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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:733-739

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:733-739.)
© 1995 American Heart Association, Inc.

Articles

Oxidation-Related Analytes and Lipid and Lipoprotein Concentrations in Healthy Subjects

Wendy Y. Craig; Sue E. Poulin; Glenn E. Palomaki; Louis M. Neveux; Robert F. Ritchie; Thomas B. Ledue

From the Foundation for Blood Research, Scarborough, Me.

Correspondence to Wendy Y. Craig, PhD, Foundation for Blood Research, PO Box 190, Scarborough, ME 04070-0190.


*    Abstract
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 *Abstract
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Abstract The relations between oxidation-related analytes and lipoprotein risk factors for coronary heart disease are poorly understood. To address this issue, ceruloplasmin, copper, iron, ferritin, cotinine, lipid peroxides, cholesterol, triglyceride, apoB, apoA-I, and lipoprotein(a) levels were measured in sera from apparently healthy subjects (51 men and 115 women). Pairwise comparisons revealed strong positive associations (P<.001) of copper and ceruloplasmin with lipid peroxides, total cholesterol, triglycerides and apoB, of transferrin with apoA-I and cholesterol, and of ferritin with triglycerides. Serum levels of oxidation-related analytes did not differ between smokers and nonsmokers. In multivariate analysis, serum copper was the major independent determinant of serum lipid peroxide level, accounting for 15% of the variability in concentration (ferritin accounted for 1.6%). Copper and ceruloplasmin accounted for 20.5% of the variation in triglyceride levels; triglycerides and apoB accounted for 12% of the variability in ferritin levels; apoB and apoA-I accounted for 9% of the variability in transferrin levels. The data suggest that serum copper contributes to lipid peroxidation in vivo. There are significant associations between lipoprotein and transition metal-related analytes, and further work is needed to elucidate the physiological basis for these relations.


Key Words: oxidation • lipids • apolipoproteins • transition metals


*    Introduction
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 *Introduction
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Lipids and lipoproteins are well known as risk factors for coronary heart disease (CHD). LDL has been identified as a major atherogenic particle1 ; however, only LDL that has been modified by oxidation or acetylation, and not native LDL, can cause cholesterol ester accumulation in macrophages (a prerequisite for foam cell formation).2 Oxidized LDL is present in atherosclerotic plaque, and autoantibodies against oxidized LDL exist in human serum, which indicate that these particles exist in vivo.3 Oxidized LDL may be atherogenic, not only by its ability to load macrophages with cholesterol, but also because it is chemotactic for circulating monocytes, is cytotoxic, and can adversely alter coagulation pathways.4

Several recent studies support the hypothesis that oxidation contributes to the development of atherosclerosis. Elevated plasma lipid peroxide levels are associated with ischemic heart disease and peripheral arterial disease,5 and increased susceptibility of LDL to oxidation is associated with the degree of coronary stenosis in young men.6 It has also been reported that antioxidant intake (in the form of vitamin E) is associated with lower risk of CHD,7 8 and that probucol, an antioxidant, inhibits the formation of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.9

Many details of the mechanism for LDL oxidation in vivo remain to be determined; however, certain serum components may influence the oxidation of serum lipids. Copper and iron oxidize LDL in vitro.10 In most instances, ferritin acts as an oxidant by virtue of its capacity to release iron11 ; however, Balla et al12 report that it can also act as an iron sequestrant and protect endothelial cells from oxidative damage. Ceruloplasmin and transferrin together have been reported to be the primary antioxidants in plasma.13 In contrast, ceruloplasmin, with ferritin, can contribute to the in vitro oxidation of LDL by stimulated neutrophils,14 catalyze hydrogen peroxide generation from homocysteine,15 and oxidize LDL in vitro.16 Samokyszyn et al17 report that in vitro ceruloplasmin can act as a pro-oxidant or antioxidant, depending on its concentration.

Consistent with the hypothesis that oxidation is involved in the development of atherosclerosis, both copper18 and ceruloplasmin19 have been shown to be positively associated with increased risk for CHD. However, the available data concerning the relation of iron and related analytes with CHD risk are conflicting. Salonen et al20 have found prospectively that high stored iron levels (as inferred from serum ferritin measurements) are associated with CHD risk. Ascherio et al21 report a relation between the dietary consumption of heme iron, such as from red meat, and the risk of myocardial infarction, particularly in subjects not taking vitamin E supplements. It has also been reported that transferrin (estimated as total iron binding capacity) has an inverse relation with CHD risk.22 Other studies, however, have found no association between iron,23 ferritin,24 percent transferrin saturation (TS),25 or the presence of iron overload disorders26 and CHD risk.

Although serum levels of analytes potentially involved with oxidation may be associated with CHD risk, little is known about the relations between these variables and serum levels of the more traditional lipid and lipoprotein risk factors, particularly in healthy individuals. In the present study, levels of ceruloplasmin, copper, iron, ferritin, transferrin, cotinine, lipid peroxides, cholesterol, triglyceride, apoB, apoA-I, and lipoprotein(a) [Lp(a)] were measured in sera from healthy subjects.


*    Methods
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 *Methods
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Samples
After informed consent, sera were collected from 186 apparently healthy volunteers, all employees at a local corporation. Samples and data were identified by code only to protect confidentiality. The study group included 60 men and 126 women, all Caucasian, aged 22 through 63 years. All 186 samples were collected (interim storage at 4°C), divided into aliquots, and frozen at -80°C within the same 8-hour period. Iron, transferrin, C-reactive protein (CRP), and ferritin were measured in fresh, unfrozen (4°C) sera within 48 hours. All other assays were performed on the stored sera 1 year after collection; aliquots were thawed only once prior to assay. Sixteen subjects (9%) with evidence of inflammation (CRP >8.5 mg/L) were not included in the study, as several of the measured variables are acute-phase proteins; furthermore, copper levels are increased,27 iron levels depressed, and ferritin levels elevated in such subjects.28 An additional 4 subjects (2%) were removed from analysis as age was missing. The final study group comprised 51 men and 115 women. Among the women, 29 were taking oral contraceptives, 9 were on hormonal replacement therapy, and none were pregnant.

To study the possible effects of freezer storage on measured analytes, 20 consecutive sera received by our laboratory for testing unrelated to the present study were assayed fresh and after being frozen at -80°C for 1, 3, and 6 months.

Lipid and Lipoprotein Assays
Total cholesterol and triglycerides were assayed by enzymatic colorimetric methods by using kits from Boehringer Mannheim and Roche, respectively. Apolipoproteins A-I and B were assayed by immunoturbidimetry on a Roche Cobas FARA using antisera and protocol from INCSTAR and apolipoprotein standard sera from Behring Diagnostics.29 The apolipoprotein assays were calibrated to the International Federation of Clinical Chemistry reference material.30 Within-run and between-run coefficients of variation (CVs) for the apolipoprotein (B and A-I) and lipid (cholesterol and triglyceride) assays were <4%. Lp(a) was assayed by enzyme-linked immunosorbent assay (ELISA) using both Macra Lp(a) kits from Terumo Medical Corp and ApoTek kits from Organon Teknika. Within-run and between-run CVs were <7.5%. The assay of Lp(a) is complicated by the existence of multiple isoforms of apo(a) that differ considerably in size31 ; thus, we used two methods to better detect assay-related confounding of data. The Macra Lp(a) ELISA detects Lp(a) by using anti-apo(a), whereas the ApoTek ELISA uses anti-apoB and is less likely to be confounded by isoform size.32

Serum Protein Assays
Transferrin was assayed by immunoturbidimetry on a Roche Cobas FARA.33 Ceruloplasmin was assayed by immunoturbidimetry on the same instrument using antisera and protocol from INCSTAR. Both assays were calibrated to RPSP3 reference material (College of American Pathologists) and had within-run and between-run CVs of <3%. CRP was measured on a Behring nephelometer using materials supplied by the manufacturer (within-run CV, 6.1%; between-run CV, 3%). Ferritin was measured by a bead enzyme immunoassay (Tandem-E) from Hybritech; within-run CV was 6.7%, and between-run CV was 9.2%.

Other Assays
Iron was measured colorimetrically on a Roche Cobas FARA using kits from Diagnostic Chemicals Ltd; within-run CV was 2%, and between-run CV was 3%. TS was calculated as the ratio of iron to transferrin; a factor of 25.2 was used to convert transferrin concentration (in grams per liter) to an equivalent total iron-binding capacity (in micromoles per liter). Cotinine was measured by radioimmunoassay34 (lower limit of detection, 10 µg/L); within-run CV was 5%, and between-run CV was 8%. Serum copper levels were assayed by using a modification of the colorimetric assay described by Kossman.35 The assay was performed on a Cobas FARA (Roche) instead of a Cobas BIO; within-run and between-run CVs were <5%. Instrument parameters were unchanged from the previous report35 ; however, bathocuproine reagent concentration was increased from 1 to 2 g/L to improve linearity. The copper assay was not contaminated by exogenous trace metal; there was no significant difference between the copper concentration of fresh deionized water (1.7±3.1 µg/L; n=10) and that of deionized water handled in the same way as the serum samples (2.5±4.9 µg/L; n=10). Serum lipid peroxide levels were estimated as levels of thiobarbituric acid–reactive substances (TBARS). TBARS were assayed immediately after the samples were thawed by using a modification of the method of Lamb and Leake.36 Specific modifications were that the standard curve range used was 0.1 to 4 µmol/L; the sample/TBA reagent (0.335% [wt/vol] thiobarbituric acid in 10% [wt/vol] trichloroacetic acid) ratio was 1:4; butylated hydroxytoluene (BHT; final concentration, 18 g/L) was added to the reaction mixture; and standards were prepared according to the method of Wong et al.37 Briefly, standards or samples were diluted 1:4 in TBA reagent, and BHT was added to a final concentration of 18 g/L. The reaction mixture was incubated for 30 minutes in a 95°C water bath; the reaction was stopped by incubating samples in an ice bath for 10 minutes. Any precipitate was removed by centrifugation at 3000 rpm for 5 minutes in a Beckman Accuspin FR microfuge, and the absorbance (at 540 nm) of the supernatant was then measured. Assay CV was 9%. Reagents for the copper and TBARS assays were from Sigma Chemical Co.

Statistical Methods
Prior to analysis, analyte data were transformed by taking the logarithm or square root, as appropriate, to fit a gaussian distribution well. Linear regression was used to adjust for age and sex and to test for trends when examining the effects of storage on analyte levels. Univariate relations between variables were examined by linear regression, and multivariate relations by stepwise linear regression. Differences between subgroups were analyzed by Student's t tests. All analyses were performed by using a statistical package from BMDP Statistical Software Inc. No adjustment of the data was made for multiple comparisons, but the significance level was set at P<.001 when such comparisons were made.


*    Results
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Serum Levels of Oxidation- and Lipoprotein-Related Analytes in Healthy Subjects
Mean, SD, and selected centiles for the serum levels of oxidation- and lipoprotein-related analytes are shown for women and men in Tables 1Down and 2Down, respectively. Serum levels of copper, ceruloplasmin, and transferrin were significantly higher and levels of iron, ferritin, and TS were lower in women than in men. Among the lipids and apolipoproteins, only the difference in apoA-I between men and women was statistically significant.


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  		Table 1. Serum Concentrations of Oxidation- and Lipoprotein-Related Variables in 115 Women


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  		Table 2. Serum Concentrations of Oxidation- and Lipoprotein-Related Variables in 51 Men

Effects of Tobacco Smoke Exposure on Serum Levels of Oxidation- and Lipoprotein-Related Analytes
Cotinine, the major metabolite of nicotine, was used to categorize subjects with active levels of exposure to tobacco smoke (cotinine >10 µg/L). Total cholesterol levels were 12% higher in subjects with cotinine levels indicative of active tobacco smoke exposure (n=15), and apoB levels were 15% higher (P=.03). No other comparisons were statistically significant (data not shown).

Relations Between Oxidation-Related Variables and Lipid and Lipoprotein Risk Factors
All pairwise correlations between variables are provided in Table 3Down. In this and in subsequent analyses, the age/sex differences have been taken into account. The Lp(a) data presented here and subsequently were obtained by using the Macra Lp(a) ELISA; similar data were obtained by using the ApoTek ELISA (not shown). Exclusion of the 38 women taking oral contraceptives or hormone replacement had no material effect on the present findings (not shown). Among the transition metal–related data, iron levels were significantly correlated with ferritin and TS, and transferrin was negatively correlated with TS and positively with ferritin. There were also significant positive associations of copper and ceruloplasmin with transferrin. Similarly, positive correlations were observed among the lipid and lipoprotein variables. Total cholesterol and triglyceride levels were significantly associated with levels of apoB, and cholesterol levels were also associated with levels of apoA-I. Significant associations were observed between oxidation-related variables and certain lipid and lipoprotein risk factors. Serum levels of copper and ceruloplasmin were positively associated with levels of TBARS, cholesterol, triglycerides, and apoB. Transferrin levels were positively correlated with apoA-I and cholesterol levels, and ferritin levels were correlated with triglycerides.


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  		Table 3. Correlation Coefficients for Relations Between Oxidation- and Lipoprotein-Related Variables

To determine which factors were independent contributors in relations between the measured analytes, stepwise regression analyses were performed. Only variables having r>=.2 (ie, each variable must univariately account for at least 4% of the variability in the dependent variable) were included in each model. As illustrated in Table 4Down, serum copper level was the major independent determinant of TBARS level, accounting for about 15% of the variability in concentration. Inclusion of ferritin accounted for a further 1.6%. In addition to the expected contributions of other lipids or lipoproteins to the variability in cholesterol, triglyceride, apoB, and apoA-I levels, oxidation-related variables were also significant contributors. In some cases, the contribution was minor; eg, copper and ceruloplasmin together accounted for only 1.6% of the variability in serum cholesterol levels. In contrast, copper and ceruloplasmin together accounted for 20.5% of the variation in serum triglyceride levels.


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  		Table 4. Multivariate Relations Between Oxidation- and Lipoprotein-Associated Variables

Conversely, when copper, ceruloplasmin, ferritin, and transferrin were examined as dependent variables, lipids and apolipoproteins contributed independently to the observed levels. Triglycerides and apoB accounted for 12% of the variability in ferritin levels. Triglyceride level also contributed significantly to variation in copper (6.5%) and ceruloplasmin (13.5%) levels. Lastly, apoB and apoA-I together accounted for 9% of the variability in transferrin levels.

Effect of Sample Storage on Relation Between Iron, Copper, and TBARS Concentrations
To determine whether correlations between serum TBARS levels and levels of copper and iron were due to increased in vitro formation of TBARS in those sera with high levels of copper or iron, these three analytes were assayed in 20 fresh sera samples that were also stored in aliquots at -80°C. Separate aliquots were thawed at 1, 3, and 6 months, and serum iron, copper, and TBARS concentrations were reassayed. There were no significant trends toward change in any of these analytes over 6 months in storage (r<.1, P>.4). Furthermore, there was no association between the change in TBARS level after 6 months of storage and the initial serum level of copper (r=.27, P=.25) or iron (r=.13, P=.57).


*    Discussion
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 *Discussion
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Normative Data
The present data for serum lipid and certain serum protein levels in healthy subjects are consistent with normative ranges.38 39 With regard to TBARS data, the possibility of in vitro oxidation must always be considered. According to Lee,40 storage at 4°C for <=8 hours would cause a minimal increase in TBARS level (<=0.007 µmol/L, or <=1% of the present mean value). Furthermore, TBARS concentrations were in the expected range.41 This indicates that our protocol for handling and storing samples was effective in minimizing auto-oxidation. Differences between men and women for the iron- and copper-related data are similar to other reported values.42 43 Although apoA-I levels were higher in women, serum levels of cholesterol, apoB, and triglycerides did not differ significantly between men and women; however, trends in median levels between men and women were in the expected direction.44

Effects of Tobacco Smoke Exposure
Tobacco smoke exposure is a known risk factor for CHD.45 In the present study, consistent with previous work,46 tobacco smoke absorption, as estimated by serum cotinine level, was associated with increases in total cholesterol and apoB levels. In agreement with results from other studies,47 48 lipid peroxide (TBARS) levels were not increased in smokers, although the power of the present study was low. These data suggest either that smoking does not cause the oxidation of serum lipids (as estimated by TBARS level) in vivo or that such lipids are removed from the circulation too rapidly to be detected by the present approach. The latter explanation is supported by in vitro work that has demonstrated that exposure to cigarette smoke extract causes LDL oxidation49 as well as increased susceptibility to oxidation50 and that such LDLs are taken up rapidly by macrophages.51

Relations Between Lipid Peroxide Level (TBARS) and Other Measured Variables
In the present study, both lipid- and transition metal–related variables were univariately associated with lipid peroxide levels; however, with the exception of copper and ceruloplasmin, these relations were weak. Our results are consistent with those of Stringer et al5 in that triglyceride but not cholesterol concentrations were significantly associated with lipid peroxide concentration. The observed association of lipid variables (triglycerides and apoB) with TBARS level is probably due to their relation with TBARS location, whereas the association of transition metal–related variables (copper, iron, ceruloplasmin, and ferritin) is most likely related to their potential as oxidizing agents.

There is considerable disagreement as to whether ferritin and ceruloplasmin are pro-oxidants or antioxidants11 12 13 14 15 16 17 ; indeed, depending on the conditions, they might be either. In the present study, we report positive bivariate correlations between lipid peroxides (as TBARS) and ceruloplasmin and ferritin but not transferrin levels in serum. These data suggest that the oxidative effects of ceruloplasmin and ferritin on serum lipids predominate, whereas the effect of transferrin is neutral.

Copper was the major independent contributor to the variability in serum TBARS concentration, suggesting that copper is not only able to oxidize LDL in vitro but that it may also perform this function in vivo. It is not possible to determine whether this relation is associated with total body copper status as, except in severe copper deficiency, serum copper level does not necessarily reflect body copper stores.52 As TBARS levels did not change in storage as a function of serum copper concentration, it is therefore unlikely that the relation between copper and TBARS levels in serum was due to auto-oxidation during sample storage.

Relations Between Transition Metal– and Lipoprotein-Related Variables
There are few studies of the relation between ferritin or copper and lipoprotein metabolism, despite reports of their possible involvement in the development of atherosclerosis (see above). Ferritin levels are reported to be associated with serum levels of apoB and triglycerides,20 cholesterol,22 53 and triglycerides.22 The current study shows that among these variables, apoB has the strongest association with ferritin levels. Likewise, our observation that serum cholesterol and apoB levels were both positively correlated with serum copper levels is consistent with findings of an association between copper concentrations and serum LDL cholesterol23 or total cholesterol54 levels. In addition, we report that serum copper levels were associated with triglyceride and apoA-I levels; indeed, these latter two variables were the only lipoprotein-related analytes to be independently associated with serum copper levels. As 60% to 70% of serum copper exists bound to ceruloplasmin,52 this result may be explained, at least in part, by the fact that some ceruloplasmin is bound tightly to apoA-I–containing lipoproteins in serum.55

High-dose dietary supplementation with copper causes increases in ceruloplasmin and cholesterol levels in adult men,56 and ceruloplasmin, apoB, and apoA-I levels are all increased by low-dose oral contraceptives.57 Consistent with these findings, Reunanen et al19 found a positive but nonsignificant (P<.1) correlation between serum ceruloplasmin and cholesterol concentrations. In the present study, ceruloplasmin was strongly correlated not only with cholesterol but also with apoB and triglyceride levels; however, only triglycerides remained a significant contributor to variation in ceruloplasmin levels when the levels of all three analytes were considered simultaneously.

We report significant associations of transferrin with cholesterol, apoB, and apoA-I but not with triglycerides. This is at least partially consistent with the finding that, as for ceruloplasmin, some transferrin is tightly bound to apoA-I–containing HDL in serum.55 In contrast, Magnusson et al22 report that transferrin was positively associated with triglycerides but not with cholesterol. It is likely that the discrepancy in triglyceride results is due to differences in statistical power between the two studies, as the data are otherwise similar (r=.144, P=.064 in the present study compared with r=.147, P<.05 in Reference 2222 ). The observed relations between transferrin and cholesterol levels, however, are too different to be explained by power (r=.279, P<.001 in the present study compared with r=.043, P>.05 in Reference 2222 ) and may be related to population differences or to differences in assay methodology (Magnusson et al22 estimated transferrin as total iron-binding capacity).

In summary, we have characterized the relations between serum lipid and lipoprotein levels and levels of certain oxidation-related variables in a healthy population. The results of diverse studies indicate the existence of certain relations between lipoprotein and transition metal metabolism, and our findings both confirm and extend these prior observations. The observed interrelations between serum concentrations of apolipoproteins and transition metal–associated proteins may be related to functional interactions between the proteins, to common regulatory pathways, or to both. There was no strong relation between lipid peroxide level and cigarette smoke exposure, suggesting that certain interactions related to oxidation status are not measurable in the serum compartment. Serum copper was the major determinant of serum lipid peroxidation status (as determined by TBARS level), indicating that it contributes to lipid peroxidation in vivo.


*    Acknowledgments
 
This work was funded by grant 9306273S from the American Heart Association, Maine Affiliate, and by the Foundation for Blood Research Development Fund. The authors would like to thank Wilfred Turgeon, MA, for assistance with database management and Nancy Healy, MLT, Angela Mackie, and Jan Cardoza, MT, for technical assistance.

Received October 6, 1994; accepted March 13, 1995.


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 *References
 

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