In this population, RBC δ15
N correlated very strongly (r ~ 0.8) with RBC polyunsaturated fatty acids EPA and DHA, which are well-established and validated biomarkers for EPA and DHA intake (9
). These relationships differed little by sex and age. Dietary EPA and DHA intake, as measured by a combined 24HR and 3DFR, were also strongly correlated with red blood cell δ15
N; however, the correlations between biomarkers were stronger.
The correlation between dietary fatty acids and RBC fatty acids was slightly but significantly stronger than the correlation between dietary fatty acids and RBC δ15
N. The difference in correlation strength may be driven by the greater coefficients of variation of RBC fatty acids (72% and 29% for EPA and DHA, respectively), compared to RBC δ15
N (19%),. Alternatively, RBC fatty acids may better capture recent diet than RBC δ15
N, thus more closely matching the diet records (collected within 1−2 weeks of the blood sample). While turnover of RBC nitrogen matches that of the cells, which live approximately 120 days, plasma fatty acids can be incorporated into RBC membranes on a shorter timeframe (39
). EPA, which is preferentially distributed in the outer leaflet of the cell membrane, turns over more rapidly than DHA, which is distributed in the inner leaflet (40
). Thus, EPA, DHA, and δ15
N in RBC may provide dietary information over different time frames. It is important to note that although both δ15
N and fatty acids were measured in RBC, they are independent markers reflecting different cellular components. Thus, the strong correlation between these markers can only derive from their having the same dietary sources.
The relationship between δ15
N and RBC DHA was nonlinear, with RBC DHA composition leveling off at ~ 9% of total membrane fatty acids. Other studies have documented this effect (9
), suggesting either that EPA displaces DHA, or DHA is converted to EPA at high levels of dietary intake. Our dataset allows the form of the relationship and the value of the asymptote to be clearly established because dietary intakes are so high.
From a practical standpoint, measurement of δ15N has many advantages over measuring EPA and DHA in red blood cell membranes. Analysis of δ15N is inexpensive, high-throughput, highly accurate, and requires no specialized sample handling. It continues to increase linearly as dietary intake increases, and does not approach detection limits at either its highest or lowest values. Thus, δ15N could provide a tool for dietary assessment that provides the advantages of a biomarker (measurement accuracy, lack of bias, low participant burden), but that is also feasible to use in large-scale studies.
Accurate information on polyunsaturated fatty acid intake is of great importance in studies of diet and health generally, but particularly in Alaska Natives and other indigenous arctic people. Like many other populations that have undergone a shift toward “westernized” diets, Alaska Natives are experiencing a rapid increase in the rates of diabetes and other chronic diseases (44
). Many researchers have suspected that high PUFA intake characteristic of the indigenous diet may be protective against chronic disease in these and other arctic populations (47
). The ability to accurately measure EPA and DHA intake without relying on self reporting is critical to understanding the rising rates of obesity and chronic disease in the Yup'ik people, and to developing effective interventions for this population.
Many of the Yup'ik participants in the CANHR study consume a large fraction of their total energy from fish, which is the major source of EPA and DHA in their diet (33
). An obvious question for further study is whether δ15
N will predict EPA and DHA intake in populations with lower levels of fish consumption. Although δ15
N is widely used as a marker of marine inputs into ancient human diets (28
) and modern ecosystem studies (e.g., 52
), relatively little is known about the relationship between δ15
N and fish intake in modern human populations. Hair δ15
N was positively correlated (r = 0.39) with the reported frequency of eating fish among 110 community-dwelling older people (mean age = 74) in Oxford, UK (16
). This relationship is suggestive, but not definitive, and further study is required in order to better understand the broader utility of δ15
N as a marker of fish, EPA and DHA intake.
N and δ13
C have also been proposed as markers for dietary intake of animal protein (15
N is typically ~3‰ higher in animals relative to their diets and thus, vegetarians can be identified by low hair δ15
). Because corn is enriched in 13
C relative to nearly all other food plants, livestock that are corn-fed also have high δ13
C values (hereafter, δ13
N and δ13
C were positively correlated with each other and with animal protein intake among German participants in the VERA nutrition study (15
). In our study, however, there was no relationship between either δ15
N or δ13
C and animal protein intake, and δ13
C was negatively correlated with δ15
N, EPA, and DHA (r = −0.16, −0.31, −0.36, respectively, all P < 0.0004). These negative relationships reflect a tradeoff between consumption of traditional subsistence (high δ15
N) vs. market (high δ13
C) foods, where market foods are largely corn-based (34
). In contrast, fingernail δ15
N and δ13
C were strongly correlated in Greenland Inuit, because available market foods were not corn-based, and marine subsistence foods had high values of both δ15
N and δ13
C compared to the rest of the diet (17
). These conflicting results demonstrate how differing complements of foods can drive different patterns of δ15
N and δ13
C, and why caution is required when applying isotopic biomarkers to nutritional studies.
This study has several limitations. It is not based on a representative random sample of the population, and the population in which it has been tested is fairly unique. Thus, whether the relationships can be generalized to the rest of the US population remains to be investigated. Physiological influences on δ15
N are not fully understood in humans, although nitrogen status and severe liver damage both are known to have effects (56
). The measure of dietary intake of EPA and DHA was based on self-report and is subject to errors in recall and potential biases associated with age, gender and other individual characteristics; therefore, the magnitudes of observed associations between dietary intake and biomarkers are underestimated. This study also has significant and unique strengths. It is based on a large sample of participants with a very large variability in dietary EPA and DHA intake, which makes the sample ideal for testing the performance of alternative biomarkers of EPA and DHA. It is also the first study to compare the performance of natural abundance stable isotope values against validated nutritional biomarkers in a human population.
In summary, we find that RBC δ15N is highly correlated with RBC EPA and DHA, and that both isotopic and fatty acid biomarkers show similar correlations with dietary intake, ranging from 0.49 to 0.65. Thus, we propose that the RBC nitrogen stable isotope ratio provides an accurate, inexpensive biomarker for dietary EPA and DHA intake that could make assessment feasible in large-scale studies. Because accurate assessment of individual dietary intake of these fatty acids is of particular interest for this population, we recommend that future studies include measurement of RBC δ15N as a proxy for RBC membrane EPA and DHA. We also suggest that δ15N would likely be an effective and accurate biomarker of EPA and DHA intake in other populations with varying levels of fish consumption.