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Over the past 50 years, increases in dietary n-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid, have been hypothesized to cause or exacerbate chronic inflammatory diseases. This study examines an individual’s innate capacity to synthesize n-6-long chain PUFAs (LC-PUFAs), with respect to the fatty acid desaturase (FADS) locus in Americans of African and European descent with diabetes/metabolic syndrome. Compared to European Americans (EAm), African Americans (AfAm) exhibited markedly higher serum levels of arachidonic acid (AA) (EAm 7.9±2.1; AfAm 9.8±1.9 % of total fatty acids, mean ± sd; p<2.29×10−9) and the AA to n-6-precursor fatty acid ratio, which estimates FADS1 activity (EAm 5.4±2.2, AfAm 6.9±2.2; p=1.44×10−5). Seven single nucleotide polymorphisms (SNP) mapping to the FADS locus revealed strong association with AA, eicosapentaenoic acid (EPA) and dihomogamma-linolenic acid (DGLA) in the EAm. Importantly, EAm homozygous for the minor allele (T) had significantly lower AA levels (TT: 6.3±1.0; GG: 8.5±2.1; p=3.0×10−5) and AA/DGLA ratios (TT: 3.4±0.8; GG: 6.5±2.3; p=2.2×10−7) but higher DGLA levels (TT: 1.9±0.4; GG: 1.4±0.4; p=3.3×10−7) compared to those homozygous for the major allele (GG). Allele frequency patterns suggest that the GG genotype at rs174537 (associated with higher circulating levels of AA) is much higher in AfAm (0.81) compared to EAm (0.46). Similarly, marked differences in rs174537 genotypic frequencies were observed in HapMap populations. These data suggest that there are likely important differences in the capacity of different populations to synthesize LC-PUFAs. These differences may provide a genetic mechanism contributing to health disparities between populations of African and European descent.
Several lines of evidence indicate that a disproportionate burden of preventable disease, death, and disability exists in racial and ethnic minority populations, especially African Americans in the US. Differences in the prevalence of metabolic syndrome have been noted in the US NHANES surveys, with prevalence notably higher among African American women (1). In addition, the profile of metabolic syndrome differs among ethnicities, with African Americans showing a smaller contribution of dyslipidemia (i.e., fewer HDL-C and triglyceride abnormalities) compared to European Americans (2). However, there is greater insulin resistance among African Americans, even during childhood (3). Likewise, cardiovascular disease risk shows significant racial/ethnic differences with the highest age-adjusted death rates observed in African Americans (4). Differences in the prevalence and severity of chronic diseases involving inflammation are further corroborated by differences in inflammatory biomarkers, including C-reactive protein (5, 6). The striking racial and ethnic differences in prevalence and/or severity of common diseases is likely explained by a complex combination of environmental, social, cultural or economic factors, and genetic factors are likely to be very important as well (7).
Agricultural and industrial revolutions have increased the quantity and variety of foods but have not necessarily improved the human diet (8, 9). In fact, >70% of the calories consumed by humans today in developed countries would not have been available to our hunter-gather ancestors (8). This rapid shift in the type of calories consumed by modern humans appears to have created a sort of malnutrition in developed countries whereby certain nutrients are not well-tolerated by our ‘hunter-gather’ genes. This problem has become a global issue as Western-derived food supplies and practices expand with global trade. The negative impact of the modern diet on health is likewise exported to developing nations.
Humans can synthesize a wide range of fatty acids, but they lack key enzymes (delta-12 and delta-15 desaturases necessary to synthesize the initial PUFAs used in the key PUFA biosynthetic pathway in mammals (10, 11). Therefore, linoleic acid (C18:2n-6; LA) and alpha-linolenic acid (C18:3n-3; ALA) are essential FAs (10). Once obtained from the diet, they are converted to LC-PUFAs by the alternate actions of two fatty acid desaturase enzymes (Δ6 and Δ5 desaturases encoded for by FADS2 and FADS1, respectively) and an elongase enzyme that introduces carbon-carbon double bonds and increase chain length by 2 carbons, respectively(12). Additionally, preformed LC-PUFAs such as arachidonic acid (C20:4n-6; AA) can also be readily obtained from human diets. AA is found in relatively high concentrations in the meats of grain-fed animals and eggs (13, 8).
Once produced, AA and its metabolic products play important roles in orchestrating immunity and inflammation (14–16) via their ability to directly impact normal and pathophysiologic responses through: (i) conversion to potent arachidonic acid (AA)-derived bioactive products (including prostaglandins, thromboxanes, leukotrienes and lipoxins); and (ii) regulation of cellular receptors (NFκB (17, 18), PPAR (19) and SREBP-1c (20, 21) ), thereby modulating the expression of many genes that control immune responses (cytokines such as IL-1, IL-6, IL-12 and TNF-α; chemokines such as IL-8, MIP-1a and MCP1; adhesion molecules such as ICAM and E-selectin; and inducer effector enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (22)).
During the 20th century in the United States, there has been a dramatic increase in LA consumption (from an estimated 2.8% to nearly 8% of energy) (13) primarily as a result of increased availability of soybean oil, margarine and poultry (13). In fact, nearly 85% of total PUFAs in a typical Western diet is LA (23). Biochemical studies using stable isotope studies largely carried out in subjects of European ancestry indicate that only a small proportion of dietary LA (~0.2%) can be converted to AA in humans(24). However, humans do appear to be able to synthesise sufficient AA or extract AA from the diet to maintain AA status (25). Nevertheless, this low rate of conversion has been assumed to apply to all human populations equally. For example, the Advisory Committee from the American Heart Association has concluded “that at least 5 to 10 % of energy from n-6 PUFAs reduces the risk of cardiovascular disease relative to lower intakes” (26). This conclusion was based on several assumptions, one of which concluded that “wide variations in dietary LA do not alter tissue AA content”. Yet the literature suggests that high LA-containing diets can reduce the LC-PUFA content of tissues when modeled from per capita food consumption data (13)or by direct analysis of tissues (27).
Studies over the past 5 years suggest that there is likely large genetic variability in the rate of conversion of LA to AA (28–32). Importantly, genetic variants that are associated with higher levels of AA are also associated with elevated levels of markers of systemic inflammation and the incidence of certain inflammatory disorders (31). The current study examines levels of LA and AA, their association with genetic variants in the FADS gene cluster, and the frequency of high converting genotypes in patients of African and European ancestry with diabetes/metabolic syndrome. The results of this study demonstrate that there is a marked increase in AA and in the frequency of alleles that favour AA synthesis in African Americans with diabetes/metabolic syndrome. This study reveals that there are likely differential effects of high concentrations of LA in African and European American populations in the US and caution should be exercised with regard to dietary recommendations that assume n-6 PUFA metabolism is uniform in all human populations.
The study population was derived from the Diabetes Heart Study (DHS; n=229) and included European American (n=166; from 89 families) and African American (n=63; from 33 families) subjects with diabetes/metabolic syndrome. Methods for ascertainment and recruitment for the DHS have been described previously (33). Briefly, siblings concordant for type 2 diabetes mellitus (T2DM) without renal insufficiency were recruited, along with unaffected siblings. Metabolic syndrome was defined using the standard definition from the Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (34). The Wake Forest University School of Medicine Institutional Review Board (IRB) approved study protocols, and all participants provided written informed consent.
Serum was isolated from fasting whole blood samples. Fatty acid methyl esters were prepared (35) in duplicate from serum samples (100 μl) in the presence of an internal standard (triheptadecanoin; NuChek Prep, Elysian, MN, USA) as previously described in detail (36). A panel of 23 fatty acids was quantified by gas chromatography with flame ionization detection. Individual fatty acids are expressed as percent of total fatty acids in a sample. For all samples, data peaks on chromatograms were examined to ensure peak quality and consistency of retention times. Fatty acids in samples were identified based on the retention times of methyl ester derivatives of authenticated fatty acids standards. These standards included Supelco 37 Component FAME Mix (Supleco, Bellefonte, PA, USA) and other individual methyl ester derivatives from Supelco (oleate, cis-11-vaccenoate, linoleate, eicosapentaenoate, n-3 docosapentaenoate), Cayman Chemicals (Ann Arbor MI, USA; stearidonate), Matreya (Pleasant Gap PA, USA; eicosadienoate, dihomogamma-linolenoate, arachidonate) and NuChek Prep (cis-eicosatrienoate, docosadienoate, docosatetraenoate, n-6 docosapentaenoate, docosahexaenoate, tricosanoate, tetracosanoate) Product-precursor ratios of circulating fatty acids, an estimate of enzymatic activity, were calculated from fatty acid mass data.
For statistical analyses of fatty acid data, normal kernel density estimation (implemented in S-Plus) was used to obtain estimates of the probability density functions. Linear mixed models were used to assess the racial difference in the fatty acids and ratios adjusting for sex and age. Family was treated as a random effect and age, sex, and race as fixed effects. Residuals were examined to assess the model assumptions.
Seven single nucleotide polymorphisms (SNP) mapping to the FADS gene cluster (rs174537, rs102275, rs174546, rs174556, rs1535, rs174576, rs174579) were selected based on previous publications (30, 37, 38). Genotypes were determined using a Sequenom Mass ARRAY SNP genotyping system (Sequenom Inc., San Diego, CA, USA) (39). Of the samples, 3.5% were genotyped in duplicate with 100% reproducibility across the SNPs. Linkage disequilibrium (LD) was assessed by calculating D′ and r2 within Haploview (40) relying on a set of independent individuals in the data (a random selection of a single individual from each pedigree, N=33 and N=89 African American and European American subjects, respectively) and haplotype blocks were defined according to the algorithm of Gabriel et al. (41).
Allele and genotype frequencies for each SNP were calculated from unrelated probands and tested for departure from Hardy-Weinberg equilibrium using a chi square goodness-of-fit test. Associations between SNPs and traits were performed using a series of variance components measured genotype models as implemented in SOLAR (Sequential Oligogenic Linkage Analysis Routines) (42). Significance was evaluated using the likelihood ratio tests based on the correlation structure suggested by the familial relationships. The additive genetic model was the primary model of interest, however, for SNPs with less than 10 individuals homozygous for the minor allele a dominant model was analyzed and all models included age and sex covariates. When necessary, phenotypes included in these analyses were transformed using the natural logarithm.
Publicly available data for ten populations from the International HapMap project (phase III, www.hapmap.org) were used to derive allele frequencies for the rs174537 SNP, which was among those that were genotyped in the DHS population. These data include 1046 samples from the following ten populations: Kenya (LWK, n=110), Nigeria (YRI, n=147), Mexican ancestry from Los Angeles, CA (MEX, n=58), African American population in the Southwest US (ASW, n =57), Gujarati (India) population in Houston TX (GIH, n=101), Japanese population in Tokyo (JPT, n=113), Chinese population in Beijing (CHB, n=137), Chinese population in Denver CO, US (CHD, n=108), Italian population in Tuscany (TSI, n=102) and a European American population in Utah (CEU, n=113).
Figure 1 shows the distribution of n-6 PUFAs (left panels, a–d) in sera of African American (n=63, 41.3% male, age = 61.0±10.1, mean±sd) and European American (n=166, 42.7% male, age = 68.2±10.5) adults with diabetes/metabolic syndrome from the Diabetes Heart Study (DHS (33)). There was a pronounced enhancement in levels of serum AA (p= 2.29×10−9) in African American compared to that in European American subjects. In contrast, no differences were observed in the levels of LA, gamma-linolenic acid (GLA) or dihomogamma-linoleic acid (DGLA), all precursors of AA. These data suggest that similar levels of LA are ingested by both populations.
Figure 1 e–g show the ratios of product to precursors (GLA/LA; DGLA/GLA and AA/DGLA) of circulating fatty acids, which are surrogates of actual enzymatic activity at each biochemical step. As suggested by fatty acid levels (Figure 1a—c), the conversion of LA to GLA (FADS2 activity) and GLA to DGLA (elongase activity) are not different in subjects of African and European descent. However, there appears to be dramatically different conversion rates for DGLA to AA in the two populations through the Δ5 desaturase (FADS1 activity) step (Figure 1g). African American subjects exhibited a markedly higher AA/DGLA ratio (p=1.44×10−5) compared to that in European American, suggesting a strikingly increased ability of the former to convert medium chain (<20 carbons) PUFAs (MC-PUFA) to LC-PUFAs.
Tests for association were performed with seven SNPs mapping to the FADS gene cluster in DHS subjects. The pattern of association in the European American subjects that was highly consistent with previous reports (43, 37). Specifically, in pattern in European Americans was in high linkage disequilibrium (LD) for this region, with a single LD block (53 Kb) that included the 7 SNPs across FADS1 and part of FADS2 (not shown). In contrast, no LD blocks were observed in the African Americans (not shown). The strength of association for AA ranged from 1.1×10−6 – 8.9×10−8 in the European American subjects (Table 1). Evidence for association was also observed (Table 1) for DGLA (7.5×10−6 – 7.1×10−7), and GLA (4.9×10−6 – 9.8×10−11).
No associations were observed in the African Americans subjects, which was likely due to the limited sample size of this sample coupled with the much lower allele frequency of the minor alleles. It is important to note that no African American individuals were found in this relatively small population that were homozygous for the minor allele of rs174537.
Figure 2 shows the distributions of DGLA, AA, and the AA/DGLA ratio by genotype at rs174537. The major allele (G) is associated with an increase in the mean serum level of AA and is consistent with an additive model in European Americans (Figure 2b). In contrast to the case for AA, the allele (G) was associated with a decrease in mean levels of DGLA, the immediate precursor of AA, (Figure 2a) and is also consistent with an additive model in European American subjects. For the ratio of AA/DGLA, an estimate of FADS1 activity (Figure 2c), it was observed that the common allele (G) appeared to be associated with an increased trait mean, i.e. increased enzymatic efficiency. As mentioned above, it was not possible to compare levels of PUFAs in homozygous minor allele with homozygous major allele as there were no African Americans found in this population that exhibited homozygous minor alleles (Figure 2d–f). The mean AA levels in the African American population (GG: 9.9±1.8; GT: 9.2±2.2; mean±sd) were significantly greater (GG: p=0.003; GT: p=0.0073; two-tailed T test)than that in the European American group (GG: 8.5±2.1; GT: 7.6±1.8).
Striking differences were observed in the allele frequencies across a majority of these SNPs in the FADS gene cluster between the African American and European American populations in the DHS Study (Table 2). The resultant genotypic frequencies for rs174537 were skewed toward the homozygous major allele (81%: GG) in the African American population with only 19% heterozygous and no homozygous minor allele genotypes observed (Table 2). In contrast, the European American population exhibited a much lower frequency of homozygous major allele (46%) with 43% heterozygous and 11% homozygous minor allele. To evaluate the genotype distribution of these alleles on global scale, patterns of genetic variation were examined within the FADS locus, and in particular in rs174537, in populations within The International HapMap Project. Figure 3 shows striking differences in genotypic frequencies between different populations around the world, with the greatest differences observed between African populations from Kenya (LWK) and Nigeria (YRI) verses populations of Mexican ancestry living in Los Angeles, CA (MEX). African Americans in the DHS study have genotypic frequencies similar to that in another African American population in the Southwest US (ASW) and a Gujarati (India) population in Houston TX (GIH). Greater than 75% of individuals in each of these populations carry the major allele homozygous GG genotype. In contrast, our DHS European American population had frequencies similar to a Japanese population in Tokyo (JPT), a Chinese population in Beijing (CHB), an Italian population in Tuscany (TSI) and a European American population in Utah (CEU). Less than 50% of individuals in these populations carry the major allele homozygous GG genotype.
Multi-factorial diseases of chronic inflammation disproportionately affect African Americans in industrialized settings such as the United States (44), yet appear to be rare in continental Africans. Only 1–2% of Africans on the African continent have type-2 diabetes, whereas the incidence is 11–13% in people of African descent in industrialized nations consuming a Western diet (45,,46). It is clear that a complex interplay between genes and environment is contributing to these differences.
Several lines of evidence from the current study suggest that this interplay is likely to be important with regard to the dietary consumption and metabolism of n-6 PUFAs. First, African Americans had higher levels of circulating AA compared to those of European descent. Importantly there were no differences in levels of the levels of fatty acid precursors (LA and GLA) to n-6 LC-PUFAs in these two populations of patients with diabetes/metabolic syndrome. To date, few studies have examined the impact of ancestry on LC-PUFA synthesis and levels. In 1991, Horrobin and colleagues (47) observed that AA levels in plasma phospholipids of nineteen subjects from Zimbabwe Africa was approximately a 2-fold higher than that in a much larger group (n= 458) of subjects with European ancestry. The current study strongly suggests that populations of African ancestry also have higher levels of circulating AA compared to those of European ancestry.
Second, there were marked differences between the African American and European American populations in our study with regard to frequencies of alleles in several SNPs in the FADS gene cluster, which have shown to be important in determining fatty acid levels. Specifically, rs174537 is the SNP near FADS1 that Tanaka and colleagues (37) have demonstrated to be most associated with AA levels (p = 5.95×10−46). They demonstrated that individuals who were homozygous for the minor allele had significantly lower AA levels compared to those who carried the homozygous major allele. The current study shows that 81% of African Americans and 46% of European Americans in the DHS population have the homozygous GG allele associated with high AA levels. In contrast, no African Americans in our study population with a homozygous TT allele were found, whereas 18 out of 159 European Americans carried the homozygous TT allele at rs174537. Additionally, as observed by Tanaka and colleagues (37), there were significant differences in AA and DGLA as well as the AA to DGLA ratio, an estimate of FAD1 activity, between GG, GT and TT in the European American populations. These differences were not seen in the African American population as the study was not powered to detect such difference in the small population, which also contained no subjects homozygous for the minor allele in the DHS data base. However, a follow-up study has demonstrated comparable (to that in DHS European Americans), highly significant genotypic differences in circulating levels of AA and DGLA and the resultant AA/DGLA ratio in an subset of a larger African American study population (GeneSTAR, Johns Hopkins University) in which the African American sample size was three times that in the current study (note added in proof: Mathias et al. (2011) BMC Genet 12:50, doi: 10.1186/1471-2156-12-50).
Third, an analysis of allele frequencies at rs174537 in HapMap populations around the world show dramatic differences in allele frequencies among populations. For example, populations from Africa have much higher frequencies of the GG alleles along with very low TT allele frequencies. At the opposite end the spectrum are endogenous populations from the Americas, which exhibit extremely low frequencies of GG alleles and much higher frequencies of the TT alleles. Other populations (a European population from Tuscany, European Americans from our study and Utah as well as Japanese and Chinese populations) lie in the middle with regard to allele frequencies of this SNP. If indeed the genotype of this SNP (rs174537) correctly predicts ‘efficient converters’ (GG), ‘modest converters’ (GT) and ‘non-converters’ (TT) with regard to Δ5 desaturase (FADS1) enzymatic efficiency, then Figure 3 suggests that a simplistic assumption that wide variations in dietary LA does not alter tissue AA content cannot be made until metabolic studies are carried out in several distinct populations around the world. This is especially important given that the current paper suggest that the efficiency of LA to AA conversion is likely population-dependent.
Finally, numerous studies have demonstrated population differences due to adaptation to pathogens (48), climate, and diet (49). However, some past adaptations (such as salt retention and hypertension) are now maladaptive, and can lead to human disease. We propose that this possibility exists for AA and inflammation. Given the elevated levels of n-6 MC-PUFAs (12–17g/day, principally LA (23)) in Western diets, a more efficient capacity to convert MC-PUFAs to LC-PUFAs could promote AA production. This hypothesis is supported by the current study showing that circulating AA in African Americans is on average higher than that in European Americans (Fig. 1), under conditions where genetic backgrounds clearly plays a role (Fig. 2). Conditions such as obesity, type-2 diabetes and hypertension are multi-factorial diseases that disproportionately affect African Americans in the United States (44). Yet for type-2 diabetes, only 1–2% of Africans on the African continent have type-2 diabetes, whereas the incidence is 11–13% in people of African descent in industrialized nations consuming a Western diet (45, 46). Although there are likely numerous genetic markers and metabolic changes that contribute to these differences, certainly polymorphisms, such as those found in the FADS gene cluster, could confer increased risk as these populations moved from traditional to Western diets. It is interesting to note that haplotypes of the FADS gene cluster, including variants associated with elevated an AA/LA ratio are related to both a higher systemic inflammation (as measured by high sensitivity-C reactive protein) and greater risk of coronary artery disease (31).
For these reasons, recommendations to increase dietary LA levels to 5 to 10% of dietary energy (26) by organizations such as the American Heart Association are particularly concerning. These recommendations have come largely as a result of registered clinical trials of mixed n-6 + n-3 PUFA diets and diets in which n-6 + n-3 PUFA have replaced trans and saturated fatty acids. Using meta-analyses that took these potential confounders into consideration, Ramsden and colleagues (50) have observed a potential risk of LA was likely missed in previous meta-analyses. Five decades of studies and the clinical impact of inhibitors of AA metabolites or metabolism (nonsteroidal anti-inflammatory drugs and leukotriene blockers) supports a central role for AA in inflammation. If circulating levels of AA are indeed important, the potential risk of elevating dietary LA would be postulated to differentially impact populations such as African Americans that have a much higher proportion of ‘efficient converters’ of LA to AA resulting in higher levels of circulating AA.
This work was supported by NIH grants P50 AT002782 (F.H.C.), R01 Hl637348 (D.W.B), R01 NS058700 (D.W.B.), F32DK083214 (C.E.H.), RO1 NR08153 (D.M.B), RO1 DK 071891 (B.I.F.) and M01 RR000052 (D.M.B). Additional support was received from Wake Forest School of Medicine General Clinical Research Center M01 RR07122 (D.W.B.) and the Wake Forest School of Medicine Center for Public Health Genomics. The authors thank the Diabetes Heart Study coordinators, Carrie Smith and Pamela Hicks for their help with obtaining specimens; and Dr. R. Mathias for access to study results prior to publication.
F.H.C. has published books with Rodale and Simon and Schuster and is a founder and consultant to GeneSmart Health, Inc., which may be partially related to his research. These potential conflicts of interest have been disclosed to Wake Forest School of Medicine and to outside sponsors and are institutionally managed. No other authors have a conflict of interest.
C.E.H. performed SNP genotyping and association analyses; M.E.R. selected SNPs and designed the genotyping assays; S.S. and P.I. performed fatty acid analyses; H.C.A. performed analyses on the HapMap data; J.T.Z., L.D.C., C.D.L. and D.V. provided statistical and genetic analyses; D.W.B. and B.I.F provided access to DHS study data; R.A.M., F.H.C. generated the hypotheses, designed the experiments; S.S, R.A.M., F.H.C. prepared the manuscript.