecke J.W. Diabetes. 1997; 46: 327-334Crossref PubMed Scopus (124) Google Scholar). of reactive might to diabetic retinopathy and elevated levels of ortho-tyrosine and in aortic proteins to hyperglycemia C. Heinecke J.W. J. Clin. 2001; PubMed Scopus Google Scholar). of amino acids with of in that glucose its protein oxidation. In vitro the end products of oxidation that hydroxyl radical both ortho-tyrosine and when These observations the possibility that the of the diabetic artery wall localized oxidized stress, which be important in the of diabetic macrovascular disease. In the we the mechanisms glucose and of and that in (6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, 15Hunt J.V. Dean R.T. Wolff S.P. Biochem. J. 1988; 256: 205-212Crossref PubMed Scopus (743) Google Scholar, 16Monnier V.M. Arch Biochem. Biophys. 2003; 419: 1-15Crossref PubMed Scopus (177) Google Scholar, 17Wells-Knecht K.J. Zyzak D.V. Litchfield J.E. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 3702-3709Crossref PubMed Scopus (558) Google Scholar, V.M. J. Clin. 2001; PubMed Scopus Google hydroxyl radical a species in Our observations suggest a pathway for protein damage. The pathway requires both polyunsaturated fatty acids and a reactive carbonyl and involves a species resembling hydroxyl of reactive by this pathway might to retinopathy and of microvascular and macrovascular disease in diabetes. amino acids. oxidized amino acids Heinecke J.W. J. 1996; Full Text PDF PubMed Scopus Google Scholar). all a to J.W. H. A. J. Clin. PubMed Scopus Google Scholar), and Low density lipoprotein was a density C. J.E. D.M. Heinecke J.W. J. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). in diethylenetriaminepentaacetic acid with RNase For of was to the reaction to by butylated a antioxidant J.W. H. A. J. Clin. PubMed Scopus Google a in and by acid C. Heinecke J.W. J. Clin. 2001; PubMed Scopus Google Scholar). For reactions fatty the fatty acid was in and in a and the was with the reaction was and the was and incubated with Model exposed to hydroxyl and C. Heinecke J.W. J. 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PubMed Scopus Google Scholar). the end of the proteins and amino acids the to to of ortho-tyrosine, meta-tyrosine, and to for ortho-tyrosine and incubated in However, the in levels of failed to promote oxidation of RNase in vitro studies have demonstrated that this pattern of protein oxidation products is by hydroxyl radical not by radical reactive species C. Heinecke J.W. J. Clin. 2001; PubMed Scopus Google Scholar, Baynes J.W. Thorpe S.R. J. 1993; Full Text PDF PubMed Google Scholar). The of ortho-tyrosine and not in increased with of to and oxidation increased a of concentrations studies have that glucose to oxidation by a pathway Med. PubMed Scopus Google Scholar). was the reaction oxidation of protein increased and RNase oxidation failed to that the oxidation The increase in protein increase in ortho-tyrosine in to protein glycoxidation Thorpe S.R. Baynes J.W. J. 1987; Full Text PDF PubMed Google Scholar). 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Med. 1995; 46: 223-234Crossref PubMed Scopus (1146) Google Scholar), and levels of a of glucose are elevated in diabetic of glucose that in and might protein oxidation. and RNase incubated with for proteins and by reactive increases in levels of ortho-tyrosine and not in a carbonyl group and group is to did not ortho-tyrosine of the oxidized RNase not observations strongly a carbonyl group in oxidation of by and by and of to not RNase that lipid might be incubated for in the absence of the and for by increased the of to protein This increase was blocked by the antioxidant the antioxidant the aminoguanidine In contrast, the the scavenger and the scavenger failed to inhibit did a of and These that of lipid in Low that with in the lipid and the reaction pathway, that are not In a of we levels of ortho-tyrosine in incubated for in the absence of and the The of ortho-tyrosine increased in exposed to with lipid peroxidation, this was blocked by BHT, aminoguanidine not by not of the a the of lipid protein oxidation when was incubated in the absence of These observations suggest that oxidation of the protein of by a pathway lipid and reactive RNase in that lipids a when we incubated RNase with polyunsaturated fatty acids and for the fatty acid was levels of ortho-tyrosine in RNase and fatty acids in the absence of was a increase in ortho-tyrosine increase in the absence of when was the fatty These that polyunsaturated not fatty acids promote protein oxidation and that the carbonyl group of the The known of polyunsaturated fatty acids to the hypothesis that a pathway protein oxidation. Hyperglycemia in the of explore the physiological relevance of the pathway, we used to quantify levels of oxidized amino acids in tissue rats hyperglycemic for by the because high levels of polyunsaturated fatty acids and is in PubMed Scopus Google Scholar, Biochem. Biophys. 48: PubMed Scopus Google Scholar). in vitro that aminoguanidine we the of aminoguanidine the oxidation of rats control diabetic control and diabetic rats. levels the levels in the control and diabetic rats and Hyperglycemia in the for the of the To proteins rats oxidized amino we the of control the tissue was and with acid, amino acids and with and of the by in the that major and to of ortho-tyrosine, meta-tyrosine, and demonstrated that the the amino acids with not These observations that acid of proteins of rats levels of oxidized amino acids. To hyperglycemia protein oxidation in diabetic in we used to quantify levels of ortho-tyrosine, meta-tyrosine, and in acid of tissue the control and hyperglycemic rats and and The the hyperglycemic ortho-tyrosine the of the levels of in the diabetic tissue In contrast, the levels of and in the control and diabetic These that hyperglycemia promote protein oxidation in and selectively proteins with ortho-tyrosine and of and in proteins control and diabetic rats by amino in a of in in lipid might protein oxidation in we used to quantify levels in lipid the of control and diabetic tissue a level of tissue control These observations that increases both protein and lipid oxidation in the retina. and in the of aminoguanidine protein oxidation in we levels of oxidized amino acids and in the tissues of control and diabetic rats with the tissue the diabetic rats failed to ortho-tyrosine, meta-tyrosine, In contrast, aminoguanidine did not the levels of ortho-tyrosine, meta-tyrosine, in the tissue of control rats. aminoguanidine failed to the levels of in the of diabetic rats in vitro and in Tilton R.G. Chang K. Hasan K.S. Ido Y. J.R. Williamson J.R. Diabetes. 1992; PubMed Google Scholar). Hyperglycemia and in the of hyperglycemia promote protein oxidation and lipid in tissues that are not known to be by the diabetic we the levels of oxidized amino acids and in the of diabetic the retina, this of the high levels of The levels of ortho-tyrosine, meta-tyrosine, and and in control and hyperglycemic rats These that hyperglycemia not promote protein lipid in the of of oxidized amino acids and in of control and diabetic amino in a of and in in Control and the between lipid and protein oxidation in we the between levels and oxidized amino acid levels in the tissue of control and rats demonstrated a between levels of ortho-tyrosine and of with a between the levels glucose and of ortho-tyrosine and did not In contrast, was between the levels of and and These observations suggest that protein oxidation and lipid oxidation increase in in the of diabetic rats and that hyperglycemia provide the of with with and in Control and is a between the levels of the oxidized amino we tissue levels of ortho-tyrosine with meta-tyrosine, and was a between levels of ortho-tyrosine and In contrast, was between the levels of ortho-tyrosine and These suggest that oxidative are to ortho-tyrosine and that mechanisms might to the formation of and to and in the of hyperlipidemia hyperglycemia increase protein oxidation and lipid in the retina, we used and to quantify the levels of oxidized amino acids and in tissue control rats and rats with diet-induced studies have that glucose levels are not in this of hyperlipidemia R. S.R. 42: Full Text PDF PubMed Scopus Google Scholar). rats control rats a low and rats a high of its the high with elevated the both the and levels increased in the the high with the low low high low high In contrast, rats the levels of high levels in both of and of the rats acids of proteins of the rats the low high and by of ortho-tyrosine and and in control and rats of lipid of control and rats in the levels of between the of in the absence of hyperglycemia not promote protein lipid of tissue in this of diet-induced hyperglycemia oxidative reactions in vitro (5Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7030) Google Scholar, 6Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-9Crossref PubMed Scopus (2140) Google Scholar, 15Hunt J.V. Dean R.T. Wolff S.P. Biochem. J. 1988; 256: 205-212Crossref PubMed Scopus (743) Google Scholar, V.M. J. Clin. 2001; PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, M. Heinecke J.W. A. J. 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Clin. 2001; PubMed Scopus Google Scholar), and we have that is not by the oxidation of and of ortho-tyrosine and was blocked by BHT, implicating reactive lipid in the reaction by was to a RNase A, a of glucose and a elevated ortho-tyrosine and of lipid this lipid peroxidation, protein and of RNase with was blocked by a carbonyl These in vitro observations that glucose and reactive promote protein oxidation by a pathway that requires and involves lipid To explore the pathophysiological relevance of this pathway, we levels of protein and lipid oxidation products in the of hyperglycemic a of diabetic increased vascular in the tissue in this Y. A. Chang K. Stramm R. Heath W.F. Williamson J.R. Science. 1997; PubMed Scopus Google Scholar, M. C. Diabetologia. 2001; PubMed Scopus Google Scholar, J. C. J. Y. Diabetologia. 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