N-3 polyunsaturated fatty acids (PUFAs) have been associated with reduced inflammation. We tested the hypothesis that high serum concentrations of the n-3 PUFAs are associated with lower serum C-reactive protein (CRP) concentrations in healthy middle-aged Finnish men. We also examined whether exposure to mercury, an environmental contaminant in fish, which is also a major source of long-chain n-3 PUFA, was associated with CRP.
Data from the prospective, population-based Kuopio Ischaemic Heart Disease Risk Factor Study were analyzed cross-sectionally to determine the associations between serum n-3 PUFAs, hair mercury and serum CRP in 1395 healthy men, aged 42–60 years. Linear regression analyses were performed to analyze the associations.
In the multivariate models, the mean serum CRP in quartiles of serum total n-3 PUFA concentration was 1.23, 1.27, 1.18 and 1.08 mg/l, P for trend=0.01. Statistically significant inverse associations were also observed with the total serum long-chain n-3 PUFA concentration and with the individual long-chain n-3 PUFAs docosapentaenoic acid and docosahexaenoic acid, but not with eicosapentaenoic acid or with the intermediate-chain n-3 PUFA alpha-linolenic acid. Hair methylmercury content was not associated with serum CRP levels and it did not modify the associations between serum n-3 PUFAs and CRP either.
Serum n-3 PUFAs and especially the long-chain n-3 PUFA concentration, a marker of fish or fish oil consumption, were inversely associated with serum CRP in men. Exposure to mercury was not associated with serum CRP.
Consumption of long-chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may decrease the risk of CVD (Mozaffarian and Rimm, 2006; Wang et al., 2006), and PUFAs have also been suggested to reduce inflammation (Simopoulos, 2002). In observational studies, fish consumption, or dietary or circulating long-chain n-3 PUFAs have been associated with lower C-reactive protein (CRP), a marker for inflammation, in most (Lopez-Garcia et al., 2004; Farzaneh-Far et al., 2009; He et al., 2009; Kalogeropoulos et al., 2010), but not all studies (Pischon et al., 2003). However, randomized controlled trials with fish oil supplementation in healthy people have found no effects on CRP (Madsen et al., 2003; Geelen et al., 2004; Skulas-Ray et al., 2011).
The relationship of the intermediate-chain length n-3 PUFA, alpha-linolenic acid (ALA) with CVD is less well known than that of EPA+DHA (Harris, 2005; Anderson and Ma, 2009). In few observational studies, higher ALA intake has been associated with reduced inflammation (Ferrucci et al., 2006; Poudel-Tandukar et al., 2009), but no effects of ALA supplements on CRP have been found in randomized controlled trials of healthy people (Geleijnse et al., 2010).
Currently, more data on the association between n-3 PUFAs and CRP are available for people with a disease than for healthy people. Therefore, the aim of this study was to test the hypothesis that high serum concentrations of the n-3 PUFAs are associated with lower serum CRP concentration in healthy middle-aged Finnish men. Fish is also a major source of methylmercury, which has previously been associated with increased risk of CVD and with attenuation of the beneficial impact of the long-chain n-3 PUFAs on the risk in this study population (Virtanen et al., 2005). High mercury exposure was also associated with increased lipid peroxidation (Salonen et al., 1995), but there is little data available on the association between mercury exposure and inflammation. Thus, we also wanted to evaluate the impact of mercury exposure on CRP.
Subjects and methods
The subjects were participants in the Kuopio Ischaemic Heart Disease Risk Factor Study (Salonen, 1988). The study was designed to investigate risk factors for CVD, atherosclerosis and related outcomes in a population-based, randomly selected sample of men from eastern Finland. The baseline examinations were performed between March 1984 and December 1989. The study sample consisted of 3235 men who were 42, 48, 54 or 60 years old at the baseline examination. Of these, 2682 (82.9%) men participated. The Kuopio Ischaemic Heart Disease Risk Factor Study protocol was approved by the Research Ethics Committee of the University of Kuopio. All subjects gave their written informed consent for participation.
Men with missing data on serum CRP (n=61) or serum n-3 PUFAs (n=148) were excluded from the analyses. To investigate the association with serum CRP in a healthy population, we excluded participants with a disease with inflammatory component.
This also reduces the possible bias that is introduced if participants had changed their diet after diagnosis, for example, increased fish consumption. In total, we excluded 1078 subjects with a history of ischemic heart disease or stroke, cardiac insufficiency, claudication, diabetes, colitis, rheumatoid arthritis, gall bladder disease, cancer, or liver or pancreas disease. These exclusions left 1395 men. Data of hair methylmercury was available for 1385 of the 1395 men.
The subjects gave hair and venous blood samples between 0800 and 1000 h at the baseline examinations. They were instructed to abstain from ingesting alcohol for 3 days, and from smoking and eating for 12 h before giving the sample. Detailed descriptions of the determination of serum lipids and lipoproteins (Salonen et al., 1992), assessments of medical history and medications (Salonen et al., 1992), family history of diseases (Salonen et al., 1992), smoking (Salonen et al., 1992), alcohol consumption (Salonen et al., 1992), physical activity (Lakka et al., 1994) and blood pressure (Salonen et al., 1992) have been published previously. Dietary intake of foods and nutrients was assessed at the time of blood sampling using a 4-day food recording (Voutilainen et al., 2001). Mercury in hair was determined between May 1992 and August 1993 by flow injection analysis, cold vapor atomic absorption spectrometry and amalgamation as described previously (Salonen et al., 1995).
Serum fatty acids
Serum esterified and non-esterified fatty acids were determined in one gas chromatographic run without preseparation as described previously (Laaksonen et al., 2002). Fatty acids were chromatographed in an NB-351 capillary column (HNU-Nordion, Helsinki, Finland) by a Hewlett-Packard 5890 series II gas chromatograph (Hewlett-Packard Company, Avondale, PA, USA) with a flame ionization detector. The coefficient of variation for repeated measurements of major esterified fatty acids was ∼5%. Because the relative degree of saturation of fatty acids varies among esterified fatty acid types (i.e., cholesterol esters, phospholipids and triglycerides), the esterified fatty acid concentrations were adjusted for serum low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglyceride concentrations. The coefficient of variation for major non-esterified fatty acids was ∼15%. No adjustment was conducted for non-esterified fatty acids. For serum total long-chain n-3 PUFAs, we used the sum of EPA (20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and DHA (22:6n-3).
Serum CRP was measured with an immunometric assay (Immulite High Sensitivity CRP Assay, DPC, Los Angeles, CA, USA). This CRP assay has been standardized against the WHO International Reference Standard for CRP immunoassay 85/506. At the level of 3.2 mg/l, the within-run coefficient of variation was 2.8% and the total coefficient of variation was 3.1%.
The relationships between baseline characteristics and serum total n-3 PUFA were explored by means and linear (for continuous variables) or logistic regression (for dichotomous variables). The associations between serum fatty acids and CRP were analyzed with linear regression models, using log-transformed values. The means and the 95% confidence intervals in quartiles of serum fatty acids were calculated of log-transformed serum concentrations and the values were then exponentiated. Correlations were estimated by the Spearman's correlation coefficients. The models were adjusted for possible confounders, selected on the basis of previously published associations with CRP or associations with exposures or outcomes in the present analysis. The basic model (Model 1) included age and examination year. The multivariate-adjusted Model 2 included the Model 1 and smoking, body mass index and income. Further adjustments for aspirin use, multivitamin use, drug for high cholesterol, hair methylmercury, years of education, leisure-time physical activity, treated hypertension or intake of alcohol, energy, fruits and vegetables, milk and milk products, meat and meat products or whole grains did not appreciably change the associations. The cohort mean was used to replace the missing values (<1.7%) of covariates. Tests of linear trend were conducted by assigning the median values for each category of exposure variable and treating those as a single continuous variable. All P-values were two-tailed (α=0.05). Data were analyzed with SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA).
The baseline characteristics of the subjects are presented in Table 1. The mean age of the cohort was 52.1 years (s.d. 5.5 years). The mean serum concentrations as a percentage of all serum fatty acids were 1.65% (s.d. 0.92%) for EPA, 0.56% (s.d. 0.10%) for DPA, 2.46% (s.d. 0.72%) for DHA and 0.75% (s.d. 0.23%) for ALA. The correlations with fish consumption were 0.43 for EPA, 0.20 for DPA and 0.44 for DHA (P for all <0.001). At baseline, men with higher serum total n-3 PUFA, compared with men with lower concentrations, were more likely to have higher education and income, higher intake of fish, fruits and vegetables and alcohol, and higher hair methylmercury concentration (Table 1). They also had lower intakes of energy, milk and milk products, whole grains and total fats, and were also less likely to be current smokers.
Serum n-3 PUFAs and CRP
The mean CRP concentration was 1.95 mg/l (s.d. 3.25 mg/l). After adjustment for age, examination year, smoking, body mass index and income (Model 2 in Table 2), we found a statistically significant inverse association between serum total n-3 PUFA, total long-chain n-3 PUFA (EPA+DPA+DHA), DPA, and DHA and CRP. Of the individual fatty acids, DPA was most strongly associated with CRP (Table 2). Serum ALA was associated with lower CRP after adjustment for age and examination year (Model 1), but further adjustments for smoking, body mass index and income attenuated the association. Serum EPA was not statistically significantly associated with CRP (Table 2).
Because subjects with acute inflammatory conditions frequently have serum CRP concentrations ⩾10 mg/l (Pepys and Hirschfield, 2003), we also repeated the analyses after excluding 29 participants with the serum CRP ⩾10 mg/l. However, the results were not appreciably different (data not shown).
Hair methylmercury and CRP
The mean hair methylmercury concentration was 1.87 μg/g (s.d. 1.91 μg/g). We did not find an association between hair methylmercury and CRP. The age, examination year, smoking, body mass index and income-adjusted (Model 2 in Table 2) CRP concentrations in the hair methylmercury quartiles were 1.08, 1.15, 1.10 and 1.02 mg/l, P for trend=0.37. Hair methylmercury concentration did not modify the associations between serum n-3 PUFAs and CRP either (P for interactions >0.10).
In this cross-sectional study among healthy middle-aged men from eastern Finland, serum total n-3 PUFA, total long-chain n-3 PUFA (EPA+DPA+DHA), DPA and DHA were inversely associated with serum CRP concentration. In contrast, serum ALA and EPA were not associated with serum CRP.
Our results are similar to most population-based studies, where consumption of non-fried fish or long-chain n-3 PUFA (He et al., 2009), or individual and total n-3 PUFA intake (Lopez-Garcia et al., 2004) has been associated with lower CRP, although not all studies have found an association (Pischon et al., 2003). In addition, higher levels of circulating long-chain n-3 PUFAs have been associated with lower CRP levels (Farzaneh-Far et al., 2009; Kalogeropoulos et al., 2010). However, randomized controlled trials with fish oil supplementation in healthy people have found no effects on CRP (Madsen et al., 2003; Geelen et al., 2004; Skulas-Ray et al., 2011). This suggests that fish consumption or higher serum long-chain n-3 PUFA concentrations may be a marker of other lifestyle factors associated with lower CRP in observational studies. The role of residual confounding cannot be wholly excluded either.
Of the individual fatty acids, DPA had the strongest association with CRP in our study. There is little prior information about DPA and CRP or inflammation. Compared with EPA or DHA, concentration of DPA in serum is low, because DPA is converted into DHA or EPA (Kaur et al., 2011). The low concentration likely contributes to the magnitude of the regression coefficient with CRP (Table 2). Serum DPA also had a weaker correlation with fish intake compared with EPA and DHA, which suggests that it is not a very good marker for fish consumption. In our previous analysis, serum DPA was not associated with the risk of atrial fibrillation in this study population (Virtanen et al., 2009). The impact of DPA on inflammation and CVD risk requires further investigation.
ALA has been associated with lower risk of CVD, but the association has not been very strong and consistent (Wang et al., 2006). ALA intake has been associated with lower CRP in some (Lopez-Garcia et al., 2004; Poudel-Tandukar et al., 2009), but not all studies (Pischon et al., 2003). However, less is known about the association between circulating ALA concentrations and inflammation. In a community-based sample from Italy, an inverse association between plasma ALA and CRP was observed, but interestingly, no significant independent correlation between ALA and interleukin-6 was found (Ferrucci et al., 2006), taken into account that the production of CRP is mainly regulated by interleukin-6 (Gabay and Kushner, 1999). The few small randomized controlled trials that have been conducted do not support the protective association between ALA and inflammation (Geleijnse et al., 2010), which suggests that ALA does not have a major role in protection against inflammation.
The anti-inflammatory properties of long-chain n-3 PUFAs may be explained by their unsaturated double bonds. These bonds may inactivate reactive oxygen species and prevent their interaction with, for example, nuclear factor-κB (De Caterina and Libby, 1996; De Caterina et al., 1999). Indeed, fish oil has been associated with anti-inflammatory gene expression, for example, with decreased expression of nuclear factor-κB (Bouwens et al., 2009).
Although we have previously shown that exposure to mercury was associated with increased risk of CVD and overall mortality in this study population (Virtanen et al., 2005), the current results do not support that this is explained by its effects on chronic low level inflammation.
Strength of this cross-sectional study is the use of serum n-3 PUFA measurements instead of dietary intakes for estimating the impact of these fatty acids on CRP. Serum long-chain n-3 PUFA concentration is a good measurement for dietary intakes of fish or fish oil (Hunter, 1998), and the serum concentrations reflect dietary intakes during the previous weeks (Nikkari et al., 1995). In contrast, a large proportion of ALA is oxidized (Burdge, 2006), so serum levels would depend more on recent intake. Serum ALA also correlates only weakly with dietary ALA intake (Hunter, 1998). We also excluded people with diseases that might have caused a diet change and biased associations. Other strengths of the study are the extensive examination of potential confounders and risk factors, and its population-based recruitment. A few limitations need to be taken into account. The study population consisted of middle-aged Finnish men, so the results may not be generalizable to other populations. Furthermore, because of the cross-sectional study design, conclusions on causality cannot be drawn. We only had data on total serum fatty acid concentrations available, although different serum compartments (e.g., phospholipids or cholesterol esters) reflect different time periods of fatty acid intake, and may have different associations with CRP (Klein-Platat et al., 2005). Theoretically, because several associations were evaluated in this study, it is possible that some may have been found due to type I error. However, although a single association could be a chance finding, the totality of evidence that supports the association of PUFAs with CRP is unlikely produced by chance alone.
In conclusion, this study suggests that higher concentrations of serum long-chain n-3 PUFA are associated with lower serum CRP concentrations. As inflammation has been suggested to increase the risk of CVD, the beneficial impact on long-chain n-3 PUFAs on the risk of CVD may partly be explained by their impact on inflammation. Further studies are needed to investigate the effect of different n-3 PUFAs on inflammation.
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The authors declare no conflict of interest.
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Cite this article
Reinders, I., Virtanen, J., Brouwer, I. et al. Association of serum n-3 polyunsaturated fatty acids with C-reactive protein in men. Eur J Clin Nutr 66, 736–741 (2012). https://doi.org/10.1038/ejcn.2011.195
- cardiovascular diseases
- C-reactive protein
- polyunsaturated fatty acids
- prospective study
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