Oxidation resistance of LDL is correlated with vitamin E status in β-thalassemia intermedia

Atherosclerosis 137 (1998) 429 – 435

Oxidation resistance of LDL is correlated with vitamin E status in b-thalassemia intermedia Luisa Tesoriere a, Daniele D’Arpa a, Aurelio Maggio c, Vita Giaccone a, Elisa Pedone b, Maria A. Livrea a,* a

Istituto di Farmacologia e Farmacognosia, Uni6ersita` di Palermo, Via C. Forlanini, 1, 90134, Palermo, Italy b Dipartimento di Chimica e Tecnologie Farmaceutiche, Uni6ersita` di Palermo, 90134, Palermo, Italy c Ser6izio Talassemia, Ospedale ‘V. Cer6ello’, 90134, Palermo, Italy Received 23 July 1997; received in revised form 21 November 1997; accepted 28 November 1997

Abstract The alteration of the oxidant/antioxidant balance may affect the susceptibility of low density lipoproteins (LDL) to oxidation in haemolytic disorders such as thalassemia. Thirty patients affected by b-thalassemia intermedia were examined, and compared with age-matched healthy controls. The mean amount of vitamin E in the thalassemic LDL was lower than control (p B0.0001), either when it was calculated on the base of LDL protein (61% decrease) or cholesterol (25% decrease). The LDL resistance to Cu2 + -induced oxidation, evaluated as the length of the lag phase before the onset of conjugated diene (CD) lipid hydroperoxide production, was 20% lower than control. Other parameters of LDL susceptibility to oxidation, such as the rate of lipid peroxidation, Rp, and the total amount of conjugated dienes produced, CDmax, were only slightly lower than control, which can be explained by a lower content of peroxidable lipids in the thalassemic LDL. Total LDL cholesterol was 1.08 × 103 and 2.07× 103 mol/mol LDL in thalassemic and in control LDL, respectively. The length of the lag phase in thalassemic LDL shows a strongly positive correlation with its vitamin E content (r =0.732; p B0.0001). The r 2-value of 0.53 provides evidence that more than 50% of the lag phase is determined by vitamin E. Oxidizability of LDL lipids may explain 22-24% of the lag phase, as calculated by the inverse correlation between the length of the lag phase and CDmax (r = −0.474; p= 0.008; r 2 = 0.22) and Rp (r= −0.499; p=0.005; r 2 =0.24). In multiple regression analysis, the lag phase was predictable to 66% by vitamin E plus CDmax, and to 60% by vitamin E plus Rp. Plasma vitamin E was 53% lower in thalassemia patients compared to control and positively correlated with vitamin E in the LDL (r=0.677; pB0.0001). None of the correlations above were observed in control subjects. In conclusion, b-thalassemia is associated with very low levels of vitamin E in plasma and in LDL, a condition that renders these particles more susceptible to in vitro oxidative modification and may account for atherogenesis-related vascular diseases described in thalassemia. The present data on a statistically significant correlation between abnormally low vitamin E and oxidizability of LDL contribute substantially to the hypothesis that vitamin E is a pathophysiologically important determinant of antioxidative protection of LDL. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Low density lipoproteins; LDL oxidizability; Oxidative stress; Thalassemia; Vitamin E

1. Introduction Precipitation of unpaired Hb chains, followed by release of hemin and redox-active iron, leading to gen* Corresponding author. Tel.: +39 91 6512262; fax: + 39 91 6512469; e-mail: [email protected]

eration of reactive oxygen species, brings about disturbance of the pro/antioxidant balance and hemolysis in disorders known as b-thalassemia [1–4]. The oxidative stress, which is further exacerbated by the secondary transfusion-dependent iron overload, depletes all plasma antioxidants and produces a marked elevation of lipid and protein peroxidation products in the severe

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Table 1 LDL vitamin E and cholesterol and oxidizability indexes in b-thalassemia intermedia patients and controls

Vit. E (nmoles/mg LDL prot) (moles/mol LDL) Cholesterol (mmol/mmol LDL) Lag time (min) Rp (nmol CD hydroperoxides/min/mg LDL prot) CDmax (nmol CD hydroperoxides/mg LDL prot) LDL vit. E/cholesterol ratio (mmol/mmol)

Patients n =30

Controls n = 30

p

6.079 3.3 3.03 91.65 1.08 90.09 61.02 920.3 7.0 91.8 181.19 9 37.6 2.80 90.3

15.38 9 2.2 7.69 91.1 2.07 90.12 76.30 98.9 7.36 90.98 210.34 9 32.7 3.70 9 0.5

B0.0001 B0.0001 B0.0001 0.004 0.088 0.002 B0.0001

Values are the mean 9S.D. of n determinations performed in duplicate on LDL samples from different subjects. Each thalassemia patient contributed the mean of two values determined during nine months of observation. p value was determined by the Student’s t-test.

b-thalassemia major [5]. Beta thalassemia intermedia is a clinical definition applied to thalassemia patients who present a milder clinical course than those with thalassemia major, with a more or less marked anemia that does not require treatment with regular blood transfusions. In spite of the ample information available on the oxidative stress to red blood cells of thalassemia patients, the oxidative status of low density lipoprotein (LDL) and its susceptibility to oxidation is not known. There is incresing evidence suggesting that oxidation of LDL is involved in the development of atherogenesis-related pathologies [6,7], although little is known about the mechanism by which the LDL becomes oxidized in vivo. Due to depletion of antioxidant defence, circulating LDL in thalassemia patients may be more susceptible to oxidation. In addition, alterations of iron homeostasis, which is peculiar to the disease, may also contribute to promote oxidative modification of LDL [8,9], thereby increasing its atherogenic potential. Atherogenesis-related vascular alteration have been reported in b-thalassemia intermedia patients [10– 12]. Recent studies have shown that plasma LDL from diabetic patients and smokers, both subjects acknowledged at risk for atherosclerosis, have increased oxidizability in vitro [13 – 16], which can be related to antioxidants such as vitamin E [17]. These observations prompted us to analyze the vitamin E status and to investigate LDL resistance to lipid peroxidation induced by Cu2 + in b-thalassemia intermedia patients, in comparison with age-matched healthy controls.

2. Experimental

2.1. Materials Alpha-tocopherol was from Sigma (St. Louis, MO). All other materials and solvents were of the highest purity

or high-performance liquid chromatography (HPLC) grade.

2.2. Subjects Thirty patients affected by b-thalassemia intermedia, who underwent occasional transfusions, 16 females and 14 males, aged 22 to 50 (36 9 9), not smokers, were recruited with consent for this study and were under observation for 9 months. All of patients had been characterized for beta globin gene mutation. The subjects underwent a complete check-up at the Servizio Talassemia of ‘V. Cervello’ Hospital in Palermo. No patient was diabetic, nor HCV positive, nor showed abnormal levels of alanine or aspartate aminotransferases. Serum ferritin was evaluated every fourth months. Patients were not under lipid altering medications. Healthy controls, habitual blood donors, 16 females and 14 males, aged 24 to 50 (329 7), were not smokers, nor were under any medical treatment. All patients and controls did not receive any vitamin supplementation and their food intake was that conceivable for a Mediterranean diet. Blood was collected by venipuncture after overnight fasting. Blood from transfused patients (n= 20, B3–4 transfusions during the period of observation) was obtained at least 30 days after transfusion, to avoid influences on the plasma level of vitamin E.

2.3. Preparation of LDL LDL (d 1.019–1.063 g/ml) was isolated from EDTA plasma by stepwise ultracentrifugation at 4°C in a Beckman L8-70 M ultracentrifuge fitted with a 50 Ti rotor using potassium bromide for density adjustments, according to Kleinveld [18]. EDTA and salts were removed from LDL by gel filtration on Sephadex G-25 Medium (Pharmacia), just before analysis. Proteins were determined by the Bio Rad colorimetric method [19]. To prevent autoxidation reactions, LDL were used immediately or after overnight storage at −70°C.

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Fig. 1. Correlation between the length of lag phase and content of vitamin E in LDL from healthy subjects (A) and thalassemia intermedia patients (B), exposed to copper oxidation. LDL samples were isolated from controls and thalassemia patients and processed as described in Methods to measure the lag phase and quantitate vitamin E concentration. Each point is the mean of two determinations performed at 3 months intervals (A: r =0.233; p= 0.21; B: r= 0.732; p B0.0001).

2.4. LDL oxidation and oxidizability indices LDL protein (0.2 mg/ml) was incubated in oxygensaturated Chelex-treated phosphate saline buffer (PBS, 5 mM, pH 7.4) supplemented with 10 mM CuCl2 as a prooxidant, in a 1-ml quartz cuvette. The oxidation was followed by continuously monitoring the formation at 37°C of conjugated diene (CD) lipid hydroperoxides at 234 nm [20], using a Beckman DU 640 spectrophotometer. The oxidation resistance of LDL was estimated in terms of the period when no oxidation occurs (lag phase), determined as the intercept of the extrapolations of the parts of the curve representing the lag and propagation phases. The rate of oxidation, Rp, was determined from the plot of CD versus time, from the slope of the peroxidation curve during the propagation phase and expressed as nanomoles of hydroperoxides formed per min and per mg of LDL protein, using a molar absorbance coefficient of 28000 [21]. CDmax is the maximal amount of dienes formed, expressed as nmoles per mg LDL prot.

2.5. Analysis of 6itamin E Alpha-tocopherol in plasma and homologous LDL was evaluated by extracting 0.2 ml plasma and 0.1 mg LDL protein, diluted to 1.0 ml with PBS, with two volumes of absolute ethanol and eight volumes of petroleum ether. The organic extracts were gathered, dried under nitrogen, resuspended in several microliters of methanol, and analyzed by a Supelco Supelcosil™ LC-18 column (0.46× 25 cm), with the same solvent at 1.0 ml/min and spectrophotometric revelation at 290 nm. Under the conditions described, a-tocopherol eluted after 12 min. Quantitation was by reference to a standard curve constructed with 1 – 100 ng amounts of a-tocopherol. All procedures were performed under

dim red light to avoid incidental photooxidation of lipids by low energy quanta of visible light and to preserve the light sensitive vitamin E.

2.6. Clinical chemistry determinations Triglycerides, total and HDL-cholesterol were evaluated from fasting individuals by commercial analytical kits (Sigma, St. Louis, MO). Concentrations of plasma LDL-cholesterol were calculated using the Friedwald formula [22]. Cholesterol in samples of isolated and desalted LDL was determined with the same method as plasma total cholesterol. Ferritin was determined by an enzyme-immunoassay (Abbott, North Chicago, IL).

2.7. Statistic analysis Conventional methods were used for calculation of means and standard deviations. Comparison between controls and thalassemia patients was performed by the unpaired Student’s t test. Correlation coefficients between covariates were calculated by Pearson’s test. Multiple regression analysis was used to investigate the influence of different variables on LDL resistance to oxidation. All statistical analyses were performed with the computer program 4.3 Statsoft for Windows.

3. Results Polyunsaturated LDL fatty acids (PUFAs) are protected against oxidation by a number of antioxidants, with vitamin E being the major antioxidant carried in these particles [23]. Amount and activity of antioxidants, concentration of cholesterol and total amount of oxidizable substrate in the LDL particle determine the overall LDL resistance to oxidation.

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Fig. 2. Correlation between the length of lag phase and rate of peroxidation (Rp, A) and maximal amount of CD produced (CDmax, B) in LDL from thalassemia patients exposed to copper oxidation. Lag phase and Rp are evaluated as described under Methods. Each point is the mean of two determinations performed at 3 months intervals (A: r = − 0.499; p = 0.005; B: r= − 0.474; p =0.008).

Vitamin E and total cholesterol content and oxidizability indices in LDL from thalassemia patients and controls are reported in Table 1. Amounts of vitamin E (1.24 –13.6 nmoles per mg LDL prot) were significantly lower in LDL from thalassemia patients than controls (11 – 19 nmoles per mg LDL prot). Similarly, cholesterol measured in LDL from thalassemia patients was about 50% with respect to control LDL (Table 1). Resistance of LDL to Cu2 + -induced oxidation was significantly different between the two groups (Table 1). Lag phases ranged from 30 – 99 min in the patients to 60 – 100 min in healthy subjects. The ratio of vitamin E to cholesterol in LDL is also a predictor of LDL susceptibility to metal ion-dependent oxidation [24]. The value calculated for thalassemia patients was significantly lower than control (Table 1). On the other hand, the rate of diene production, as well as the maximal amount of CD produced after Cu2 + oxidation was slightly lower in LDL from thalassemia patients than in controls (Table 1). No correlation was observed in healthy subjects between the amount of vitamin E in LDL and its resistance to oxidative stress, as measured by the duration of lag phase (Fig. 1A). On the contrary, the resistance of LDL from thalassemia patients to Cu2 + -induced oxidation was strongly correlated with the endogenous vitamin E content (r =0.732; p B 0.0001, Fig. 1B). An inverse correlation was found between the length of lag phase and either the rate of diene production in LDL from thalassemia patients (r = − 0.499; p = 0.005, Fig. 2A), or the maximal amount of conjugated dienes produced (r = −0.474; p= 0.008, Fig. 2B). Table 2 shows the plasma level of vitamin E, the lipid status and the level of ferritin of patients and controls. The amount of vitamin E in plasma from b-thalassemia patients was significantly lower than in healthy subjects. The lowest and the highest amounts were 2.35 and 19.1 mM, whereas in controls they ranged 12 – 27.5 mM. The

total, HDL and LDL-cholesterol in the patients were 50%, 60% and 36% of the control value, respectively, whereas triglycerides did not differ significantly (Table 2). A similar pattern of plasma lipids and lipoproteins has been observed by other researchers [25,26]. Although both vitamin E and cholesterol were markedly lower than control, the ratio vitamin E to cholesterol was slightly, but significantly, decreased (Table 2). As the main result of increased intestinal iron absorption, ferritin levels of the patients were significantly higher than controls (range 90–950 ng/ml; mean 6509308, Table 2). The plasma vitamin E of b-thalassemia patients was significantly correlated with the vitamin E content of LDL (r= 0.677; pB 0.0001, Fig. 3A), whereas such a correlation did not occur in controls (r= − 0.126; p= 0.506, Fig. 3B).

4. Discussion Oxidation of LDL is considered an important factor in the onset and progression of atherosclerotic lesions [9]. This prompted development of assays aimed at assessing the oxidizability of LDL, both in healthy people, to eventually predict atherosclerotic risk, and also in subjects with clinical evidence of atherosclerotic lesions, where the LDL proneness to oxidation has been taken as an index of severity of the lesions [27] or a potential marker of antioxidant therapies [28]. Among the methods used to quantify the susceptibility of LDL to oxidation, the determination of the lag phase before the rise of formation of CD lipid hydroperoxides in isolated LDL exposed to copper ions [20] is widely used. It can be argued that Cu2 + -induced oxidation of LDL is unlikely to occur in vivo, since physiological copper concentrations are much lower than those used in in vitro studies. However, it has been

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Table 2 Hematological data of b-thalassemia intermedia patients and healthy controls

Vit. E (mmol/l) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Vit. E/Cholesterol ([mmol/l]/[mmol/l]) Triglycerides (mmol/l) Ferritin (ng/ml)

Patients n =30

Controls n =30

p

8.959 4.06 2.8 9 0.5 0.85 9 0.16 1.32 9 0.10 3.19 9 0.4 1.15 90.16 650 9308

18.9 9 4 5.5 90.17 1.40 9 0.13 3.60 9 0.4 3.43 90.44 1.17 9 0.12 85 910

B0.0001 B0.0001 B0.0001 B0.0001 0.031 n.s. B0.0001

Values are the mean 9 S.D. of n determinations performed in duplicate on LDL samples from different subjects. Each thalassemia patient contributed the mean of two values determined during nine months of observation. p value was determined by the Student’s t-test. n.s., non-significant, with p = 0.568

recently shown that the susceptibility of LDL to oxidation by copper ions is closely related to its proneness to biological modification [29]. On the other hand, higher serum concentrations of copper ions are associated with accelerated progression of atherosclerosis [30] and detectable levels of redox active transition metals, including copper and iron, in human atherosclerotic lesions [31] support a role for these metals in vivo. In thalassemia patients, where an imbalance of iron metabolism occurs, a metal ion-dependent mechanism may effectively contribute to LDL oxidation in vivo. As evaluated by the length of the lag phase, LDL from thalassemia patients showed an increased susceptibility to Cu2 + -induced formation of conjugated dienes. Consistent with this finding, the decreased ratio LDL-vitamin E/LDL-cholesterol was also predictive of increased susceptibility of LDL to oxidation. Nevertheless, the mean overall rate of formation of peroxides during the propagation phase, Rp, and the maximum amount of peroxides formed, CDmax, were paradoxically slightly lower than control. It has been shown that the total amount of oxidable substrate in the LDL particle is the major determinant of Rp and CDmax [32], while an inverse correlation occurs between Rp and cholesterol [24], in Cu2 + -oxidized LDL. Although we did not measure the amount of unsaturated lipids, the marked depletion of total cholesterol in LDL, which can also be taken as a reliable index of low levels of PUFAs [32], may account for the low Rp value in thalassemic LDL, with respect to controls containing about twice of cholesterol. Several studies addressed the question of how important vitamin E, the major antioxidant in the LDL particle [23] is in preventing or limiting its oxidative damage. In spite of the essential role of this lipophilic antioxidant in protecting against lipid peroxidation, no correlation has been found in healthy subjects in ours, as well as in previous studies from other laboratories, between the resistance of LDL, measured as lag time, and its basal amount of vitamin E [18,23,33 – 35]. Thus, at ‘normal’ range of vitamin E in LDL, individual differences of LDL oxidizability are primarily due to components other than vitamin E such as content of PUFAs, cholesterol

to protein ratio, distribution of vitamin E in plasma lipoproteins and the amount of other LDL antioxidants, i.e. the so-called vitamin E-independent component of lag phase [36]. On the other hand, the role of vitamin E emerges by eliminating the influences of other factors, either by keeping them constant [37] or when, after massive vitamin E supplementation, leading to increase of vitamin E in LDL, individual factors become relatively less important [35,36]. In this context our thalassemia intermedia patients offer the opportunity to make observations in an inadequately poor range of vitamin E. The etiopathogenesis of the disease entails a remarkable oxidative stress associated with the precipitation of haemoglobin chains inside red blood cells, which brings about a marked depletion of all blood antioxidants, including vitamin E. At the same time, vitamin E content of LDL is much lower than control and strongly correlates with the resistance of LDL to Cu2 + -induced oxidation. Under these conditions, all individual factors involved in the initial chain reaction appear of minor importance and vitamin E is the last and long-lasting defence. Vitamin E appears to play a prominent, but not exclusive, role in the resistance of LDL to oxidation. The coefficient of correlation between the duration of lag phase and vitamin E content in LDL was 0.732. This r-value correspond to a r 2-value of 0.53, indicating that more than 50% of the lag time depends on the vitamin E amount. Taking into account that total peroxidable lipids determine the rate of diene production [24,32], 20–24% of the lag period may be accounted for by the lipid composition and amount, as calculated from the correlation between the duration of lag phase and the maximal amount of conjugated dienes produced (r= − 0.474, i.e. r 2 = 0.22) or the rate of diene production Rp (r= −0.499, r 2 = 0.24). When considered in conjunction with vitamin E, the latter variables predict 66 and 60%, respectively, of the lag phase. According to Frei and Gaziano [24], the amount of endogenous or seeding lipid hydroperoxides in LDL may affect the lag phase in copper-oxidated LDL. Other results from this laboratory show high levels of

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Fig. 3. Correlation between plasma vitamin E and vitamin E in homologous LDL from thalassemia patients (A) and healthy controls (B). Extraction and analysis of vitamin E was as described in Methods. Each point is the mean of two determinations performed at 3 months intervals (A: r=0.677; p B0.0001; B: r= − 0.126; p= 0.506).

CD hydroperoxides in thalassemic LDL [38]. Utilizing those values we calculated that no more than 4% of the lag phase may be accounted for by endogenous hydroperoxides in thalassemic LDL. It is not possible to predict from the a-tocopherol content of a normal LDL the length of its lag phase and its oxidation resistance. In addition, previous studies [39] and our own suggest that in normal non-supplemented individuals the a-tocopherol content of LDL does not correlate with plasma vitamin E. One reason is the interindividual variability in the distribution of vitamin E among all plasma lipoprotein fractions. The latter appears to be dependent on plasma LDL-cholesterol concentrations [40] and is affected by the HDL/ LDL ratio [41]. All these factors concur to a different pattern in thalassemia patients, the lipid status of whom is markedly varied compared to controls. We found that plasma level of vitamin E is strongly correlated with the level of the vitamin in LDL, suggesting that it may also be a suitable marker of LDL susceptibility to oxidative stress. Peroxidizability of LDL by copper ions per se could be not predictive of the individual proneness to atherogenic processes [42] of apparently healthy subjects, however studies in patients with cardiovascular diseases report increased LDL peroxidizability [27,34,43,44], concomitant with low levels of vitamin E in LDL [43,45] and/or in plasma [45 – 47]. In some of these studies a correlation was demonstrated between severity of coronary artery disease and LDL susceptibility to oxidation [27], or with the amount of LDL vitamin E [45]. The enhanced LDL susceptibility to oxidation and the strong vitamin E depletion observed in thalassemia patients could be of relevance in the development of atherosclerotic lesions in these patients. Autoptic or epidemiological data on a large scale, confirming the proneness of thalassemia patients to atherosclerosis, as related to vitamin E depletion, are not available at this

time. 20 of the patients participating in the present study are now checked to disclose evidence of coronary or peripheral atherosclerosis.

Acknowledgements The cooperation of the staff of the ‘Servizio Talassemia’, Ospedale V. Cervello di Palermo is gratefully acknowledged. This research has been funded by Assessorato Sanita` Regione Sicilia.

References [1] Chiu DT-Y, Van den Berg J, Kuypers FA, Hung I-J, Wei J-S, Liu T-Z. Correlation of membrane lipid peroxidation with oxidation of hemoglobin variants: possibly related to the rates of hemin release. Free Rad Biol Med 1996;21:89. [2] Van Dyke BR, Saltman P. Hemoglobin: a mechanism for the generation of hydroxyl radicals. Free Rad Biol Med 1996;20:985. [3] Grinberg LN, Rachmilewitz EA, Kitrossky N, Chevion M. Hydroxyl radical generation in b-thalassemic red blood cells. Free Rad Biol Med 1995;18:611. [4] Scott MD, Van den Berg JJM, Repka T, Rouyer-Fessard Ph, Hebbel RP, Beuzard Y, Lubin B. Effect of excess a-hemoglobin chains on cellular and membrane oxidation in model b-thalassemic erythrocytes. J Clin Invest 1993;91:1706. [5] Livrea MA, Tesoriere L, Pintaudi AM, Calabrese A, Maggio A, Freisleben H-J, D’Arpa D, D’Anna R, Bongiorno A. Oxidative stress and antioxidant status in b-thalassemia major: iron overload and depletion of lipid soluble antioxidants. Blood 1996;88:3608. [6] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modification of low density lipoprotein that increase atherogenicity. N Engl J Med 1989;320:915. [7] Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Rad Biol Med 1996;20:707. [8] Heinecke JW, Rosen H, Chait A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest 1984;74:1890.

L. Tesoriere et al. / Atherosclerosis 137 (1998) 429–435 [9] Araujo JA, Romano EL, Brito BE, Parthe´ V, Romano M, Bracho M, Montano RF, Cardier J. Iron overload augments the development of atherosclerotic lesions in rabbits. Arterioscler Thromb Vasc Biol 1995;15:1172. [10] Butthep P, Bunyaratvej A, Funahara Y, Kitaguchi H, Fucharoen S, Sato S, Bhamarapravati N. Alterations in vascular endothelial cell-related plasma proteins in thalassaemic patients and their correlation with clinical symptoms. Thromb Haemost 1995;74:1045. [11] Wong V, Yu Y, Laing RHS, Tso WK, Li AMC, Chan TK. Cerebral thrombosis in b-thal/Hb E disease. Stroke 1990;21:812. [12] Sonakul D, Suwanagool P, Sirivaidyapong P, Fucharoen S. Distribution of pulmonary thromboembolism lesions in thalassemic patients. Birth Defects 1988;23(5A):375. [13] Harats D, Ben-Naim M, Daback Y, Hollander G, Stein O, Stein Y. Cigarette smoking renders LDL susceptible to peroxidative modification and enhanced metabolism by macrophages. Atherosclerosis 1989;79:245. [14] Scheffler E, Huber L, Fruhbis J, Schulz I, Ziegler R, Dresel HA. Alteration of plasma low density lipoprotein from smokers. Atherosclerosis 1990;82:261. [15] Nishigaki I, Haghara M, Tsunekawa H, Maseki M, Yagi K. Lipid peroxide levels of serum lipoprotein fractions of diabetic patients. Biochem Med 1981;25:373. [16] Hunt JV, Dean RT, Wolff SP. Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochem J 1988;256:205. [17] Scheffler E, Wiest E, Woehrle J, Otto I, Schulz I, Huber L, Ziegler R, Dresel HA. Smoking influences the atherogenic potential of low-density lipoprotein. Clin Invest 1992;70:263. [18] Kleinveld HA, Hak-Lemmers HLM, Stalenhoef AFH, Demaker 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. [19] Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248. [20] Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Rad Res Commun 1989;6:67. [21] Pryor WA, Castle L. Chemical methods for detection of lipid hydroperoxides. In: Packer L, editor. Methods in Enzymology. Orlando: Academic Press, 1984;105:293. [22] Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of LDL-cholesterol in plasma without use of preparative ultracentrifuge. Clin Chem 1972;18:499. [23] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992;13:341. [24] Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human low density lipoprotein to metal-ion dependent and independent oxidation. J Lipid Res 1993;34:2135. [25] Maioli M, Cuccuru GB, Pranzetti P, Pacifico A, Cherchi GM. Plasma lipids and lipoproteins pattern in beta-thalassemia major. Acta Haemat 1984;71:106. [26] Maioli M, Pettinato S, Cherchi GM, Giraudi D, Pacifico A, Pupita G, Tidore MGB. Plasma lipids in beta-thalassemia minor. Atherosclerosis 1989;75:245. [27] Regnstrom J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low density lipoprotein oxidation and coronary atherosclerosis in men. Lancet 1992;340:858. [28] O’Leary V, Tilling L, Fleetwood G, Stone D, Darley-Usmar V. The resistance of low density lipoprotein to oxidation promoted by copper and its use as an index of antioxidant therapy. Atherosclerosis 1996;119:169.

435

[29] Frostegaard J, Regnstrom J, Tornvall P, Hamsten A, Nilsson J. The susceptibility of low density lipoprotein to chemical oxidation is closely related to proneness to biological modification. Free Rad Res 1995;23:581. [30] Salonen JT, Salonen R, Seppanen K, Kantola M, Suntioinen S, Korpela H. Interactions of serum copper, selenium, and low density lipoprotein cholesterol in atherogenesis. Br Med J 1991;302:756. [31] Smith C, Mitchinson MJ, Aruoma O, Halliwell B. Stimulation of lipid peroxidation and hydroxyl radical generation by the contents of human atherosclerotic lesions. Biochem J 1992;286:901. [32] Kontush A, Hubner C, Finckh B, Kohlschutter A, Beisiegel U. How different constituents of low density lipoprotein determine its oxidizability by copper: a correlational approach. Free Rad Res 1996;24:135. [33] Babiy AV, Gebicki JM, Sullivan DR. Vitamin E content and low density lipoprotein oxidizability induced by free radicals. Atherosclerosis 1990;81:175. [34] Cominacini L, Garbin U, Pastorino AM, Davoli A, Campagnola M, De Santis A, Pasini C, Faccini GB, Trevisan MT, Bertozzo L. Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease. Atherosclerosis 1993;99:63. [35] Reaven PD, Khoiow A, Beltz WF, Parthasarathy S, Witztum JL. Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin E but not by beta carotene. Arterioscler Thromb 1993;13:590. [36] Esterbauer H, Schmidt R, Hayn M. Relationships among oxidation of low-density lipoprotein, antioxidant protection, and atherosclerosis. In: Sies H. editors. Antioxidants in disease mechanisms and therapy, Advances in Pharmacology. San Diego: Academic Press, 38;1997:425 [37] Pricen HM, van Duyvenvoorde W, Buytenhek R, van der Laarse A, van Poppel G, Gevers Leuven JA, van Hinsberg VW. Supplementation with low doses of vitamin E protects LDL from lipid peroxidation in men and women. Arterioscler Tromb Vasc Biol 1995;15:325. [38] Livrea MA, Tesoriere L, Maggio A, D’Arpa D, Pintaudi AM, Pedone E. Oxidative modification and cytotoxicity of LDL in b-thalassemia. Blood, 1998, in press. [39] Ziouzenkova O, Winklhofer-Roob BM, Puhl R, Roob JM, Esterbauer H. Lack of correlation between the a-tocopherol content of plasma and LDL, but high correlation for g-tocopherol and carotenoids. J Lipid Res 1996;37:1936. [40] Kostner GM, Oettl K, Jauhiainen M, Ehnholm C, Esterbauer H, Dieplinger H. Human plasma phospholipid transfer protein accelerates exchange-transfer of alpha-topherol between lipoproteins and cells. Bioch J 1995;305:659. [41] Traber MG, Lane JC, Lagmay NR, Kayden HJ. Studies on the transfer of tocopherol between lipoproteins. Lipids 1992;27:657. [42] Arshad MAQ, Bhadra S, Cohen RM, Subbiah MTR. Plasma lipoprotein peroxidation potential: a test to evaluate individual susceptibility to peroxidation. Clin Chem 1991;37:1756. [43] Miwa K, Miyagi Y, Fujita M. Susceptibility of plasma low density lipoprotein to cupric ion-induced peroxidation in patients with variant angina. J Am Coll Cardiol 1995;26:632. [44] Chiu H-C, Jeng J-R, Shieh S-M. Increased oxidizability of plasma low density lipoprotein from patients with coronary artery disease. Biochim Biophys Acta 1994;1225:200. [45] Regnstrom J, Nilsson J, Moldeus P, Strom K, Bavenholm P, Tornvall P, Hamsten A. Inverse relation between the concentration of low density lipoprotein vitamin E and severity of coronary artery disease. Am J Clin Nutr 1996;63:377. [46] Riemersma RA, Wood DA, Macintyre CC, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentrations of vitamins A, C and E and carotene. Lancet 1991;337:1. [47] Miwa K, Miyagi Y, Igawa A, Nakagawa K, Inoue H. Vitamin A deficiency in variant angina. Circulation 1996;94:14.