Limitations of this study include a relatively small sample size and its short-term nature (12 weeks only). In addition, strict activity diaries kept by subjects might better record confounders such as daily sun exposure during the study.
Changes in the metabolomic profile and clinical changes observed are associations and not necessarily causative. It has been reported that increases in free fatty acids induce oxidative stress by generating reactive oxygen species and inflammation by increasing levels of NF-kB [14–16]. Since high levels of fatty acids both promote and indicate a high level of oxidative stress and inflammation, the antioxidants in the supplement could alleviate these stressors. Future studies into histologic or molecular changes might better characterize the precise changes occurring in the skin. While we are able to detect particular orally ingested antioxidants such as carotenoids in the skin, quantitative measures of other antioxidants (such as catechins) in the skin can be difficult. Measurable increased levels of these antioxidants in the skin would bolster the contention that orally ingested supplements reach the skin and can effect change.
Non-smokers in our study experienced fewer overall changes in metabolite levels, but an increase in metabolites within the vitamin/cofactor pathways. One explanation for this observation is that, upon antioxidant and vitamin supplementation, non-smokers may have had a surplus in vitamins and cofactors that remained unconsumed. Previous studies have shown that cigarette smoke directly depletes the levels of circulating antioxidants and vitamins, such as ascorbate, alpha-tocopherol, beta-carotene, lycopene, and retinol [17, 18]. When taking these supplements, therefore, antioxidants and vitamins might be consumed in smokers, but left as excess in non-smokers. This supports a model in which smokers would benefit more from supplements than non-smokers.
We acknowledge that the number of cigarettes smoked per day may affect the metabolites present . The total pack-years smoked varied widely in our study population: the amount ranged from 4.2 to 37.5 pack-years. Although there were variations in the amount of pack-years, we instructed the participants to not change their habits, especially the amount they smoked. From the metabolomic analysis, the levels of cotinine, a well-known metabolite of nicotine , did not significantly change in either group after 12 weeks of supplementation. This demonstrates that the smokers did not have drastic changes in smoking habit, suggesting that the observations we observed may have been related to the concentration of free radical oxygen species entering the circulatory system from supplementation. Further studies in which the amount of smoking are controlled could explore whether a minimum amount of smoking is required to detect significant metabolomic changes as well as define maximum levels of smoking beyond which supplementation might have limited benefit.
The decrease in the mScore of the study population toward a normal distribution (especially in the supplemented smokers) and the alteration of metabolomic heat maps in supplemented smokers suggests that the study supplement may be able to reduce the amount of metabolites generated in pathways affected by smoking. Smokers displayed more changes in their metabolome than non-smokers after study supplementation, most notably significant decreases in their LCFAs.
Of note, cigarette smoking not only introduces free radicals that promote atherosclerosis and increase the risk of cardiovascular disease [5, 6], it has also been linked to changes in lipid metabolism and increased levels of free fatty acids in blood [13, 21, 22]. An additional explanation for the change in fatty acid composition with study supplementation is that peripheral lipolysis is attenuated and possibly accompanied by improved mitochondrial function. Lipolysis is a hallmark of insulin resistance, and has been shown to be elevated in smokers [23, 24]. Any compounds within the study supplement that affect insulin sensitivity could result in decreased lipolysis and decreases in levels of LCFAs, as detected in this study. Also, components in the supplement that might improve mitochondrial function could allow for fatty acids and amino acids to be consumed more efficiently. It is possibly a combination of improved insulin sensitivity and mitochondrial function that causes the decline in fatty acids and amino acids. Evidence that supports this idea is that two markers of excessive carbon flow into the tricarboxylic acid cycle, beta-hydroxybutyrate and 2-hydroxybutyrate, decline with supplementation. These arise when either too much carbon is produced relative to tricarboxylic acid cycle capacity or the tricarboxylic acid cycle is not operating at full capacity. It also can be seen that these markers changed (only significantly for beta-hydroxybutyrate) for the non-smokers as well. Overall, these observations would suggest that the supplement improves energy metabolism for both groups but that the non-supplemented smokers simply have a more pronounced issue of higher levels of peripheral lipolysis and the fatty acids that accompany this. These observations suggest a direct link between smoking and the fatty acid composition of our sample set.
Further studies will be required to identify the specific components of oral supplementation contributing to the metabolomic changes seen in this study. In addition, further studies will need to delineate whether the decreases in LCFAs are beneficial or detrimental to overall health status. In our current study, for instance, smokers' skin improved with respect to elasticity measurements and clinical appearance of fine wrinkles, glow and hydration but displayed worsened deep wrinkling appearance after 12 weeks of study supplementation. It has been observed previously that increased fatty acid content in the skin regulates the healing process through cell-surface interactions . In addition, longer-chained fatty acids have garnered a significant amount of attention for their role in skin health, which has revealed that animal models with deficient essential fatty acids experienced increased water loss through the skin . These results suggest that fatty acids are a critical component of skin cell morphology and health and further supports a link between our metabolic observations and our clinical observations.
While smoking can be regarded as a 'disease condition' worthy of possible 'treatment' with oral supplementation, it is unclear whether healthy individuals such as non-smokers would benefit from oral supplementation. In fact, there are large epidemiologic studies suggesting that over-supplementation of particular nutrients can be detrimental to health [27–29]; hence, further exploration into the metabolic alterations that occur due to supplementation and their role in organ phenotypes is needed.