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Relationship of Plasma Polyunsaturated Fatty Acids to Circulating Inflammatory Markers

Longitudinal Studies Section (L.F.), Clinical Research Branch, National Institute on Aging, Baltimore, Maryland 21225; Department of Clinical and Experimental Medicine (A.Ch., U.S.), Institute of Gerontology and Geriatrics, Perugia University Medical School, 06100 Perugia, Italy; Department of Geriatric Rehabilitation (S.B.), Tuscany Regional Health Agency (A.Co., F.L.), 50125 Florence, Italy; Division of Nutritional Sciences (B.B.), Cornell University, Ithaca, New York 14853; Human Nutrition Research Center on Aging (A.M.), Tufts University, Boston, Massachusetts 02111; Department of Nutrition and Food Science (C.A.-L.), CeRTA, University of Barcelona, 08028 Barcelona, Spain; and Laboratory of Epidemiology, Demography, and Biometry (J.M.G.), National Institute on Aging, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Luigi Ferrucci, M.D., Ph.D., Longitudinal Studies Section, Clinical Research Branch, National Institute on Aging, National Institutes of Health, Harbor Hospital, 5th Floor, 3001 Hanover Street, Baltimore, Maryland 21225. E-mail: ferruccilu{at}grc.nia.nih.gov.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Aims: Persons with high intake of polyunsaturated fatty acids (PUFAs) have lower cardiovascular morbidity and mortality. The protective effect of PUFAs is mediated by multiple mechanisms, including their antiinflammatory properties. The association of physiological PUFA levels with pro- and antiinflammatory markers has not been established.

Methods and Results: In 1123 persons (aged 20–98 yr), we examined the relationship between relative concentration of fatty acids in fasting plasma and level of inflammatory markers. Adjusting for age, sex, and major confounders, lower arachidonic and docosahexaenoic acids were associated with significantly higher IL-6 and IL-1ra and significantly lower TGFß. Lower -linolenic acid was associated with higher C-reactive protein and IL-1ra, and lower eicosapentaenoic acid was associated with higher IL-6 and lower TGFß. Lower docosahexaenoic acid was strongly associated with lower IL-10. Total n-3 fatty acids were associated with lower IL-6 (P = 0.005), IL-1ra (P = 0.004), and TNF (P = 0.040) and higher soluble IL-6r (P < 0.001), IL-10 (P = 0.024), and TGFß (P = 0.0012). Lower n-6 fatty acid levels were significantly associated with higher IL-1ra (P = 0.026) and lower TGFß (P = 0.014). The n-6 to n-3 ratio was a strong, negative correlate of IL-10. Findings were similar in participants free of cardiovascular diseases and after excluding lipids from covariates.

Conclusions: In this community-based sample, PUFAs, and especially total n-3 fatty acids, were independently associated with lower levels of proinflammatory markers (IL-6, IL-1ra, TNF, C-reactive protein) and higher levels of antiinflammatory markers (soluble IL-6r, IL-10, TGFß) independent of confounders. Our findings support the notion that n-3 fatty acids may be beneficial in patients affected by diseases characterized by active inflammation.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THERE IS EVIDENCE that a diet rich in polyunsaturated fatty acids (PUFAs) and, in particular, the omega-3 family (n-3), is associated with lower cardiovascular morbidity and mortality and reduced risk of sudden death, independent of other known cardiovascular risk factors (1, 2, 3, 4, 5). Studies have suggested that the protective effects of n-3 PUFA are mediated by multiple mechanisms, including their antiinflammatory properties (6).

Preclinical studies have shown that fatty acids modulate the inflammatory response by multiple mechanisms, including transcriptional down-regulation of proinflammatory cytokines and the vascular surface expression of endothelial leukocyte adhesion molecules (7, 8). In particular, in experimental and animal models, n-3 fatty acids inhibit the production of IL-1 and TNF (7). An antiinflammatory effect of n-3 fatty acids is supported by the beneficial effect of n-3 fatty-acid supplementation in patients affected by diseases characterized by active inflammation, such as rheumatoid arthritis and Crohn’s disease (9). In small groups of healthy volunteers, dietary supplementation with n-3 fatty acids was associated with reduced levels of IL-1ß, thromboxane ß₂, and prostaglandin E2 (10, 11) but not C-reactive protein (CRP) (12).

A recent study showed that dietary intake of n-3 and omega-6 (n-6) fatty acids in American men and women was inversely associated with plasma levels of soluble TNF receptors 1 and 2 but not with other cytokines (13). This observation is important because it suggests that physiological levels of fatty acids modulate inflammation. However, studies on dietary intake should be complemented by studies that investigate the relationship between fatty acid plasma levels and serum levels of multiple inflammatory markers. In fact, although some studies found a good correlation between dietary intake of fatty acids and blood levels (14), others did not confirm this finding, reporting only a modest association (15), likely due to the fact that circulating fatty acid levels reflect the interplay among dietary intake, absorption, and metabolism.

Using data from a representative sample of the general population, we tested the hypothesis that circulating levels of selected PUFAs are associated with lower concentrations of proinflammatory cytokines and, possibly, higher levels of antiinflammatory cytokines. This information is important in furthering our understanding of the mechanisms by which fatty acids modulate cardiovascular risk and other clinical conditions characterized by a proinflammatory state.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study population and collection of blood samples.

Invecchiare in Chianti, aging in the Chianti area (InCHIANTI) is an epidemiological study conducted in two small towns of Tuscany, Italy. The rationale, design, and data collection methods of InCHIANTI are described elsewhere (16). In brief, in August 1998, 1270 persons aged 65 yr or older were randomly selected from the population registry of the two sites. Additionally, men and women randomly sampled from the age strata 20–29, 30–39, 40–49, 50–59, and 60–64 yr were sequentially invited to participate in the study, until at least 30 men and 30 women in each decade, ages 20–69 yr, had been enrolled.

Of the 1714 eligible persons, 640 men and 813 women (84.8%) agreed to participate and were interviewed. Of those, 595 men and 748 women (92.4%) provided blood samples. Data on plasma fatty acid and serum cytokine composition were obtained for 1180 participants (87.9%). Because poor cognitive status is associated with inflammatory conditions and strongly affects dietary intake (17), we also excluded 57 participants in whom we established a dementia diagnosis, based on the Diagnostic and Statistical Manual of Mental Disorders, version III-R criteria (18). Thus, the final study population included 1123 participants, none of whom had dietary supplementation of fatty acids.

The study protocol complies with the Declaration of Helsinki and was approved by the Italian National Institute of Research and Care on Aging Ethical Committee. Participants received an extensive description of the study and signed an informed participation consent that included permission to conduct analyses on the biological specimens collected and stored.

Laboratory analysis

Blood samples were collected in the morning after a 12-h fast. Aliquots of serum and plasma were immediately obtained and stored at –80 C. The samples used to measure circulating levels of cytokines and fatty acids had not been previously thawed.

Fatty acids were measured using a fasting plasma sample. A known amount of heptadecanoic acid (17:0) (Sigma Chemical Co., St Louis, MO) was added to each sample as an internal standard, and total lipids were extracted from 0.15 ml of plasma (19). In a pilot study, we had found that no traces of heptadecanoic acid were detectable in 25 plasma samples from InCHIANTI participants. Fatty acid methyl esters (FAME) were prepared through transesterification using Lepage and Roy’s method (20), modified according to Rodriguez-Palmero et al. (21). Separation of FAME was carried out on an HP-6890 gas chromatograph (Hewlett-Packard, Palo Alto, CA) with a 30-m fused silica column (HP-225; Hewlett-Packard). FAMEs were identified by comparison with pure standards (NU Chek Prep, Inc., Elysian, MA). For quantitative analysis of fatty acids as methyl esters, calibration curves for FAME (ranging from C14:0 to C24:1) were prepared by adding six increasing amounts of individual FAME standards to the same amount of internal standard (C17:0; 50 µg). The correlation coefficients for the calibration curves of 20 fatty acids were in all cases higher than 0.998 in the range of concentrations studied. Fatty acid concentration was expressed as a percentage. Fatty acid percent area/area was also calculated. The coefficient of variation for all fatty acids was on average 1.6% for intraassay and 3.3% for interassay.

In the present analysis, we examined data on the concentration of PUFAs, which are characterized by two or more double bonds in the hydrocarbon chain. The n-3 and n-6 families of fatty acids account for more than 95% of total PUFAs and are named from the position of the first double bond, located on the third or sixth carbon, respectively, from the terminal methyl group (22). The total n-6 fatty acids included linoleic (LA) (C18:2-n6), eicosadienoic (C20:2n6), dihomo-g-linolenic (C20:3n6), and arachidonic (AA) (C20:4n6) acids, whereas the total n-3 fatty acids included -linolenic (ALA) (C18:3n3), eicosapentaenoic (EPA) (C20:5n3), and docosahexaenoic (C22:6-n3) (DHA) acids.

Serum levels of IL-6, soluble IL-6 receptor (sIL-6r, 80 kDa), IL-1ß, IL-1 receptor antagonist (IL-1ra), and TNF were measured by ELISAs using commercial kits (BIOSOURCE International, Camarillo, CA). TGFß and IL-10 levels were measured in duplicate using highly sensitive quantitative sandwich assays (Quantikine HS, R&D Systems, Minneapolis, MN). The lowest detectable concentrations were 0.1 pg/ml for IL-6, 8 pg/ml for sIL-6r, 0.01 pg/ml for IL-1ß, 0.09 pg/ml for TNF, 4 pg/ml for IL-1ra, 7 pg/ml for TGFß, and 1.5 pg/ml for IL-10. The interassay coefficient of variation was 4.5% for IL-1ra and less than 8.0% for the other cytokines.

CRP was measured in duplicate using an ELISA colorimetric competitive immunoassay that used purified protein and polyclonal anti-CRP antibodies. The minimum detectable concentration was 0.03 mg/liter and the interassay coefficient of variation was 5.0%. Total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were assessed using commercial enzymatic tests (Roche Diagnostics, Mannheim, Germany).

Covariates

Participants were classified as nonsmokers or former smokers vs. current smokers based on self-report. Weight was measured using a high-precision mechanical scale. Standing height was measured to the nearest 0.1 cm. Body mass index (BMI) was calculated as weight (kilograms)/height (square meters). Average daily intake of energy (kilocalories) and carbohydrates, proteins, total lipids and unsaturated, and monosaturated and polyunsaturated fatty acids (grams) were estimated by administering the European Prospective Investigation into Cancer and Nutrition food frequency questionnaire, which has been extensively validated in the Italian population (23).

Study participants responded to an extensive questionnaire on habitual physical activity and were classified as sedentary if they reported being completely inactive or performing low-intensity physical activity, such as short walking or light housekeeping activities totaling less than 2 h/wk.

A physician evaluated all participants. Diseases were ascertained according to standard, preestablished criteria that combined information from self-reported physician diagnoses, current pharmacological treatment, medical records, clinical examinations, and blood tests. Diseases included in the current analysis were coronary heart disease (including angina and myocardial infarction), congestive heart failure, cerebrovascular disease (including transient ischemic attack and stroke), diabetes, and hypertension. Diagnostic algorithms were modified versions of those created for the Women’s Health and Aging Study (24). An ankle-arm index of 0.9 or less was considered indicative of peripheral arterial disease (25). Participants were asked to report all drugs taken at least once over the last 15 d. Using this information, we created a dichotomous variable indicating whether the participant received treatments that may affect circulating levels of fatty acids and/or inflammatory markers, including statins, other drugs aimed at reducing circulating lipids, steroids, nonsteroidal antiinflammatory drugs, and angiotensin-converting enzyme inhibitors. This condition is defined in the text as potentially confounding drug treatment.

Statistical analysis

Continuous variables are reported as mean ± SD and categorical variables as percentages. Log-transformed values of cytokines, except for TGFß, were used in the analysis.

The relationships of specific fatty acids with potential covariates were explored by computing age- and sex-adjusted partial Pearson correlations. Further analyses were performed to test the mutually independent effects of total n-3 and n-6 fatty acids on inflammatory markers, after adjusting for age; sex; education; intake of energy, proteins, and carbohydrates; physical activity; BMI; smoking; low-density lipoprotein (LDL) cholesterol; HDL cholesterol; triglycerides; hypertension; diabetes; coronary heart disease; congestive heart failure; stroke; peripheral arterial disease; and potentially confounding drug treatment. All analyses were performed using the SAS statistical package (version 9.1; SAS Institute, Inc., Cary, NC) with a statistical significance level set at P < 0.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The principal characteristics of the study population are reported in the first column of Table 1. Only data on total n-3 and n-6 PUFAs are shown. Both total n-3 and n-6 were negatively correlated with age (n-3: r = –0.13; n-6: r = –0.33) but not sex (n-3: r = 0.02; n-6: r = 0.03). Independent of age and sex, total n-3 PUFAs were positively correlated with education and HDL cholesterol and negatively correlated with triglycerides. Total n-6 PUFAs were positively correlated with total, LDL, and HDL cholesterol and negatively correlated with most of the other cardiovascular risk factors and cardiovascular diseases (Table 1). LA was positively and independently correlated with LDL cholesterol. LA (r = 0.31), AA (r = 0.28), EPA (r = 0.23), and DHA (r = 0.14) were positively correlated with HDL cholesterol. LA (r = 0.51) was positively correlated (r = 0.51), whereas AA (r =–0.34), EPA (r = –0.21), and DHA (r = –0.20) were negatively correlated with triglycerides. Other correlations of specific fatty acids with the variables reported in Table 1 were small (<0.09) and generally not statistically significant. In particular, neither n-3 nor n-6 fatty acids were independently correlated with parameters of dietary intake.

Of the plasma n-3 fatty acids, 13.6 ± 6.1% were ALA, 18.8 ± 5.0% were EPA and 67.7 ± 4.9% were DHA. Of the n-6 fatty acids, 75.3 ± 5.0% were LA and 24.4 ± 4.9% were AA. Levels of inflammatory markers according to quartiles of specific fatty acids are reported in Table 2. All mean values and statistical tests are adjusted for age and multiple confounders. Lower AA and DHA were associated with higher IL-6 and IL-1ra and lower TGFß. Lower DHA was also associated with lower IL-10. Lower ALA was associated with higher CRP and IL-1ra, and lower EPA was associated with higher IL-6, lower TGFß, and lower IL-10. Participants in the two lower quartiles of LA had significantly lower sIL-6r than those in the two upper quartiles. AA/EPA ratio was not associated with any of the different inflammatory markers. After removing lipids as covariates from these models, these results were substantially unchanged, except that the inverse association of IL-1ra with LA (adjusted values across quartiles: 149, 129,and 127 pg/ml; P = 0.0003) and EPA (143, 132, 128, and 130; P = 0.0076) became stronger and highly statistically significant (cf. Table 2). Restricting the analysis to the 432 men and 569 women free of prevalent cardiovascular disease, all the associations that were statistically significant in Table 2 remained statistically significant, and in addition, the inverse relationship between LA and sIL-6r and between DHA and TNF that were borderline statistically significant became statistically significant (respectively, P = 0.020 and P = 0.040).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Median serum levels of inflammatory markers according to total n-3 and n-6 PUFAs quartiles. Comparisons between groups and tests for trend are based on age- and sex-adjusted nonparametric ANOVA. The thresholds for quartile definition were 2.72, 3.27, and 3.93 for total n-3 fatty acids and 30.4, 33.13, and 36.26 for total n-6 fatty acids.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The role of sIL-6r in inflammation is still unclear. In general, a specific IL-6 receptor is expressed by the membranes of hepatocytes, monocytes/macrophages, and some leukocytes. However, the sIL-6r/IL-6 complex can stimulate a much wider range of cell types that have the gp130 protein, the inner portion of the IL-6 receptor, on their membrane. Recent literature suggests that this mechanism is active only when plasma levels of soluble gp130 are low, which is probably a rare condition. When high levels of gp130 and sIL-6r are present, a hexameric complex is created (2*IL-6 + 2*sIL-6r + 2*gp130), which tends to precipitate and has no biological activity (35). Thus, in this condition, sIL-6r levels are antiinflammatory (36). IL-6 and sIL-6r have shown opposite biological activity in several instances. Recently we reported that IL-6 is associated with insulin resistance, whereas sIL-6r has the opposite effect (37).

Similarly to Pischon et al. (13), we found some evidence that n-6 fatty acids may be antiinflammatory with no evidence of the proinflammatory activity previously suggested by many authors. However, our data suggest that the immunomodulatory effect of PUFAs may be influenced by the n-6 to n-3 ratio, which in our study was the strongest negative correlate of IL-10 and TGFß, two powerful antiinflammatory cytokines. Pischon et al. found no significant association between plasma PUFA concentrations and serum CRP levels, and in our study only ALA was an independent negative correlate of CRP. This particular finding is somewhat puzzling and difficult to interpret because the production of CRP is mainly regulated by IL-6 (38), and ALA was not a significant independent correlate of IL-6. Interestingly, in a recent trial, supplementation of ALA vs. LA for 2 yr significantly reduced serum CRP but had no effect on other inflammatory markers (39).

Our findings show that plasma levels of AA and omega-3 PUFAs, which probably reflect higher dietary intake, are associated with lower serum concentrations of certain proinflammatory cytokines and lower concentrations of certain antiinflammatory cytokines. Therefore, these findings support the view that AA and n-3 PUFAs may modulate the inflammatory response by acting both on the proinflammatory and antiinflammatory arms of the cytokine network. Such a modulatory effect on multiple signaling pathways suggests a direct regulatory effect on gene expression. Interestingly, short-term infusion of n-3 lipid emulsion markedly suppresses monocytic generation of TNF, IL-1, IL-6, and IL-8 in response to endotoxin (40).

The mechanism by which AA and n-3 fatty acids may inhibit the production of proinflammatory cytokines has been intensively investigated. Fatty acids can bind to the peroxisome proliferator activated receptors (PPaR) and PPaR, which regulate the transcription of target genes (7, 41). PPaRs can also repress gene transcription by interfering with signaling molecules, such as nuclear factor-B, therefore inhibiting the production of proinflammatory cytokines (42, 43). De Caterina and colleagues (8, 44) found that polyunsaturated fatty acids have antiinflammatory properties and hypothesized that they exert this effect because they have an unsaturated double bond, which, regardless of the n-3 or n-6 position, inactivates reactive oxygen species and prevents their interaction with nuclear factor-B. This hypothesis is consistent with our findings suggesting that n-3 and n-6 fatty acids have both antiinflammatory properties.

The most important limitation of this study is the cross-sectional nature of our analysis. Although the consistency of the effect across multiple inflammatory markers is suggestive of causality, the correlation reported in this study does not prove the link between PUFAs and inflammatory markers but suggests that the physiological concentration of PUFAs reflects the severity of inflammation independently of other risk factors. In addition, although our analysis was adjusted for a number of potential confounders, we cannot exclude the possibility that other factors affect both n-3 fatty acids and cytokine concentrations. The diet of the Tuscany population is particularly poor of polyunsaturated fatty acids (45). In the InCHIANTI study, the average estimated daily intake of PUFAs was 7.4 g, which is lower than the intake reported for other populations (46) and even compared with other Italian populations (47). On the contrary, the intake of monounsaturated fatty acids in our population tended to be high (>50% of total lipids), likely the result of the large consumption of olive oil in Italy and, in particular, in the Tuscany region. The generalizability of our findings to other populations with different dietary intake should be confirmed by other studies.

Our finding that n-3 and n-6 account for a significantly lower percentage of total fatty acids in older persons may explain the mild proinflammatory state that is often found in the elderly and is not completely accounted for by cardiovascular risk factors and morbidity (48). If this hypothesis is correct, nutritional intervention may contrast this age-related trend to a proinflammatory state.

This study also has several strengths. To our knowledge, this is the first investigation of the relationship between plasma concentrations of fatty acids and multiple proinflammatory and antiinflammatory cytokines based on a representative sample of the general population. Different fatty acids were directly measured in plasma and not estimated from dietary reports. Because no participants were using dietary supplements, our findings are based on physiological plasma concentrations; this information is more precise and provides a more objective measure of fatty acid exposure, which depends on both intake and metabolism. Finally, information on multiple potential confounders, including dietary intake, was available for all participants.

Because serum levels of specific fatty acids can be easily modified by a different selection of foods in the diet or dietary supplementation, physicians should consider dietary interventions to suppress production of proinflammatory compounds as part of the prevention and treatment of diseases in which inflammation exerts adverse effects on clinical progression.

	Acknowledgments

 
The authors are indebted to Chiara Ceccucci for integrating fatty acids peaks in the chromatograms and for data entry.

	Footnotes

 
This work was supported as a targeted project by the Italian Ministry of Health (ICS110.1/R597.71) and an unrestricted grant by Bracco Imaging SpA, Italy (to A.Ch.). C.A.-L. was supported by the Ramon Cajal program by the Ministry of Science and Technology, Spain.

First Published Online October 18, 2005

Abbreviations: AA, Arachidonic acid; ALA, -linolenic acid; BMI, body mass index; CRP, C-reactive protein; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; HDL, high-density lipoprotein; IL-1ra, IL-1 receptor antagonist; InCHIANTI, Invecchiare in Chianti, aging in the Chianti area; LA, linoleic acid; LDL, low-density lipoprotein; n-3, omega-3 family; n-6, omega-6 family; PPaR, peroxisome proliferator activated receptor; PUFA, polyunsaturated fatty acid; sIL-6r, soluble IL-6 receptor.

Received June 16, 2005.

Accepted October 7, 2005.

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