Release of superoxide radicals by mouse macrophages stimulated by oxidative modification of glycated low density lipoproteins

ATHEROSCLEROSIS ELSEVIE;R

Atherosclerosis I I8 (1995) I-8

Release of superoxide radicals by mouse macrophages stimulated by oxidative modification of glycated low density lipoproteins Hirokazu Kimura”“, Hisanori Minakamib, Satsuki Kimura”, Tamiko Sakuraid, Tadashi Nakamurac, Satonori Kurashige”, Minoru Nakanof, Akira Shojif “Gunmu

Pr&~ral

‘Photon

Institute bDepartment

of Public Health and Emironmentrrl Science.\. 3-21- 19 Iwagami, Maebushi qf’ Obstetrics and Gvnecolog~~. Jic,hi Medical School. Tochigi. Japan ‘Institute of Immunological Dtjtww. Yokohama. Japan d TokJ*o College of Pharmac:v. Tokjv. Japan

‘College of’ Medical Care and Tec,hnolog.v. Gumna Unicersity. Gunma, Jupan Medical Reseurch Center, Humamatsu Urzicer.srt~. S1~l7ool of Medicine, Shizuoka. “Focult~~ of Enginrwi~~g, Gunmtr C~nirrr.tit~,. Gunmtr. Jupcm

371, J~XIII

Jupun

Received 6 December 1994; revision received I4 March 1995; accepted 31 March 1995

Abstract Diabetic patients have high levels of glycated LDL. Although glycated LDLs are implicated in the development of atherosclerosis in such patients, convincing data are lacking. We observed release of superoxide radicals (0; ) from mouse resident peritoneal macrophages stimulated by an oxidized/glycated LDL by using a highly sensitive and specific chem.iluminescence method. Oxidized/glycated LDL was achieved by an addition of low concentration of Fe’+ to glycated LDL. Macrophages took up an appreciable amount of the glycated LDL oxidized by iron. leading to the development of foam cells, while they did not take up untreated glycated LDL or the native LDL. These observations clearly indicate that the oxidized/glycated LDL reacts well with macrophages. Since an oxidation of glycated LDL may occur in vivo, the oxidized/glycated LDL might play an important role in atherogenesis. Kqwordsr

Arherosclerosis:

Glycated LDL; Diabetes mellitus; Phagocytosis; Chemiluminescence

Abbreuiation~: CL, chemiluminescence; G-LDL, glycated LDL; HBSS, Hank‘s lipoprotein: MCLA, 2-methyl-(p-methoxyphenyl)-3.7.dihydromidazo[l ,2-alpyrazin-j-one; myristate acetate; U-LDL, untreated (not glycated) LDL. * Corresponding author. Tel.: + 81 272 32 4881; Fax: + 81 272 34 8438.

0021-9150,95,‘$39.50cs 1995 Etlsevier Science Ireland Ltd. All rights reserved SSDI

0021-9150(95)05587-M

balanced salt solution: LDL. low densit) OZ. opsonized zymosan; PMA. phorbol

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1. Introduction

2. Materials

Attention is being directed to the role of modified lipoproteins, especially of low density lipoprotein (LDL), in the initiation and propagation of atherosclerosis [l]. The LDL modified by reagents such as CuSO,, malondialdehyde, and acetate is taken up by macrophages, recognized by scavenger receptors, but not by the cell’s LDL receptors [ 11. Consequently, the oxidatively modified LDL accumulates in the macrophages, which develop into foam cells [2]. However, it seemsunlikely that such changes would occur in vivo, since the physiological concentrations of these substances may not allow the modification of LDL. Patients with diabetes mellitus have high levels of glycation in their LDL collagen, lens, crystallin, and hemoglobin [3,4]. However, those glycated LDLs are not taken up by macrophages [5]. We previously showed that glycated polylysine treated with Fe’+ -ADP complex led to the generation of superoxide radicals and induced the lipid peroxidation of rat liver microsomal phospholipids [6,7] and of native human LDL [8]. The native human LDL peroxidized by the glycated polylysine- Fe3+ -ADP system was easily taken up by resident peritoneal macrophages of the rat [8]. The oxidative modification of glycated human LDL occurred in the presence of low concentrations of Fe’ + -ADP complex, which suggeststhat the oxidation of glycated LDL may occur in vivo 191. Active oxygen species have been implicated in the development of atherosclerosis [lo]. Nonenzymatically-produced active oxygen speciesdenature LDL to be taken up by macrophages [8]. The leukocyte may be an intrinsic source of active oxygen species [l 11.Macrophages, which are key cells in the development of atherosclerosis, also produce active oxygen species [ 12,131. However, the relationship between the production of superoxide radicals and the development of foam cells is poorly understood. Accordingly, we studied the release of superoxide radicals (0;) from mouse resident peritoneal macrophages that were stimulated by an oxidatively modified glycated LDL.

2.1. Prepuration and properties of LDL

and methods

Human LDL having a density between 1.019 and 1.063 g/ml was isolated by the sequential flotation ultracentrifugation of plasma containing 0.04% ethylenediaminetetracetic acid (EDTA), according to the method of Schumaker and Puppione [14]. Three milliliters of isolated LDL was dialyzed overnight against 4 1 of 67 mM potassium phosphate buffer (pH 7.4) at 4°C saturated with N, gas. This is referred to as native LDL. Whole protein concentration was determined by Lowry’s method. The purity of LDL was verified by electrophoresis on agarose gel (1%) and immunoassay of apolipoprotein [ 151.The LDL sample showed a single band on an electrophoretic plate after staining with Fat Red 7B. This LDL contained about 0.8 mg of apolipoprotein B/mg of protein. 2.2. Preparation und properties of glycated LDL

Glycated LDL was prepared by the method previously described [9]. In brief, LDL was sterilized by passagethrough a 0.22 pm Millipore filter and divided into two portions. One portion of LDL (2 mg of protein) in 2 ml of 67 mM potassium phosphate buffer containing 200 mM glucose and 0.01% gentamycin was incubated for 3 days at 37°C with gentle agitation. At the end of incubation, the glucose-treated LDL was dialyzed overnight against 67 mM potassium phosphate buffer containing 1 mM EDTA at 4°C. This sample is referred to as glycated LDL (G-LDL). The other portion of LDL was incubated without glucose and dialyzed, and is referred to as untreated LDL (U-LDL). Before being used, both samples were dialyzed overnight against 4 1 of 67 mM potassium phosphate buffer at 4°C under N, to remove EDTA. Those procedures were performed under sterile conditions. The amounts of glucose incorporated into apolipoprotein B in GLDL, which had been assayed by the thiobarbituric acid method after hydrolysis of LDL by oxalic acid [7], were calculated to be approximately 10 mol/mol of apolipoprotein B [9]. This corresponds to modification of 2.8% of lysine residues in apolipoprotein B.

H. Kimura

2.3. Prepanrtion of oxidatively Fe 3 + - ADP complex

et al. / Atherosclerosis

modified G-LDL by

The procedure for the oxidative modification of G-LDL was as previously described [9]. The standard reaction mixture contained 500 pg of protein of G-LDL (or U-LDL or native LDL), 60 PM Fe3+ - 1 mM ADP complex, and 67 mM potassium phosphate buffer at pH 7.4, in a total volume of 500 ~1. The reaction was initiated by addition of Fe’ + -ADP and was continued for 24 h at 37°C with gentle agitation [9]. 2.4. Chemicals and enzymes

The Fe3‘~-ADP complex was prepared by mixing Fe (NO,), with ADP (1:16.6 M/M) in water [ 161.Bovine erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, 3500 units/mg protein), desferrioxamine mesylate, zymosan A, and phorbol myristate acetate (PMA) were purchased from Sigma. RPM1 1640 and Hank’s solution (without phenol red) were purchased from Nissui Phaumaceutical Co. (Tokyo). Opsonized zymosan (OZ) was prepared by the method previously described [17], but mouse serum was used. Other chemicals were of reagent grade and were obtained from Wako Pure Chemicals (Tokyo). 2.5. Preparation of mouse resident peritoneal macrophages

ICR male mice weighing about 50 g were used. Resident peritoneal macrophages were prepared as follows. Chilled 5 ml of RPM1 1640medium at pH 7.4 was injected intraperitoneally while the animal was anesthetized with phenobarbital. The peritoneal fluid obtained from 30 treated mice was pooled. Cells were collected by centrifugation at 700 x g at 4°C then washed twice with chilled RPM1 1640 medium. Macrophages were then collected according to Kumagai et al.‘s method [18]. More than 95% of cells were confirmed morphologically to be macrophages. The viability of these macrophages was more than 97% by the trypan blue exclusion test. Macrophages were suspended to I x IO’ cells/ml in Hank’s balanced salt solution (HBSS) without Ca* + and Mg” + , and stored at 4’C for no longer than 3 h prior to use. 2.6. Assay ,for 0;

based on chemiluminescence

The chemiluminescence (CL) probe for O;- de-

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tection, Cypridina luc@rin analog, 2-methyl-6-(pmethoxyphenyl)-3,7-dihydroimidazo [ 1,2-a] pyrazin-3-one (MCLA), was synthesized according to a previous report [17]. MCLA was dissolved in twice-distilled water and its concentration was calculated based on the following: E~~~~,,, = 9600/ M per cm [16]. MCLA-dependent CL was measured with a luminescence reader (BLR-102, Aloka, Tokyo). The CL assay mixtures contained O-2 x lo6 cells, 1 p M MCLA, 150 PM desferrioxamine mesylate, 20 ,ug of protein of G-LDL (or 1 118of PMA or 1.6 mg of mouse serum opsonized zymosan (OZ)) in a total volume of 2 ml of HBSS. Desferrioxamine is added to this system to inhibit the production of 0, caused by the interaction between glycated lysine residues and Fe’ + -ADP complex [7]. Since a protein concentration over 10 ,fcg/ml interferes with the accuracy of the method used in this study for detection of superoxide radicals [19,20], a fixed concentration of 10 /lg/ml of LDL was used throughout the study. This concentration of LDL corresponds to about one-thirtieth of that seen in the serum of normal men. The experiment with the same mixture but in which the G-LDL (or PMA or OZ) was omitted served as control. All components except for the macrophages were incubated for 3 min at 37°C. The reaction was initiated by adding macrophages that were preincubated at 37°C. During the measurement of CL, the incubation mixture in the luminescence reader was agitated by rotation at 37°C. The maxima1 intensity of light obtained by MCLA-dependent CL was corrected for each control [21]. 2.7. Uptoks of oxidutiwiy mcrcrophuges

rnod$rd

G-LDL AJ%

Macrophages ( 106)were incubated with 100 pg of protein of G-LDL that had been exposed to the Fe?+-ADP complex, G-LDL, the U-LDL exposed to the Fe3+ - ADP complex, U-LDL, or native LDL in 2 ml of RPM1 1640 at pH 7.4 for 6 h at 37°C with gentle shaking. Following incubation, the cells were stained with Sudan III and hematoxylin. 2.8. Electrophoretic

method

Agarose gel electrophoresis was carried out on gel plates (Ciba Corning Diagnosis Corp.) using 0.065 M barbital buffer (pH 8.6) according to the

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Fig. 1. Electrophoretic mobilities of various LDLs. G-LDL, U-LDL, and native LDL were incubated with or without 60 FM Fe”+ -1 mM ADP in 67 mM potassium phosphate buffer at pH 7.4 at 37°C for 24 h. At the end of incubation, an aliquot (1 ~1) of reaction mixture was run on an agarose gel, and stained by Fat Red 7B. Lanes I and 2, native LDL incubated without or with Fe’+ -ADP; lanes 3 and 4, U-LDL incubated without or with Fe-‘+-ADP. , lanes 5 and 6, G-LDL incubated without or with Fe’+ -ADP, 0, origin.

manufacturer’s instructions. LDL bands were visualized by staining with Fat Red 7B [9]. 3. Results 3.1. Denaturation complex

of G-LDL

by Fe-7+-ADP

To compare the electrophoretic mobility of the Fe3+ -ADP-treated G-LDL with that of untreated G-LDL or native LDL, all samples were run on agarose gel and stained with Fat Red 7B. Only the G-LDL exposed to Fe3+ -ADP complex moved more rapidly to the anode than the other LDLs, suggesting the denaturation of G-LDL. The peroxidation of G-LDL is involved in such denaturation [9]. 3.2. 0, production by mouse resident peritoneal macrophages

The production of superoxide radicals by mouse resident peritoneal macrophages is shown in Figs. 2 and 3. The reaction mixture contained 1 p M MCLA, 150 PM desferrioxamine mesylate, 0.0-2.0 x lo6 mouse resident peritoneal macrophages, and a stimulus (20 pg of LDL variously modified, 1.6 mg of OZ, or 1 pug of PMA) in 2 ml of HBSS. The addition of macrophages to the reaction mixture produced a prompt and marked chemiluminescent curve (CL) (Fig. 2, curves l-7). This CL was completely

abolished in the presence of 0.5 PM SOD (Fig. 2, curve 9). The production of superoxide monitored by the intensity of CL increased with the increase in number of macrophages (Fig. 2, curves 1-6, and Fig. 3). Also, 0; production, in the presence of 1 x lo6 mouse resident peritoneal macrophages, was dose-dependently enhanced with increasing concentration of oxidized G-LDL, in the range 2-20 pg (data not shown). The U-LDL exposed to the Fe3+ -ADP complex elicited a small amount of CL. The U-LDL, G-LDL, native LDL exposed to Fe3+ -ADP complex, or native LDL did not affect the macrophages with respect to superoxide generation (Fig. 2, curves 7 and 8). In the presence of 5 x lo5 macrophages, the maximal CL elicited by 20 pg of oxidized G-LDL was 2.6 x lo4 counts. It was about one fifth of that, 1.3 x 10’ counts, elicited by 1.6 mg of OZ, and was equal to that elicited by 1 pg of PMA (Fig. 3). Nonspecific metal catalyzed CL of MCLA [20] was investigated. Desferrioxamine mesylate, an iron chelating agent, did not affect the intensity of the MCLA-dependent CL evoked by the addition of macrophages (data not shown). 3.3. Uptake of oxidatively macrophages

modijied G-LDL

by

The macrophages that had been incubated with G-LDL exposed to the iron complex developed

H. Kbnuro e/ al. , Arl~ero,sc~l~~o.\ir 118 (IY95) I 8

into foam cells containing lipids (Fig. 4). Little or no endocytic lipid storage was observed in the macrophages incubated with G-LDL, the U-LDL exposed to iron complex, U-LDL, or native LDL (data not shown). 4. Discus&m

We dernonstrated that mouse peritoneal macrophages took up the G-LDL that was denatured by exposure to a low concentration of Fe’ + ADP complex. Whereas body iron is believed to be so tightly bound that there may not be free iron available in vivo under physiological conditions, oxidative stress can free iron from ferritin [22,23]. This finding suggeststhat G-LDL may be oxidized in vivo. Macrophages did not take up the untreated G-.LDL, the U-LDL exposed to Fe3+ ADP complex, U-LDL, or the native LDL. The denaturation of G-LDL is achieved by the oxida-

01234567 INCUBATION

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Fig. 2. Chemiluminescence (CL) curves produced by superoxide radicals released from macrophages. All systems contained 1.25 x lOh mouse resident peritoneal lf3’--2.0 x macrophages. I /‘M MCLA, 150 AIM desferrioxamine mesylate, and one of various LDLs (20 jig of protein) in a total volume of 2 ml of HBSS. The reaction was initiated by the addition of macrophages at 0 min. Curves I --6 indicate CL in the presence of 2.0 x 10h. I.5 x 106. I.0 x IOh, 5.0 x IO’, 2.5 x IO‘, or 1.25 x IO” of macrophages, respectively, and Fe’+ -ADP treated G-LDL. Curves 7 and 8 indicate the CL in the presence of 2.0 x 10” of macrophages and Fe’ + - ADP treated U-LDL or other LDLs (native LDL, native LDL exposed to Fe’ + ADP complex, G-LDL, or U-LDL), respectively. Curve Y is that produced by adding 0.5 /IM SOD to the conditions used for curve I.

CELLS

~10’

Fig. 3. Interrelationships between the maximal intensity of CL. number of macrophages, and various stimuli. Maximal intensity of light is indicated in the presence of various numbers of macrophages stimulated by 20 /(g of protein of 60 11M Fe’ ’ 1 mM ADP treated G-LDL (0) or 60 /rM Fe’+ -I mM ADP treated U-LDL (m). Inset shows the CL stimulated by 1.6 mg of 02 (0) or I /lg of PMA (m). All reaction mixtures contained I HIM MCLA and I50 /IM desferrioxamine mesylate in a total volume of 2.0 ml of HBSS at pH 7.4 and 37°C.

tive modification of G-LDL [9]. The nonenzymatic glycation of peptides or proteins via condensation between glucose and lysine or free amino groups of N-terminus is a well known phenomenon [24]. We previously reported that a low concentration of Fe’ + could be coordinated with endiol group in the Amadori compound derived from glycated peptide, and could be converted to ferry1 iron with a high redox potential [S]. These reaction products initiate the lipid peroxidation of native LDL. causing the simultaneous denaturing of apolipoproteins [8]. This lipid peroxidation and denaturing of apolipoproteins is inhibited by such antioxidants as x-tocopherol or probucol, which suggests that the Fe’ -+-~ADP complex-treated G-LDL produces ferry1 type complex to initiate lipid peroxidation. The resulting products of lipid peroxidation modify apolipoprotein [8,9]. Recently, it has been reported that glucose reacts directly with phospholipids to form advanced glycosylation end products [25]. This compound can initiate lipid peroxidation [25]. It is to be studied whether glucose liked with lysine residues in LDL or with phospholipids is mainly involved in initiation of lipid peroxidation.

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Fig. 4. Appearance of lipid-laden macrophages on light microscopy

We also demonstrated in this study that the oxidized G-LDL present in a low physiological concentration stimulated the macrophages to produce an appreciable amount of superoxide radicals. The untreated G-LDL, U-LDL, native LDL exposed to Fe3+ -ADP complex, and native LDL had no effect on macrophages in this respect. These results clearly indicate that the oxidized G-LDL, which may occur in vivo, is important in the uptake of G-LDL by macrophages, and that the oxidized G-LDL itself stimulates the macrophages to release superoxide radicals which will further free iron from ferritin [22]. We have reported that the oxidized G-LDL was negatively charged strongly [9]. Scavenger receptors of macrophage react well with negatively charged products [26]. Although we did not examine whether uptake of oxidized G-LDL was inhibited competitively by ‘251-labeled LDLs variously modified which were known to be taken up by scavenger receptors, negatively charged the oxidized G-LDL may be recognized and taken up by scavenger receptors of macrophages. At present it

is uncertain whether stimulation of scavenger receptor is directly related to 0, production. A small amount of CL was induced by the U-LDL exposed to Fe3+ -ADP complex. It cannot be ruled out that the U-LDL exposed to the Fe3+ -ADP complex may contain some denatured LDL that can stimulate macrophages; however, we do not know whether such CL was derived from the macrophages. Active oxygen specieshave been linked causally to various diseases, including atherosclerosis. In the development of atherosclerosis, the oxidation or some modification of native LDL is important in its recognition by scavenger receptors of macrophages [l]. Active oxygen species may be required for the oxidation of LDL [lo]. The leukocytes (macrophages, monocytes, and granulocytes) are possible sources of active oxygen species. Leukocytes that are stimulated by chemoattractants injure the adjacent cells and tissue by the release of active oxygen species [27]. Our first observation that the macrophages that were stimulated by low physiological concentra-

H. Kimura ef al. / Athrrosclrrosis

tions of denatured LDL released active oxygen species strongly suggests that the macrophage itself injures the endothelial cells. Our highly sensitive and specific method for detecting 0, [ 191 allowed us to observe generation of superoxide by macrophages. The release of active oxygen species from the macrophages may also be involved in the development of atherosclerosis. Although we used a supraphysiological 200 mM concentration of glucose to obtain G-LDL, the G-LDL used in this study contained about 2.8% of gl:ycated lysine residues in apolipoprotein B [9]. This concentration of glycated lysine residues in G-LDL has been reported in diabetic patients with hyperglycemia [28]. Thus, conditions of our study appear to simulate those reported in diabetic patients. It will be interesting to examine whether LDL from diabetic patients with hyperrelease from glycemia can stimulate 0; macrophages. We found evidence that the oxidatively modified G-LDL, which may be present in vivo, reacts with macrophages. This observation may help to explain why diabetic patients are highly prone to developing atherosclerosis. Acknowledgments

We thank Drs. K. Otsuki, T. Nakajima, K. Funada and K. Kuwabara for helpful discussion. We also thank Y. Kobayashi and M. Hoshino for skillful assistance. References [l] Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223. [2] Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells. J Biol Chem '980;25!?9344. [3] Bunn HF, Gabbay KH, Gallop PM. The glycosylation of hemoglclbin: relevance to diabetes mellitus. Science '987;200:2'. [4] Lyons 1-J. Baynes JW, Patrick JS, Colwell JA, LopesVirella MF. Glycosylation of low density lipoprotein in patients with type 1 (insulin-dependent) diabetes: corre’ations with other parameters of glycaemic control. Diabetologia 1986;29:685.

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[5] Witztum JL, Branks MJ, Mahoney EM, Steinberg D. Glycosylation of low density. lipoprotein (LDL) alters high affinity receptor uptake by fibroblasts but not macrophages. Clin Res 1981;29:427A (abstract). [6] Sakurai T. Tsuchiya S. Superoxide production from nonenzymatically protein. FEBS Lett glycated 1988;236:406. [I] Sakurai T, Sugioka K, Nakano M. 0; generation and lipid peroxidation during the oxidation of a glycated polypeptide, glycated polylysine, in the presence of ironADP. Biochim Biophys Acta 1990;1043:27. PI Sakurai T. Kimura S, Nakano M, Kimura H. The oxidative modification of low density lipoprotein by nonenzymatically glycated peptide-Fe complex. Biochim Biophys Acta 1991;1086:273. (91 Sakurai T, Kimura S, Nakano M, Kimura H. Oxidative modification of g’ycdted low density lipoprotein in the presence of iron. Biochem Biophys Res Commun '99';177:433. [‘Ol Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocytej macrophages during atherosclerosis. Proc Nat’ Acad Sci USA 1987;84:2995. [“I Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms. J Clin Invest 1973;52:741. [‘21 Johnston RB Jr, Godzik CA, Cohn ZA. Increased superoxide anion production by immunologically activated and chemically elicited macrophages. J Med Exp '978;'48:"5. [I.31Sugioka K, Nakano M, Kurashige S. Akuzawa Y, Goto T. A chemiluminescent probe with a cypridina luciferin analog, 2-methyl-6-phenyl-3, I-dihydroimidazo [I ,2-a] pyrazin-3-one, specific and sensitive for 0; production in phagocytizing macrophages. FEBS Lett 1986;197:27. ['41 Schumaker VN, Puppione DL. Sequential flotation ultracentrifugation. Methods Enzymol 1986:128: 155. of serum s51 Noma A, Hata Y, Goto Y. Quantitation apolipoprotein A-l, A-II, B, C-II, C-III, and E in healthy . . Japanese by turbldlmetrlc Immunoassay: reference value, and age- and sex-related differences. Clin Chim Acta ‘991;199:147. [16] Sugioka K, Nakano M. Mechanism of phospholipid peroxidation induced by ferric iron-ADP-adriamycin-co-ordination complex. Biochim Biophys Acta 1982;713:333. [II] Nishida A, Kimura H, Nakano M. Goto T. A sensitive and specific chemiluminescence method for estimating the ability of human granulocytes and monocytes to generate 0;. Clin Chim Acta 1989;179:177. [I81 Kumagai K, ltoh K, Hinuma S, Tada M. Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation. J Immunol Methods 1979:29: 17. [I91 Kimurd H, Nakano M. Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes. FEBS Lett 1988:239:347.

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[20] Nakano M, Kimura H, Hara M, Kuroiwa M, Kato M, Totsune K, Yoshikawa T. A highly sensitive method for determinig both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells. Anal Biochem 1990;181:277. [21] Nakano M, Sugioka K, Ushijima Y, Goto T. Chemiluminescence probe with cypridina luciferin analog, 2methyl-6-phenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3one, for estimating the ability of human granulocytes to generate 0;. Anal Biochem 1986;159:363. [22] Biemond P, Swaak AJ, Beindorff CM, Koster JF. Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Biochem J 1986;239:169. [23] Koster JF, Slee RG. Ferritin, a physiological donor for microsomal lipid peroxidation. FEBS Lett 1986;199:85.

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[24] Robins SP, Bailey AJ. Age-related chages in collagen: The identification of reducible lysine-carbohydrate condensation products. Biochem Biophys Res Commun 1972;48:76. [25] Bucala R, Makita Z, Koschinsky T, Cerami A, Vlassara H. Lipid advanced glycosylation: pathway for lipid oxidation in vivo. Proc Nat1 Acad Sci USA 1993;90:6434. [26] Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Nat1 Acad Sci USA 1979;16:333. [27] Sacks T, Moldow CF, Craddock PR, Bowers TK. Oxygen radicals mediate endothehal cell damage by complement-stimulated granulocytes. J Clin Invest 1978;61:1161. [28] Steinbrecher UP, Witztum JL. Glucosylation of low-density lipoproteins to an extent comparable to that seen in diabetes slows their catabolism. Diabetes 1984;33:130.