Pyridoxamine Traps Intermediates in Lipid Peroxidation Reactions in Vivo
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m304292200
ISSN1083-351X
AutoresThomas Metz, Nathan L. Alderson, Mark E. Chachich, Suzanne R. Thorpe, John Baynes,
Tópico(s)Alcohol Consumption and Health Effects
ResumoMaillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5–10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of α-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases. Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5–10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of α-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases. Non-enzymatic chemical modification of protein by reducing sugars, known as the Maillard reaction, is implicated in the development of pathology during aging and in chronic diseases such as diabetes, atherosclerosis, and Alzheimers disease (1Thorpe S.R. Baynes J.W. Drugs Aging. 1996; 9: 69-77Crossref PubMed Scopus (269) Google Scholar, 2Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-8Crossref PubMed Scopus (2143) Google Scholar, 3Ulrich P. Cerami A. Recent Prog. Horm. Res. 2001; 56: 1-21Crossref PubMed Scopus (685) Google Scholar). The Maillard reaction between sugar and protein proceeds through a labile Schiff base, which isomerizes to a ketoamine adduct, the Amadori compound. Oxidative decomposition and further reaction of the Amadori compound produce advanced glycation end products (AGEs), 1The abbreviations used are: AGE, advanced glycation end product; AA, arachidonic acid; AG, aminoguanidine; ALE, advanced lipoxidation end product; ESI+-LC/MS/MS, positive ion electrospray ionization liquid chromatography/mass spectrometry/mass spectrometry; GO, glyoxal; LA, linoleic acid; MGO, methylglyoxal; MRM-LC/MS/MS, multiple reaction monitoring-LC/MS/MS; PM, pyridoxamine; PUFA, polyunsaturated fatty acid; RP-HPLC, reversed phase high performance liquid chromatography; STZ, streptozocin; Db, diabetic; HAPM, N-hexanoyl-PM; NDAPM, N-nonanedioyl-PM; PDAPM, N-pentanedioyl-PM; FAPM, N-formyl-PM; PyPM, N-pyrrolo-PM; FPyPM, N-(2-formyl)-pyrrolo-PM; GOPM, glyoxal-PM; ZDF/Gmi-fa, male Zucker diabetic fatty rat; +/fa, lean Zucker diabetic fatty rat; fa/fa, Zucker non-diabetic fatty rat. such as pentosidine and vesperlysines (2Baynes J.W. Thorpe S.R. Diabetes. 1999; 48: 1-8Crossref PubMed Scopus (2143) Google Scholar). Similarly, advanced lipoxidation end products (ALEs), such as the malondialdehyde and 4-hydroxy-2-nonenal adducts to lysine, are formed on protein during lipid peroxidation reactions (4Baynes J.W. Thorpe S.R. Free Radic. Biol. Med. 2000; 28: 1708-1716Crossref PubMed Scopus (473) Google Scholar, 5Miyata T. Kurokawa K. Van Ypersele De Strihou C. J. Am. Soc. Nephrol. 2000; 11: 1744-1752PubMed Google Scholar). N ϵ-(Carboxymethyl)lysine and N ϵ-(carboxyethyl)lysine, which are major products of both glycoxidation and lipoxidation reactions (4Baynes J.W. Thorpe S.R. Free Radic. Biol. Med. 2000; 28: 1708-1716Crossref PubMed Scopus (473) Google Scholar, 5Miyata T. Kurokawa K. Van Ypersele De Strihou C. J. Am. Soc. Nephrol. 2000; 11: 1744-1752PubMed Google Scholar, 6Fu M. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. J. Biol. Chem. 1996; 271: 9982-9986Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar), are termed AGE/ALEs. Through effects on protein structure, function, and turnover, the accumulation of AGEs and ALEs in tissue proteins is thought to contribute to the development of diabetic complications. AGE/ALE inhibitors are designed to limit the accumulation of AGE/ALEs in protein and thereby protect against the development of diabetic complications. Hudson and colleagues (7Khalifah R.G. Baynes J.W. Hudson B.G. Biochem. Biophys. Res. Commun. 1999; 257: 251-258Crossref PubMed Scopus (252) Google Scholar, 8Khalifah R.G. Todd P. Booth A.A. Yang S.X. Mott J.D. Hudson B.G. Biochemistry. 1996; 35: 4645-4654Crossref PubMed Scopus (75) Google Scholar, 9Booth A.A. Khalifah R.G. Todd P. Hudson B.G. J. Biol. Chem. 1997; 272: 5430-5437Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) reported that PM was a potent inhibitor of the formation of AGEs from Amadori adducts in vitro. Onorato et al. (10Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. J. Biol. Chem. 2000; 275: 21177-21184Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) later reported that PM traps intermediates in lipid peroxidation and protects proteins from chemical modification during lipid peroxidation reactions (lipoxidation) in vitro. Degenhardt et al. (11Degenhardt T.P. Alderson N.L. Arrington D.D. Beattie R.J. Basgen J.M. Steffes M.W. Thorpe S.R. Baynes J.W. Kidney Int. 2002; 61: 939-950Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and Alderson et al. (12Alderson N.L. Chachich M.E. Youssef N.N. Beattie R.J. Nachtigal M. Thorpe S.R. Baynes J.W. Kidney Int. 2003; 63: 2123-2133Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) demonstrated that PM also inhibited the formation of AGE/ALEs in vivo and retarded the development of nephropathy in both streptozocin (STZ)-induced diabetic and Zucker (obese, hyperlipidemic) rats. The presence of severe hyperlipidemia in the STZ diabetic rats and the reno-protective effects of PM in both the diabetic and the Zucker rat suggested that lipids, rather than carbohydrates, might be the primary source of chemical modification of proteins in diabetes. In the present study, we have extended earlier work on the reaction of PM with the polyunsaturated fatty acids (PUFAs), linoleate and arachidonate, to identify other intermediates trapped by PM during lipid peroxidation reactions and to determine whether PM functions as an inhibitor of advanced lipoxidation reactions in vivo. We show that several lipid-derived PM adducts formed in in vitro reactions are also detected in the urine of PM-treated control, diabetic, and hyperlipidemic rats and that these adducts are present at substantially higher concentrations in the urine of diabetic and hyperlipidemic animals, compared with control rats. Our results support a role for lipoxidation reactions in the chemical modification of proteins and development of complications in diabetes and prediabetic hyperlipidemic states and demonstrate that the protective effects of PM are consistent with its role in reducing plasma lipids and trapping intermediates in lipoxidation reactions in vivo. Materials—Except where indicated, all chemicals were purchased from Sigma-Aldrich or Fisher Scientific. Reaction of PM with Fatty Acids—PM (1 mm) and either linoleic acid (LA) (5 mm) or arachidonic acid (AA) (5 mm) were incubated in 200 mm phosphate buffer, pH 7.4, in a shaking water bath at 37 °C, and phosphate buffers were sterilized by ultrafiltration through 0.2-μm syringe filters (Costar Corp., Acton, MA). Aliquots were taken at 0, 1, 3, and 6 days, quenched with a final concentration of 1 mm diethylenetriamine-pentaacetic acid, and frozen at –70 °C until analyzed. Samples were diluted 1:10 in 0.1% heptafluorobutyric acid (HFBA) (Acros Organics, Morris Plains, NJ) before analysis by reversed phase high performance liquid chromatography (RP-HPLC) and/or positive ion electrospray ionization liquid chromatography/mass spectrometry/mass spectrometry (ESI+-LC/MS/MS). Control incubations consisted of either PM or PUFA alone. Synthesis of PM Adducts—All synthesized PM adduct standards were purified by RP-HPLC and characterized by ESI+-LC/MS/MS and/or by 1H and 13C NMR, as described below. The structures of synthesized PM adducts that were also detected in urine are shown in Fig. 1. N-Hexanoyl-PM (HAPM) and N-nonanedioyl-PM (NDAPM) were synthesized from PM and hexanoyl chloride or nonanedioic acid monomethyl ester, respectively, as described (10Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. J. Biol. Chem. 2000; 275: 21177-21184Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). For ESI+-MS of HAPM, m/z = 267 [M+H]+, and for NDAPM, m/z = 339 [M+H]+. Heavy labeled N-hexanoyl-PM (d 11-HAPM) was synthesized from PM·(HCl)2 and d 11-hexanoic acid according to the method of Kato et al. (13Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. J. Biol. Chem. 1999; 274: 20406-20414Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Equimolar amounts (0.6 mmol) of d 11-hexanoic acid (Cambridge Isotope Laboratories, Inc., Andover, MA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysulfosuccinimide (Pierce) were dissolved in 15 ml of dimethylformamide and incubated for 24 h at room temperature. PM·(HCl)2 (0.6 mmol) in 1 ml of 200 mm borate buffer, pH 9, was added, and the reaction mixture was incubated for an additional 4 h. The reaction mixture was diluted to 30 ml with water, and the pH was adjusted to 8.1 with 1 m NaOH. The mixture was extracted three times with an equal volume of diethyl ether, and the pooled extracts were dried under nitrogen. The resulting white powder was reconstituted in 1% HFBA, and d 11-HAPM was purified in 20% yield by semi-preparative RP-HPLC. For ESI+-MS, m/z = 278 [M+H]+. N-Pentanedioyl-PM (PDAPM) was synthesized from PM and glutaric anhydride. PM·(HCl2) (0.24 mg; 1 μmol) was dissolved in 1 ml of 250 mm NaHCO3, pH 10, and placed in a shaking water bath at 37 °C. A total of 3.4 mg (30 μmol) of glutaric anhydride was added in three aliquots at 10-min intervals, and the reaction was continued for an additional 10 min after the last addition. The reaction mixture was then dried overnight in vacuo (Savant Speed Vac, Savant Instruments Inc., Farmingdale, NY). The residue was reconstituted in 1% HFBA, and PDAPM was purified (20% yield) by semi-preparative RP-HPLC. For ESI+-MS, m/z = 283 [M+H]+. N-Formyl-PM (FAPM) was synthesized according to the method of Yamada and Okamoto (14Yamada H. Okamoto T. Yakugaku Zasshi. 1975; 95: 487-492Crossref PubMed Scopus (1) Google Scholar). Briefly, pyridoxine·HCl (1.14 g, 10 mmol) was added to 4.8 ml of formamide (120 mmol) and incubated in a heating block at 100 °C for 24 h. The resulting red oil was diluted 1:100 in 1% HFBA, and a red precipitate formed immediately and was removed by centrifugation. The supernatant was fractionated by RP-HPLC, and a region containing FAPM was collected and dried overnight in vacuo. FAPM was subsequently purified in 50% yield upon re-injection of the collected fraction. For ESI+-MS, m/z = 197 [M+H]+. Structure conformation was provided by 1H NMR on a Varian (Palo Alto, CA) Inova 500 MHz instrument using deuterated dimethyl sulfoxide (d 6-Me2SO) as solvent. The chemical shifts were: δ 2.49 (s, 3H), 4.37 (d, 2H, J = 5.8 Hz), 4.72 (s, 2H), 5.20 (s, 1H), 5.75 (s, 1H), 8.01 (s, 1H), 8.92 (t, 1H, J = 5.8 Hz), and 11.2 (s, 1H). N-Pyrrolo-PM (PyPM) was synthesized by the method of D'Silva and Walker (15D'Silva C. Walker D.A. J. Org. Chem. 1998; 63: 6715-6718Crossref Google Scholar). Briefly, PM·(HCl)2 (0.27 g, 1 mmol) and 2,5-dimethoxytetrahydrofuran (0.16 g, 1 mmol) were dissolved in 4.25 ml of pyridine: acetic acid:water (1.9:1.35:1), and the mixture was reacted in a heating block at 100 °C for 2 h. The resulting brown liquid was diluted 1:100 in 1% HFBA, and PyPM was purified in 95% yield by semi-preparative RP-HPLC. For ESI+-MS, m/z = 219 [M+H]+. Structure conformation was provided by 500 MHz 1H and 13C NMR using d 6-Me2SO as solvent. The chemical shifts were: 1H NMR δ 2.60 (s, 3H), 4.55 (s, 2H), 5.30 (s, 2H), 5.97 (s, 2H), 6.75 (s, 2H), and 8.20 (s, 1H). The chemical shifts were: 13C NMR δ 15.8 [–CH3 (PM)], 42.3 [–CH2– (PM)], 57.5 [–CH2– (PM)], 107.9 [–CH=(pyrrole)], 121.2 [–CH=(pyrrole)], and 130.6–152.2 [5 pyridinyl (PM)]. N-(2-Formyl)-pyrrolo-PM (FPyPM) was synthesized from PM and xylose based on the method of Hayase and Kato (16Hayase F. Kato H. Agric. Biol. Chem. 1985; 49: 467-473Google Scholar). PM·(HCl)2 (2.4 g, 10 mmol) and xylose (1.5 g, 10 mmol) were dissolved in 5 ml of water and heated at 95 °C for 2 h, after adjusting the pH to 4 with 6 n NaOH. The resulting brown liquid was extracted three times with equal volumes of diethyl ether, and the pooled ether extracts were evaporated under nitrogen. The residue was reconstituted in 1% HFBA, and FPyPM was purified in 0.1% yield by semi-preparative RP-HPLC. For ESI+-MS, m/z = 247 [M+H]+. Structure conformation was provided by 500 MHz 1H NMR using d 6-Me2SO as a solvent. The chemical shifts were: δ 2.35 (s, 3H), 4.38 (d, 2H, J = 4.7 Hz), 5.20 (t, 1H, J = 4.7 Hz), 5.60 (s, 2H), 6.15 (dd, 1H, J = 2.5, 3.9 Hz), 6.82 (unresolved dd, 1H), 7.00 (dd, 1H, J = 1.7, 3.9 Hz), 7.99 (s, 1H), and 9.61 (s, 1H). Quantification of PM Adduct Standards—The concentrations of FAPM, HAPM, NDAPM, and PDAPM standards were determined by acid hydrolysis and subsequent quantification of released PM, as described (10Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. J. Biol. Chem. 2000; 275: 21177-21184Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Briefly, aliquots of standard were hydrolyzed in 2 n HCl for 2 h at 95 °C, dried in vacuo, and then reconstituted in 1% HFBA. The concentrations of standards were determined, based on the yield of PM measured by RP-HPLC. Because FPyPM and PyPM were stable to acid hydrolysis and did not exhibit the fluorescence characteristic of compounds containing PM, the concentrations of these standards were determined by RP-HPLC using absorbance detection as described below. FPyPM and PyPM concentrations were estimated using the molar extinction coefficient of PM (92,000 m–1 cm–1). RP-HPLC—Samples were analyzed on a Waters (Milford, MA) 626/600 HPLC system, using an Aquasil (Thermo Hypersil-Keystone, Bellefonte, PA) C-18 column (5 μm; 250 mm × 4.6 mm) at a flow rate of 0.95 ml/min. Solvent A was 0.1% HFBA in water and Solvent B was 90% acetonitrile in water. The gradient was as follows: 0–2 min, 15% B; 2–37 min, linear ramp to 90% B; 37–40 min, hold at 90% B; 40–50 min, return to 15% B; 50–65 min, hold at 15% B. PM and PM adducts were detected by fluorescence (λex = 295 nm, λem = 393 nm) and/or by absorbance at 294 nm. Mass Spectrometry—ESI+-LC/MS/MS was performed on a Micromass (Beverly, MA) Quattro LC mass spectrometer equipped with an Agilent (Palo Alto, CA) 1100 series HPLC system and an Aquasil C-18 column (5 μm, 250 × 2 mm) at a flow rate of 0.2 ml/min. Solvent A was 0.1% HFBA in water and Solvent B was 90% acetonitrile in water; all solvents including water were of HPLC grade. The gradient was as follows: 0–5 min, 15% B; 5–37 min, linear ramp to 90% B; 37–42 min, hold at 90% B; 42–52 min, return to 15% B; 52–60 min, hold at 15% B. The capillary was held at 3.11 kV, and the sampling cone and collision cell were held at 14 and 20 V, respectively. The source block and desolvation temperatures were maintained at 100 and 350 °C, respectively. The identity of all synthesized PM adducts detected in urine was unequivocally established by mixing experiments and by ESI+-LC/MS/MS, including precursor-ion and product-ion spectrum scans. PM and PM adducts generate m/z = 152 as the predominant product-ion, corresponding to the deaminated ion (PM), deamidated ion (FAPM, HAPM, NDAPM, and PDAPM), or loss of the pyrrole functional group (FPyPM and PyPM), when subjected to collision-induced dissociation. In addition to m/z = 152, unidentified intramolecular rearrangement ions of PM (m/z = 140 and 134) were consistently observed in production analyses of all PM adducts, as well as the [M+H]+ ion of PM, m/z = 169. Animal Studies—All studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of South Carolina. Female Sprague-Dawley rats were purchased at 5 weeks of age from Harlan Industries (Indianapolis, IN). Male Zucker diabetic fatty (ZDF/Gmi-fa), lean Zucker diabetic fatty (+/fa), and Zucker non-diabetic fatty (fa/fa) rats were purchased at 5 weeks of age from Genetic Models, Inc. (Charles River Laboratories, Wilmington, MA). Rats were housed in the Animal Resource Facility for 1 week before studies were started; all animals had free access to food and water throughout the study. Plasma glucose and plasma triglycerides were measured with Sigma Kit No. 315 and No. 339, respectively. Sprague-Dawley rats were maintained on Harlan Teklad (Indianapolis, IN) rodent diet (W). Diabetes was induced by a single tail-vein injection of 45 mg/kg streptozocin in 0.1 m sodium citrate buffer, pH 4.5, as described previously (11Degenhardt T.P. Alderson N.L. Arrington D.D. Beattie R.J. Basgen J.M. Steffes M.W. Thorpe S.R. Baynes J.W. Kidney Int. 2002; 61: 939-950Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Non-diabetic animals were sham-injected with buffer only. Animals with plasma glucose above 16 mm were classified as diabetic and assigned to either an untreated diabetic control group (STZ-Db, n = 16) or a diabetic group receiving PM (STZ-Db+PM, n = 16) in drinking water at 1 g/liter. Non-Db (n = 12) animals received PM (non-Db+PM) in drinking water at 2 g/liter, to compensate, in part, for the lower water consumption of these animals. Diabetic animals received insulin (3–5 IU; Humulin; Eli Lilly, Indianapolis, IN) 3 times/week to maintain body weight and limit hyperglycemia. Plasma glucose in STZ-Db animals was ∼25 mm throughout the study, whereas plasma glucose in non-Db animals remained at ∼5 mm. Further experimental details are provided elsewhere (11Degenhardt T.P. Alderson N.L. Arrington D.D. Beattie R.J. Basgen J.M. Steffes M.W. Thorpe S.R. Baynes J.W. Kidney Int. 2002; 61: 939-950Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Zucker rats were maintained on Purina (St. Louis, MO) 5008 rat chow (16.7% of total calories from fat). The ZDF/Gmi-fa rat develops non-insulin-dependent diabetes on this diet. Plasma glucose increased to 20 mm by week 5 of the study and rose gradually to 40 mm during the course of the 23-week study. Animals were assigned to either untreated or treated groups (n = 10/group) designated as follows: +/fa control (L), +/fa receiving PM (L+PM) in drinking water at 2 g/liter, fa/fa control (F), fa/fa receiving PM (F+PM) in drinking water at 2 g/liter, ZDF/Gmi-fa control (ZDF-Db), and ZDF/Gmi-fa receiving PM (ZDF-Db+PM) in drinking water at 1 g/liter. Plasma glucose in +/fa and fa/fa rats was ∼5 and 8 mm, respectively. Urine Preparation and Analysis—Rats were housed in metabolic cages for 24 h to collect urine; urine collection beakers contained several drops of toluene to prevent microbial growth. Urine samples were stored at –70 °C, and aliquots of urine from each group of rats were pooled for analysis. For analyses of NDAPM and FPyPM, either 8 ml of diabetic or 1.5 ml of non-diabetic urine, containing ∼1 mg of creatinine, was adjusted to pH 1 with 6 m HCl and centrifuged (Marathon, Fisher Scientific) at 2000 rpm for 10 min at 4 °C to remove particulate matter. The supernatant was then applied to a 60-mg Oasis™ MCX column (Waters), which was washed sequentially with 4 ml each of 0.1 n HCl and methanol and then eluted with 4 ml of 10% concentrated NH4OH in methanol. The eluted fractions were dried in vacuo, reconstituted in 1% HFBA, and ultrafiltered prior to analysis. For analyses of FAPM, HAPM, PDAPM, and PyPM, aliquots of urine were diluted 1:20 in 1% HFBA, ultrafiltered as described above, and analyzed directly without further purification. PM adducts were quantified using d 11-HAPM as the internal standard, which was added to urine samples prior to sample preparation. Standard curves were constructed with mixtures of serially diluted authentic standards and constant amounts of d 11-HAPM. Total daily excretion of PM adducts was calculated using average 24-h urine output volumes for each animal group: non-Db+PM and STZ-Db+PM animals, ∼30 and 145 ml of urine, respectively; L+PM, F+PM, and ZDF-Db+PM animals, ∼15, 38, and 158 ml of urine, respectively. Reaction of PM with PUFAs—We have shown previously that PM traps intermediates formed during lipid peroxidation reactions in vitro (10Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. J. Biol. Chem. 2000; 275: 21177-21184Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). As an extension of these studies and to identify additional PM-lipid characteristic of each of these fatty acids, PM was incubated with either LA or AA in phosphate buffer at physiological pH for 6 days. Approximately 60% of PM was consumed in both of the PM-PUFA incubations after 6 days. As shown in Fig. 2, products containing PM were observed in both the LA and AA systems. Product characterization, described below, focused on those PM derivatives that were subsequently detected in the plasma and urine of PM-treated animals. Eight PM adducts were observed in the incubation of PM with LA (Fig. 2A). Six of these (m/z = 267, 305, 323, 339, 377, and 479) had been observed previously, of which two had been identified, the hexanoate amide HAPM (m/z = 267) and the nonanedioate amide NDAPM (m/z = 339) (10Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. J. Biol. Chem. 2000; 275: 21177-21184Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The PM derivative with m/z = 197 has now been identified as the formic acid amide FAPM, based on MRM-LC/MS/MS analysis of mixing experiments with authentic standard. The structure of the PM adducts having m/z = 305, 321, 323, 377, and 479, which were not detected in urine, have not been determined. However, the product with m/z = 479 is consistent with a PM-LA dioxo derivative, and the products with m/z = 323 and 305 are consistent with a hydrated furan and a furan derivative of PM. Although formed in different yields than in reactions with LA, four adducts (FAPM, HAPM, and unknowns with m/z = 305 and 377) were also detected in incubations of PM with AA (Fig. 2B). Four new compounds were also observed in the PM-AA incubation. Three of these were identified as pyrrole (PyPM), formylpyrrole (FPyPM), and pentanedioate (PDAPM) derivatives of PM, respectively, by MRM-LC/MS/MS analysis of mixing experiments with authentic standards. None of the other products in either the LA or AA reactions were detected in the urine of animals treated with PM, possibly because they are early or transient intermediates or were labile during urine collection or storage. Detection of PM Adducts in Vivo—To determine whether PM also traps intermediates formed in lipid peroxidation reactions in vivo, we treated diabetic and obese animals with PM in drinking water. Urine samples (24 h) were collected at monthly intervals, and urine pools collected at the 7th month of the studies were analyzed for PM derivatives. Several compounds identified in incubations of PM with PUFA in vitro were detected in urine, and in each case, authentic standards were added to the urines to confirm the presence of the PM adduct in question by MRM-LC/MS/MS. A typical spiking experiment is shown in Fig. 3. Six PM adducts observed in reactions of PM with LA (Fig. 4A) and AA (Fig. 4B) were also detected in the urine of animals treated with PM. NDAPM (derived from LA), PDAPM, PyPM, FPyPM (derived from AA), and FAPM and HAPM (derived from either PUFA) were readily detected in urine from STZ-Db+PM (Fig. 4C) and F+PM (Fig. 4D) animals by MRM-LC/MS/MS. None of these PM adducts was observed in the urine of untreated, control animals (data not shown). Other PM derivatives, which have not yet been characterized, were also detected in the urine of PM-treated rats (Fig. 4, C and D), and each of these products had fragment ions characteristic of PM when analyzed by daughter-ion LC/MS/MS. Most are more hydrophobic than PM, which elutes between FAPM and NDAPM, and may represent products from other lipid (or carbohydrate) precursors trapped during PM inhibition of oxidative modification of proteins in vivo. Quantification of PM Adducts in Vivo—PM adducts in pooled urine of treated animals were quantified by MRM-LC/MS/MS, using d 11-HAPM as the internal standard. As shown in Fig. 5, the levels of these adducts in the urine of PM-treated diabetic animals were on average 5–10-fold higher than those in the urine of PM-treated non-diabetic animals. FAPM (Fig. 5A), which may be derived from both LA and AA, was the major adduct in the urine of control (non-Db) and STZ-Db animals (22.9 ± 0.8 and 132 ± 12 nmol/24 h, respectively), whereas PDAPM (Fig. 5A) derived exclusively from AA was the major product in the urine of Zucker lean (L+PM), Zucker obese (F+PM), and Zucker type 2 diabetic (ZDF-Db) rats (9.1 ± 0.2, 68.9 ± 7.0, and 97.0 ± 4.5 nmol/24 h, respectively). Although FAPM and HAPM were produced in similar yield from both LA and AA in vitro, FAPM was detected in much larger relative amounts in urine (Fig. 5, A versus B), suggesting alternative sources of FAPM in vivo. HAPM excretion (Fig. 5B) was greatest in STZ-Db animals, about 800 pmol/24 h, and was 10-fold higher than in the urine of control (non-Db) animals. HAPM excretion in ZDF-Db animals was also 10-fold higher than in the urine of lean Zucker rats (L) and 7-fold higher than in the urine of obese (F) animals. The level of NDAPM (Fig. 5B), which is produced only from LA peroxidation (or other Δ9 PUFAs) was typically the lowest of any of the measured adducts in the urine of animals, averaging about 7 pmol/24 h in non-diabetic animals and 50–100 pmol/24 h in diabetic animals. In general, PDAPM, FPyPM, and PyPM, products of AA oxidation, were more abundant than the LA-derived NDAPM (Fig. 5), suggesting that despite the higher concentration of LA in plasma and membrane lipids, AA contributes to the majority of PM adduct formation in vivo. This is consistent with the chemistry of these PUFAs, i.e. that AA is more readily oxidized than LA. Similar results were obtained during Cu2+-catalyzed oxidation of human low density lipoprotein in the presence of PM (data not shown), i.e. the yield of AA-derived products was greater than that of LA-derived products, despite the higher concentration of LA compared with AA in low density lipoprotein. Metabolic Analyses—Plasma glucose and triglyceride levels in various groups of rats are summarized in Fig. 6. As noted in several previous studies (11Degenhardt T.P. Alderson N.L. Arrington D.D. Beattie R.J. Basgen J.M. Steffes M.W. Thorpe S.R. Baynes J.W. Kidney Int. 2002; 61: 939-950Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 12Alderson N.L. Chachich M.E. Youssef N.N. Beattie R.J. Nachtigal M. Thorpe S.R. Baynes J.W. Kidney Int. 2003; 63: 2123-2133Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Stitt A. Gardiner T.A. Alderson N.L. Canning P. Frizzell N. Duffy N. Boyle C. Januszewski A.S. Chachich M.E. Baynes J.W. Thorpe S.R. Diabetes. 2002; 51: 2826-2832Crossref PubMed Scopus (311) Google Scholar, 18Nagaraj R.H. Sarkar P. Mally A. Biemel K.M. Lederer M.O. Padayatti P.S. Arch. Biochem. Biophys. 2002; 402: 110-119Crossref PubMed Scopus (152) Google Scholar), PM had no effect on plasma glucose concentrations in any group (Fig. 6A). However, PM significantly decreased levels of plasma triglycerides in both STZ-Db and ZDF animals (Fig. 6B) by ∼50 and 35%, respectively, although the effects on hyperlipidemia in the obese rats, when fed a high fat diet (used to induce diabetes in the ZDF rat), were not statistically significant. Both groups of diabe
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