The Impact of Age, Body Mass Index, and Fish Intake on the EPA and DHA Content of Human Erythrocytes Scott A. Sands, Kimberly J. Reid, Sheryl L. Windsor, and William S. Harris* Mid America Heart Institute, Saint Luke’s Hospital, Kansas City, Missouri 64111, and the Department of Medicine, University of Missouri–Kansas City, Kansas City, Missouri 64108
ABSTRACT: n-3 FA are beneficial for cardiovascular health, reducing platelet aggregation, TG levels, and the risk of sudden death from myocardial infarction. The percentage of EPA + DHA in red blood cells (RBC), also known as the Omega-3 Index, has recently been proposed as a risk marker for death from coronary heart disease (CHD). The purpose of this study was to begin to explore the factors that can influence RBC EPA + DHA. We collected information on the number of servings of tuna or nonfried fish consumed per month, as well as on age, gender, ethnicity, smoking status, the presence of diabetes, and body mass index (BMI) in 163 adults in Kansas City who were not taking fish oil supplements. The average RBC EPA + DHA in this population was 4.9 ± 2.1%. On a multivariate analysis, four factors significantly and independently influenced the Omega-3 Index: fish servings, age, BMI, and diabetes. The Index increased by 0.24 units with each additional monthly serving of tuna or nonfried fish (P < 0.0001), and by 0.5 units for each additional decade in age (P < 0.0001). The Index was 1.13% units lower in subjects with diabetes (P = 0.015) and decreased by 0.3% units with each 3-unit increase in BMI (P = 0.001). Gender or smoking status had no effect, and the univariate relationship with ethnicity vanished after controlling for fish intake. Given the importance of n-3 FA in influencing risk for death from CHD, further studies are warranted to delineate the nondietary factors that influence RBC EPA + DHA content. Paper no. L9662 in Lipids 40, 343–347 (April 2005).
vascular disease (1). This would equate to approximately 500 mg/d of EPA + DHA. The percentage of EPA + DHA in the red blood cell (RBC) membranes is a well-known biomarker of long-chain n-3 FA intake. Recently termed the “Omega-3 Index,” it has been proposed as a new risk marker for CHD death (8). The RBC EPA + DHA has been shown to be responsive to increased intakes of n-3 FA (8), a valid surrogate for human myocardial n-3 content (9), and to be strongly associated with reduced risk of sudden cardiac death (10,11). Based on these previous studies, an RBC EPA + DHA of 8% or above has been proposed as a target cardioprotective level (8). The EPA + DHA content of RBC membranes varies across populations, presumably as a function of fish intake. Levels are generally low in Europeans (12,13), Americans (14,15), and Canadians (16), and high in Japanese (17) and Norwegians (18). Factors other than diet that may influence the incorporation of EPA + DHA in RBC have not been systematically investigated. The purpose of this study was to estimate average values for RBC EPA + DHA in a sample of adults from a Midwestern U.S. city, and to begin exploring the effects of gender, age, body mass index (BMI), ethnicity, smoking status, and the presence of diabetes on RBC EPA + DHA independent of fish intake. MATERIALS AND METHODS
The n-3 FA EPA and DHA are known to have cardioprotective effects (1). Some of these effects include a reduction of platelet aggregation (2,3) and serum TG levels (4), and an increased heart rate variability (5), the latter being predictive of a lower risk of mortality due to cardiac arrhythmia (6). In the GISSI-Prevenzione study (7), n-3 FA supplementation produced a 20% reduction in total mortality and a 45% reduction in relative risk for sudden cardiac death, consistent with a reduced risk for arrhythmias. Because of these and other findings, the American Heart Association (AHA) now recommends that patients with known coronary heart disease (CHD) consume about 1 g/d of EPA + DHA. For individuals without known CHD, the AHA recommends at least two servings/wk of (preferably oily) fish to reduce the risk of cardio*To whom correspondence should be addressed at 4320 Wornall Rd., Suite 128, Kansas City, MO 64111. E-mail:
[email protected] Abbreviations: AHA, American Heart Association; ANCOVA, analysis of covariance; BMI, body mass index; CHD, coronary heart disease; RBC, red blood cells.
Copyright © 2005 by AOCS Press
Volunteer selection. Healthy men and women between the ages of 20 and 80 from the Kansas City Metropolitan area were invited to participate in this study. Although generally healthy individuals were sought, those with self-reported, well-controlled type 1 or type 2 diabetes mellitus qualified as well. Individuals reporting consumption of fish oil supplements were excluded. The demographic information collected included age, gender, height, weight, ethnicity, smoking, diabetes, and the number of servings of tuna or nonfried fish consumed per month (hereafter referred to as “fish intake”). Weekly fish intake was calculated based on the reported monthly intake. The intake of tuna or nonfried fish was specifically queried because the answer to this question proved to be a powerful predictor of risk for death from ischemic heart disease in the Cardiovascular Health Study (19). Hence, we sought to determine whether it would correlate with RBC EPA + DHA. The protocol was approved by the Saint Luke’s Hospital Institutional Review Board, and informed, written consent was obtained.
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Laboratory methods. The sum of the EPA and the DHA, expressed as a percentage of total RBC FA, was measured as described previously (8). Briefly, blood was collected in EDTA, and the RBC pellet was isolated by centrifugation. After discarding the plasma and buffy coat, RBC were frozen at –70°C. The RBC pellet was warmed to room temperature, and an aliquot was extracted with isopropanol and hexane (each containing 50 mg/L of the antioxidant BHT). The lipid extract was dried under nitrogen and methylated for 10 min at 100°C with BF3–methanol (Sigma, St. Louis, MO). After cooling, the FAME were extracted with hexane after the addition of water. The sample was again dried under nitrogen, reconstituted with hexane, and analyzed by flame ionization GC (Shimadzu GC-14A; Shimadzu Corporation, Kyoto, Japan) using a capillary column (SUPELCOWAX 10, 30 m length, 0.32 mm i.d., 0.25 µm film thickness; Supelco, Bellefonte, PA). FA were identified by comparison with known standards, and FA composition was reported as the weight percentage of total FA. Statistical analysis. Univariate regression and one-way ANOVA models were used to determine the effects of each patient characteristic on RBC EPA + DHA. Analysis of covariance (ANCOVA) was used to determine the effect of fish consumption on RBC EPA + DHA after considering other possible influences (age, BMI, diabetes, gender, race, smoking). Nonlinear relationships were explored by testing quadratic terms in the ANCOVA models. Model assumptions were checked and verified. A critical alpha level of 0.05 was used to determine significance, and all significance tests were two-tailed. All data were analyzed using SAS, version 8.02 (SAS Institute, Cary, NC).
month of tuna and nonfried fish, 4.2 ± 4.2 (range 0–30); smokers, 13 (8%); persons with diabetes, 13 (8%). The mean (±SD) RBC EPA + DHA was 4.9 ± 2.1%, with a range of 1.7 to 12.4% (Fig. 1). RBC EPA + DHA was below 8% in 91% of the sample and below 4% in 44% of the sample (Fig. 1). Of the seven patient characteristics examined, five (all but gender and smoking status) proved to be significant predictors of RBC EPA + DHA on a univariate analysis (Table 1). On a multivariate analysis, ethnicity ceased to be a predictor, leaving BMI, age, diabetes, and fish intake as independent predictors. From Table 1, one can see that every additional 10 yr of age increased RBC EPA + DHA by 0.5 units, that each 3-unit rise in BMI decreased the Omega-3 Index by 0.3 units, and that the presence of diabetes was associated with a 1.13unit decrease in RBC EPA + DHA. For every reported monthly serving of tuna or other nonfried fish, RBC EPA + DHA increased by 0.24 units. The relationships between reported fish intake (by frequency category and continuously) and the Omega-3 Index are presented in Figures 2 and 3. In both cases, there was a highly significant positive relationship between intake and this biomarker. Although overall, men and women did not have significantly different unadjusted values for RBC EPA + DHA (5.0 ± 1.9 vs. 4.7 ± 2.2, P = 0.25), an examination of the male– female difference by decade suggested that women tended to have slightly higher levels than men (except for the 30s; Fig.
RESULTS The study population included 163 individuals, 74 males and 89 females [Caucasian, 134 (82%); African American, 13 (8%); Hispanic, 10 (6%); Native American, Asian, East Indian, Arabic, 6 (3.6%)], with an average (±SD) age of 48 ± 15 yr (ages 20–29, 24; ages 30–39, 27; ages 40–49, 36; ages 50–59, 35; 60 and above, 41). Other study population characteristics were as follows: BMI, 26.2 ± 4.8 (range 18–47); servings per
FIG. 1. Distribution of the percentage of red blood cell (RBC) EPA + DHA values (Omega-3 Index) in the study population. Lines at 8% and 4% indicate proposed low- and high-risk horizons, respectively, and the dotted line at 4.9% is the population average.
TABLE 1 Red Blood Cell EPA + DHA Model Results Univariatea Lower
Upper
Multivariateb Lower
Upper
Independent variable
Estimate
95% CI
95% CI
P-value
Estimate
95% CI
95% CI
P-value
Fish servings Age (10 yr) BMI (3 units) Diabetes Female Caucasian Smoker
0.30 0.53 −0.40 −1.15 0.37 1.07 −0.20
0.24 0.33 −0.59 −2.32 −0.27 0.25 −1.38
0.36 0.73 −0.21 0.01 1.01 1.89 0.98
