Oxidation of low density lipoprotein in hemodialysis patients: effect of dialysis and comparison with matched controls

Atherosclerosis 129 (1997) 199 – 205

Oxidation of low density lipoprotein in hemodialysis patients: effect of dialysis and comparison with matched controls Justin Westhuyzen a,*, David Saltissi b, Helen Healy b a

Department of Pathology, Conjoint Renal Laboratory, Royal Brisbane Hospital, Herston 4029, Brisbane, Australia b Department of Renal Medicine, Royal Brisbane Hospital, Herston 4029, Brisbane, Australia Received 6 June 1996; received in revised form 21 October 1996; accepted 23 November 1996

Abstract End stage renal failure is associated with lipoprotein abnormalities and a high prevalence of premature atherosclerosis. Oxidative modification of low density lipoprotein (LDL) may be promoted by hemodialysis increasing its atherogenicity. The oxidative status of LDL was therefore examined in female subjects before and after routine hemodialysis (HD; n=10) and compared with women of similar age without significant renal disease (n =19). There were no significant differences between the groups in the LDL fatty acid composition, or in the content of reactive amino acid groups (lysine) before or after exposure to Cu2 + . The kinetics of LDL oxidation by Cu2 + showed no significant differences between the groups with respect to the lag time, the level of conjugated dienes before and after oxidation, or the maximal rate of oxidation during the propagation phase. No acute effects of HD were demonstrated. The present study provides no evidence that circulating LDL isolated from HD patients is more extensively modified or more susceptible to oxidation in vitro than gender-matched controls without renal failure. © 1997 Elsevier Science Ireland Ltd. Keywords: Atherosclerosis; Fatty acids; Hemodialysis; Lipid peroxidation; Lipoproteins; Low density lipoprotein; Lysine

1. Introduction Dyslipidemia has been incriminated in the high cardiovascular morbidity and mortality in patients with end stage renal failure (ESRF) [1 – 4]. Hypertriglyceridemia is the most common lipid abnormality with triglyceride enrichment of all lipoprotein fractions: very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) [5,6]. Plasma cholesterol may be within the normal range, but tends to be elevated in patients with marked hypertriglyceridemia [6]. More subtle qualitative changes in lipid composition are increasingly being reported, including a redistribution of cholesterol from HDL to VLDL and IDL, * Corresponding author. Tel.: + 61 7 32531084; fax: +61 7 32521324.

a variety of changes in lipoprotein structure and function, plus increased concentrations of the atherogenic lipoprotein(a) (Lp(a)) in the serum [7]. Recent evidence suggests that modifications of LDL may greatly increase its atherogenicity [8–10]. Oxidative modification is of particular interest, and may involve the apolipoprotein, cholesterol, triglyceride and fatty acid moieties of LDL. Oxidised LDL (Ox-LDL) is taken up more avidly than native LDL by the scavenger receptor of macrophages and is less readily degraded, resulting in cholesterol accumulation and foam cell formation [9]. Ox-LDL can promote many early events in atherogenesis and the presence of foam cells in the intima of blood vessels is an early feature of atherosclerotic plaque both in human vessels and in animal models [8–11]. It is still unclear how LDL with its endogenous antioxidants (principally a-tocopherol, b-carotene and

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phytofluene) undergoes significant oxidation in vivo [8,12,13]. It has been suggested that LDL may be subject to increased oxidative stress during its passage through the arterial wall, particularly during residence in the interstitial matrix [8]. In ESRF patients undergoing hemodialysis (HD), whole blood is regularly exposed to extracellular membranes for prolonged periods, raising the concern of oxidative stress in this patient group [14,15]. Evidence of oxidative stress in HD patients and in particular, lipid peroxidation and decreased antioxidant reserve has been reported [14,16– 27]. It is still unclear whether LDL is oxidatively modified in ESRF patients on HD [28 – 30], and whether HD contributes significantly to the modification of LDL. In the present study we examined the oxidation of LDL in ESRF patients treated with HD. To minimise artefactual changes during the isolation procedure, LDL was isolated by a short-run ultracentrifugation method and compared with LDL isolated from controls of the same gender and age without significant renal disease. The susceptibility of LDL to oxidation in the presence of copper ions was examined in vitro before and after HD. Concentrations of moieties susceptible to oxidation, specifically polyunsaturated fatty acids (PUFA) and reactive amino groups (lysine) on apolipoprotein B were also determined.

2. Methods

2.1. Subjects In view of the known differences in lipids [31] and in vitro susceptibility of LDL to oxidation [30] between the genders, only female patients with ESRF and established on HD for more than 9 months [32] were studied (mean 68.5 months; n =10). Subjects were clinically stable, aged 52–75 years (mean 65.5 years), and not receiving corticosteroids, immunosuppressive treatment or lipid lowering therapy. All patients received erythropoietin and supplemental iron. Serum ferritin concentrations were 5659265 mg/l and transferrin saturation 18.8 95.0% (mean9 S.D.). Patients with documented diabetes, nephrotic syndrome or liver disease were excluded. Three patients with hypothyroidism (2 HD and 1 control) who were on a stable dose of thyroxine ( \ 12 months) were included in the study. None of the subjects received drugs known to directly affect antioxidant status or LDL oxidisability. HD subjects received dietary advice in line (as far as possible) with the National Heart Foundation of Australia. Cholesterol intake was limited and a ratio of polyunsaturated:monounsaturated:saturated fatty acids of 1:1:1 was targeted. Fat comprised 35% of total energy in HD subjects.

Active female members of the community of a similar age (mean 62.8 years) and without documented renal, cardiovascular or cerebrovascular disease or of elevated lipids, served as controls (n= 19). No dietary advice was given. Characteristics of the subjects are summarised in Table 1. The study was approved by the Human Ethics Committee of the Royal Brisbane Hospital.

2.2. Procedure After an overnight fast, blood samples were collected into sequestrene tubes containing 1 mg EDTA/ml from the blood lines of patients prior to the commencement of dialysis, and from the antecubital vein of controls. Routine lipid analysis was carried out on serum samples. To investigate the acute effect of HD treatment, subjects were also sampled on completion of dialysis at least 1 h after the discontinuation of heparin.

2.3. Laboratory methods LDL was isolated within 3 h of collection by short run high speed ultracentrifugation [33]. Briefly, 1.7 ml plasma was adjusted to a density of 1.24 g/ml with potassium bromide and layered under two volumes phosphate buffered saline in 5.1 ml polyallomer centrifuge tubes. LDL was separated by centrifugation at 100 000 rpm (543 000×g) using the Optima TL100 ultracentrifuge and 100.4 rotor (Beckman). The LDL fraction was withdrawn by aspiration through the wall of the centrifuge tube and stored in microfuge tubes at 4°C. The susceptibility of LDL to oxidation in vitro was determined within 24 h by the method of Kleinveld et Table 1 Characteristics of HD subjects and age-matched controls

Gender Age (years) Body weight (kg) BMI (kg/m2) Current smokers Duration of HD (months) Plasma creatinine (mmol/l) HBA1c (% total hemoglobin) Plasma Lipids (mmol/l) Total cholesterol HDL cholesterol LDL cholesterol VLDL cholesterol Triglycerides

HD (n = 10)

Controls (n =19)

Female 65.5 98.7 60.5 97.0 25.1 9 3.9 2/10 68.5 966.1 0.874 90.143 0.083 9 0.009

Female 62.8 9 7.1 64.6 9 9.2 26.7 94.3 0/19 not applicable 0.073 90.012*** 0.0769 0.006*

5.52 91.21 1.11 90.29 3.49 90.98 0.98 90.46 2.44 91.34

6.21 9 1.16 1.97 9 0.84** 3.50 9 1.21 0.73 9 0.20* 1.57 9 0.44*

Values are mean9S.D. Significant difference, * PB0.05. ** PB0.01. *** PB0.001.

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al. (1992) [34]. LDL samples were first desalted and EDTA removed by gel chromatography using PD10 columns (bed volume 9.1 ml; Pharmacia) and the final concentration in the assay adjusted to 0.05 mg LDL protein/ml. Oxidation was initiated by the addition of freshly prepared CuCl2.2H2O solution (final concentration 6 mmol/l). The kinetics of LDL oxidation were monitored at 30°C by the change in absorption at 234 nm in a Beckman DU650 spectrophotometer equipped with a water-jacketed six-position sample changer. The absorbance was recorded every 3 min for 300 min. The oxidation profiles were characterised by the lag time (min), the level of conjugated dienes (CD; mmol/g protein) before and after oxidation and the maximal rate attained during the propagation phase (mmol/min/ g protein) as described previously [34]. Within-run CVs determined on a pool of plasma were 6.3% for lag time, 3.3 and 2.0% for dienes before and after oxidation, and 5.2% for maximal propagation rate. The lysine content (free amino groups) in LDL was determined by the TNBS method as described by Stembrecher et al. [35], and lysine reactivity following incubation with copper ions as described by Liu et al. [36]. Fatty acid composition was determined by gas-liquid chromatography using the one-step lipid extractiontransesterification method of Lepage and Roy [37]. Fatty acid methyl esters were separated on a moderately polar BP225 capillary column (25 m×0.53 mm; SGE, Ringwood, Victoria, Australia) using a Varian 3500 gas chromatograph with on-column injection, flame ionisation detection and hydrogen as the carrier gas (35 cm/s). Column temperature was programmed to rise from 80 to 180°C at 50°C/min and then 1°C/min to 205°C. The final hold time was 3 min. Injector temperatures of 80°C ramping to 230°C at 200°C/min and a detector temperature of 250°C were employed. Fatty acid profiles were recorded on a LDC/Milton Roy thermal printer (Riviera Beach, FL) and identified by comparison with known fatty acid standards (Alltech Associates, Deerfield, IL). Relative peak areas were quantitated using a LDC/Milton Roy Cl-10B integrator and the fatty acid composition expressed as areas percent. Routine serum lipids and creatinine were analysed on a Hitachi 747 autoanalyser using reagents supplied by Boehringer Mannheim (Germany). HBA1c was determined by agar gel electrophoresis using the REP System (Helena Laboratories, TX).

2.4. Statistical analysis Comparisons between groups were made using Student’s t-test or one way analysis of variance (ANOVA) followed by Student-Newman-Keuls method for multiple comparisons. Non-parametric data was analysed by the Mann-Whitney rank sum test or Kruskal-Wallis

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Table 2 Fatty acid composition of LDL Fatty acid

14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 20:5 22:0 22:6 24:0 24:1 16:1, 18:1, 24:1 PUFA

Controls

Hemodialysis patients

ANOVA

Before HD

After RD

P

0.9 9 0.6 20.2 91.2 1.8 9 0.6 6.8 9 0.4 18.3 9 1.7 33.6 9 3.4 0.5 90.3 8.2 91.2 0.6 9 0.5 0.7 90.6 2.2 90.6 0.3 90.3 6.0 91.8 26.1 92.6

0.8 90.2 20.6 91.7 1.1 90.3* 6.7 90.6 20.6 9 3.2 33.2 94.9 0.2 90.3* 7.7 91.1 0.5 90.3 0.4 90.3 1.9 9 0.5 0.4 9 0.3 5.8 9 1.6 27.6 93.4

0.7 9 0.2 20.6 9 1.6 1.1 9 0.3* 6.6 9 06 20.6 9 2.9 33.4 9 5.2 0.2 9 0.2* 7.8 91.3 0.6 90.4 0.8 90.7 2.0 90.6 0.4 90.3 5.0 91.0 26.7 9 3.1

NS NS 0.001 NS NS NS 0.01 NS NS NS NS NS NS NS

45.1 93.33

43.6 94.34

44.1 9 4.4

NS

Areas percent, mean9S.D. * Significantly different to controls, PB0.05.

one way ANOVA on ranks. Relationships between two variables were sought using Pearson’s product-moment correlation coefficient (r). PB0.05 (two-tailed) was considered significant.

3. Results Subject profiles are summarised in Table 1. Two of the HD group smoked (20%) compared to none of the controls. There were no significant differences between the two groups with respect to age, body weight and body mass index (BMI). While there were no differences in the concentration of total cholesterol and LDL cholesterol, HDL cholesterol was lower and VLDL cholesterol and triglycerides significantly higher in the HD group. Uraemic states share many features of insulin resistance syndrome with subtle degrees of glucose intolerance, and this is reflected in the higher HBA1c level in the patient group. The fatty acid composition of LDL is summarised in Table 2. Two fatty acids, namely 16:1 (palmitoleic acid) and 18:3 (linolenic acid) were present in significantly lower concentrations in the LDL of HD subjects. There were no differences in the other PUFA, nor in the sum of the PUFAs 18:2, 18:3, 20:4, 20:5 and 22:6. The percentage of monounsaturated fatty acids (16:1 + 18:1+24:1) was similar in the two groups. The LDL fatty acid composition was not measurably affected by HD. Concentrations of reactive amino groups (lysine content) were similar in all groups, and were unaffected by

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Table 3 Lysine content of LDL and susceptibility of lysine residues to coppercatalysed oxidation Component

Controls

Hemodialysis patients

ANOVA

Before HD

After HD

P

0.599 0.06

0.609 0.07

NS

0.45

0.489 0.1**

NS

lag phase and the LDL content of linolenic acid (C18:3) or arachidonic acid (C20:4).

4. Discussion

Lysine 0.58 90.07 (mmol/mg protein) After 0.42 9 0.10* Cu2+ incubation

9 0.11**

Mean 9 S.D. * Significantly different to pretreatment samples, PB0.001; **PB 0.01.

HD (Table 3). Exposure to Cu2 + ions resulted in a reduction in lysine reactivity in all subject groups. The kinetics of LDL lipid peroxidation in the presence of copper ions, is summarised in Table 4. There were no significant differences between the groups with respect to the duration of the lag phase, nor in the level of conjugated dienes initially present. The maximal oxidation rates were similar in all groups, as was the peak concentration of conjugated dienes formed. When the data from all the subjects was combined (n= 29), the length of the lag phase correlated positively (r= 0.562, P=0.002), and the maximal propagation rate negatively (r= − 0.426, P =0.021) with the concentration of oleic acid (C18:1) in LDL. The lag phase correlated negatively (r = − 0.504, P = 0.005), and the maximal propagation rate (r= 0.555, P =0.002), and the amount of conjugated diene formed during oxidation (r =0.465, P = 0.011) positively with linoleic acid (C18:2), the primary PUFA in LDL (Fig. 1A–C). There was no relationship between the duration of the

Increased levels of the lipid peroxidation product malondialdehyde (MDA) [14,16,18,22,24–26] and decreased levels of the primary lipid-soluble antioxidant a-tocopherol [14,21–23] suggest that HD is associated with oxidative stress. These changes have also been described in LDL in relation to atherosclerosis in the non-ESRF population [8,38]. It has therefore been postulated that oxidative modification of LDL may have a role in the increased susceptibility of ESRF patients to atherosclerosis. The evidence for this hypothesis however, is conflicting [28–30]. Modification of LDL may involve the protein and/or the lipid moieties. The apolipoprotein of LDL, apo B may be post-translationally glycosylated or desialylated (removal of sialic acid groups) [39], or may react with products of lipid peroxidation [9]. Oxidation of the protein moiety can be detected by changed concentrations of the reactive amino acid lysine [36]. In our study, lysine reactivity was similar in HD patients and controls, suggesting there was no difference in degree of apo B oxidation in ESRF. Oxidative modification of lipids has been detected by determining PUFA, formation of conjugated dienes, and increased negative charge and density [9,36]. We determined the fatty acid composition of LDL, since higher PUFA content enhances the susceptibility of LDL to lipid peroxidation [40]. Monounsaturates on the other hand are less susceptible to oxidation [41,42]. Dietary lipids are thus an important consideration in studies of LDL oxidisability [43]. Our HD patients, but not controls, were given dietary advice on fat intake as recommended by the National Heart Foundation (Australia). Calories from fat amounted to about 35% of

Table 4 Susceptibility of isolated LDL to copper-induced oxidation in vitro: lag phase and formation of conjugated dienesa Parameter

Lag time (min) CDb (mmol/g protein) Initial Peak Max oxidation rate (mmol/min/g protein) a b

Controls

Hemodialysis patients

ANOVA

Before HD

After HD

P

91.4 (82.7 – 101.0)

105.2 (90.9 – 112.0)

102.0 (89.7 – 137.6)

NS

211 (191 – 224) 803 (758 – 842)

205 (200 – 231) 806 (749 – 892)

205 (195 – 222) 845 (777 – 934)

NS

9.5 9 2.6

8.89 2.9

9.59 2.3

NS

Medians (25 – 75 percentiles), except oxidation rate (means9 S.D.). CD, conjugated dienes.

NS

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Fig. 1. Correlations between LDL content of linolenic acid (C18:2) and parameters of copper-induced oxidation of LDL in vitro. Since there was no significant difference between the HD patients and controls, the data have been combined (n= 29). A, lag phase; B, the maximal rate; C, the conjugated diene peak formed during oxidation.

total calories. Compared with the study of Sutherland et al. (1995) [30] in which dietary advice on fat intake was not specifically given [30], the sum of LDL monounsaturated fatty acids was higher in our HD group (26.4 versus 22.6%) but PUFAs were lower (43.6 versus 48.4%). Despite its limitations [44], the copper oxidation method has been widely used as a convenient measure of LDL susceptibility to oxidation, and hence atherogenicity. In contrast with the findings of Sutherland et al. who demonstrated significantly slower maximal oxidation rates, lower concentrations of conjugated dienes formed in dialysis patients compared with healthy controls, but no difference in the lag phase [30], we were unable to detect any significant differences in oxidation

203

kinetics between our two groups. It should be noted however that different assay temperatures may have been used in the two studies: 30°C in our study and unstated in Sutherland’s [30], which would affect both lag time and maximal rate of diene production [34]. By further contrast, Maggi et al. [29] found a shorter lag phase and increased propagation rate in their dialysis group compared to controls. Interestingly in the light of our study, Loughrey et al. [28] found an increased resistance to oxidation in their HD patients compared to controls when copper but not when the free radical generator 2,2%-azobis-(2-amidinopropane hydrochloride) (AAPH) was used to initiate oxidation. Copper promotes oxidation of LDL by catalysing the cleavage of preformed lipid peroxides, which generate further lipid peroxides in a chain reaction [45]. LDL which has been depleted of lipid peroxides in vitro is reportedly resistant to oxidation by copper [13,45]. AAPH on the other hand induces oxidation by the generation of peroxyl radicals in the aqueous rather than lipid phase and is not dependent on preformed lipid peroxides [46]. Differences in the findings of the above-mentioned studies may reflect a variety of confounding factors such as differences in the amount of preformed lipid peroxides in the LDL substrate used in the oxidation test [28], the elevating effect of smoking on lipid peroxide levels, and the presence of peripheral vascular disease [47]. Importantly, lengthy isolation procedures for LDL may affect the degree of in vitro lipid peroxidation [48]. We used a rapid 2 h ultracentrifugation method in conjunction with the removal of EDTA by gel filtration. LDL samples were thus subjected to copper oxidation within 24 h of blood collection, minimising the opportunity for artefactual formation of lipid peroxides and degradation of endogenous antioxidants. Regarding the latter, we have previously found serum concentrations (means 9 S.D.) of a-tocopherol (289 6 mmol/l) and b-carotene (0.469 0.21 mmol/l) in ESRF patients to be similar to levels in apparently healthy controls (31 9 9 mmol/l, n= 86 and 0.45 90.35 mmol/l, n= 73 respectively) (unpublished data). Serum retinol concentrations (8.592.2 mmol/l) were 2–3 times higher than in controls (3.2 9 1.0 mmol/l, n=73, PB0.001). In our patient population therefore, concentrations of the LDL-associated antioxidants are ‘normal’ or raised, which may explain the lack of difference in LDL susceptibility to oxidation between the ESRF patients and controls. Since HD is most probably associated with some degree of oxidative stress, the significance of this stress to the integrity and biological function of LDL on the one hand, and the propensity of these subjects to develop atherosclerosis and vascular disease on the other, needs to be further elucidated. Indeed, it may well be that in vitro circulatory studies are inappropriate here and that techniques for investigating the prop-

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erties of modified LDL directly in vascular tissue are required.

Acknowledgements Colleen Morgan RN, Senior Research Assistant, is thanked for her work in subject recruitment and liaison. The authors also thank the dialysis patients and bowlers at the Everton Park Bowls Club (Brisbane) for their willing participation in this study.

References [1] Brown JH, Hunt LP, Vites NP, Short CD, Gokal R, Mallick NP. Comparative mortality from cardiovascular disease in patients with chronic renal failure. Nephrol Dial Transplant 1994;9:1136 – 1142. [2] Losowski MS, Kenward DH. Lipid metabolism in acute and chronic renal failure. J Lab Clin Med 1968;71:736 – 745. [3] Lowrie E, Lazarus M, Mocelin A, Bailey G, Hampers C, Wilson R, Merril J. Survival of patients undergoing chronic hemodialysis and renal transplantation. N Engl J Med 1973;288:863 –868. [4] Senti M, Romero R, Pedro–Botet J, Pelegri A, Nogues X, Rubies-Prat J. Lipoprotein abnormalities in hyperlipidemic and normolipidemic men on hemodialysis with chronic renal failure. Kidney Internat 1992;41:1394–1399. [5] Appel G. Lipid abnormalities in renal disease. Kidney Int 1991;39:169 – 183. [6] Attman P-O, Alaupovic P. Lipid abnormalities in chronic renal insufficiency. Kidney Int 1991;39(Suppl 31):516–523. [7] Webb AT, Reaveley DA, O’Donnell M, O’Connor B, Seed M, Brown EA. Lipids and lipoprotein (a) as risk factors for vascular disease in patients on renal replacement therapy. Nephrol Dial Transplant 1994;5:354–357. [8] Witztum JL. Susceptibility of low-density lipoprotein to oxidative modification. Am J Med 1993;94:347–349 (Editorial). [9] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915 –924. [10] Witztum JL. Role of oxidised low density lipoprotein in atherogenesis. Br Heart J 1993;69(Suppl):12S–18S. [11] Aviram M. Oxidative modification of low density lipoprotein and its relation to atherosclerosis. Isr J Med Sci 1995;31:241 – 249. [12] Sies H. Oxidative stress: introductory remarks. In: Sies H (ed). Oxidative stress. London: Academic Press 1985:18. [13] Halliwell B. Oxidation of low-density lipoproteins: questions of initiation, propagation, and the effect of antioxidants. Am J Clin Nutr 1995;61(Suppl):670S–677S. [14] Loughrey CM, Young IS, Lightbody JH, McMaster D, McNamee PT, Trimble ER. Oxidative stress in haemodialysis. Q J Med 1994;87:679–683. [15] Westhuyzen J, Adams CE, Fleming SJ. Evidence for oxidative stress during in vitro dialysis. Nephron 1995;70:49 – 54. [16] Miguel A, Miguel A, Linares M et al. Evidence of an increased susceptibility to lipid peroxidation in red blood cells of chronic renal failure patients. Nephron 1988;50:64–65. [17] Schmidtmann S, V. Baehr R, Precht K. Free radicals induce increase lysis of red bloodcells after haemodialysis. Nephrol Dial Transplant 1990;5:600–603.

[18] Richard MJ, Arnaud J, Jurkovitz C et al. Trace elements and lipid peroxidation abnormalities in patients with chronic renal failure. Nephron 1991;57:10 – 15. [19] Toborek M, Wasik T, Drozdz M, Klin M, Magner-Wrobel K, Kopieczna-Grzebieniak E. Effect of hemodialysis on lipid peroxidation and antioxidant system in patients with chronic renal failure. Metabolism 1992;41:1229 – 1232. [20] Kuroda M, Asaka S, Tofuku Y, Takeda R. Serum antioxidant activity in uraemic patients. Nephron 1985;41:293–298. [21] Ono K. Effects of large dose vitamin E supplementation on anaemia in haemodialysis patients. Nephron 1985;40:440–445. [22] Taccone-Gallucchi M, Giardini O, Lubrano R et al. Red blood cell peroxidation in predialysis renal failure. Clin Nephrol 1987;27:238 – 241. [23] Taccone-Gallucci M, Lubrano R, Bandino D et al. Discrepancies between serum and erythrocyte concentrations of vitamin E in hemodialysis patients: role of HDL-bound fraction of vitamin E. Artif Organs 1988;12:379 – 381. [24] Fillit H, Elion E, Sullivan J, Sherman R, Zabriskie JB. Thiobarbituric acid reactive material in uraemic blood. Nephron 1981;29:40 – 43. [25] Dasgupta A, Hussain S, Alunad S. Increased lipid peroxidation in patients on maintenance hemodialysis. Nephron 1992;60:56– 59. [26] Taylor JE, Scott N, Bridges A, Henderson IS, Stewart WK, Belch JJF. Lipid peroxidation in continuous ambulatory dialysis patients. Peritoneal Dial Int 1992;12:252 – 256. [27] Giardini O, Taccone-Gallucci M, Lubrano R et al. Evidence of red blood cell membrane lipid peroxidation in haemodialysis patients. Nephron 1984;36:235 – 237. [28] Loughrey CM, Young IS, McEneny J et al. Oxidation of low density lipoprotein in patients on regular haemodialysis. Atherosclerosis 1994; 110:185 – 193. [29] Maggi E, Bellazzi R, Falaschi F et al. Enhanced LDL oxidation in uremic patients: an additional mechanism for accelerated atherosclerosis? Kidney Int 1994;45:876 – 883. [30] Sutherland WHF, Walker RJ, Ball MJ, Stapley SA, Robertson MC. Oxidation of low density lipoproteins from patients with renal failure or renal transplants. Kidney Int 1995;48:227–236. [31] Schaefer EJ, Lichtenstein AH, Lamon-Fava S, McNamara JR, Ordovas JM. Lipoproteins, nutrition, aging, and atherosclerosis. Am J Clin Nutr 1995;61(Suppl):726S – 740S. [32] Avram MM, Fein PA, Antignani A et al. Cholesterol and lipid disturbances in renal disease: the natural history of uraemic dyslipidaemia and the impact of haemodialysis and continuous ambulatory peritoneal dialysis. Am J Med 1989;87:55N–60N. [33] Sattler W, Bone P, Stocker R. Isolation of human VLDL, LDL, HDL and two subclasses in the TL-100 tabletop centrifuge using the TLA-100.4 rotor. Beckman Tech Inf 1992. [34] Kleinveld HA, Hak-Lemmers HLM, Stalenhoef AFH, Demacker PNM. Improved measurement of low-density-lipoprotein susceptibility to copper-induced oxidation: application of a short procedure for isolating low-density lipoprotein. Clin Chem 1992;38:2066 – 2072. [35] Stembrecher UP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism. Arteriosclerosis 1987;7:135–143. [36] Liu K, Cuddy E, Pierce GN. Oxidative status of lipoproteins in coronary disease patients. Am Heart J 1992;123:285–290. [37] Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 1986;27:114–120. [38] Esterbauer H, Wa¨g G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull 1993;49:566 – 576. [39] Sobenin IA, Tertov VV, Orekov AN, Smirnov VN. Synergistic effect of desialylated and glycated low density lipoproteins on cholesterol accumulation in cultured smooth muscle intimal cells. Atherosclerosis 1991;89:151 – 154.

J. Westhuyzen et al. / Atherosclerosis 129 (1997) 199–205 [40] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LFL. Free Radic Biol Med 1992;13:341–390. [41] Kleinveld HA, Naber AHJ, Stalenhoef AFH, DeMacher PNM. Oxidation resistance, oxidation rate and extent of oxidation of human low-density lipoprotein depend on the ratio of oleic acid content to linoleic acid content: studies in vitamin E deficient subjects. Free Radical Biol Med 1993;15:273–280. [42] Reaven P, Parthasarathy S, Grasse BJ, Miller E, Steinberg D, Witztum JL. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoproteins to oxidative modification in mildly hypercholesterolemic subjects. J Clin Invest 1993;91:668 – 676. [43] Abbey M, Belling GB, Noakes M, Hirata F, Nestel PJ. Oxidation of low-density lipoproteins: intraindividual variability and the effect of dietary linoleate supplementation. Am J Clin Nutr 1993;57:391 – 398.

.

205

[44] Stocker R. Lipoprotein oxidation: mechanistic aspects, methodological approaches and clinical relevance. Curr Opin Lipidol 1994;5:422 – 433. [45] Thomas CE, Jackson RL. Lipid hydroperoxide involvement in copper-dependent and independent oxidation of low density lipoproteins. J Pharmacol Exp Ther 1991;256:1182–1188. [46] Stocker R, Bowry VW, Frei B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does a-tocopherol. Proc Natl Acad Sci USA 1991;88:1646 – 1650. [47] Sanderson KJ, Van Rij AM, Wade CR, Sutherland WHF. Lipid peroxidation of circulating low density lipoproteins with age, smoking and in peripheral vascular disease. Atherosclerosis 1995; 118:45 – 51. [48] Menasche P, Pasquier C, Bellucci S, Lorente P, Jaillon P, Piwnica A. Deferoxamine reduces neutrophil-mediated free radical production during cardiopulmonary bypass in man. J Thorac Cardiovasc Surg 1988;96:582 – 589.