High Levels of Inflammatory Biomarkers Are Associated with ...

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agnostic coronary arteriography either at Harlem Hospital Center in. New York ..... M.D., Department of Medicine, University of California, Davis, UCD. Medical ...

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ARTICLE R e s e a r c h

High Levels of Inflammatory Biomarkers Are Associated with Increased Allele-Specific Apolipoprotein(a) Levels in African-Americans Erdembileg Anuurad, Jill Rubin, Alan Chiem, Russell P. Tracy, Thomas A. Pearson, and Lars Berglund Department of Medicine (E.A., A.C., L.B.), University of California, Davis, Sacramento, California 95817; Department of Medicine (J.R.), Columbia University, New York, New York 10027; Department of Pathology (R.P.T.), University of Vermont, Burlington, Vermont 05405; Department of Community and Preventive Medicine (T.A.P.), University of Rochester, Rochester, New York 14627; and VA Northern California Health Care System (L.B.), Sacramento, California 95655

Background: A role of inflammation for cardiovascular disease (CVD) is established. Lipoprotein(a) [Lp(a)] is an independent CVD risk factor where plasma levels are determined by the apolipoprotein(a) [apo(a)] gene, which contains inflammatory response elements. Design: We investigated the effect of inflammation on allele-specific apo(a) levels in AfricanAmericans and Caucasians. We determined Lp(a) levels, apo(a) sizes, allele-specific apo(a) levels, fibrinogen and C-reactive protein (CRP) levels in 167 African-Americans and 259 Caucasians. Results: Lp(a) levels were increased among African-Americans with higher vs. lower levels of CRP [⬍3 vs. ⱖ3 mg/liter (143 vs. 108 nmol/liter), P ⫽ 0.009] or fibrinogen (⬍340 vs. ⱖ340 mg/liter, P ⫽ 0.002). We next analyzed allele-specific apo(a) levels for different apo(a) sizes. No differences in allele-specific apo(a) levels across CRP or fibrinogen groups were seen among African-Americans or Caucasians for small apo(a) sizes (⬍22 kringle 4 repeats). Allele-specific apo(a) levels for medium apo(a) sizes (22–30 kringle 4 repeats) were significantly higher among African-Americans, with high levels of CRP or fibrinogen compared with those with low levels (88 vs. 67 nmol/liter, P ⫽ 0.014, and 91 vs. 59 nmol/liter, P ⬍ 0.0001, respectively). No difference was found for Caucasians. Conclusions: Increased levels of CRP or fibrinogen are associated with higher allele-specific medium-sized apo(a) levels in African-Americans but not in Caucasians. These findings indicate that proinflammatory conditions result in a selective increase in medium-sized apo(a) levels in AfricanAmericans and suggest that inflammation-associated events may contribute to the interethnic difference in Lp(a) levels between African-Americans and Caucasians. (J Clin Endocrinol Metab 93: 1482–1488, 2008)

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he role of inflammation in the pathogenesis of cardiovascular disease (CVD) is well recognized. Inflammation contributes to all phases of atherosclerosis, from fatty streak initiation, growth, and complication of the atherosclerotic plaque to CVD events (1). Thus, virtually every step in atherosclerosis is believed to involve cytokines, bioactive molecules, and proinflammatory cells. The inflammatory response is mediated by acute-phase reactants such as C-reactive protein (CRP) and fibrinogen, and considerable attention has recently been paid to the association of such factors with coronary artery disease

(CAD) (2–5). Notably, many prospective studies have demonstrated positive associations between the risk for CAD and plasma levels of CRP (6, 7) or fibrinogen (8, 9). In view of these results, a recent statement from the Centers of Disease Control and Prevention and the American Heart Association concluded that it is reasonable to measure CRP as an adjunct to established cardiovascular risk factors (10). Lipoprotein(a) [Lp(a)] is an independent CVD risk factor where plasma levels are largely determined by apolipoprotein(a) [apo(a)] gene size (11–13). The size of the apo(a) gene is highly

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Abbreviations: Apo(a), Apolipoprotein(a); CAD, coronary artery disease; CRP, C-reactive protein; CVD, cardiovascular disease; HDL, high-density lipoprotein; K4, kringle 4; LDL, low-density lipoprotein; Lp(a), lipoprotein(a).

Printed in U.S.A. Copyright © 2008 by The Endocrine Society doi: 10.1210/jc.2007-2416 Received October 31, 2007. Accepted January 25, 2008. First Published Online February 5, 2008

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J Clin Endocrinol Metab. April 2008, 93(4):1482–1488

J Clin Endocrinol Metab, April 2008, 93(4):1482–1488

variable, resulting in a size variation of the apo(a) protein, as manifested in a variable number of kringle 4 (K4) repeats (11, 14). There is an inverse relationship between apo(a) size and Lp(a) levels, because smaller apo(a) sizes in general are associated with higher plasma Lp(a) levels. However, even for a defined apo(a) size, there is considerable variability in Lp(a) levels (15– 18). The use of size allele-specific apo(a) levels offers opportunity to more accurately assess the relationship between apo(a) size and Lp(a) levels. Allele-specific apo(a) levels are higher among African-Americans compared with Caucasians, and the most pronounced interethnic difference is seen for medium-sized apo(a) alleles (15, 18, 19). Although genetic polymorphisms in the apo(a) gene have been reported to be contributory, the differences in allele-specific apo(a) levels between AfricanAmericans and Caucasians has not been fully explained (16, 17, 20). It is well known that the apo(a) gene contains response elements for inflammatory factors such as IL-6 (21, 22). However, the role of Lp(a) as an acute-phase reactant is controversial, and the effect of inflammation on allele-specific apo(a) levels is unknown. The objectives of our study were to investigate the potential effect of inflammation measured by two acute-phase reactants (CRP and fibrinogen) on allele-specific apo(a) levels in African-Americans and Caucasians.

Subjects and Methods Subjects Subjects were recruited from a patient population scheduled for diagnostic coronary arteriography either at Harlem Hospital Center in New York City or at the Mary Imogene Bassett Hospital in Cooperstown, NY. The clinical characteristics of the study population and the study design including inclusion and exclusion criteria have been described previously, and notably, exclusion criteria included use of lipid lowering drugs (19, 23, 24). Briefly, a total of 648 patients, self-identified as Caucasian (n ⫽ 344), African-American (n ⫽ 232), or other (n ⫽ 72) were enrolled. The apo(a) allele sizes, circulating apo(a) isoforms, and allele-specific apo(a) levels were available on 426 subjects (167 AfricanAmericans, 259 Caucasians). The study was approved by the Institutional Review Boards at Harlem Hospital, the Mary Imogene Bassett Hospital, Columbia University College of Physicians and Surgeons, and University of California, Davis, and informed consent was obtained from all subjects.

Clinical and biochemical assessment Fasting blood samples were drawn approximately 2– 4 h before the catheterization procedure, and plasma samples were stored at ⫺80 C before analysis. High-sensitivity CRP levels were measured using an ELISA, standardized according to the World Health Organization First International Reference Standard (25, 26). Fibrinogen was measured by the clot-rate method of Clauss (27). Plasma total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined by standard enzymatic procedures. Low-density lipoprotein (LDL) cholesterol levels were calculated in subjects with triglyceride levels of less than 400 mg/dl using the formula of Friedewald et al. (28). All analyses were carried out shortly after completion of the study.

Measurement of plasma Lp(a) levels Lp(a) levels were measured by using a method insensitive to size variation in apo(a) by a sandwich ELISA (Sigma Diagnostics, St. Louis,

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MO). In our hands, the interassay coefficient of variation was 8.4% at an apo(a) level of 19.9 nM and 9.0% at an apo(a) level of 67.1 nM (19).

Apo(a) allele and isoform size determination To determine apo(a) allele sizes, we performed genotyping using pulsed-field electrophoresis of DNA from leukocytes embedded in agarose plugs, essentially as described previously (29, 30). Apo(a) isoform sizes were analyzed by SDS-agarose gel electrophoresis of plasma samples, followed by immunoblotting. Briefly, the apo(a) bands were visualized with the ECL Amersham technique using a second, labeled antibody (Pierce, Rockford, IL) (19, 31, 32).

Determination of allele-specific apo(a) levels and dominance The protein dominance was determined by optical analysis of the apo(a) protein bands on the Western blots, and the visual estimations were validated by computerized scanning. For each of the apo(a) protein bands, levels were apportioned according to the degree of intensity of the bands on the Western blot, using 10% increments (30). For example, an individual with plasma Lp(a) level of 100 nmol/liter, carrying apo(a) proteins with 25 K4 and 35 K4 repeats, with the smaller protein dominating by 80%, would have allele-specific apo(a) level of 80 nmol/liter for 25 K4 and allele-specific apo(a) level of 20 nmol/liter apportioned to the 35-K4 repeat protein.

Statistics Analyses of data were done with SPSS statistical analysis software (SPSS Inc., Chicago, IL). Results are expressed as means ⫾ SD. Triglyceride and CRP levels were logarithmically transformed, and Lp(a) levels and allele-specific apo(a) levels were square root transformed to achieve normal distributions. Proportions were compared between groups using ␹2 analysis and Fisher exact test where appropriate. Group means were compared using Student’s t test. The CRP and fibrinogen levels were dichotomized as high and low groups (CRP, ⬍3 vs. ⱖ3 mg/liter; fibrinogen, ⬍340 vs. ⱖ340 mg/dl) based on common practice from previous studies (3, 10, 33–36). The distribution of apo(a) alleles for each CRP and fibrinogen group was analyzed using the nonparametric KolmogorovSmirnov test. Unless otherwise noted, a nominal two-sided P value ⬍ 0.05 was used to assess significance.

Results The characteristics of the study population are shown in Table 1. Compared with Caucasians, African-Americans were younger and had significantly higher levels of mean and median Lp(a). Also in agreement with previous studies based on the National Health and Nutrition Examination Survey, African-Americans had higher levels of CRP and fibrinogen compared with Caucasians (37–39). There was no difference in the levels of total and LDL cholesterol between the two ethnic groups, whereas African-Americans had significantly higher levels of HDL cholesterol and lower levels of triglycerides compared with Caucasians. We next analyzed the effect of increased levels of CRP (ⱖ3 mg/liter) or fibrinogen (ⱖ340 mg/dl) on plasma Lp(a) levels in each ethnic group. As mentioned above, cutoff levels of CRP and fibrinogen were chosen based on previous studies (3, 10, 33–36). As seen in Table 2, Lp(a) levels were increased among AfricanAmericans with higher vs. lower CRP (143 vs. 108 nmol/liter, P ⫽ 0.009) or fibrinogen (146 vs. 105 nmol/liter, P ⫽ 0.002), whereas no differences were found in Lp(a) levels across CRP or

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TABLE 1.

Inflammation and Allele-Specific Apo(a) Levels

Characteristics of study population

Clinical characteristics

African-Americans (n ⴝ 231)

Caucasians (n ⴝ 336)

Men/women (n) Diabetes (n) Hypertension (n) Postmenopausal (n) Age (yr) Lp(a) (nmol/liter) CRP (mg/liter) Fibrinogen (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglyceride (mg/dl)

132/99 68 (29.4%) 167 (72.3%) 66 (66.7%) 68 (29.4%) 107.5 (59.3–179.9) 4.0 (1.9 –10.6) 384 ⫾ 8 196 ⫾ 3 125 ⫾ 2 49 ⫾ 1 105 (79 –143)

218/118 69 (20.5%)a 188 (55.9%)b 94 (79.7%)a 69 (20.5%)a 24.3 (7.4 –78.9)b 2.9 (1.4 –7.8)a 328 ⫾ 5b 198 ⫾ 2 123 ⫾ 2 41 ⫾ 1b 153 (113–221)b

Data are means ⫾ SEM or for nonnormally distributed variables as median (interquartile range). Group means were compared using Student’s t test. Values for triglyceride and CRP were logarithmically transformed, and values for Lp(a) were square root transformed before analyses. a

P ⬍ 0.05.

b

P ⬍ 0.001.

fibrinogen groups for Caucasians. We hypothesized that the different pattern of Lp(a) levels in African-Americans across CRP and fibrinogen groups might be due to either 1) a different distribution of apo(a) alleles or 2) a difference in allele-specific apo(a) levels. To test the first possibility, we compared the distribution of apo(a) alleles for each CRP (⬍3 vs. ⱖ3 mg/liter) or fibrinogen (⬍340 vs. ⱖ340 mg/dl) group using the KolmogorovSmirnov test. However, there were no significant differences in cumulative frequency distribution curves of apo(a) alleles for either CRP or fibrinogen levels in both ethnic groups (Fig. 1). Because we did not observe any difference in the apo(a) allele distribution across either CRP and/or fibrinogen levels, we next analyzed allele-specific apo(a) levels for different apo(a) sizes. Further analysis for allele-specific apo(a) levels was done on 426 subjects (167 African-Americans, 259 Caucasians). First we dichotomized apo(a) sizes by using the median apo(a) size (26 K4 repeats), as in our previous studies (16, 17). As seen in Fig. 2A, allele-specific apo(a) levels for apo(a) sizes less than 26 K4 were significantly higher among African-Americans with high levels of CRP (ⱖ3 mg/liter) compared with those with low levels (125 vs. 84 nmol/liter, P ⫽ 0.034). Increased allele-specific apo(a) levels were also seen among African-Americans with high fibrinTABLE 2. Plasma levels of Lp(a) in African-American and Caucasian subjects with low and high levels of CRP and fibrinogen

Lp(a) CRP ⬍ 3 mg/liter CRP ⱖ 3 mg/liter Lp(a) fibrinogen ⬍ 340 mg/dl Fibrinogen ⱖ 340 mg/dl

African-Americans (n ⴝ 231)

Caucasians (n ⴝ 336)

107.6 ⫾ 8.7 143.3 ⫾ 8.8a 104.8 ⫾ 8.6 145.6 ⫾ 8.8b

64.0 ⫾ 6.8 54.3 ⫾ 6.0 56.8 ⫾ 5.5 63.9 ⫾ 7.8

Data are means ⫾ SEM. Group means were compared using Student’s t test. Values for Lp(a) were square root transformed before analyses. a

P ⬍ 0.005 compared with low CRP (⬍3 mg/liter) group.

b

P ⬍ 0.005 compared with low fibrinogen (⬍340 mg/dl) group.

J Clin Endocrinol Metab, April 2008, 93(4):1482–1488

ogen levels (ⱖ340 mg/dl) carrying apo(a) sizes less than 26 K4 compared with those with lower fibrinogen levels (125 vs. 81 nmol/liter, P ⫽ 0.019) (Fig. 3A). For African-American subjects with larger apo(a) sizes (ⱖ26 K4), there was no difference in allele-specific apo(a) levels between the two CRP or fibrinogen groups (data not shown). Notably, no difference was observed across CRP or fibrinogen groups for either apo(a) size range among Caucasians. Because a cutoff of 22 K4 commonly has been used to define small-size apo(a), we repeated our analysis using this cutoff. We did not detect any significant differences in allele-specific apo(a) levels across CRP (⬍3 vs. ⱖ3 mg/liter) (Fig. 2B) or fibrinogen groups (⬍340 vs. ⱖ340 mg/dl) (Fig. 3B) among either AfricanAmericans or Caucasians for small apo(a) sizes (⬍22 K4). These results suggested that an increase in CRP or fibrinogen was not associated with any change of allele-specific small-size apo(a) levels. However, because our finding was limited to AfricanAmericans and because the major difference in allele-specific apo(a) levels between African-Americans and Caucasians have been found for the medium-sized apo(a) range, we extended our approach to include medium apo(a) sizes (22–30 K4). As seen in Fig. 2C, allele-specific apo(a) levels for medium apo(a) sizes were significantly higher among African-Americans with high levels of CRP compared with those with low levels (88 vs. 67 nmol/ liter, P ⫽ 0.014). Similar results were observed for fibrinogen, where higher allele-specific medium-size apo(a) levels were found among subjects with higher compared with lower fibrinogen levels (91 vs. 59 nmol/liter, P ⬍ 0.0001) (Fig. 3C). In contrast, there were no significant differences in allele-specific apo(a) levels across CRP or fibrinogen groups among Caucasians with medium-sized apo(a) alleles. Finally, we divided our study population into quartiles and repeated the analyses. In agreement with our findings, allelespecific apo(a) levels for the two middle quartiles, i.e. 23–25 K4 and 26 –28 K4 repeats representing medium-sized apo(a), were higher among African-American subjects with high levels of CRP or fibrinogen (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Notably, no difference was observed across CRP or fibrinogen groups for either quartile among Caucasians.

Discussion The main novel finding in our study was that increased levels of inflammatory markers such as CRP or fibrinogen were associated with higher allele-specific apo(a) level for medium apo(a) sizes (22–30 K4 repeats) in African-Americans but not in Caucasians. No difference in allele-specific apo(a) levels was seen in African-Americans or Caucasians with higher vs. lower CRP or fibrinogen for small (⬍22 K4) apo(a) sizes. Our findings suggest that inflammation-associated events may selectively affect apo(a) levels representing specific apo(a) genotypes in African-Americans and provide a potential explanation for differences in Lp(a) levels between African-Americans and Caucasians.

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Lp(a) levels have been reported (47– 49). Recently, it was shown that a combination of high Lp(a) levels with a high level of ei75% ther CRP or fibrinogen was associated with an increased risk for CAD (50). Further50% more, Tsimikas et al. (51) have shown that Lp(a) levels increased significantly during CRP

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