Low denfity lipoprot¢ins (LDL) oxidati~ly modified by maeropha~s have been shown to tm atherogeni¢ in ~ vivo studio, W= studied the potential role of nitric oxide (NO), a free r-adi~l produt:ed by maerophag=;, in LDL modifi~tion, Human LDL (I mg/ml) were incubated with mouse Ixritom:al ma¢rophag~ in Ham's F-IO medium. The ¢¢1ls w=r¢ then stimulat~ by interferon.y and tumor n¢¢rosi= fa=or-¢ to inerea~ their production of NO from t.3 to 12.2 tam in 24 h. us mcatsured by nitrite. Lipid l~roxidation of LDL, as m~sured by thio~rbitudc acid.tractive materials (";'BARS). was reduexd in stimulated c¢{l~ in a time.dependent manner. At 24 h, the d=rcar¢ ~s about 2"/%. In tl;¢ preu:n¢¢ of an NO syntha~ inhibitor (N".aminophomoarginin¢). the Ilcncration of NO was diminished and tim proration a~init LDL lipid pcroaidation wa~ reverted, The extent of LDL protein modifiemtion was al~ assessed by examining its electropho~tic mobility. It wall found that macrophage NO reduced the change in LDL ¢lectromobility, These data ladS=ate that the production of NO may inlfibit the oxidative modification of LDL with cytokine,stimulatcd macrophag¢*, We suggest that NO plays a protective role in limiting macrophage.indutu=d LDL modifl=ation, [15,16], In the presence of an antioxidant, such as probucol, the formation of foam ~lls is inhibited both in vitro and in vivo [17][18][19][20][21]. However, the sources and species of those free radicals responsible for the oxidation of LDL are not well established, The superoxid¢ anion (Of) produced by macrophages is known to be involve, sin~ the pretreatment of macrophages with superoxid¢ dismuta~e (SOD) attenuates LDL lipid peroxidation [22]. Macrophages have been shown to produce the nitric oxide (NO) radical via nitric oxide synthase. This enzyme converts arginine into citrulline and is indued by cytokin, treatment [23,24], Although NO can neutrali~ Oi-to form a stable peroxynitrit¢ anion (ONOO') [25], under some conditions th¢ peroxynitrit¢ can also rapidly decompose to form a strong oxidant with reactivity similar to the hydroxyl radical In addition. NO generation in vivo may lead to the mobilization of iron with a subsequent increase in the level of reactive oxygen slzt¢ics [2?]. These observation= could ¢~plain the cytotoxi¢ action of NO that has been demonstrated in a numL~r of studies [28,29]. The purpose of the present study was to dctermin, the ,ff~t of NO production by macrophagcs on LDL lipid [mroxi. datioa. To this end we incubated LDL with macrophages in which the synthesis of NO was induced with interf¢ron-7 and tumor necrosis factor-~, and followed the extent of oxidative modification of LDL. ,i, Nitric oxide .¢k.tztha~'e inhibitor (MDL lO0.24tt) $-Mcthylisothio~emi~ar "b~tde hydroio,Jid¢ (17,8 8. 0.0?fi tool) was added to L-ly£n¢ monohydrate (9,3 $, 0,076 tool) in water (t00 ml) and adjusted to pH 9.5 with I N NaOH, The r~gtion mi~tu~ wa= h~t~ and stirred at 40"C for 30 h. The solution was then cooled to room teml~rature and acidified to pH 4.0 with acetic acid. F~vianic acid (...
