Increased F₃-Isoprostanes in the Canadian Inuit Population Could Be Cardioprotective by Limiting F₂-Isoprostane Production

Abstract

Context:

F₃-isoprostanes (F₃-IsoPs), derived from peroxidation of eicosapentaenoic acid (C20:5n-3), could be cardioprotective by limiting production of F₂-isoprostanes (F₂-IsoPs), a cardiovascular disease risk factor.

Objective:

The objective of the study was to determine whether the n-3-polyunsaturated (PUFA)-rich Inuit diet is associated with a lower plasma ratio of F₂-IsoPs to F₃-IsoPs.

Design:

This was a cross-sectional observational study.

Setting:

The study was conducted in 36 Canadian Arctic Inuit communities.

Participants:

Participants included a random subset (n = 233) of Inuit adults taken from a population-based survey.

Main Outcome Measures:

Plasma F₂-IsoPs and F₃-IsoPs, cardiometabolic risk factors (blood lipids, C-reactive protein, blood pressure, fasting glucose) and markers of dietary exposure (erythrocyte n-3 and n-6 PUFA, blood levels of Se, mercury, polychlorinated biphenyls) were measured.

Results:

Inuit aged 40 years old and older vs younger Inuit showed higher concentrations of plasma F₃-IsoPs and erythrocyte n-3 PUFA and lower plasma F₂-IsoPs concentrations despite having higher blood lipids, fasting glucose, systolic blood pressure, and percentage body fat. Plasma F₃-IsoPs were not associated with any cardiometabolic measures. When subjects were categorized into tertiles according to total n-3 PUFA erythrocyte concentrations, F₃-IsoPs increased with increasing tertiles, whereas the F₂-IsoP to F₃-IsoP ratio was lowest at the highest n-3 tertile. The F₂-IsoP to F₃-IsoP ratio was significantly predicted by C20:5n-3 (β= −.365, P = .002); C20:4n-6:C20:5n-3 (β = .056, P = .006), blood mercury (β = −.812, P =.015), blood Se (β = −1.95, P = .015), and smoking (β = .745, P = .025).

Conclusions:

Plasma F₃-IsoPs were not associated with cardiometabolic risk factors previously seen with F₂-IsoPs. Higher n-3 fatty acid status was associated with lower plasma F₂-IsoPs and higher plasma F₃-IsoPs, which provides partial explanation to the cardioprotective effects of the n-3 PUFA-rich Inuit diet.

Abstract

Plasma F3-Isoprostanes were measured in relation to F2-Isoprostanes in Canadian Inuit. F3-IsoPs levels provide partial explanation to the cardioprotective effects of the n-3 PUFA-rich Inuit diet.

Numerous epidemiological and interventional studies have shown that regular consumption of fish containing a high content of the n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3), is associated with reduced cardiovascular risk and mortality (1, 2). Several traditional and nonconventional cardiovascular risk factors are influenced favorably by n-3 PUFA intake including plasma triglyceride levels (3), arrhythmias and thrombosis (4), proinflammatory eicosanoids, and endothelial function (5). It is well established that the 3-, 5-series eicosanoids derived from EPA are less biologically active as compared with the 2-, 4-series eicosanoids generated from the n-6 PUFA arachidonic acid (AA; C20:4n-6) and thus are considered as less inflammatory (6). More recently there is evidence that due to the high degree of unsaturation of EPA, it can readily undergo oxidation to several bioactive compounds that may exert cardioprotective properties (7, 8). In that regard, F₃-isoprostanes (F₃-IsoPs), formed from free radical-induced peroxidation of EPA, may exert antiatherogenic and antiinflammatory effects by acting as competitive inhibitors of cyclooxygenase, reducing the production of the proinflammatory 2-series prostaglandins, and thromboxane generated from AA (7). Also, oxidized EPA, but not native EPA, significantly inhibits human neutrophil and monocyte adhesion to endothelial cells, which is linked to the development of atherosclerosis and inflammation (8). Dietary EPA may also diminish the formation of proinflammatory F₂-isoprostane (F₂-IsoPs) derived from n-6 PUFAs by channeling the free radical pathway away from F₂-IsoPs. Mice receiving EPA supplementation showed markedly increased heart tissue levels of F₃-IsoPs together with reduced levels of the proinflammatory F₂-IsoPs by up to 64% (6).

The Inuit diet is high in n-3 PUFAs, which is thought to contribute to the historically low prevalence of chronic diseases in this population (9). In recent years, however, a shift away from the traditional diet toward an increasingly Western-style based diet has been observed that could increase the burden of chronic diseases including obesity, cardiovascular disease, and type 2 diabetes (10). The health benefits of the traditional Inuit diet are complicated by presence of prooxidant contaminants such as methylmercury and polychlorinated biphenyls (PCBs), which could be partly counteracted by the high n-3 PUFA and selenium (Se) intake of the Inuit (11). We have demonstrated that plasma levels of isofurans, formed by free radical-mediated peroxidation of arachidonic acid, were inversely related to Se intake from traditional Inuit foods (12). Additionally, F₂-IsoPs and isofurans correlated positively to the proinflammatory marker high sensitivity C-reactive protein (CRP) and systolic blood pressure (SBP) (12).

Previous studies have highlighted a generational gap with traditional food use, ie, the elderly consume more traditional foods compared with the younger generation (13). With the current trend of decreased consumption of traditional foods in the younger generation, it is important to examine the impact of such dietary change as assessed via red blood cell (RBC) fatty acid profiles and plasma oxidative stress parameters. We hypothesized that a decline in traditional food intake commonly observed between age groups (young Inuit younger than 40 y vs older than 40 y) would be exhibited by an altered RBC membrane fatty acid composition that would be associated with a lower plasma ratio of F₂-IsoPs to F₃-IsoPs.

The objectives of the present study were to: 1) determine F₃-IsoP levels and their relationship to F₂-IsoPs and to metabolic and dietary parameters; 2) investigate the association of RBC fatty acid composition with F₂-IsoPs and F₃-IsoPs; and 3) compare the metabolic and lipid profiles of two age categories (younger than 40 y vs older than 40 y) in relation to plasma isoprostanes.

Subjects and Methods

Study population

The current study is based on a random subsample of participants of a population-based International Polar Year Inuit Health Survey, details of which are available elsewhere (14). A cross-sectional survey was conducted in the summer and fall 2007 and 2008 for 33 coastal communities and for three noncoastal communities, which represented all communities in Inuvialuit Settlement Region (Northwest Territories), Nunavut, and Nunatsiavut Region (Northern Labrador). Trained interviewers and nurse staff collected information on subjects' dietary habits, physical activity, psychosocial factors, medical history, blood pressure (BP), anthropometric indices, fasting lipids, and various clinical indices. Medical files were used to obtain information on study participants with regard to chronic disease conditions and medication usage. Age, sex, and smoking and alcohol consumption were collected by questionnaires. Territorial research licenses were obtained and the Ethical Review Board of the McGill University Faculty of Medicine approved the study. Informed consent was obtained from all participants prior to enrollment. The study was performed in accordance with ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Anthropometric, physiological measures, and definitions

Height, weight, and waist circumference (WC) and BP were measured during a clinical session, performed by a trained research nurse according to the same standard protocol in the survey as has been previously reported (14). A body mass index (BMI) of 25.0–29.9 kg/m² was considered overweight, and a BMI of 30 kg/m² or greater was considered obese.

Blood collection and lipid and fatty acid analyses

All samples were taken under fasting conditions and stored at −80°C for future analysis. Fasting serum total cholesterol (T-Chol), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were determined using enzymatic colorimetric tests, and low-density lipoprotein cholesterol (LDL-C) was calculated by Nutrasource Diagnostics (Life Laboratories-Gamma Dynacare). The interassay coefficients of variation (CVs) were 0.76%, 1.13%, and 0.76%, respectively, for T-Chol, high-density lipoprotein, and TGs. Serum concentrations of high-sensitivity CRP were determined using an immunoturbidimetric assay with SYNCHRON high-sensitivity CRPH reagent in conjunction with SYNCHRON Systems CAL 5 Plus (Beckman Coulter Inc) in the Centre for Indigenous Peoples' Nutrition and Environment at McGill University. The interassay CV was less than 10%. According to the American Heart Association recommendations (12), CRP levels of 10 mg/L or greater represent evidence of active infection, systemic inflammatory processes, or trauma, so those individuals were excluded. Fasting plasma glucose was measured by a hexokinase enzymatic assay using the Roche Modular system. Lipids were extracted from the blood samples according to the method of Folch et al (15). Fatty acid composition of RBCs was determined based on previous studies (9). The fatty acid methyl esters were prepared by the method of Morrison and Smith (16) and were analyzed on a Varian 2400 gas-liquid chromatograph with a 60-m DB-23 capillary column (0.32 mm internal diameter). The interassay CVs for fatty acid analysis were 2%, 3%, and 3% for the major saturated fatty acids (SFAs; C16:0 and C18:0), n-6 (C18:2 n-6, C20:3 n-6 and C20:4 n-6), and n-3 fatty acids (C20:5 n-3 and C22:6 n-3), respectively.

Analysis of total blood mercury (Hg) and Se levels

Analyses of total blood Hg and Se were performed at the Laboratoire de Toxicologie, Institut National de Santé Publique du Quebéc, which participates in the quality assurance/quality control programs of the Canadian Northern Contaminants Program and the Arctic Monitoring Assessment Program. Whole-blood samples were diluted in a basic solution containing octylphenol ethoxylate and ammonia followed by inductively coupled plasma mass spectrometric analysis. Matrix matched calibration was performed using blood from a nonexposed individual. The interassay CVs for blood Hg and Se measurements were 2.1% and 6.1%, respectively. The Laboratoire de Toxicologie partakes in the quality assurance/quality control programs of the Canadian Northern Contaminants Program and the Arctic Monitoring Assessment Program.

Analysis of PCBs

Analysis of PCBs was performed at the laboratory of the Centre de Toxicologie du Québec (Québec, Canada), which is accredited by the Canadian Association for Environmental Analytical Laboratories. Sixteen PCB congeners (International Union for Pure and Applied Chemistry; numbers 28, 52, 99, 101, 105, 118, 128, 138, 153, 156, 163, 170, 180, 183, and 187) and Aroclor 1260 were measured in the purified extracts with an HP 5890 high resolution gas chromatography (GC) equipped with dual capillary columns (HP Ultra I and Ultra II) and dual Ni63 electron capture detectors (Hewlett Packard). The CVs for PCBs ranged from 3.9% to 18.5% apart from PCB 105, which had a CV of 31.6%. PCBs were reported on a standardized lipid-adjusted basis when relying on blood specimens for quantifying concentration of lipophilic environmental contaminants (17). Estimates of total serum lipids were calculated by the following formula: total lipids = 0.9 + 1.3(cholesterol + triglycerides) (18). Lipids were measured using the standard enzymatic procedures as explained above.

Isoprostane analysis

Plasma samples were prepared from blood samples and stored at −80°C until time of analysis. Purification, derivatization, and analysis of F₂-IsoPs and F₃-IsoPs by stable isotope dilution GC/negative ion chemical ionization mass spectrometry were performed as previously described (19). An Agilent 5973 mass spectrometer coupled to an Agilent 6890N GC using a 15 mDB 1701 GC column was used with an inlet temperature of 260°C. The helium carrier gas flow rate was 2 mL/min. For sample injection, the GC oven was programmed to run from 190°C to 300°C at 20°C/min for 9 minutes. Selective ion GC/negative ion chemical ionization mass spectrometry monitoring was 569 m/z for F₂-IsoPs, 567 m/z for F₃-IsoPs, and 573 m/z for the internal standard (2H⁴) 15-F₂t-IsoP. Values are expressed in picograms per milliliter of plasma. The precision of the assay is ±6% and the accuracy is 96%.

Statistical analysis

Anthropometrics, clinical, and biochemical measures for all subjects were reported as mean ± SD. Prevalence of preexisting medical conditions was determined based on self-reports and/or medication intake for the existing condition. The primary outcomes in terms of the isoprostane variables were treated on a continuous scale in the statistical analyses. Skewed variables were logarithmically transformed. Results were described as geometric mean ± 95% confidence interval (CI) for log-transformed data. Testing for normality was performed by looking at the histogram and normal probability plots of the residuals in addition to skewness and kurtosis measures in the descriptive statistics. Smoking status and alcohol consumption were considered as categorical variables (smokers vs nonsmokers; alcohol drinkers vs nondrinkers). Fatty acid content in erythrocyte membranes was expressed as a percentage of total fatty acids. The n-3 PUFAs and n-6 PUFAs were calculated by summing the concentrations of individual fatty acids from the same class if they were detectable, and the ratio between the two parameters was calculated. Fatty acid data are presented as mean and 95% CI. The plasma levels of F₂-IsoPs and F₃-IsoPs were compared using a Student's t test between the two age groups (<40 y and ≥40 y).

Plasma F₂-IsoPs and F₃-IsoPs, the ratio of F₂-IsoPs to F₃-IsoPs and fatty acid concentrations as a percentage of total fatty acids were evaluated by two age categories using age 40 years as a cutoff point, which has been consistently used as the age cutoff in previous Inuit studies (9, 10, 13). Correlation analysis was performed using Pearson's correlation analysis to assess the relationship between plasma concentrations of measures of oxidative stress (F₂-IsoPs and F₃-IsoPs) and other study parameters including obesity measures and cardiovascular risk factors. A partial correlation analysis was performed accounting for age, sex, and WC and for total lipids, Hg, and Se. An ANOVA was used to compare the characteristics of cardiometabolic risks including oxidative stress among tertiles of erythrocyte total n-3 PUFA concentrations. If the overall difference was significant, pairwise comparisons were conducted with Bonferroni correction.

All P values were two tailed, and P < .05 was considered significant for all tests performed. To estimate final predictors of the individual isoprostane biomarker variability and examine the influence of priori selected confounding variables (obesity, smoking, medical history) (5, 6, 9, 19), a multivariable analysis with stepwise regression was used. For the stepwise regression, an adjusted α value of .01 was used to exclude variables that had little or no influence on the biomarker under analysis. Cases with missing values were excluded pairwise. Goodness of fit of the model was tested by observing the normality of the residuals and checking the coefficient of determination (The R² measure of goodness of fit). All statistical analysis was performed using SPSS version 13.0 software (SPSS Inc).

Results

The current study is based on 294 subjects aged 18 years and older chosen randomly from the International Polar Year Inuit Health Survey from which specimens were available for full analysis. Of the 294 individuals available for analyses, CRP was elevated (≥10 mg/L) for 20.8% of subjects (n = 60), who were excluded from the study. In addition, one outlier with F₂-IsoP levels with much lower levels than the lower limit of 95% CI was also excluded, decreasing the sample size to 233 subjects. The mean age of the participants was 42.6 ± 15.4 years. Of the 233 participants (56% women), the mean BMI was 27.78 kg/m²; 33.5% were overweight and 30.4% were obese. Based on medical histories of participants the prevalence was 5.7% for diabetes, 28.6% for hypertension, 13.7% for dyslipidemia, 8.5% for cancer, 5.3% for episodes of myocardial infarction, and 3.1% for stroke. Seventy percent of participants were current smokers, with 41.8% of smokers reporting more than 10 cigarettes/d, and 65% of all participants reported drinking alcohol in the past year. The mean isoprostane (log transformed) values of F₂-IsoPs, F₃-IsoPs, and the F₂-IsoP to F₃-IsoP ratio are presented in Table 1. Sixty-six subjects (28.3%) of our population subsample had plasma F₂-IsoP levels of 35 pg/mL or greater, which is considered to be the upper value in the range for normal human plasma levels (30 ± 6 pg/mL) (20).

Table 1.

Plasma Concentrations of Isoprostanes and Relative Concentrations of Erythrocyte Fatty Acids (Percentage of Total Fatty Acids) of Study Population

	n	Mean	95% CI
F2-IsoPs, pg/mL	230	27.31	25.73–28.64
F3-IsoPs, pg/mL	184	3.71	3.23–4.23
F2-IsoP to F3-IsoP ratio	183	2.74	2.50–2.97
Total n-3a	233	6.05	5.61–6.48
EPA	233	1.51	1.36–1.66
DHA	233	2.57	2.38–2.76
EPA + DHA	233	4.07	3.77–4.38
Total n-6b	233	24.95	24.12–25.77
AA	233	7.88	7.47–8.28
n-3:n-6	233	.25	.23–.27
n-6:n-3	233	6.27	5.42–7.11
AA to EPA ratio	233	8.65	7.62–9.68
PUFAc	233	30.99	29.97–32.01
SFAd	233	43.04	42.36–43.72
MUFAe	233	24.60	24.20–25.00
TFAf	233	1.37	1.28–1.46

^a

C18:3 + 18:4 + 20:3 + 20:4 + 20:5 + 22:5 + 22:6.

^b

C18:2 + 18:3 + 20:2 + 20:3 + 20:4 + 22:2 + 22:4 + 22:5.

^c

(n-3 + n-6 series).

^d

C14:0 +16:0 + 17:0 + 18:0 + 20:0 + 22:0 + 24:0.

^e

C14:1 + 16:1 + 18:1 + 20:1 + 22:1 + 24:1.

^f

C16:1t + 18:1t + 18:2t.

F₃-IsoP relationship to F₂-IsoPs, Se, and contaminants

Pearson's correlations showed that F₃-IsoPs were positively correlated with F₂-IsoPs (r = 0.191, P = .01). In terms of blood Se, the F₃-IsoP relationship was the opposite to that observed with F₂-IsoPs (blood Se [r = −.146, P = .044]) because they were positively correlated with Se (r = 0.276, P < .001). In terms of contaminants, F₃-IsoP was positively correlated with Hg (r = 0.358, P < .001) and with the Hg to Se ratio (r = 0.305, P < .001). The ratio of F₂-IsoPs to F₃-IsoPs was highly correlated to F₃-IsoPs (r = −.809, P < .001), which indicates that it was modulated by F₃-IsoPs. F₃-IsoPs were positively associated with age (r = 0.251, P = .001) but were not associated with any metabolic measures including blood lipids, CRP, BP, or fasting glucose (FG).

IsoPs and PCBs

The concentrations of the 16 PCBs measured in plasma samples from the 233 subjects are shown in Supplemental Table 1. Total PCBs as represented by Aroclor 1260 was detected in about 50% of the samples. Samples that were below the detection limit were left as empty cells, but they were not removed from the analyses but were treated as missing values to not bias the analysis. F₃-IsoPs positively correlated with all of the congeners of PCBs measured; however, when correlations were adjusted for age, gender, WC, and percentage body fat (%BF), they did not persist (data not shown).

IsoPs and relative erythrocyte fatty acid concentrations

With regard to the fatty acids, F₃-IsoPs were positively associated with total n-3 PUFAs (r = 0.269, P < .001) and positively to the ratio of n-3 to n-6 (r = 0.262, P < .001), whereas F₂-IsoPs correlated negatively with n-3 PUFAs (r = −.138, P = .036) and with n-3:n-6 (r = −.184, P = .005). F₃-IsoPs were negatively correlated with AA to EPA ratio (r = −.329, P < .001) and the ratio of F₂-IsoPs to F₃-IsoPs was positively correlated with AA to EPA (r = 0.357, P < .001); however, F₂-IsoPs were not associated with the AA to EPA ratio. Further correlational analysis performed with individual fatty acids revealed that F₃-IsoPs were correlated positively with AA (r = 0.355, P < .001) and DHA (r = 0.218, P = .003) and negatively to C20:0 (r = −.232, P = .002) and C20:2n-6 (r = −.246, P = .001). F₂-IsoPs were positively correlated to C16:0 (r = 0.132, P = .046) and C16:1 (r = 0.150, P = .023), whereas the F₂-IsoP to F₃-IsoP ratio correlated positively with C16:1 (r = 0.148, P = .016), trans-C16:1 (r = 0.197, P = .008), C18:0 (r = 0.212, P = .004), C20:0 (r = 0.229, P = .002), and C20:2n-6 (r = 0.198, P = .007) and correlated negatively with EPA (r = −.365, P < .001).

Comparison between n-3 tertiles

Participants were subgrouped into tertiles according to the relative total n-3 PUFA concentrations of the entire group: less than 4.36%; 4.36% or greater and 6.90% or less; and 6.90% or greater (Table 2) as the indicator of traditional food intake. For the remainder of the analysis, the congener PCB 153 was used as a proxy measure for PCB exposure because it is a major component of environmental PCBs (21), and among our study population, it was the highest in concentration. Contaminant levels in terms of Hg and the Hg to Se ratio were significantly higher in the third and second tertiles in comparison with the first tertile, whereas levels of PCB 153 were significantly (P < .001) higher in the third tertile in comparison with the two lower tertiles that were not different between each other. Even though the Se level increased with increasing n-3 tertiles, the Hg to Se ratio also increased. F₃-IsoP concentrations increased with increasing tertiles, whereas the F₂-IsoPs to F₃-IsoPs ratio was lowest at the highest n-3 tertile. In addition, monounsaturated fatty acids (MUFAs), SFAs, and trans-fatty acids (TFAs) were all lowest levels at the two higher n-3 tertiles.

Table 2.

Unadjusted Plasma Isoprostanes, Blood Contaminants, Se, and Relative Erythrocyte Fatty Acid Concentrations According to Tertiles of n-3 PUFA Level

	T1 (<4.36) (n = 77) Mean (95% CI)	T2 (≥4.36 and <6.90) (n = 76) Mean (95% CI)	T3 (≥6.90) (n = 77) Mean (95% CI)	P Value
F2-IsoPs, pg/mL	27.35 [24.96–29.96]	28.85 [26.31–31.64]	25.37 [23.05–27.94]	NS
F3-IsoPs, pg/mL	3.69 [2.70–4.54]a	4.93 [4.07–5.97]a,b	5.56 [4.77–6.49]b	.005
F2-IsoP to F3-IsoP ratio	3.39 [2.92–3.86]a	2.60 [2.26–2.94]b	2.30 [1.94–2.65]b	<.001
Blood Hg, μg/L	10.40 [7.71–14.02]a	17.56 [14.00–21.78]b	28.44 [23.74–34.06]b	<.001
Blood Se, μg/L	241.71 [222.95–262.12]a	270.46 [244.51–299.16]a	421.02 [367.62–482.06]b	<.001
Hg to Se ratio	.42 [.37–.48]a	.51 [.47–.55]b	.56 [.53–.59]b	<.001
Adjusted PCB 153d	109.60 [72.58–165.50]a	153.36 [111.48–211.01]a	646.99 [463.77–905.61]b	<.001
n-3	2.58 [2.29–2.87]a	5.78 [5.62–5.95]b	9.69 [9.14–10.25]c	<.001
n-6	21.74 [19.95–23.53]a	27.37 [26.23–28.50]b	25.72 [24.71–26.73]b	<.001
n-6:n-3	11.25 [9.10–13.40]a	4.81 [4.57–5.05]b	2.84 [2.63–3.04]b	<.001
AA:EPA	12.95 [10.52–15.39]a	8.71 [7.48–9.94]b	4.40 [3.61–5.19]c	<.001
SFA	47.19 [45.77–48.61]a	41.75 [40.98–42.52]b	40.24 [39.66–40.82]b	<.001
MUFA	26.91 [26.21–27.62]a	23.91 [23.34–24.47]b	23.03 [22.53–23.53]b	<.001
PUFA	24.31 [22.31–26.31]a	33.14 [31.98–34.30]b	35.41 [34.51–36.32]b	<.001
TFA	1.58 [1.40–1.76]a	1.20 [1.09–1.32]b	1.31 [1.15–1.48]b	<.001

Abbreviation: NS, not significant. Unmatched superscript letters a b and c in the same row indicate significant statistical difference as determined by one-way ANOVA with Bonferroni adjusted multiple comparisons.

^d

Adjusted for total serum lipids, which were calculated by the following formula: total lipids = 0.9 + 1.3 (cholesterol + triglycerides) (23).

Comparison between age categories

A Student's t test assessing the difference between the two age categories (<40 and ≥ 40 y) showed that the older Inuit had lower levels of F₂-IsoPs, a lower ratio of F₂-IsoPs to F₃-IsoPs, higher Hg, and a deregulated cardiometabolic profile evident by higher SBP, FG, T-Chol, and LDL-C (Table 3). In addition, older Inuit have higher F₃-IsoPs and n-3 PUFA and total PUFA intake (Tables 3 and 4). The younger Inuit show higher F₂-IsoP concentrations and significantly higher AA to EPA ratio and lower n-3:n-6 (Tables 3 and 4).

Table 3.

Comparison of Study Characteristics According to Age Categories (<40 and ≥40 y)

	N1/Na	Age <40 y Mean ± SD (or 95% CI)b	Age ≥40 y Mean ± SD (or 95% CI)b	P Value
Age, y	107/126	29.17 ± 5.70	53.94 ± 11.33	<.001
Plasma F2-IsoPs, pg/mL	106/124	28.89 (26.67–31.12)	25.77 (23.79–31.21)	.038
Plasma F3-IsoPs, pg/mL	84/100	3.13 (2.49–3.90)	4.25 (3.59–34.99)	.027
Plasma F2-IsoP to F3-IsoP ratio	84/99	3.15 ± .21	2.39 ± .17	.001
Blood Hg, μg/L	104/123	11.39 (9.18–14.14)	24.68 (20.68–29.47)	<.001
Blood Se, μg/L	104/123	250.49 (232.22–270.15)	355.14 (320.25–393.92)	<.001
Blood Hg to Se ratio	104/123	0.44 ± 0.21	0.55 ± 0.17	<.001
Serum CRP, mg/L	107/126	1.44 (1.13–1.72)	1.85 (1.57–2.16)	.042
SBP, mm Hg	103/110	112.44 ± 13.99	122.00 ± 17.54	<.001
DBP, mm Hg	103/110	75.43 ± 9.95	77.41 ± 11.20	.173
Plasma FG, mmol/L	107/125	4.71 ± .51	5.18 ± .69	<.001
Serum T-Chol, mmol/L	107/125	4.60 ± .91	5.40 ± 1.22	<.001
Serum LDL-C, mmol/L	106/122	2.58 ± .77	3.22 ± 1.09	<0.001
Serum HDL-C, mmol/L	107/125	1.42 ± .47	1.51 ± .49	.128
Serum TG, mmol/L	107/123	1.32 ± .80	1.53 ± 1.30	.143
WC, cm	104/118	91.13 ± 14.91	93.61 ± 13.96	.205
BMI	105/120	27.13 ± 5.60	28.34 ± 5.65	.111
%BF	105/120	28.13 ± 10.63	30.92 ± 10.53	.049

Abbreviation: DBP, diastolic blood pressure.

^a

N1 = (n) age younger than 40 years; N2 = (n) age 40 years or older.

^b

SD reported for nonlog-transformed variables, and 95% CIs are reported for log-transformed variables.

Table 4.

Comparison of Relative Erythrocyte Fatty Acid Concentrations According to Age Categories (<40 and ≥40 y)

	N1/N2a	Age <40 y Mean ± SD	Age ≥40 y Mean ± SD	Significance (P < .05)
n-3	107/126	4.49 ± 2.31	7.37 ± 3.55	<.001
n-6	107/126	25.38 ± 7.17	24.58 ± 5.67	.132
n-3:n-6	107/126	.179 ± .102	.308 ± .178	<.001
AA to EPA ratio	107/126	11.24 ± 9.37	6.45 ± 5.79	<.001
SFA	107/126	43.75 ± 5.78	42.43 ± 4.75	.06
MUFA	107/126	24.99 ± 3.34	24.28 ± 2.86	.08
PUFA	107/126	29.88 ± 8.69	31.94 ± 7.06	.047
TFA	107/126	1.38 ± .678	1.35 ± .729	.162

^a

N1 = (n) age younger than 40 years; N2 = (n) age 40 years or older.

Final predictors of F₃-IsoPs

Multivariable analyses presented in Table 5 show that the variance of F₃-IsoP concentrations was significantly predicted by Hg (β = .294, P = .002), smoking (β = −.211, P = .020), n-3 PUFA (β = .203, P = .036), AA to EPA ratio (β = −.235, P = .017), and EPA (β = .282, P = .003). None of the other variables that were tested persisted as predictors in the final model (R² = 0.238).

Table 5.

Stepwise Regression

Dependent Variable F3-Isoprostanes
	Standardized Coefficients β	t	P Value	95% CI for β
				Lower Limit	Upper Limit	R2	F Value
Model 1
Mercury	.320	3.37	.001	0.086	.334	.203	8.33
n-3	.203	2.13	.036	0.001	0.037
Smoking	−.220	−2.40	.018	−.277	−0.026
Model 2
Mercury	.292	3.01	.003	0.065	.318	.214	8.87
AA/EPA	−.235	−2.43	.017	−0.017	−0.002
Smoking	−.205	−2.27	.025	−.265	−0.018
Model 3
Mercury	.294	3.15	.002	0.071	.314	.238	10.20
C20:5n3	.282	3.04	.003	0.023	.110
Smoking	−.211	−2.38	.020	−.267	−0.024

All models included the following independent variables: Hg, Se, PCB 153, WC, age, sex, FG, T-Chol, SBP or CRP, smoking (1 = yes, 2 = no); and alcohol intake (1 = yes, 2 = no).

Discussion

In this cross-sectional study of adult Inuit, the highest tertile of erythrocyte concentrations of n-3 PUFA had the lowest plasma F₂-IsoPs to F₃-IsoPs ratio (Table 2). This finding coincides with animal studies suggesting that formation of F₃-IsoPs from dietary EPA may channel free radical-mediated oxidation away from production of the highly inflammatory F₂-IsoPs (22). Plasma F₃-IsoPs were directly related to erythrocyte EPA concentrations that accounted for 28% of total n-3. Importantly, our data indicated no correlation of F₃-IsoPs with any cardiometabolic derangements, which contrasts to the strong associations of F₂-IsoPs and isofurans with SBP and CRP we previously showed in this Inuit population (12). Similar to previously reported observations in northern Aboriginal populations (13, 23, 24), our data showed that older Inuit had higher erythrocyte concentrations of n-3 PUFA than younger Inuit (Table 4), reflecting their higher intakes of traditional foods. Despite older Inuit having a less favorable cardiometabolic profile (ie, higher T-Chol, LDL-C, FG, %BF and SBP), they showed lower concentrations of F₂-IsoPs, a nontraditional cardiovascular disease risk factor positively related to T-Chol and LDL-C (25).

Plasma F₂-IsoP values correlate with coronary disease events and severity and are predictors of vascular events and cardiovascular mortality (26). The F₂-IsoP to F₃-IsoP ratio was more sensitive than F₂-IsoPs to variations in essential fatty acid status because plasma F₂-IsoPs were not related to erythrocyte fatty acid concentrations of any essential fatty acids. Similarly, a urinary measurement of F₂-IsoPs, 8-iso-prostaglandin F₂, was inversely associated with high habitual intake of fatty fish in healthy women but was unrelated to serum EPA concentrations (27). Previous human supplementation studies with DHA and EPA showed that decreases in tissue F₂-IsoPs were not related to changes in tissue content of EPA, DHA, AA, total n-3, or n-6 PUFAs (28–30). The higher F₃-IsoPs concentrations in older Inuit could provide metabolic protection because in vivo and in vitro studies have demonstrated that F₃-IsoPs exert antiinflammatory activities as opposed to the potent proinflammatory effects of F₂-IsoPs implicated in cardiovascular pathophysiology (26). Unlike F₂-IsoPs, F₃-IsoPs do not affect adversely platelet shape or aggregation (26) nor cause an increase in systemic arterial BP (31). Several animal studies have shown that supplementation with fish oil is associated with increased tissue F₃-IsoPs concentrations and suppression of the proinflammatory F₂-IsoPs. Yin et al (32) reported that fish oil intake increased F₃-IsoPs and suppressed F₂-IsoPs in lung tissue in a dose-related manner in a murine model of ovalbumin-induced lung inflammation. An increase in F₃-IsoPs and a decrease in F₂-IsoPs were demonstrated both in vitro and in vivo from heart tissues of CCl₄-treated mice fed EPA (22).

Regardless of their higher blood concentrations of PCBs and Hg, older Inuit showed no increase in F₂-IsoPs, which could be partly related to their higher Se status as shown via whole-blood Se. In that regard, we have reported protective effects in the same cohort from their high Se intake against Hg-mediated oxidative stress measured via plasma isofurans (12). Previous studies have reported increased oxidative stress in animals exposed to PCBs (33). PCB-induced lipid peroxidation has been associated with decreased hepatic Se liver content and diminished Se-dependent glutathione peroxidase activity (21). Because blood Se was positively and significantly associated with all PCBs (P < .001; data not shown), it is conceivable that the Inuit are protected from PCB-induced lipid peroxidation by their high Se status.

A study limitation is that it appears that plasma F₃-IsoPs are highly modulated at the same level by the simultaneous presence of n-3 PUFAs, Se, PCBs, and Hg in the food matrix. Thus, these results should be interpreted with caution because all the above variables were highly correlated to each other and at same level to F₃-IsoPs. The positive association of EPA to F₃-IsoPs is consistent with this substrate-product relationship, but the positive associations between F₃-IsoPs and PCBs, Hg, or Se in the multivariable model may reflect associations of F₃-IsoPs to the common food source of EPA in the form of marine mammals (23). Also, the consistent positive relationship of F₃-IsoPs to PCBs, Hg, and WC might indicate that these proinflammatory factors are promoting F₃-IsoP formation. More studies are needed to determine the causal mechanisms for these observations.

In summary, plasma F₃-IsoPs were related to the high erythrocyte EPA concentrations in Inuit who are regular consumers of n-3 PUFA-rich seafood. Notably, no positive associations were noted in the Inuit between F₃-IsoPs and any cardiometabolic derangements, which could be related to the antiinflammatory characteristics attributed to F₃-IsoPs. Thus, the elevated concentrations of F₃-IsoPs demonstrated in Inuit plasma could be a significant factor to be considered in future evaluations of the health benefits implicated with the traditional Inuit high-n-3 PUFA diet.

Acknowledgments

We acknowledge the assistance of all members of the International Polar Year Inuit study staff, coordinators, nurses, interviewers, Coast Guard crew, and especially the members of the community. We also thank Mr William Zackert (Vanderbilt University Medical Center) and Ms Donna Leggee (McGill University's Centre for Indigenous Peoples' Nutrition and Environment) for their intensive laboratory training and their technical expertise.

Author contributions included the following: D.A. and S.K. conceived and designed the experiments. D.A. and L.J.R. performed the experiments. D.A., L.J.R., and S.K. analyzed the data. D.A. and S.K. cowrote the paper. L.J.R. and H.M.C. contributed to the editing of the manuscript. All authors read and approved the final manuscript.

This paragraph lists the sources of funding. No funding was obtained from NIH or other US federal agency.

This work was based on support by the Government of Canada Federal Program for International Polar Year, Canadian Institutes of Health Research, Canadian Foundation for Innovation, Canada Research Chair Program, Health Canada, Aboriginal Affairs, and Northern Development Canada, Government of Nunavut, University of Toronto, and Arctic Net.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations

 
• AA

  arachidonic acid

 
• %BF

  percent body fat

 
• BMI

  body mass index

 
• BP

  blood pressure

 
• CI

  confidence interval

 
• CRP

  C-reactive protein

 
• CV

  coefficient of variation

 
• DHA

  docosahexaenoic acid

 
• EPA

  eicosapentaenoic acid

 
• FG

  fasting glucose

 
• F₂-IsoP

  F₂-isoprostane

 
• F₃-IsoP

  F₃-isoprostane

 
• GC

  gas chromatography

 
• HDL-C

  high-density lipoproteins cholesterol

 
• Hg

  mercury

 
• LDL-C

  low-density lipoprotein cholesterol

 
• MUFA

  monounsaturated fatty acid

 
• PCB

  polychlorinated biphenyl

 
• PUFA

  polyunsaturated fatty acid

 
• RBC

  red blood cell

 
• SBP

  systolic blood pressure

 
• Se

  selenium

 
• SFA

  saturated fatty acid

 
• T-Chol

  total cholesterol

 
• TFA

  transfatty acid

 
• TG

  triglyceride

 
• WC

  waist circumference.