The present study examined the association between circulating concentrations of ascorbic acid, α-tocopherol, and 25(OH)D, and the concentrations of five cytokines in healthy young adults. Previous intervention studies have assessed the effects of antioxidant-rich dietary preparations on various markers of inflammation [10, 37]. However, to our knowledge, this is the first study to examine the relationship of each of these micronutrients with pro-inflammatory cytokines in healthy young adults while controlling for the other micronutrients. Our findings indicate that, when accounting for circulating concentrations of the other two micronutrients, α-tocopherol concentrations are inversely associated with biomarkers of inflammation in healthy young adults. However, ascorbic acid and 25(OH)D were not associated with decreased concentrations of inflammatory biomarkers in this population after accounting for relevant covariates, including the concentrations of the other micronutrients.
Recently, a cross-sectional study of a small population of primarily overweight and obese Mexican women assessed the association between Zinc and vitamins A, C, and E and various pro-inflammatory cytokines, although none of the cytokines examined in the present study were included in that one . In that study, only higher concentrations of Zinc were associated with reduced risk of higher inflammatory cytokine concentrations. However, our study population is overall very lean, and different cytokines were assessed. Therefore, the lack of association between vitamins C and E and any of the cytokines observed in that study does not contradict the findings of the present study, since other mechanisms may be at play.
Vitamin E, and in particular α-tocopherol, plays an important role in immune regulation and anti-inflammatory processes . Vitamin E exists as several types of tocopherol, of which α-tocopherol is the major circulating form in the body . Intake of vitamin E results in increased T cell division and lymphocyte proliferation , and research in animal models has shown that vitamin E supplementation increased IL-2 and IL-1RA, and decreased IL-4 gene expression . In addition, supplementation with α-tocopherol resulted in decreased circulating IL-1β, IL-6, TNF-α, and CRP in individuals with diabetes . These anti-inflammatory effects of vitamin E are thought to result partly through inhibition of 5-lipoxygenase, an enzyme involved in inflammatory prostaglandin synthesis , as well as through down-regulation of NFκB . Furthermore, vitamin E is a potent lipophilic antioxidant that protects cell membranes, prevents low density lipoprotein (LDL) lipid peroxidation, and is thought to decrease the oxidative stress associated with hyperglycemia, dyslipidemia, and other cardiometabolic dysregulations .
A previous study identified an inverse association between concentrations of several cytokines, including RANTES, and consumption of an antioxidant-rich juice powder preparation in a population of healthy adults . Intake of this preparation also led to increases in circulating α-tocopherol, as well as ascorbic acid. However, this previous study did not examine the individual impact of each micronutrient on these cytokines. In the present study, high circulating α-tocopherol, but not ascorbic acid, was inversely associated with RANTES, and this association remained after adjusting for the other two micronutrients. This observation suggests that α-tocopherol in particular may play a role in modulating RANTES concentrations. RANTES is a pro-inflammatory cytokine that plays an important role in various immune processes, such as recruitment of leukocytes to sites of inflammation and mediating T cell and monocyte traffic . In addition, it also promotes angiogenesis . Elevated RANTES concentrations are observed in several inflammatory conditions, including atherosclerosis .
We also observed an inverse association between α-tocopherol and IFN-γ after adjusting for the other micronutrients. IFN-γ is a pro-inflammatory cytokine that plays a central role in modulating Th1-mediated immune cascades and, together with other cytokines, contributes to pancreatic β cell destruction, thus playing a potentially detrimental role in glucose metabolism [27, 28]. A recent study reported decreased α-tocopherol plasma and tissue concentrations and increased expression of the gene encoding IFN-γ in breast cancer cases . On the other hand, α-tocopherol supplementation in healthy Asian individuals did not elicit changes in production of IFN-γ or other cytokines in leukocytes . The discrepancy between findings of the latter study and findings from the present study may stem partly from the differences in ethnic distribution of the populations assessed. However, all our analyses were adjusted for ethnicity, thus minimizing the possibility of confounding by ethnicity.
Overall, the results of the present study indicate that, in this population of young adults, circulating concentrations of α-tocopherol are inversely associated with certain pro-inflammatory cytokines, and these associations remain after adjusting for circulating ascorbic acid and 25(OH)D. These findings suggest that ensuring an adequate vitamin E status in healthy young adults may have a potential beneficial effect on inflammation, which may partly contribute to chronic disease prevention later in life [47, 48].
A wealth of research using cell lines and animal models suggests a key role for vitamin D in modulating immune responses . However, human studies have yielded inconsistent results on the association between vitamin D and inflammatory biomarkers [16, 50–52]. In the present study, we observed no association between 25(OH)D and any cytokine under any of the models assessed. It is possible that none of the cytokines measured are modulated by vitamin D. Additionally, our study population consisted of healthy young adults with low concentrations of inflammatory biomarkers. The potential effects of 25(OH)D on inflammatory biomarkers may be more apparent in populations with a higher degree of inflammation, such as older or diseased groups.
Ascorbic acid plays important roles in immune regulation and oxidative stress through scavenging of reactive molecular species, protection against protein glycation, and prevention of lipid peroxidation [4, 19]. In addition, ascorbic acid interacts with α-tocopherol, restoring its antioxidant potential through reduction of the oxidized form of α-tocopherol . Findings from epidemiologic studies, including one previously conducted in the TNH population, show an inverse association between ascorbic acid and CRP, suggesting an anti-inflammatory effect of this micronutrient at the systemic level [12, 13]. However, several intervention trials reported no association between vitamin C intake, circulating ascorbic acid, and various inflammatory biomarkers [17, 53, 54]. The present study found no inverse associations between ascorbic acid and any cytokine, and, furthermore, identified an unexpected positive association between this micronutrient and IFN-γ. Given that ascorbic acid did not influence concentrations of other cytokines and is inversely associated with CRP in this population , it seems unlikely that ascorbic acid negatively affects inflammation in these healthy individuals. Nevertheless, the observation of a positive association between ascorbic acid and IFN-γ warrants further study.
We observed weak correlations between the examined micronutrients and cytokines. These correlations were in a positive direction, except for those between ascorbic acid, α-tocopherol and RANTES. Considering the multitude of factors that affect circulating cytokine concentrations, the finding of poor crude correlations between the vitamins and cytokines is not surprising. As our other results using ANCOVA indicate, adjusting for recognized covariates brought out robust associations between some of the micronutrients and cytokines. Finally, we observed some strong positive associations between certain cytokines, namely IL-1RA and IFN-γ, IL-1RA and PDGF-bb, and IFN-γ and PDGF-bb. These observations are in agreement with previous in vitro work reporting that IFN-γ up-regulates production of both IL-1RA and PDGF-bb [55, 56].
One limitation of the present study is the cross-sectional nature of the analyses, which precludes making any inferences about causality. In addition, we did not consider dietary contributions to the circulating concentrations of the micronutrients assessed here and, therefore, we cannot comment on the effects of specific food items or dietary patterns on cytokine concentrations. However, the primary focus of our study was to understand the relationship between circulating micronutrient concentrations and inflammatory markers. Focusing on circulating micronutrient concentrations provides a more direct measure of exposure than reported dietary intake because, due to factors such as limitations of dietary measurement tools, genetic variation between individuals, and, in the case of vitamin D, endogenous production after sun exposure, dietary intake may not always reflect physiological concentrations of a given micronutrient.
An alternate approach would have been to examine the association between the cytokines and the total antioxidant capacity of the diet. Indeed, the vitamins examined in the present study all possess antioxidant and anti-inflammatory properties . However, these effects may occur through different pathways, as discussed previously . While examining the total antioxidant capacity of the diet may help characterize the overall efficacy of the studied vitamins, it may preclude identifying the individual associations for each vitamin, which is the main scope of the present study.
The associations reported here may be confounded by various unaccounted factors. We attempted to minimize the probability of confounding by adjusting all our analyses for available anthropometric, demographic, and biochemical variables known to affect concentrations of any of the assessed vitamins and cytokines. Nevertheless, it is possible that other biological, demographic and lifestyle factors may affect the identified associations. Finally, the study population consists of young adults whose inflammation status is generally lower than those of older individuals, in whom inflammation-related chronic disease processes may be ongoing. Indeed, of the 27 cytokines initially measured in a subset of the population, the concentrations of many were too low to detect accurately, which prevented us from examining other important inflammatory biomarkers such as IL-6 and TNFα. However, studying healthy individuals provides a picture of inflammatory status before the onset of disease, and understanding how inflammation may be modulated by various micronutrients at this stage provides the potential for developing nutrition-based strategies to prevent later disease development.