Chem. Res. Toxicol. 1997, 10, 875-879
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Efficient Scavenging of Fatty Acid Oxidation Products by Aminoguanidine Yousef Al-Abed and Richard Bucala* The Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030 Received March 4, 1997X
Lipid oxidation leads to the formation of reactive aldehydes that may play an important role in atherogenesis by altering the normal pathway of lipoprotein metabolism and by exerting toxic effects on vascular wall components. Recent studies indicate that advanced glycation end products, which form spontaneously from the reaction of reducing sugars with amino groups, may promote oxidative damage in vivo. Moreover, the pharmacological inhibitor of advanced glycation aminoguanidine has been shown to lower circulating low-density lipoprotein levels in human subjects and to inhibit certain oxidative reactions in vitro. To define more precisely the potential interaction of AG with oxidized lipids, we have studied and identified the major products that form from the reaction of AG with the oxidation products 4-hydroxynonenal and malondialdehyde. AG was found to be an efficient scavenger of R,β-unsaturated aldehydes when compared to nucleophilic amino acids (Cys, Lys, His), suggesting that one of its mechanisms of action in vivo is to protect tissue constituents from the damaging effects of oxidative stress.
Introduction The nonenzymatic, oxidative degradation of lipids has been linked to several pathological processes in vivo, including atherosclerosis (1, 2). In the case of polyunsaturated lipids, which are more susceptible to oxidation than saturated lipids, oxidation proceeds by a complex rearrangement and fragmentation pathway that leads to the formation of R,β-unsaturated aldehydes such as the 4-hydroxy-2-alkenals: 4-hydroxynonenal (HNE)1 and 4-hydroxyhexenal (HHE), and malondialdehyde (MDA) (3-5). The damaging effects of lipid oxidation in vivo have been proposed to be due in large part to cellular dysfunction induced by the reaction of these low molecular weight degradation products with critical macromolecules (1-5). Although the precise mechanism for the formation of these reactive aldehydes is not entirely known, they appear to arise by cleavage of alkoxyl radical intermediates (3-5). Accordingly, HNE forms by the peroxidation of fatty acids of the n-6 series, and HHE forms by the peroxidation of a member of the n-3 fatty acid series. Malondialdehyde (MDA) is considered to form mainly from the oxidative degradation of fatty acids with at least two methylene-interrupted double bonds; however, a significant portion that forms in vivo also arises as a byproduct of the action of thromboxane synthetase on the prostaglandins PGH2, PGH3, and PGG2 (6). The 4-hydroxy-2-alkenals react with the nucleophilic amine and sulfhydryl groups of proteins. Michael addition to the CdC bond is a major reaction pathway, and the resulting adducts are stabilized by formation of a cyclic hemiacetal (Scheme 1) (7, 8). Pyrroles have been isolated from model reactions and arise by dehydration of alkenal-derived Schiff bases; however, their formation * Corresponding author. Tel: 516-365-4200. Fax: 516-365-5090. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, July 1, 1997. 1 Abbreviations: AG, aminoguanidine; AGE, advanced glycation end product; LDL, low-density lipoprotein; MDA, malondialdehyde; HHE, 4-hydroxyhexenal; HNE, 4-hydroxynonenal.
S0893-228x(97)00035-0 CCC: $14.00
appears less favored than Michael addition products (9). A cyclic hemiaminal, or 1:2 4-HNE-lysine adduct, also has been isolated from incubations by trapping with borohydride reduction (10). Model studies with proteins in vitro suggest that most adducts that form from these unsaturated aldehydes are reversible. Nevertheless, it is conceivable that within certain protein microenvironments, dehydration reactions may occur to produce stable adducts such as alkenal-derived pyrroles (10). Among the chemical modifications that affect lowdensity lipoprotein (LDL) in vivo are the covalent addition products that result from advanced glycation reactions (11). These products, termed advanced glycation end products or AGEs, arise in vivo by the rearrangement of glucose-derived Amadori products and have the capacity to cross-link the proximate amino groups of proteins or lipids. AGEs accumulate as a consequence of hyperglycemia and age and appear to contribute to many of the structural and functional alterations that occur in tissues as a result of diabetes or aging (12, 13). AGEs are present in appreciable levels in LDL, particularly in the LDL of patients with diabetes or end-stage renal disease, and are covalently linked both to the aminecontaining, polar head groups of phospholipids and to the free amino groups of apolipoprotein B (apoB) (11, 14). In the case of apolipoprotein B, the predominant site of advanced glycation has been mapped to a 67-amino acid domain, and modification at this site interferes with the uptake of LDL by high-affinity, fibroblast LDL receptors (15). Of particular importance, however, is the observation that the advanced glycation of LDL is accompanied by the formation of lipid oxidation products, such as the hydroxyalkenals HNE and HHE. In model studies performed with either purified LDL or phospholipid, AGE-mediated oxidative modification occurs in the absence of exogenously added transition metals, to be dependent on the presence of primary amino groups within the phospholipid and to be inhibited by aminoguanidine, which blocks the late stages of advanced glycation reaction (11, 16). The observation that these AGE-induced oxidative reactions occur in the absence of © 1997 American Chemical Society
876 Chem. Res. Toxicol., Vol. 10, No. 8, 1997
Al-Abed and Bucala
Scheme 1. Reaction between r,β-Unsaturated Aldehydes and the Nucleophilic Amino Acids Cysteine, Lysine, and Histidinea
a
R ) C5H11 for HNE (7-10).
exogenously added, free transition metals has led to the proposal that lipid-advanced glycation may represent an important and previously unrecognized mechanism for oxidative modification in vivo (11). The role of advanced glycation in the development of hyperlipidemia and atherosclerosis has been affirmed both by experimental animal studies and by early clinical trials of aminoguanidine (14, 17, 18). Aminoguanidine (AG) inhibits AGE formation by reacting with carbonylcontaining intermediates, such as protein-bound dideoxyosones, and preventing the further rearrangement reactions that lead to covalent cross-linking (19). In human subjects, AG has been found to decrease both circulating hemoglobin-AGE levels (a hemoglobin subfraction modified by advanced glycation) and total circulating LDL levels (14, 20). AG is a nucleophile and might be expected to react with certain unsaturated aldehydes that form as a result of lipid oxidation. Evidence for an interaction between AG and MDA has been reported previously based on the ability of AG to block the reaction of MDA with thiobarbituric acid (11, 21, 22). In the present study, we describe the major products formed by the reaction of AG with HNE and MDA and provide evidence that AG is an efficient scavenger of R,β-unsaturated aldehydes.
Materials and Methods General Methods. NMR spectra were recorded on a JOEL 270 instrument. HPLC was performed on a Hewlett Packard Model 1090 or Waters Associates instrument. The protected amino acids were obtained from Advanced ChemTech (Louisville, KY), and aminoguanidine-HCl was provided by Alteon, Inc. (Ramsey, NJ). All other materials were of reagent grade. Electrospray ionization (ESI) samples were run on a Quattrotriple quadrupole mass spectrometer. Loop injection samples were performed using an ABI Model 140B syringe pump employing H2O/CH3CN (1:1) at a flow of 15 µL/min, a Rheodyne Model 7125 valve with a 10 µL loop, and a Micromass Megaflow ESI probe using nitrogen for the nebulizer/drying gas. LC/MS samples were run employing the ABI pump and Rheodyne valve with a 20 µL loop at a flow of 50 µL/min. A binary solvent gradient consisting of 0.05% TFA in H2O (solvent A) and CH3CN (solvent B) was delivered as follows: 0-45 min, linear gradient from A:B (95:5) to A:B (15:85); 45-50 min, linear gradient from A:B (15:85) to A:B (95:5). Preparation of HNE. HNE-diethyl acetal (HNE-DEA), as a solution in chloroform, was provided by Prof. H. Esterbauer (University of Graz, Graz, Austria) and was hydrolyzed by a standard method (23). The chloroform was evaporated from 0.5 mL of this solution (equivalent to 23 mg of HNE-DEA) under a
stream of nitrogen. The remaining oily residue was suspended in 5 mL of 1 mN HCl. A turbid solution first was obtained which then became clear as the hydrolysis of HNE-DEA to HNE proceeded to completion over 60 min. Reaction between HNE and AG. To a freshly prepared solution of HNE (0.01 mmol) in 5 mL of 0.2 M phosphate buffer (pH 7.4) was added 0.012 mmol of AG-HCl. The reaction mixture was stirred at 25 °C for 24 h. The solvent then was evaporated and the residue redissolved in methanol, and aliquots were subjected to preparative, reversed-phase HPLC using a C18 column (250 × 21 mm, 5 µm; Primesphere, Phenomenex Inc., Torrance, CA). The elution was performed with the following profile: 1 min of 100% solvent A followed by a linear gradient of 0-100% solvent B over 40 min (solvent A, 0.05% TFA in ddH2O; solvent B, methanol). The flow rate was 9 mL/min, and detection was by UV absorbance at λmax 210 nm. Fractions were collected and analyzed further by 1H-NMR spectroscopy. A Schiff base adduct between HNE and AG was isolated (λmax 254 nm) in 87% yield (18 mg). Positive FAB-MS: m/z 213 [MH]+. 1H-NMR (CD3OD, 270 MHz): δ 0.91 (t, 3H, J ) 6.4 Hz), 1.2-1.6 (m, 8H, H-5,6,7,8), 4.21 (m, 1H, H-4), 6.26 (dd, 1H, J ) 15.8, 5.2 Hz, H-3), 6.42 (dd, 1H, J ) 15.8, 9.0 Hz, H-2), 7.75 (d, 1H, J ) 8.9 Hz, H-1). 13C-NMR (CD3OD, 67.5 MHz): δ 13.1 (C-9), 22.3 (C-8), 24.9 (C-7), 31.6 (C-6), 36.6 (C-5), 70.9 (C-4), 125.0 (C-3), 146.9 (C-2), 149.4 (C-1), 160.0 (H2NC(dNH)-). Reaction between MDA and AG. MDA was obtained by hydrolysis of 1,1,3,3-tetraethoxypropane with 1% H2SO4 for 1 h. AG-HCl (14 mM) was added to a solution of MDA (13.8 mM) in 0.2 M phosphate buffer (pH 7.4). The pH was corrected to 7.4 with 2 N NaOH and the reaction mixture stirred overnight at 25 °C. The reaction products were dried in vacuo, and the residue was extracted with methanol. The methanolic extract was dried (89% yield), redissolved in D2O, and subjected to 1HNMR spectroscopy. 1H-NMR (D2O, 270 MHz): δ 2.93 (dd, 1H, J ) 19.8, 1.2 Hz, H-4), 3.35 (dd, 1H, J ) 19.8, 7.9 Hz, H-4′), 5.85 (d, 1H, J ) 7.9 Hz, H-5), 7.34 (bs, 1H, H-3). 13C-NMR (D2O, 67.5 MHz): δ 44.3 (C-4), 78.93 (C-5), 151.7 (C-3), 160.0 (H2NC(dNH)-). MS: m/z 229 [MH]+. Reaction of Nr-Blocked Amino Acids with HNE. To a solution of HNE (0.01 mmol) in 0.7 mL of aqueous 0.2 M phosphate buffer (pH 7.4) were added NR-acetylLysOMe-HCl, NR-acetylCysOH, and NR-acetylHisOH (each at 0.01 mmol). The mixture was shaken for 8 days at 25 °C and the reaction progress monitored over time by 1H-NMR spectroscopy and LC/ MS. For kinetic studies of the reactivity of HNE with NRblocked amino acids versus AG, a solution of HNE (0.01 mmol) in 0.7 mL of aqueous 0.2 M phosphate buffer (pH 7.4) was added to NR-acetylLysOMe-HCl, NR-acetylCysOH, NR-acetylHisOH or AG (each 0.01 mmol). The reaction mixture was shaken for 8 days at 25 °C and the reaction progress followed over time by 1H-NMR spectroscopy. The downfield protons of HNE at δ 9.40, 7.15, and 6.25 served as a useful diagnostic tool to quantify the
Aminoguanidine Reacts with Unsaturated Aldehydes
Chem. Res. Toxicol., Vol. 10, No. 8, 1997 877
Scheme 2. Proposed Reaction for the Formation of 1H-Pyrazole-1-carboxamide, a Stable AG-MDA Addition Product
loss of the HNE starting material. This loss was measured by a decrease in the integration of these protons in time course experiments upon incubation of HNE with NR-blocked amino acids or aminoguanidine. Reaction of Nr-Acetylcysteine with HNE-AG Adduct. To a solution of HNE-AG adduct (5 mg, 0.024 mmol) in 1 mL of aqueous (D2O) 0.2 M phosphate buffer (pH 7.4) was added NRacetylcysOH (0.024-0.24 mmol). The reaction mixture was stirred at ambient temperature (25 °C) and the reaction progress monitored over time by 1H-NMR measurement.
Results and Discussion AG was found to react with HNE in aqueous 0.2 M phosphate buffer (pH 7.4) at 25 °C to yield a stable adduct that was isolated by reversed phase HPLC in 87% yield. The mass spectrum of the purified product showed a molecular ion at m/z 213 [MH+], and the 1H-NMR spectrum showed three downfield-shifted protons that resonate as an ABX system at δ 7.75, 6.42, and 6.26. Taken together, these data are consistent with the structure of a resonance-stabilized Schiff base between AG and HNE. We next investigated the chemical interaction between MDA and AG. A reaction product was identified to form after incubating equimolar amounts of MDA with AG in 0.2 M phosphate buffer (pH 7.4). Lyophilization of the water and extraction of the residue with MeOH produced a stable MDA-AG adduct in 89% yield. The 1H-NMR spectrum showed one Schiff base proton at 7.34 ppm and a hemiaminal proton at 5.85 ppm. The mass spectrum of the adduct displayed a molecular ion of m/z 229 [MH+]. Overall, these data are consistent with the assignment of the structure as 5-hydroxy-2-pyrazoline1-carboxamidine (Scheme 2). We did not find evidence by either NMR or MS analyses for an expected 1Hpyrazole-1-carboxamidine structure. Once purified, however, the 5-hydroxy-2-pyrazoline-1-carboxamidine adduct was found to dehydrate very slowly at pH 7.4, and lowering the pH to 4.0 markedly accelerated this reaction (data not shown). 4-Hydroxyalkenals react with the nucleophilic side chain residues of cysteine (Cys), lysine (Lys), and histidine (His) to form stable addition products that may account for certain toxicities of these aldehydes in vivo (Scheme 1) (5). We tested the ability of AG to protect amino acids from hydroxyalkenal addition reactions by establishing model incubations in which HNE was mixed with equimolar quantities of NR-acetylCys, NR-acetylLysOMe, and NR-BocHis under physiological conditions (pH 7.4, 37 °C). The reaction products then were analyzed over time by HPLC, LC/MS, and 1H-NMR. The downfield protons of HNE at δ 9.40, 7.15, and 6.25 in 1H-NMR served as a useful diagnostic tool to quantify the loss of the HNE starting material. The reaction between HNE and NR-blocked amino acids was studied for up to 8 days (Figure 1). LC/MS analyses
Figure 1. HNE loss as analyzed by 1H-NMR produced by the reactions of HNE with Cys (9), Lys (1), His (2), or AG (b) as described in Materials and Methods. Triplicate measurements were obtained for each time point, and the values shown are the mean of duplicate experiments. The SD for each point shown is e10%.
performed at this time showed the total disappearance of NR-acetylcysteine and the appearance of a major product that eluted as diastereomers at 29 min in the LC/MS chromatogram (Figure 2B, peak 1). The molecular ion of the major new adduct was determined by electrospray mass spectrometry to be 465 Da, consistent with the structure of a cyclic monothioacetal adduct (HNE-(Cys)2) comprised of two molecules of acetylcysteine and one molecule of HNE minus one molecule of water (Scheme 3). Interestingly, a stable 1:1 adduct between Cys and HNE was not detected at any time during the incubation period. It would appear therefore that the rate-determining step in the formation of HNE(Cys)2 is Michael addition of the sulfhydryl group of cysteine to HNE. The initial 1:1 adduct is in equilibrium with the hemiacetal, and the addition of a second cysteine produces the monothioacetal that is then stably trapped by cyclization. Further analysis of these incubations by LC/MS provided evidence for a 1:1 histidine-HNE adduct, although only in trace amounts (Figure 2B, peak 2; also data not shown). No stable adducts involving Lys and HNE could be identified in this model incubation system. Overall, these data affirm the high reactivity of the cysteine side chain toward HNE when compared to Lys and His (5). In the presence of 1 equiv of AG, the formation of HNE(Cys)2 was decreased, as assessed by HPLC-UV, 1H-NMR, and LC/MS (Figure 2C; also data not shown). In the presence of a 3-fold excess of AG, no HNE-(Cys)2 could be detected. By 1H-NMR analysis, a marked decrease in the intensity of the HNE protons was apparent after 3 days, and the appearance of new signals at 7.75 ppm (d, J ) 8.9 Hz) suggested the formation of a resonancestabilized Schiff base adduct between HNE and AG. The LC/MS profile of this incubation mixture showed a major
878 Chem. Res. Toxicol., Vol. 10, No. 8, 1997
Al-Abed and Bucala Scheme 3. Proposed Reaction for the Formation of HNE-AG, HNE-Cys, and HNE-AG-Cys Adducts
Figure 2. LC/MS analyses of products formed by the reaction of NR-acetylcysteine, NR-acetyllysine-OMe, and NR-Boc-histidine with HNE or with HNE plus AG. (A) LC/MS spectrum of a mixture of the NR-acetylCys, NR-acetylLysOMe, and NR-BocHis starting material. (B) LC/MS spectrum of a mixture of the NRacetylCys, NR-acetylLysOMe, and NR-BocHis with HNE for 8 days at 37 °C as described in Materials and Methods. (C) LC/ MS spectrum of products formed by incubating NR-acetylCys, NR-acetylLysOMe, and NR-BocHis with HNE plus 1 equiv of AG for 8 days at 37 °C. Peaks 1, Cys-derived, cyclic hemithioacetal diastereoisomers; 2, HNE-His adduct; 3, HNE-Cys-AG adduct; 4, HNE-AG adduct.
hydrophobic peak with a molecular ion at m/z 214 (Figure 2C, peak 4). Both the LC/MS and 1H-NMR data are consistent with the structure of a resonance-stabilized HNE-AG adduct. A product (peak 3) with a mass (m/z 376.0) that was consistent with a stable HNE-AG-Cys adduct also was detected. To identify the reaction pathway leading to the HNEAG-Cys adduct, we tested the ability of the HNE-AG
Schiff base to act as a Michael acceptor for the -SH group by adding NR-acetylcysteine (1-10 equiv) to HNE-AG in 0.2 M phosphate buffer in D2O and following the progress of the reaction over time by 1H-NMR. No new products were detected, suggesting that HNE-AG cannot undergo Michael addition by Cys under these conditions. Accordingly, the formation of the substituted, cyclic hemiaminal involving HNE, AG, and Cys may be explained best by the direct addition of AG to the HNE-Cys adduct (Scheme 3). The resulting Schiff base then undergoes cyclization via addition of the hydroxyl group to the CdN bond to form a stable hemiaminal. In summary, these studies indicate that the advanced glycation inhibitor AG can form stable addition products with HNE or MDA under physiological conditions. Among the nucleophilic amino acids, Cys was found to be most reactive in a model Michael acceptor system involving the R,β-unsaturated aldehyde, HNE. AG in equivalent amounts was observed to compete effectively with Cys and reduce the formation of HNE-(Cys)2. AG, which is known to interfere with advanced glycation chemistry in human subjects (20), may function additionally in vivo to scavenge certain reactive carbonyl and R,β-unsaturated aldehydes that arise from lipid oxidation.
Acknowledgment. This work was supported by NIH Grant DK19655-15.
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