In this study, we stratified healthy Japanese individuals over 20 years of age by sex and age and found multiple significant associations between serum carotenoid levels and VFA. The associations are discussed in the following sections, considering the results of a dietary survey conducted using BDHQ.
4.1. Relationship between Serum Carotenoid Levels and VFA
In the simple correlation analysis, only in females, total carotenoid concentrations in the serum were significantly and inversely associated with VFA. These relationships were kept in the lifestyle-adjusted multiple regression analysis or even age-stratified analysis. In our previous study [8
], a significant negative relationship between total carotenoid levels in the serum and BMI was found only in females, which is consistent with the results of the present study. Sex differences in the serum concentrations of carotenoids may be one of the factors that contributed to this difference between males and females. In the present study, the mean total carotenoid concentration in females was significantly higher than that in males (male: 1.15 ± 0.53 μg/mL and female: 1.67 ± 0.75 μg/mL). If a certain threshold level of carotenoids was required to affect VFA, and many males do not meet the threshold, it may be possible that no significant relationship was found between carotenoid levels and VFA in males. It might also be possible that there are some differences in the carotenoid metabolism between the sexes. However, there is no concrete evidence for differences in carotenoid absorption or metabolism based on sex. In addition, differences in lifestyle between males and females may have influenced the results. Males are more likely than females to smoke, exercise, and consume alcohol. These lifestyles might be strongly associated with VFA in males. In a previous report [14
], a relationship between waist circumference and carotenoids was found only in females, and the study speculated that smoking could be a contributing factor. In this study, we adjusted for these lifestyle factors in multiple regression analysis, but the effect of some of them on VFA is so large that it may not have been fully adjusted for males. Thus, although the causes of sex differences are considered diverse, there is no definitive evidence to support either of the hypotheses, and further research is needed. However, at least in females, higher serum total carotenoid levels may be beneficial for preventing cardiovascular disease, considering that VFA, a better predictor of cardiovascular disease than BMI, was also negatively associated with total carotenoid levels.
There are four hypotheses for the possible mechanisms by which carotenoids may seem to be useful in preventing cardiovascular disease. First, some physiological effects of carotenoids are hypothesized to be responsible. Carotenoids are known to have a variety of physiological effects [24
]. A typical example is the ability to quench singlet oxygen, one of the ROS. ROS have been reported to be the cause of obesity [25
], suggesting that reduction in their levels may inhibit obesity. One of the major factors for the increase in VFA is the accumulation of adipocytes. There are also reports that fucoxanthin, a carotenoid, suppresses the accumulation of adipocytes and, thereby, suppresses the accumulation of visceral fat [26
]. In other words, carotenoids may directly inhibit the accumulation of adipocytes, which causes obesity and the accumulation of visceral fat. In addition, it has been reported that β-carotene concentrations are 50% lower in adipocytes of obese individuals compared to non-obese individuals [27
], suggesting that carotenoids could be consumed under obese conditions to protect against obesity.
The second hypothesis is that it is because of the physiological action of vegetable components other than carotenoids. As previously reported, serum carotenoid levels are positively correlated with vegetable intake [8
]. In addition, in the present study, serum levels of many carotenoids were positively associated with vegetable intake. In addition to vegetable intake, supplements are also assumed to be a source of carotenoids, but there was no data on the consumption of dietary supplements in this study. However, carotenoids supplements were reported to be taken by 2.4% of men and 4.4% of women in Japan [28
], and we believe that the impact of those supplements on carotenoid intake was limited. Thus, individuals with high carotenoid levels may also have higher intakes of other vegetable ingredients. Dietary fiber is one of the components that is assumed to inhibit various chronic diseases. Dietary fiber has been previously suggested to reduce obesity and visceral fat accumulation [29
]. However, the inverse associations between carotenoid levels and VFA were observed even though we added dietary fiber intake as an adjustment factor in the multiple regression analysis. Therefore, the associations are considered independent of the effect of dietary fiber. However, it cannot be denied that the influence of vegetable components other than dietary fiber may be indirectly observed.
Third, it is hypothesized that high vegetable intake leads to a reduction in energy intake. If this hypothesis is correct, it is thought that persons with a high intake of dietary vegetables and high serum carotenoid levels have a relatively low intake of nutrients, such as carbohydrates and lipids, and inhibit obesity and visceral fat accumulation. However, there was no significant correlation between total energy intake and total carotenoid concentration (male: r = 0.08, p = 0.172; female: r = 0.03, p = 0.56). Therefore, the inverse association between carotenoid concentrations and VFA observed in this study is unlikely to be due to a decrease in energy intake associated with high vegetable intake.
The fourth hypothesis is that it is caused by other healthy lifestyle aspects that are strongly associated with vegetable intake. Individuals with high health literacy and high vegetable intake may also have other healthy lifestyle habits, such as exercise habits and low breakfast absenteeism [30
]. Thus, the inverse association observed in this study may be indirectly influenced by some healthy lifestyle habit that correlates with vegetable intake, rather than by direct effects of carotenoids or other vegetable components. However, the relationship between carotenoid levels and VFA is unlikely to be due to the presence or absence of smoking or exercise habits because exercise and smoking habits are included as adjustment factors in the multiple regression analysis. However, the influence of healthy eating habits, especially eating habits other than vegetable intake, cannot be denied. To clarify the dietary habits associated with high serum levels of these carotenoids, a simple correlation analysis was performed between various dietary groups calculated using BDHQ and serum carotenoid levels. The results suggested that the intake of soybean foods (natto and tofu) was also high in individuals with high serum levels of lutein and β-carotene. Several previous reports have shown that aggressive intake of soy food has an inhibitory effect on cardiovascular disease [31
]. On the contrary, serum levels of lutein and β-carotene were inversely associated with the intake of hamburgers, fried foods, ramen, and beer. It is possible that diets high in vegetables and soybean foods and lifestyle habits that do not lead to excessive intake of carbohydrates and lipids could have inhibited the accumulation of VFA. However, more detailed studies evaluating the relationship between dietary intake and VFA are needed to confirm this.
To investigate the first hypothesis mentioned above, which proposes the direct effect of carotenoids, we analyzed the relationship between serum concentrations of individual carotenoids and VFA.
4.2. Relationship between Serum Concentrations of Lutein, β-Carotene, and β-Cryptoxanthin and VFA
In females, higher serum levels of lutein and β-carotene were associated with lower levels of VFA. This association and total carotenoid concentrations were maintained significantly in all age groups when stratified by age. The mean serum concentration of these two carotenoids is higher than that of the other carotenoids, and the sum of the two carotenoids is almost 60% of the total carotenoid concentration. These two carotenoids are ingested through the intake of many foods (mainly vegetables). For example, serum lutein levels were positively correlated not only with the intake of abundant green leafy vegetables [32
] but also with that of raw vegetables, cooked cabbage, and pickles. β-Carotene was also positively correlated with the intake of all vegetable groups except raw vegetables and could be ingested through vegetables, in general.
The antioxidant effect common to carotenoids (including these two types of carotenoids [24
]) was thought to have an inhibitory effect on the accumulation of VFA and obesity by inhibiting chronic inflammation. For example, β-carotene has been reported to be negatively associated with obesity and serum triglycerides [8
]. It has been reported that β-carotene and its metabolites by β-carotene-9′,10′-dioxygenase control lipid metabolism within adipocytes, inhibit adipocyte maturation transformation, and suppress cell differentiation [33
]. However, further studies are needed to elucidate the mechanisms by which lutein and β-carotene affect VFA.
Serum levels of β-cryptoxanthin were significantly negatively associated with VFA in females of all ages. The main source of β-cryptoxanthin was fruits, especially citrus fruits, similar to that in the Mikkabi cohort study conducted in Japan [34
]. β-Cryptoxanthin has also been shown to be negatively associated with the markers of chronic cardiovascular disease [35
]. Although the mechanisms by which β-cryptoxanthin prevents obesity have not been entirely clarified, it was suggested that β-cryptoxanthin might induce PPAR-α [36
], antagonize PPAR-γ [37
], and upregulate Keap1/Nrf2 pathway [38
]. In the present study, lower BMI, but not VFA, was significantly associated with higher serum levels of β-cryptoxanthin in middle-aged females. The absence of this relationship with VFA could be due to the relatively low serum concentration of β-cryptoxanthin in citrus fruits (this study was conducted in May and June when the citrus fruits are not in season). Therefore, we speculate that a significant relationship between β-cryptoxanthin and VFA could have been observed if the studies would have been conducted during winter when the intake of citrus fruits is high. However, in-depth studies are needed to demonstrate the reduction in VFA by β-cryptoxanthin ingestion.
4.3. Relationship between Lycopene Levels and VFA
In males, a direct association was found between serum lycopene concentrations and VFA. The relationship was maintained even after stratification by age. However, lycopene has not been reported to increase VFA. Conversely, lycopene, as well as lutein and β-carotene, have potent antioxidant effects and may prevent obesity through the elimination of ROS, and there has been a report showing an inverse association between lycopene and VFA [17
]. Takagi et al. showed that taking a beverage containing high lycopene reduced abdominal circumference in obese Japanese men by a small intervention study [39
]. In the present analysis, a significant relationship was found, especially in the old male group, but the serum levels of lycopene were significantly lower in the old than in the other age groups. In addition, it has been reported that the low lycopene levels in Japanese individuals do not correlate with obesity [17
]. Thus, the positive association between serum lycopene concentration and VFA is likely not a direct effect of lycopene. In addition, serum levels of lycopene were significantly positively correlated with the frequency of consumption of hamburgers and pastas. These foods are representative of tomato dishes, but they are also rich in carbohydrates and lipids. Previous reports [19
] have also shown that monounsaturated fatty acids, which are abundant in olive oil, are positively correlated with VFA and may have been influenced by other nutrients in tomato dishes.