Lipid Advanced Glycosylation: Pathway for Lipid Oxidation in Vivo
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6434-6438, July 1993 Medical Sciences Lipid advanced glycosylation: Pathway for lipid oxidation in vivo (low density lipoprotein/atherosclerosis/aminoguanidine/lipofuscin) RICHARD BUCALA*t, ZENJI MAKITA*, THEODOR KOSCHINSKYt, ANTHONY CERAMI*, AND HELEN VLASSARA* *The Picower Institute for Medical Research, Manhasset, NY 11030; and tDiabetes Research Institute, Dusseldorf, Germany Contributed by Anthony Cerami, March 18, 1993 ABSTRACT To address potential mechanisms for oxida- unpaired electrons at the ir antibonding level (31g). Thus, the tive modification oflipids in vivo, we investigated the possibility reaction ofoxygen with molecules of singlet multiplicity such that phospholipids react directly with glucose to form advanced as unsaturated fatty acids is spin forbidden. Singlet oxygen glycosylation end products (AGEs) that then initiate lipid (1Ag and 1lg) may add directly to unsaturated bonds to oxidation. Phospholipid-linked AGEs formed readily in vitro, produce allylic hydroperoxides; however, this reaction does mimicking the absorbance, fluorescence, and immunochemical not initiate the abstraction of allylic hydrogens (6, 7). Fur- properties of AGEs that result from advanced glycosylation of thermore, there is insufficient singlet oxygen available under proteins. Oxidation of unsaturated fatty acid residues, as normal, physiological conditions to initiate lipid peroxida- assessed by reactive aldehyde formation, occurred at a rate that tion. Significant amounts of the superoxide anion (O -) do paralleled the rate of lipid advanced glycosylation. Ami- form in vivo (0.1-1.0 AM), but superoxide also is incapable of noguanidine, an agent that prevents protein advanced glyco- abstracting bisallylic hydrogens from unsaturated bonds. sylation, inhibited both lipid advanced glycosylation and oxi- Under acidic conditions, superoxide may initiate oxidative dative modification. Incubation of low density lipoprotein modification by forming perhydroxyl radicals (HOO ) or by (LDL) with glucose produced AGE moieties that were attached reacting with transition metals to form reactive hydroxyl to both the lipid and the apoprotein components. Oxidized LDL radicals (HO') (8, 9). Nevertheless, low trace metal concen- formed concomitantly with AGE-modified LDL. Of signifli- trations, the high availability of ligands that form tight cance, AGE ELISA analysis of LDL specimens isolated from coordination complexes with metals, and the abundant an- diabetic individuals revealed increased levels of both apopro- tioxidant capacity of plasma suggest that metal-catalyzed tein- and. lipid-linked AGEs when compared to specimens autoxidation and reactive oxygen species play little, if any, obtained from normal, nondiabetic controls. Circulating levels role in mediating lipid oxidation in vivo (10-12). of oxidized LDL were elevated in diabetic patients and corre- In the present study, we investigated the possibility that lated significantly with lipid AGE levels. These data support the glucose-mediated advanced glycosylation reactions initiate concept that AGE oxidation plays an important and perhaps oxidative modification in vivo. Advanced glycosylation is a primary role in initiating lipid oxidation in vivo. major pathway for the posttranslational modification oftissue proteins and begins with the nonenzymatic addition of sugars The oxidative modification of lipids in vivo has been pro- such as glucose to the primary amino groups of proteins (13, posed to play a central role in atherogenesis and to contribute 14). These early glucose-denrved Schiff base and Amadori to the diverse vascular sequelae of diabetes and aging (1). products then undergo a series of inter- and intramolecular Oxidation of the lipid component of low density lipoprotein rearrangement, dehydration, and oxidation-reduction reac- (LDL), for example, leads to the loss of LDL recognition by tions to produce the "late" products termed advanced gly- cellular LDL receptors and in the preferential uptake of cosylation end products (AGEs). Excessive accumulation of oxidized LDL by macrophage scavenger receptors (2-4). The AGEs on tissue proteins has been implicated in the patho- enhanced endocytosis of oxidized LDL by vascular wall genesis of many of the sequelae of diabetes and normal aging macrophages transforms them into the lipid-laden foam cells (13-15). Protein-linked AGEs act to crosslink connective that characterize early atherosclerotic lesions. This is fol- tissue collagen (16) and to chemically inactivate nitric oxide lowed progressively by the development of fatty streaks and activity (17); they also act as recognition signals for AGE the complex, proliferative lesions that ultimately cause arte- receptor systems that are present on diverse cell types rial insufficiency and occlusion (1-5). (18-20). Despite increased investigation into the biological role of The presence of reactive, primary amino groups on phos- lipid oxidation, there has been little insight into the biochem- pholipids such as phosphatidylethanolamine or phosphati- ical processes that initiate lipid oxidation in vivo. In vitro dylserine led us to consider the hypothesis that glucose also studies have demonstrated that metal-catalyzed peroxidation reacts with lipids to initiate advanced glycosylation. Inter- reactions occur readily at the unsaturated bonds within fatty molecular oxidation-reduction reactions might then occur to acid residues. Polyunsaturated fatty acids are particularly oxidize fatty acid residues-in the absence ofexogenous, free sensitive to peroxidation because bisallylic hydrogens are radical-generating systems. more easily abstracted by free radical processes. Diene conjugation then occurs and hydroperoxides form. Once lipid MATERIALS AND METHODS oxidation is initiated, fatty acids decompose readily to a variety ofreactive aldehydes that rapidly propagate oxidative Reagents. Glucose, EDTA, butylated hydroxytoluene (2,6- reactions (6, 7). di-t-butyl-p-cresol) (BHT), L-a-phosphatidylethanolamine, It is important to note that in the absence of transition metals or free radicals, oxygen itself is a poor oxidant. The Abbreviations: AGE, advanced glycosylation end product; LDL, electronic structure of triplet (ground state) oxygen has two low density lipoprotein; MDA, malonaldehyde bis(diethyl acetal); PC, phosphatidylcholine; PE, phosphatidylethanolamine; TBA, thiobarbituric acid; BHT, butylated hydroxytoluene. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: The Picower payment. This article must therefore be hereby marked "advertisement" Institute for Medical Research, 350 Community Drive, Manhasset, in accordance with 18 U.S.C. §1734 solely to indicate this fact. NY 11030. 6434 Downloaded by guest on September 24, 2021 Medical Sciences: BucalaMedicalet al. Sciences: Bucala et al. ~Proc. Nati. Acad. Sci. USA 90 (1993) 6435 dioleoyl {1,2-di[(cis)-9-octadecenoyl]-sn-glycero-3-phos- at 370C. Aliquots then were subjected to AGE-specific phoethanolamine} (PE), and L-a-phosphatidylcholine, dio- ELISA (23). leoyl {1,2-di[(cis)-9-octadecenoyl]-sn-glycero-3-phosphocho- Analyses. Lipid samples were diluted in methanol (125-0.5 line} (PC) were obtained from Sigma. Malonaldehyde bis(di- mg/ml) and absorbance and fluorescence spectra were re- ethyl acetal) (MDA) was purchased from Aldrich and corded as described (23). aminoguanidine-HCI was from Alteon (Northvale, NJ). Lipid oxidation was assessed by formation of thiobarbitu- Glucose-Lipid Incubations. PE or PC dissolved in chloro- ric acid (TBA)-reactive substances (24). After reaction with form/methanol (1:1) was aliquoted into sterile scintillation TBA, samples (1 ml) were extracted into 1 ml of 1-butanol vials and the solvent was evaporated under nitrogen. One prior to fluorescence measurement (emission at 553 nm upon milliliter of deaerated buffer containing 0.1 M sodium phos- excitation at 515 nm) (25). TBA-reactive substanfces were phate (pH 7.4), 1 mM EDTA, and various concentrations of quantitated by comparison of duplicate samples to a MDA gl'ucose and test reagents then was added under nitrogen. The standard curve (expressed as pmol of MDA equivalents per vials were sonicated in an ice water bath for 30 min to produce Ag of lipid). lipid suspensions and these were incubated in the dark at 370C Protein-linked AGEs were measured by competitive (21). At different time intervals (0-50 days), 1 ml of chloro- ELISA (23). One unit of AGE activity was defined as the form/methanol (2:1) was added to extract the lipid-soluble amount of antibody-reactive material equivalent to 1.0 ji&g of products from unreacted glucose. The vials were gently an AGE bovine serum albumin (BSA) standard (23). Lipid rocked for 10 min, the interface was cleared by centrifuga- AGEs were measured in a direct, noncompetitive ELISA. tion, and the extraction was then repeated two additional Triplicate 100-/4l aliquots of lipid-soluble material (dissolved times. In various experiments, the content of in methanol) were added to round-bottomed, 96-well plates lipid-soluble and the was were material was quantitated by weighing 1-ml aliquots that were solvent evaporated. The wells then washed evaporated in preweighed microcentrifuge tubes, by reaction three times with phosphate-buffered saline (PBS)/0.05% with and visualization Tween 20. Antiserum (final dilution, 1:1000) was added, the 1,6-diphenyl-1,3,5-hexatriene (21), by were incubated for 1 hr at room and with