Atherosclerosis and Lipoproteins |
From the Department of Clinical Nutrition (A.T.E., M.I.J.U.), the A.I. Virtanen Institute of Molecular Sciences (O.N., S.Y.-H.), and the Department of Medicine (S.L., S.Y.-H.), University of Kuopio, Kuopio, Finland.
Correspondence to Arja Erkkilä, Department of Clinical Nutrition, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland. E-mail Arja.Erkkila{at}uku.fi
| Abstract |
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Key Words: coronary disease autoantibodies oxidized low density lipoprotein anti-cardiolipin antibodies
| Introduction |
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Antiphospholipid antibodies, which contain antibodies against cardiolipin, phosphatidylserine, and phosphatidylethanolamine, have traditionally been linked with inflammatory and autoimmune diseases, such as systemic lupus erythematosus.5 The epitopic sites detected by the conventional anti-cardiolipin antibody assays include ß2-glycoprotein 1, cardiolipin, or a complex of ß2-glycoprotein 1 and cardiolipin.5 6 7 8 The major source of ß2-glycoprotein 1 in the assay is bovine ß2-glycoprotein 1 from the bovine serum or bovine serum albumin used in blocking and the sample diluent, and a minor source is human ß2-glycoprotein 1 from the test serum or plasma. It is also suggested that neoepitopes of oxidized phospholipids or adducts of oxidized phospholipids and associated proteins, including ß2-glycoprotein 1, could account for the antigenicity.9 Cross-reactivity between autoantibodies against cardiolipin and against oxLDL has been shown in patients with systemic lupus erythematosus.10
Several studies have been conducted to investigate the role of autoantibodies against oxLDL (reviewed by Ylä-Herttuala11 in 1998) and cardiolipin in atherogenesis. However, the results are conflicting. In some prospective studies, antibodies against oxLDL have predicted myocardial infarction12 13 and progression of carotid atherosclerosis.4 There are also studies that have not found any association between anti-oxLDL antibodies and the extent of atherosclerosis14 15 16 or restenosis after balloon angiography.17 In addition to antibodies against oxLDL, those against cardiolipin have also predicted myocardial infarction in men13 18 and have been associated with ischemic heart disease,19 thrombosis,20 21 recurrent myocardial infarction,22 and restenosis after coronary bypass graft.23 On the contrary, some investigators24 25 26 have not found an association between anti-cardiolipin antibodies and atherosclerosis or myocardial infarction.
Because the presentation and severity of coronary heart disease (CHD) could affect the levels of antibodies against oxLDL and cardiolipin, we determined their levels at least 6 months after discharge from the hospital in 4 groups of patients with CHD: patients with (1) coronary artery bypass surgery, (2) balloon angioplasty, (3) myocardial infarction, and (4) myocardial ischemia. We also evaluated the effect of dietary variables on the levels of these antibodies.
| Methods |
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For each diagnostic category, 125 consecutive patients (with the exception of 156 patients for the AMI category) were identified. Patients hospitalized before November 1, 1994, were invited for an interview and examination at least 6 months after hospitalization. From the respective CABG, PTCA, AMI, and AMIS groups, 109, 106, 101, and 99 patients participated; 1, 4, 20, and 0 patients were dead; and 15, 15, 35, and 26 patients did not participate (there was no response to invitation, they refused, or travel was impractical). The median time interval between hospital admission and the interview and examination for the respective patient groups was 1.0 (range 0.8 to 1.3) years, 1.9 (range 0.9 to 4.0) years, 2.3 (range 0.9 to 3.7) years, and 2.2 (range 1.0 to 3.8) years . Extensive background information, including weight, height, smoking (breath carbon monoxide measurement), blood pressure, and diabetes, was collected as described.27 Fasting blood samples were collected for the analysis of serum lipids, fatty acid profile of serum lipids, and autoantibodies against oxLDL and cardiolipin. Standardized enzymatic methods were used for the analysis of serum total cholesterol, HDL cholesterol, and triglycerides (Boehringer GmbH kits 237574 and 701904). The study was approved by the Ethics Committee at the University of Kuopio. All patients gave their informed consent for the study.
Autoantibodies Against oxLDL and Cardiolipin
LDL was isolated from pooled plasma of 2 healthy donors as
described earlier, and oxLDL was prepared by oxidation of LDL with
20 µmol/L copper at 37°C for 48 hours.2 A
modified ELISA was used to determine autoantibodies against oxLDL. One
half of a flat-bottomed microtiter plate (Polysorp, Nunc) was coated
with native LDL (2 µg/mL in PBS), and the other half was coated with
copper-oxidized LDL (2 µg/mL in PBS) at 100 µL per well. To prevent
oxidation of native LDL, PBS contained 0.27 mmol/L EDTA and
20 µmol/L BHT throughout the analysis, excluding the
washing buffer. Coated plates were incubated overnight at 4°C and
then were washed 3 times with PBS containing 0.05% Tween 20 by use of
an automatic washer (Wellwash 4 MK II, Labsystems Oy). Plates were
blocked with PBS containing 1% human serum albumin (150 µL
per well, Finnish Red Cross) at 4°C for 2 hours and washed as
described above. Serum samples were diluted (1:50) in PBS containing
0.2% human serum albumin and 0.05% Tween 20 and pipetted on
plates at 100 µL per well. Plates were incubated overnight at 4°C
and washed as described above. Horseradish peroxidaseconjugated
anti-human IgG (Silenus) diluted 1:8000 in the previous buffer was
placed on the plates (100 µL per well) and incubated at 4°C for 4
hours. After the washing, the plates were incubated with peroxidase
substrate (3,3',5,5'-Tetramethylbenzidine [Riedel de
Haën] as chromogen) at 100 µL per well for 30 minutes in the
dark. Color development was stopped with 0.5 mol/L
H2SO4 at 100 µL per well.
Absorbances were measured at 450 nm (Multiscan). The results are
expressed as the ratio of binding to oxLDL to binding to native LDL
(oxLDL/native LDL) after subtracting the mean background binding to the
wells. On each plate, 2 standard serum samples were analyzed,
and the ratio of binding to oxLDL to binding to native LDL should be at
a limit of mean±25% to accept the results of unknown samples on the
plate.
Autoantibodies against cardiolipin were also determined by ELISA. One half of a flat-bottomed microtiter plate (Maxisorp, Nunc) was coated with absolute ethanol, and the other half was coated with cardiolipin (Sigma Chemical Co, 50 µg/mL in absolute ethanol) at 25 µL per well. Ethanol was evaporated to dryness under a stream of nitrogen, and plates were incubated at room temperature for 24 hours to oxidize the cardiolipin. The plates were blocked with PBS containing 2% BSA, 0.27 mmol/L EDTA, and 20 µmol/L BHT at 150 µL per well for 2 hours. Plates were washed 3 times with PBS containing 0.05% Tween 20 (Wellwash 4 MK II, Labsystems Oy). Serum samples were diluted (1:50) in PBS containing 1% BSA, 0.05% Tween 20, 0.27 mmol/L EDTA, and 20 µmol/L BHT and pipetted on plates at 50 µL per well. Plates were incubated for 2 hours and washed as described above. Horseradish peroxidaseconjugated anti-human IgG (Cappel) diluted 1:4000 in the sample buffer was placed on the plates at 50 µL per well and incubated for 2 hours. After the plates were washed, adding the substrate (3,3',5,5'-Tetramethylbenzidine [Riedel de Haën] as chromogen), stopping the color reaction, and measuring the absorbances were performed as described for the oxLDL ELISA, but the reaction volume was 50 µL per well. All the incubations were carried out at room temperature. The results were calculated by subtracting the binding to ethanol-coated wells from the binding to cardiolipin-coated wells after subtracting the mean background binding to the wells. On each plate, 2 standard serum samples were analyzed, and their absorbances should be at a limit of mean±10% to accept the results of unknown samples on the plate. A narrower acceptance limit was used for the anti-cardiolipin than for the anti-oxLDL antibody assay because of the more homogeneous antigen.
Dietary Assessment
Dietary intake was measured by a 4-day food record. The
nutrient intake was calculated by using the Micro-Nutrica dietary
analysis program (version 2.0, Finnish Social Insurance
Institute, Turku, Finland), which is based on the national database of
the Finnish Social Insurance Institute.
The fatty acid profile of serum cholesteryl esters (CEs) was used as a biomarker of dietary fat quality. Lipids were extracted from the serum sample with chloroform-methanol (2:1, vol/vol). Lipid fractions were separated with an aminopropyl column.28 Fatty acids were analyzed with a gas chromatograph (Hewlett-Packard 5890 series II, Hewlett-Packard Co) equipped with an FFAP column (Hewlett-Packard), with helium as a carrier gas. Fatty acids are presented as molar percentages of total fatty acids.
Statistical Analyses
Statistical analyses were performed by use of the
SPSS for Windows program (version 6.0.1, SPSS Inc). Normal distribution
of variables was checked with the Kolmogorov-Smirnov (Lilliefors)
test, and logarithmic transformation was used for those not normally
distributed. The basic characteristics, nutrient intakes, and fatty
acids among the groups were tested by ANOVA. Differences in the
autoantibody levels among the groups and among the quartiles of dietary
intake were analyzed by ANOVA. When differences in
autoantibodies between 2 groups (eg, men versus women) were
analyzed, a t test for independent samples was used.
Pearson correlation coefficients were calculated between autoantibodies
and dietary and clinical variables. The
2
test was used when categorical variables were compared. The results
for continuous variables are expressed as mean±SD, except for
Figure 2
, for which mean±SEM is used. A value of
P<0.05 (2-tailed) was considered statistically
significant.
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| Results |
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-linolenic, and arachidonic acids in CEs
were similar in all groups. Of the whole study group, 18% used vitamin
supplements, and the use did not differ among the groups.
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The distributions of autoantibodies against cardiolipin and oxLDL in
the study population were skewed to the right (Figure 1
). Antibodies against oxLDL were higher
in men with AMI than in other groups (P=0.022, Table 3
). Controlling for age, serum
cholesterol, diastolic blood pressure, smoking,
and diabetes in the ANOVA did not change the statistical significance
of the difference in anti-oxLDL antibodies among the groups in men
(P=0.023). In women, there was no difference in antibodies
against oxLDL among the groups. The titers of anti-cardiolipin
antibodies did not differ among the groups in men or in women. The
relation of recurrent coronary events after the index
hospitalization to autoantibody titers was also analyzed. Of
the patients with CABG, 5 had experienced CABG, PTCA, AMI, or AMIS
after the hospitalization; the corresponding figures were 35 in
patients with PTCA, 31 in patients with AMI, and 43 in patients with
AMIS. Antibody titers against oxLDL and cardiolipin did not differ
between those patients who had experienced recurrent events and those
patients who had not (data not shown). The autoantibodies against
cardiolipin and oxLDL did not differ between diabetic and nondiabetic
patients or between patients with and without elevated blood pressure
(systolic blood pressure >140 mm Hg and/or
diastolic blood pressure >90 mm Hg; data not
shown).
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Correlation coefficients were calculated between antibody titers, serum
lipids, and fatty acids in CEs for men and women separately (Table 4
). Autoantibodies against cardiolipin
and oxLDL were not correlated with each other (r=0.082,
P=0.097). There were no significant correlations between
autoantibodies and serum lipids, except that triglyceride
concentration was positively correlated with autoantibodies against
oxLDL in women (Table 4
). Dietary intake of polyunsaturated fat
(PUFA) was inversely correlated with autoantibodies against oxLDL and
cardiolipin in men but not in women. Also, dietary intake of vitamin E
was inversely correlated with anti-cardiolipin autoantibodies in men.
The intakes of vitamin E and PUFA were highly correlated
(r=0.588, P<0.001). The proportion of linoleic
acid in CE was inversely related and that of
-linolenic acid
was positively related to autoantibodies against cardiolipin.
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The relations of PUFA and vitamin E with autoantibody levels were
further studied by analyzing the antibody titers against oxLDL and
cardiolipin in sex-specific quartiles of intakes of vitamin E and PUFA
(Figure 2
). The autoantibody levels
against cardiolipin were higher in men in the lowest quartiles of
vitamin E or PUFA intake than in men in the highest quartiles of
intakes.
| Discussion |
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Men with AMI had higher antibody titers against oxLDL than did men in the other groups, even though the analysis was controlled for known cardiovascular risk factors. The time interval between the hospitalization and autoantibody measurement was 0.8 to 4.0 years, and it is possible that the severity and presentation of the disease could had changed before the measurement of antibodies. However, recurrent events after the index hospitalization were not associated with autoantibodies against oxLDL and cardiolipin.
To date, normal values for anti-oxLDL antibody measurements have not been determined in any studies, but based on an unrelated control population (n=341) analyzed independently in our laboratory, the mean value for anti-oxLDL antibody titer was 1.83 (SD 1.17), and titers >4.17 (mean+2 SD) could be regarded as high (O.N. et al, unpublished data, 1999). Similarly, the mean value for anti-cardiolipin antibodies was 0.48 (SD 0.24) in the unrelated control population (n=110), and titers >0.96 (mean+2 SD) could be regarded as high. The distribution of the antibody titers in the control population did not differ from that observed in the CHD patients.
As analyzed together, all men had higher anti-oxLDL antibody titers and a tendency to higher anti-cardiolipin antibodies than did all women (oxLDL 2.13±1.87 [men] versus 1.73±1.05 [women], P=0.026; cardiolipin 0.49±0.26 [men] versus 0.44±0.23 [women], P=0.073). However, this finding needs to be confirmed in other studies, because in most previous studies, there is no mention of sex differences in antibody levels, and either sex may be included in the results.12 18 19 20 29 30 31
There were no significant correlations between serum lipids and autoantibodies, except for a positive one between serum triglycerides and antibodies against oxLDL in women. Results of earlier studies have been contradictory but suggest an inverse relation of HDL cholesterol12 14 and a positive relation of total or LDL cholesterol32 and antibodies against oxLDL, but several studies have not found any association between lipid and lipoprotein concentrations and antibodies against oxLDL.4 13 33 Based on the present study and earlier studies, serum lipid concentrations do not seem to be a major determinant of autoantibodies against oxLDL and cardiolipin.
The dietary intakes of PUFA and vitamin E were inversely associated
with antibodies against cardiolipin in men. Fats and oils are the major
source of vitamin E in the average diet of the Finnish
population34 ; thus, the intakes of vitamin E and PUFA were
highly correlated in the present study. The intake of vitamin E in
the present study corresponded well to the average intake in the
Finnish population (men, 10 mg/d; women, 8 mg/d).35
Autoantibodies against malondialdehyde-derivatized LDL or copper oxLDL
have been inversely associated with
-tocopherol
concentrations in serum or in LDL,15 29 36 although 1
study found no correlation between antibodies against oxLDL and plasma
or LDL vitamin E concentration.37 In a supplementation
trial, autoantibodies against oxLDL were decreased after 4 months of
vitamin E treatment in hypercholesterolemic
smokers.38 To our knowledge, there are no studies
reporting an association between antibodies against cardiolipin and
dietary vitamin E.
Dietary fatty acids are reflected in the fatty acid profile of serum lipids.39 In the present study, the inverse correlation between the proportion of linoleic acid in CEs and anti-cardiolipin antibodies is consistent with the inverse association between dietary intake of PUFA and anti-cardiolipin antibodies. In previous studies, it has been shown that the dietary fatty acids affect the susceptibility of LDL to oxidation, with the linoleic acidenriched diet enhancing the susceptibility of LDL to oxidation compared with the oleic acidenriched diet.40 However, high intake of vitamin E was connected to high intake of PUFA in the present study and may affect the associations of PUFA and antibodies.
Autoantibodies against cardiolipin and oxLDL were not correlated with each other, which is corroborated by an earlier follow-up study in 50-year-old men.13 On the other hand, significant correlations between antibodies against oxLDL and cardiolipin18 33 and cross-reactivity between the 2 antibodies10 have also been reported. It may imply that in addition to possible differences in the ELISA assays used in different laboratories, the antibodies could partly be directed against common epitopes in the lipid part,9 41 but oxLDL could also have other epitopes (eg, formed in the modification of apoB) not common with cardiolipin.
The present study was undertaken to find out whether autoantibodies against oxLDL and cardiolipin would discriminate patients with different manifestations of CHD. Thus, these results cannot be generalized to populations without CHD. On the basis of the results, there seems to be no major clinical value to measure these antibodies in CHD patients in clinical practice, although men with myocardial infarction had higher titers of antibodies against oxLDL than did men in other patient groups. The antibodies also showed no association with recurrent coronary events.
| Acknowledgments |
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Received February 16, 1999; accepted August 11, 1999.
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