(Circulation. 1997;95:840-845.)
© 1997 American Heart Association, Inc.
Articles |
Lipoprotein Oxidation and Progression of Carotid Atherosclerosis
the Research Institute of Public Health (J.T.S., K.N., R.S., E.P.-S., T.-P.T.), University of Kuopio, Finland, and Karolinska Institute (U.D., I.B.), Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Huddinge University Hospital, Stockholm, Sweden.
Correspondence to Prof Jukka T. Salonen, Research Institute of Public Health, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland.
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Abstract |
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Background Epidemiological studies and animal experiments have provided evidence supporting the role of lipid peroxidation in atherogenesis and cardiovascular diseases. Direct evidence linking lipid oxidation to atherosclerotic progression in humans, however, has been lacking. We investigated the association of lipid oxidation products with the progression of early carotid atherosclerosis in hypercholesterolemic men from eastern Finland.
Methods and Results Twenty subjects with a fast progression and 20 with no progression of carotid atherosclerosis in 3 years were selected from >400 participants in the Kuopio Atherosclerosis Prevention Study. Progression of carotid atherosclerosis was assessed by high-resolution B-mode ultrasonography. Serum 7ß-hydroxycholesterol, a major oxidation product of cholesterol in membranes and lipoproteins, and seven other cholesterol oxidation products were measured by isotope dilution–mass spectrometry, lipid hydroperoxides in LDL fluorometrically as thiobarbituric acid–reactive substances (TBARS) and oxidation susceptibility of LDL and VLDL kinetically. High concentrations of serum 7ß-hydroxycholesterol (ß=.47, P=.0005), cigarette smoking (ß=.35, P=.0167), and LDL TBARS (ß=.23, P=.0862) and an increased oxidation susceptibility of VLDL+LDL (ß=.22, P=.1114) were the strongest predictors of a 3-year increase in carotid wall thickness of more than 30 variables tested in step-up least-squares regression models. A 10-variable model explained 60% of the atherosclerotic progression. In a multivariate logistic model, the risk of experiencing a fast progression increased by 80% (P=.013) per unit (µg/L) of 7ß-hydroxycholesterol.
Conclusions The findings of this study provide further evidence to support an association between lipid oxidation and atherogenesis in humans.
Key Words: arteriosclerosis • carotid arteries • cholesterol • oxysterols
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Introduction |
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Steinberg and coworkers1 formulated the hypothesis that oxidative modification increases the atherogenicity of LDL.1 Several authors2 3 4 have suggested that some form of modification of LDL is involved in atherogenesis and in the progression of early atherosclerotic plaques. The original theory is based on the concept that the oxidative modification promotes the uptake and retention of circulating LDL in the arterial wall. However, lipid peroxidation could promote atherogenesis through other pathways as well. These include the oxidation of membrane lipids of platelets as well as arterial wall endothelial cells and smooth muscle cells. These effects could enhance the intercellular interactions that promote atherogenesis. Some of the oxidation products of cholesterol are directly cytotoxic and have been implicated in atherogenesis.5 These include 7ß-hydroxycholesterol.5 6
There is evidence that supports the hypothesis concerning the role of lipid peroxidation in atherogenesis, but all of this evidence is indirect. Experiments in rabbits have shown that atherogenesis can be inhibited by supplementation with antioxidants such as probucol,1 7 BHT,8 9 and vitamin E.10 Epidemiological follow-up studies suggest that a high intake of vitamin E is associated with a reduced risk of coronary events.11 12 Elevated body stores of the transition metals iron and mercury, which catalyze lipid peroxidation, have been associated with excess risk of myocardial infarction.13 14 In a prospective, nested, case-control study,15 we observed an association between a high titer of autoantibodies against oxidized LDL and accelerated progression of carotid atherosclerosis. Regnstrom and coworkers16 reported an association between a reduced oxidation resistance of LDL and the severity of coronary atherosclerosis in a small, cross-sectional study.
Direct evidence linking lipid peroxidation to atherosclerotic progression in humans, however, has been lacking. For this reason, we performed a study concerning the association of lipid oxidation products with the progression of carotid atherosclerosis in humans.
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Methods |
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The present study was conducted in a subset of the participants of KAPS, a randomized, double-masked, placebo-controlled trial concerning the effect of LDL cholesterol lowering with pravastatin on the progression of ultrasonographically assessed atherosclerosis.17 The design of the present study is case-control nested in a cohort. Some of the lipid oxidation measurements were done prospectively and some retrospectively from frozen samples. For the statistical analysis comparing cases and control, "case" and "control" were defined before measurement of the most important indicator of lipid peroxidation, the cholesterol oxidation products. The laboratory (Karolinska Institute) in which these measurements were done was blinded with regard to case-control status and all other measurements.
The KAPS subjects were recruited from an observational population study, the KIHD.18 All eligible KIHD participants were invited to LDL cholesterol rescreening. Of these, 557 eligible men were enrolled in KAPS. After a 2½-month placebo lead-in/dietary advice period, 447 men whose serum LDL cholesterol remained 
4.0 mmol/L and whose total serum cholesterol was <7.5 mmol/L were randomized in 1990 to receive either 40 mg pravastatin or placebo once daily at bedtime, and 410 men were examined at the 3-year follow-up in 1993.
For the present study, 30 subjects with the fastest and 30 with the slowest 3-year progression of common carotid atherosclerosis were selected from the KAPS participants with complete lipid peroxidation measurements. The subjects with slow progression were matched to the fast-progressing subjects with regard to randomized treatment group (pravastatin versus placebo) and month of entry into the trial. To increase the precision of the assessment of atherosclerotic progression, the progression was reevaluated by having another observer reread the video recordings of arterial wall ultrasonography using different software. On the basis of this rereading, 20 subjects with fast progression were designated as case subjects and 20 subjects with slow or no progression were designated control subjects. Cholesterol oxidation products were measured only for these 40 subjects.
High-resolution B-mode ultrasonography was used to image the carotid arteries.17 19 Briefly, the examination involved scanning of the right and left common carotid arteries and the area of the carotid sinus (bulb). The ultrasound system used was the Biosound phase 2 scanner equipped with a 10-MHz annular array probe.
The PCVISION Plus Frame Grabber digitizer board (Imaging Technology Inc) and image-measure morphometry software (Microscience Inc) were used to measure wall thickness in KAPS. In the rereading of the videotapes, the Data Translation DT 2861 video frame grabber interfaced to a Panasonic AG 7355 VCR and Prosound20 software with automated boundary detection were used.
In KAPS, one measurement of IMT was performed of both the right and left arteries, separately in (1) the distal subbulbar common carotid artery and (2) the carotid bulb area (the locally dilated part), each at the site of the greatest IMT at baseline. All measurements were always done consecutively in the same session for each subject after the subject had completed the study.17 In the rereading, IMT was determined as the average difference of some 100 points between the intima/lumen and media/adventitia interfaces in the distal 1.0 to 1.5 cm below the bulb. The mean of the measurements in the subbulbar common carotid artery was used in the statistical analysis.
The 7ß-hydroxycholesterol concentrations were determined at the Karolinska Institute by isotope dilution–mass spectrometry and the use of a deuterated internal standard.21 In addition, 7-oxocholesterol, 7
-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, cholesterol 5,6-epoxide, cholesterol 5ß,6ß-epoxide, and cholestane-3ß,5,6ß-triol were measured. This method involves mild alkaline hydrolysis of serum containing BHT in an argon atmosphere, followed by solid-phase extraction of the steroids on silica columns and elution of the oxysterols. After conversion into trimethylsilyl ethers, the oxysterol fraction is analyzed by gas chromatography–mass spectrometry. The coefficient of variation of this procedure for 7-oxygenated steroids, 24-hydroxycholesterol, and 27-hydroxycholesterol was between 2% and 8% depending on the concentration. The coefficient of variation for 25-hydroxycholesterol was 11% (at 9 ng/mL) and somewhat higher for the 5,6-oxygenated oxysterols.21 Serum oxysterols were measured in BHT serum samples taken at the final follow-up visit and were kept for 20 to 28 months at -70°C. The deep freezer was kept oxygen-free by intermittent addition of dry ice. At the time of the measurement, the laboratories had no knowledge of the values of any of the other measurements.
The LDL fraction was separated from fresh EDTA plasma by one ultracentrifugation step followed by gel permeation chromatography.22 Separated LDL fractions were frozen at -70°C, and TBARS were determined within a few weeks in batches. TBARS were measured fluorometrically23 after incubation of the LDL fraction with trichloroacetic acid and thiobarbituric acid for 1.5 hours at 75°C (modified from the method of Babiy et al24 ). This procedure was calibrated with 1,1,3,3-tetraethoxypropane used as a standard.25 The protein content of the LDL fraction was determined by a sensitive pyrogallol reagent (Labport). TBARS were expressed per unit of protein in LDL.
VLDL and LDL were separated together from 3 mL EDTA plasma by use of one-step gradient ultracentrifugation.26 The oxidation reaction was started by adding hemin and hydrogen peroxide solutions to the VLDL+LDL fraction, yielding final concentrations of 0.26 mmol/L VLDL+LDL cholesterol, 2.5 µmol/L hemin, and 50 µmol/L hydrogen peroxide in the reaction mixture. Degradation of the hemin ring during the reaction was followed photometrically.27 The length of the lag phase and the maximum oxidation velocity were determined by fitting least-squares slopes.26 28 The oxidation resistance assays were performed in fresh samples.
Separated VLDL+LDL fraction (250 µL) fatty acids were extracted with 6 mL methanol-chloroform (1:2) including eicosane as internal standard, and 1.5 mL water was added. The chloroform phase was evaporated under nitrogen. For methylation, the remainder was treated with 1.5 mL sulfuric acid–methanol (1:50) at +85°C for 2 hours. The mixture was diluted with 1.5 mL water and extracted with light petroleum. Fatty acids from the ether phase were determined by use of a gas chromatograph (Hewlett Packard 5890) with a flame ionization detector and an NB-351 capillary column (HNU-Nordion). For retention time and quantitative standardization, we used fatty acids purchased from Nu-Chek-Prep Inc.
Serum LDL-C was determined after precipitation with polyvinyl sulfate (Boehringer Mannheim) and serum HDL-C concentrations after precipitation with magnesium chloride dextran sulfate. Serum total cholesterol, triglycerides, and plasma phospholipids were measured enzymatically (Kone Specific, Kone Ltd).17 Smoking was assessed by an interview by a nurse, and medical history was assessed by a physician.
Statistical Analysis
The present study concerned the associations of lipoprotein oxidation measurements with the 3-year progression of carotid atherosclerosis. In the primary statistical analysis, lipid oxidation measurements were in continuous form. Least-squares correlation and regression coefficients were used to describe the strength of associations. For the secondary analysis, SPSS multivariate logistic regression analysis was used to estimate the strength of associations of lipid oxidation indexes with the probability of an individual being classified as a case subject, defined above. The distribution and a normal fit of the main outcome variable (linear annual change in the mean common carotid IMT) is presented separately for case and control subjects in the Figure
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All multivariate models were constructed with step-up procedures. A total of >30 variables were tested for entry. The variables of age, treatment (pravastatin versus placebo), month of examination, and serum LDL cholesterol were forced into all least-squares models, because the purpose was to estimate the impact of lipid oxidation indexes on atherosclerotic progression, independently of serum LDL cholesterol level.
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Results |
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The distributions of the lipid oxidation indexes are presented in Table 1
. Average serum LDL-C was relatively high: 4.1 mmol/L (159 mg/dL). Of the 40 subjects, 6 had smoked daily, 1 had diabetes, 2 had taken antihypertensive medication, and none had a history of myocardial infarction.
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Levels of serum 7ß-hydroxycholesterol correlated with increases in both common carotid wall-thickness measurements (Pearson's r=.555, P<.001 for the mean IMT; r=.260, P=.126 for the mean maximal IMT; and r=.420, P=.007 for the mean of these two values). These correlations were higher when the distribution was normalized with the use of a logarithmic transformation. Of all variables tested for entry into the step-up regression models, serum 7ß-hydroxycholesterol concentration was the strongest single predictor of the progression of carotid atherosclerosis, expressed as the mean of all common carotid measurements (Table 2). It had consistent associations with all atherosclerotic progression measurements, even when adjustment was made statistically for other important predictors. None of the other oxysterols measured had any consistent and statistically significant associations with the progression indexes. However, all oxysterols except cholesterol-5ß,6ß-epoxide had positive associations (unadjusted correlation coefficient between .05 and .27) with the main outcome variable.
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The next strongest predictors of the main outcome variable were the number of cigarettes smoked daily, TBARS content of LDL, and the oxidation resistance of VLDL+LDL, measured as the lag time to oxidation in vitro (Table 2
). A short lag time, indicating reduced oxidation resistance, was nonsignificantly associated with accelerated atherosclerotic progression. The regression models accounted for as much as 55% to 63% of the variation in the progression of common carotid atherosclerosis. None of the variables included in the models had any notable intercorrelation (r<.10) that could have caused multicollinearity problems. Of the variables tested for entry, neither serum triglyceride, plasma phospholipid, or blood glucose concentrations, systolic blood pressure, or diabetes (presence versus absence) entered any of the models.
We repeated the linear regression analyses so that 15 variables concerning molar concentrations of fatty acids in VLDL+LDL were added into the step-up models. None of the fatty acids had a significant independent association with any of the outcome variables. For the main outcome variable (the mean common carotid wall thickness), the model including eicosapentaenoic acid (ß=-.19, P=.1233) explained 63% of the variation. The coefficient and probability values for 7ß-hydroxycholesterol remained unchanged (ß=.468, P=.0004). Serum 7ß-hydroxycholesterol concentration had no important or significant correlations with any of the fatty acids (highest r=.12 with linoleic acid). The regression coefficient for 7ß-hydroxycholesterol also did not change after logarithmic normalization of its distribution (ß=.469, P=.0003).
The regression analyses were also repeated after excluding either the 6 current smokers, 1 diabetic, or 2 subjects with antihypertensive medication. The results were similar. For instance, the regression coefficient for 7ß-hydroxycholesterol was .607 (P=.0001) among 34 nonsmokers, .462 (P=.0006) in 39 nondiabetics, and .458 (P=.0008) in 38 subjects who had no antihypertensive medication in models with the same covariates as in the main analysis.
A second statistical analysis concerned the probability of being a predefined case rather than a control subject. The same four variables were also the strongest predictors of fast atherosclerotic progression in this analysis, but in a different order (Table 3). All three lipid oxidation measurements provided statistically significant (P<.05) additional information into the model. None of the fatty acid variables were entered into the step-up model.
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Discussion |
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The findings of this study show that three different markers for lipid oxidation were consistently associated with accelerated progression of carotid atherosclerosis in hypercholesterolemic men. These findings are consistent with observations concerning the association between elevated titers of antibodies against oxidatively modified LDL and accelerated progression15 or increased severity29 of atherosclerosis. Antibodies are an indirect measurement of lipid peroxidation and also have the problem of cross-reactivity between various antigens and epitopes.30 These problems do not concern lipid oxidation products and the oxidation resistance of lipoproteins.
7-Oxygenated oxysterols (7- and 7ß-hydroperoxycholesterol, 7- and 7ß-hydroxycholesterol, and 7-oxycholesterol) are the major oxysterols formed in connection with lipid peroxidation in biological membranes and lipoproteins.31 The two hydroperoxides are the primary labile products that are rapidly reduced to the corresponding hydroxy derivative or dehydrated to the oxo derivative.31 There is substantial formation of 7-oxygenated cholesterol products during oxidation of LDL particles, in particular 7-oxocholesterol and 7ß-hydroxycholesterol.32 33
Most of the 7ß-hydroxycholesterol in the circulation is present in esterified form in the LDL fraction,21 34 and it is generally believed that most, if not all, of this oxysterol is of nonenzymatic origin. 7-Hydroxycholesterol in plasma may be derived both from nonenzymatic oxidation and from the hepatic cholesterol 7-hydroxylase.35 In view of this, 7ß-hydroxycholesterol can be expected to be a better marker for lipid peroxidation than 7-hydroxycholesterol.
Sevanian and coworkers34 pointed out the difficulty in distinguishing between oxysterols that represent dietary intake of oxidized lipids versus oxidation of cholesterol occurring in vivo. Our work is concerned with the etiologic role of oxysterols and other markers of lipid peroxidation in atherosclerotic progression, and we do not attempt to separate the impacts of dietary and endogenous oxysterols.
The main difficulty in the measurement of in vivo lipid oxidation products is the possibility that lipids oxidize ex vivo after drawing the blood sample. According to a report by Kudo and coworkers,36 a substantial amount of 7ß-hydroxycholesterol found in blood samples may be formed during sample processing. Using an in vivo 18O2-labeling technique in rats, we recently showed that at least a major portion of 7ß-hydroxycholesterol in the circulation in rats is formed in vivo.37 Although this does not exclude the possibility that part of the 7ß-hydroxycholesterol may have an enzymatic origin, it seems likely that most of this compound in the circulation is formed in connection with the oxidative modification of LDL. Also, treatment with an antioxidant led to a reduction of the levels of circulating 7-oxygenated oxysterols in rabbits.8 We took several precautions to prevent the formation of oxysteroids ex vivo. First, a powerful lipophilic antioxidant, BHT, was added immediately after serum separation. Second, cholesterol was removed from serum immediately after thawing a sample. Third, further treatment of the samples was done in an argon atmosphere in which oxidation of cholesterol is not possible. Thus, we believe that the measured concentration of 7ß-hydroxycholesterol is an indicator of cholesterol oxidation in vivo or of dietary intake of cholesterol oxides, because some of the cholesterol oxides are also found in the diet.34
In a small cell-culture study, 7ß-hydroxycholesterol caused a time- and concentration-dependent perturbation of endothelial cells.38 7ß-Hydroperoxycholesterol, the labile precursor of 7ß-hydroxycholesterol, has been found to possess a pro-oxidative effect on the copper-initiated oxidation of LDL.39 Chisolm and coworkers40 recently reported the presence of 7ß-hydroperoxycholesterol in human atherosclerotic lesions and in oxidized LDL. They estimated that this compound accounts for 
90% of the cytotoxicity of the lipids of oxidized LDL and proposed that 7ß-hydroperoxycholesterol may be the primary cytotoxin in LDL. Our present observation is consistent with this hypothesis and with the need for further studies of the role of 7-oxygenated oxysterols in atherogenesis.
TBARS are the most traditional lipid peroxidation measurement. They measure the formation of dialdehydes that are thought to result from the breakdown of lipid hydroperoxides.2 3 Part of the dialdehydes are produced in vivo and part during sample preparation. Our assay was standardized using a precursor of malondialdehyde, the oxidation product of arachidonic acid. The main problem with TBARS is their limited specificity. This problem is overcome to some extent by measuring TBARS in LDL, which has a particularly high arachidonate content. The use of LDL excludes sources of malondialdehyde other than lipid peroxidation. To prevent the formation of TBARS ex vivo, we separated LDL from fresh EDTA plasma samples. EDTA was used to inhibit lipid oxidation catalyzed by transition metals. In spite of this, high TBARS could reflect high transition metal content of the sample to some extent.
We used hemin- and hydrogen peroxide–induced in vitro oxidation for the measurement of VLDL and LDL oxidation resistance. Hemin is a ubiquitous iron-containing compound that together with hydrogen peroxide catalyzes LDL oxidation, reducing the absorbance of the heme ring.27 In our antioxidant supplementation study,26 change in lag time to heme degradation was associated with change in antioxidant content of the VLDL+LDL fraction. In another study,28 the reduction of body iron stores by bloodletting increased lag time in hemin- and hydrogen peroxide–induced oxidation of VLDL+LDL fraction. Those findings showed that the oxidation susceptibility of the atherogenic lipoproteins correlates with the changes in the antioxidant and pro-oxidant status of the body. The results of the present study provide support to the idea that increased susceptibility to the oxidation of VLDL and LDL may play a role in the process of atherogenesis.
In a previous study of all KAPS participants,41 we have shown that a high dietary intake of linoleic acid is associated with reduced oxidation resistance of VLDL+LDL. In the present data, concentration of linoleic acid in VLDL+LDL was associated with an accelerated increase in common carotid wall thickness (Pearson's r=.331, P=.037). When linoleic acid concentration was entered into the multivariate models, however, serum 7ß-hydroxycholesterol remained a strong and significant predictor of atherosclerotic progression. The impacts of oxidation resistance and TBARS on progression were weakened due to their association with the lipoprotein linoleic acid content. However, the fatty acid content of VLDL and LDL is a major determinant of the oxidation resistance of these lipoproteins, and thus a statistical control for the fatty acid content may represent an overadjustment. Fatty acids are not confounding factors but rather a part of the causal chain.
The linear regression model of 10 variables accounted for as much as 60% of the variation of the main outcome variable (progression of common carotid artery wall thickness). More than half of this prediction was due to the three lipid peroxidation measurements. Although the high attributable fraction speaks in favor of strong relationships, these quantitative estimates are probably somewhat exaggerated by the bimodal distribution of the outcome variable (see Figure) due to the case-control design. On the other hand, measurement imprecision tends to dilute the observed relationships,42 and no correction for the regression dilution bias was used in the present study.
In conclusion, the findings of this study provide further evidence to support an association of lipid oxidation and atherogenesis. 7ß-Hydroxycholesterol could be an extremely useful marker in assessing this hypothesis. These observations need to be tested in other epidemiological and clinical studies with preplanned measurements of products of cholesterol oxidation and other lipid peroxidation.
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Acknowledgments |
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This work was supported by research grants from the Academy of Finland, the Swedish Medical Research Council, and the Marianne and Marcus Wallenberg Foundation. Dr J.T. Salonen is an Academy Professor of the Academy of Finland.
Received April 9, 1996; revision received August 28, 1996; accepted September 6, 1996.
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References |
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1. 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.[Medline] [Order article via Infotrieve]
2. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793-795.[Medline] [Order article via Infotrieve]
3.
Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A. 1994;91:10771-10778.
4. Salonen JT. Epidemiological studies on LDL oxidation, pro- and antioxidants and atherosclerosis. In: Bellomo G, Finardi G, Maggi E, Rice-Evans C, eds. Free Radicals, Lipoprotein Oxidation and Atherosclerosis. London, UK: Richelieu Press; 1995:27-52.
5.
Kandutsch AA, Chen HW, Heiniger HJ. Biological activities of some oxygenated sterols. Science. 1978;201:498-501.
6. Hietter H, Bischoff P, Beck JP, Ourisson G, Luu B. Comparative effects of 7ß-hydroxycholesterol towards murine lymphomas, lymphoblasts and lymphocytes: selective cytotoxicity and blastogenesis inhibition. Cancer Biochem Biophys. 1986;9:75-83.[Medline] [Order article via Infotrieve]
7.
Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks slowing the progression of atherosclerosis in the WHHL rabbit. Proc Natl Acad Sci U S A. 1987;84:7725-7729.
8.
Bjorkhem I, Henriksson-Freyschuss A, Breuer O, Diczafalusy U, Berglund L, Henriksson P. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arterioscler Thromb. 1991;11:15-22.
9. Freyschuss A, Stiko-Rahm A, Swedenborg J, Henriksson P, Bjorkhem I, Berglund L, Nilsson J. Antioxidant treatment inhibits the development of intimal thickening after balloon injury of the aorta in hypercholesterolemic rabbits. J Clin Invest. 1993;91:1282-1288.
10. Williams RJ, Motteram JM, Sharp CH, Gallagher PJ. Dietary vitamin E and the attenuation of early lesion development in modified Watanabe rabbits. Atherosclerosis. 1992;94:153-160.[Medline] [Order article via Infotrieve]
11.
Stampfer MJ, Hennekens CH, Manson JE, Coditz GA, Rosner B, Willett WC. Vitamin E consumption and risk of coronary disease in women. N Engl J Med. 1993;328:1444-1449.
12.
Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willet WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med. 1993;328:1450-1456.
13.
Salonen JT, Nyyssonen K, Korpela H, Tuomilehto J, Seppanen R, Salonen R. High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men. Circulation. 1992;86:803-811.
14.
Salonen JT, Seppanen K, Nyyssonen K, Korpela H, Kauhanen J, Kantola M, Tuomilehto J, Esterbauer H, Tatzber F, Salonen R. Intake of mercury from fish, lipid peroxidation, and the risk of myocardial infarction and coronary, cardiovascular, and any death in eastern Finnish men. Circulation. 1995;91:645-655.
15. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL. Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet. 1992;339:883-887.[Medline] [Order article via Infotrieve]
16. Regnstrom J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet. 1992;339:1183-1186.[Medline] [Order article via Infotrieve]
17.
Salonen R, Nyyssonen K, Porkkala E, Rummukainen J, Belder R, Park J-S, Salonen JT. Kuopio Atherosclerosis Prevention Study (KAPS): a population-based primary preventive trial of the effect of LDL lowering on atherosclerotic progression in carotid and femoral arteries. Circulation. 1995;92:1758-1764.
18. Salonen JT. Is there a continuing need for longitudinal epidemiologic research: the Kuopio Ischaemic Heart Disease Risk Factor Study. Ann Clin Res. 1988;20:46-50.[Medline] [Order article via Infotrieve]
19. Salonen JT, Salonen R. Ultrasound B-mode imaging in observational studies of atherosclerotic progression. Circulation. 1993;87(suppl II):II-55-II-65.
20.
Blankenhorn DH, Selzer RH, Crawford DW, Barth JD, Liu CR, Liu CH, Mack WJ, Alaupovic P. Beneficial effects of colestipol-niacin therapy on the common carotid artery: two- and four-year reduction of intima-media thickness measured by ultrasound. Circulation. 1993;88:20-28.
21. Dzeletovic S, Breuer O, Lund E, Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. Anal Biochem. 1995;225:73-80.[Medline] [Order article via Infotrieve]
22. Nyyssonen K, Salonen JT. Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol. J Chromatogr. 1991;570:382-389.[Medline] [Order article via Infotrieve]
23. Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med. 1976;15:212-216.[Medline] [Order article via Infotrieve]
24. Babiy AV, Gebicki JM, Sullivan DR. Vitamin E content and low density lipoprotein oxidizability induced by free radicals. Atherosclerosis. 1990;81:175-182.[Medline] [Order article via Infotrieve]
25. Gutteridge JMC. The use of standards for malonyldialdehyde. Anal Biochem. 1975;69:516-526.
26. Nyyssonen K, Porkkala E, Salonen R, Korpela H, Salonen JT. Increase in oxidation resistance of atherogenic serum lipoproteins following antioxidant supplementation: a randomized double-blind placebo-controlled clinical trial. Eur J Clin Nutr. 1994;48:633-642.[Medline] [Order article via Infotrieve]
27.
Balla G, Jacob HS, Eaton JW, Belcher JD, Vercellotti GM. Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury. Arterioscler Thromb. 1991;11:1700-1711.
28. Salonen JT, Korpela H, Nyyssonen K, Porkkala E, Tuomainen T-P, Belcher JD, Jacobs DR Jr, Salonen R. Lowering of body iron stores by blood letting and oxidation resistance of serum lipoproteins: a randomized cross-over trial in male smokers. J Intern Med. 1995;237:161-168.[Medline] [Order article via Infotrieve]
29.
Maggi E, Chiesa R, Melissano G, Castellano R, Astore D, Grossi A, Finardi G, Bellomo G. LDL oxidation in patients with severe carotid atherosclerosis: a study of in vitro and in vivo oxidation markers. Arterioscler Thromb. 1994;14:1892-1899.
30. Aho K, Seuri M, Leikola J, Alfthan G, Vaarala O, Palosuo T. Serum antibodies to oxidised low-density lipoprotein. Lancet. 1994;344:199-200.
31. Smith LL. Cholesterol Autooxidation. New York, NY: Plenum Publishing Corp; 1981.
32. Bhadra S, Arshad MA, Rymaszewski Z, Norman E, Wherley R, Subbiah T. Oxidation of cholesterol moiety of LDL in the presence of endothelial cells and Cu++ ions: identification of major products and their effects. Biochem Biophys Res Commun. 1991;176:431-440.[Medline] [Order article via Infotrieve]
33. Malavasi B, Rasetti MF, Roma P, Fogliatto R, Allevi P, Catapano AL, Galli G. Evidence for presence of 7-hydroperoxycholest-5-en-3ß-ol in oxidized human LDL. Chem Phys Lipids. 1992;62:209-214.[Medline] [Order article via Infotrieve]
34. Sevanian A, Seraglia R, Traldi P, Rossato P, Ursini F, Hodis H. Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry. Free Radic Biol Med. 1994;17:397-409.[Medline] [Order article via Infotrieve]
35.
Bjorkhem I, Reihner E, Angelin B, Ewerth S, Åkerlund J-E, Einarsson K. On the possible use of serum levels of a 7-hydroxylase in human liver. J Lipid Res. 1987;28:889-894.[Abstract]
36. Kudo K, Emmons GT, Casserly EW, Via DP, Smith LC, Pyrek J, Schroepfer GJ Jr. Chromatography of acetate derivatives of oxygenated sterols. J Lipid Res. 1989;30:1097-1111.[Abstract]
37.
Breuer O, Bjorkhem I. Use of an 18O2-inhalation technique and isotopomer distribution analysis to study oxygenation of cholesterol in rat: evidence for in vivo formation of 7-oxo-, 7ß-hydroxy-, 24-hydroxy and 25-hydroxycholesterol. J Biol Chem. 1995;270:20278-20284.
38.
Pettersen JS, Boberg KM, Stabursvik A, Prydz H. Toxicity of oxygenated cholesterol derivatives toward cultured human umbilical vein endothelial cells. Arterioscler Thromb. 1991;11:423-428.
39. Blanche D, Rodriguez C, Davignon J. Pro-oxidant effects of 7-hydroperoxycholest-5-en-3ß-ol on the copper-initiated oxidation of low density lipoprotein. FEBS Lett. 1995;357:135-139.[Medline] [Order article via Infotrieve]
40.
Chisolm GM, Ma G, Irwin KC, Martin LL, Gunderson KG, Linberg LF, Morel DW, DiCorleto PE. 7-Beta-hydroperoxycholest-5-en-3-beta-ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein. Proc Natl Acad Sci U S A. 1994;91:11452-11456.
41.
Louheranta AM, Porkkala-Sarataho EK, Nyyssonen MK, Salonen RM, Salonen JT. Linoleic acid intake and susceptibility of very-low-density and low-density lipoproteins to oxidation in men. Am J Clin Nutr. 1996;63:698-703.
42. Salonen JT. Statistical correction for measurement imprecision. Lancet. 1992;339:1116. Letter.
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