title: Glycosylation enhances oxygen radical-induced modifications and decreases acetylhydrolase activity of human low density lipoprotein.
abstract: BACKGROUND
  Posttranslational nonenzymatic glycosylation of native low-density lipoprotein (n-LDL) occurs both in vitro and in vivo in diabetic patients. Glycosylated LDL (glc-LDL) behave similarly to oxidized LDL in some respects. In fact, unlike n-LDL, uptake the glc-LDL can occur in part by the "scavenger" receptor(s), as also demonstrated for oxidized LDL. The enzyme acetylhydrolase, carried by LDL, catabolizes platelet activating factor (PAF). This enzymatic activity is inhibited in oxidized LDL. However, it is unknown whether glc-LDL have reduced acetylhydrolase activity.
  
  
  OBJECTIVE
  The first aim of the study was to investigate whether glc-LDL were more susceptible than n-LDL to oxidative modification, and which different oxygen radical species were involved in the phenomenon. Moreover, in order to investigate whether glycosylation may affect acetylhydrolase, we also measured this enzymatic activity in both n- and glc-LDL.
  
  
  METHODS
  In vitro glc-LDL and n-LDL were exposed to the oxidants xanthine/xanthine oxidase (X/XO; 2 mM and 100 mU/ml, respectively), or CuSO4 (10 microM) for 18 hs at 37 degrees C. Parallel experiments were done in the presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), the hydrogen peroxide scavenger catalase (1000 U/ml), or the hydroxyl radical scavenger dimethylthiourea (10 mM) or dimethylsulfoxide (1 mM). Standards of PAF and lyso-PAF were visualized with iodine vapors after separation by thin layer chromatography. The distribution of label was determined by an imaging scanner. Labeled products were then isolated from the chromatography plate, and the amount of 3H-lyso-PAF formed was determined by liquid scintillation counting.
  
  
  RESULTS
  Glc-LDL were more susceptible than n-LDL to lipid peroxidation (n-LDL 22.9 +/- 3.4 vs 34.8 +/- 4.2* nmoles/MDA/mg of protein in glc-LDL oxidized by X/XO and n-LDL 28.9 +/- 4.2 vs 40.4 +/- 4.1* in glc-LDL oxidized by CuSO4, *p < 0.05 vs n-LDL). SOD, but not other scavengers, prevented peroxidation, indicating an obligatory role for superoxide radicals. Oxidation of glc-LDL also induced a higher degree of apolipoprotein-B100 modifications than n-LDL, with increased electrophoresis mobility and decreased TNBS reactivity. These effects were similarly prevented by SOD. Finally, acetylhydrolase activity was significantly lower in glc-LDL than in n-LDL.
  
  
  CONCLUSIONS
  Glycosylation increases LDL oxidation due to superoxide radicals, and also reduces acetylhydrolase activity. These phenomenona may contribute to enhance and/or accelerate the progression of atherosclerosis in diabetic patients.
authors.affiliation: Division of Clinical Immunology, Federico II University of Naples, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Napoli
authors.forename: M
authors.initials: M
authors.lastname: Triggiani
authors.forename: G
authors.initials: G
authors.lastname: Palumbo
authors.forename: M
authors.initials: M
authors.lastname: Condorelli
authors.forename: M
authors.initials: M
authors.lastname: Chiariello
authors.forename: G
authors.initials: G
authors.lastname: Ambrosio
mesh_terms: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000704:Analysis of Variance; D019327:Copper Sulfate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016166:Free Radical Scavengers; D017127:Glycation End Products, Advanced; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood* / Q000302:isolation & purification; D010741:Phospholipases A / Q000097:blood*; D010972:Platelet Activating Factor / Q000378:metabolism; D012016:Reference Values; D012044:Regression Analysis; D013481:Superoxides / Q000378:metabolism*; D019820:Xanthine; D014969:Xanthine Oxidase; D014970:Xanthines; D000093844:Glycated Proteins
chemical_list: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D019327:Copper Sulfate; D016166:Free Radical Scavengers; D017127:Glycation End Products, Advanced; D008077:Lipoproteins, LDL; D010741:Phospholipases A; D010972:Platelet Activating Factor; D013481:Superoxides; D019820:Xanthine; D014969:Xanthine Oxidase; D014970:Xanthines; C031797:glycated lipoproteins, LDL; D000093844:Glycated Proteins