In this case-control study, we showed that a high intake of total nitrate-containing vegetables and low-nitrate-containing vegetables was related to decreased odds of NAFLD, independent of potential confounding variables. In contrast, there was no significant association between intakes of medium- and high nitrate-containing vegetables and the odds of NAFLD. Also, our finding reported that one SD increment in nitrate content of vegetables and fruits was linked inversely with odds of NAFLD; however, a positive association was observed between each one SD increments in nitrate content of dairy products and meats and processed meats with risk of NAFLD. Furthermore, each one SD increments in the intakes of all dietary nitrate had no association with odds of NAFLD.
To date, this study was the first study that examines the relationship of NCVs with the odds of NAFLD; however, several studies have previously assessed the association of dietary nitrate intake or NCVs with the risk of other chronic diseases such as CKD , atherosclerosis and ischemic cerebrovascular disease , blood pressure , and CVD , which all of them have the characteristics of metabolic disorders similar to NAFLD. A prospective study conducted on Tehranian adults has reported that high intake of high-NCVs was associated with increased the risk of CKD at baseline, but, after 3 years of follow-up, no significant association was observed between intakes of total NCVs and its categories (high-, medium-, and low NCVs) with the risk of CKD . However, in a population-based cohort study, Golzarand et al. have shown that dietary nitrate from vegetable sources can significantly decrease the risk of hypertension (HTN) and consequent CVD complications, independent of dietary fiber and potassium . Also, Bondonno et al. have revealed that older women who had higher intakes of vegetable nitrate had a significantly lower risk of carotid atherosclerosis and ischemic cerebrovascular disease event . Furthermore, the Australian Longitudinal Study has provided evidence on the protective role of vegetable nitrate intakes on developing CVD-related disorders, including HTN, thrombosis, heart disease, and stroke among middle-aged Australian women over a 15-year follow-up period . Although studies conducted on total dietary nitrate intake or NCVs have examined their role in the risk of developing various chronic diseases, which may also be methodologically different, the findings of most of these studies provided evidence that the intakes of nitrate from vegetable sources can have a beneficial impact on the prevention of developing chronic diseases such as NAFLD, HTN, CVD complications.
Convincing evidence has suggested that individuals with higher consumption of vegetables had a lower risk of NAFLD [25, 26] . This process may be associated with higher intakes of fiber, antioxidant vitamins, and bioactive compounds, as well as the low energy content of vegetables. According to interesting findings of the current study, we also suggested that the high consumption of total NCVs has been remarkably associated with a reduction in the odds of NAFLD, which can be due to increased intake of dietary nitrate; because, in the current study, the inverse association between NCVs and risk of NAFLD was reported and confirmed independently of other dietary factors such as dietary intakes of energy, refined grains, high-fat dairy, red and processed meats, fruits, saturated fatty acids, vitamin E, fiber, and fructose, each of which could affect the risk of NAFLD. So, these results of our study show that nitrate intake from vegetables not only does not increase the risk of NAFLD but may also be a protective factor in the form of vegetables against risk of chronic diseases such as NAFLD. It should be noted that, according to the classification of vegetables into three groups based on nitrate content, although the odds of NAFLD is reduced with increasing intake of vegetables in all three groups, the results are significant only in the group of low nitrate vegetables. The main reason that the results of the group of low-nitrate vegetables have been remarkable compared to the other two groups (medium-nitrate vegetables and high-nitrate vegetables groups) is that, according to the definition of NCVs, most of the total vegetables intakes in our study population was low nitrates vegetables (more than 90% of all vegetables intakes based on the results of Table 2), and so the individual’s dietary intakes of medium-nitrate vegetables and high-nitrate vegetables was very low. Also, there is no high dispersion in the dietary intake of vegetables in participants across tertiles of the medium-nitrate vegetables and high-nitrate vegetables groups, as a result, the power of medium-nitrate vegetables and high-nitrate vegetables has not been significant in predicting the odds of NAFLD compared to low-nitrate vegetables group.
Also, we showed that differences in dietary sources of dietary nitrate could also play a significant role in the risk of liver disease; our results reported that higher intake of nitrate from plant-based food sources, including vegetables and fruits, have a protective role against onset or progression of NAFLD, whereas, higher intake of nitrate from animal-based food sources including dairy products and meats and processed meats have worsened the development of NAFLD. Therefore, our finding in Table 3 suggested that the nature of food groups and their related characteristics can cause nitrate intake to play a different role in predicting the risk of NAFLD. In other words, it seems that differences in food sources of nitrate have an influential role in selecting the target pathway for nitrate to enter the metabolic cycle.; this in turn subsequently causes dietary nitrate intake to play a dual role in (increasing or reducing) the odds of NAFLD. Based on recent documents, the potential physiological and pathological role of nitrate intakes from various food sources in the body are not fully understood, and also there are controversial views on the possible adverse impact vs. health benefits of nitrate intake [11, 12, 27]; although, further studies are needed to understand how dietary nitrate intake is metabolized within the food matrix, it seems that on a metabolic level this paradoxical role of nitrate intakes is possibly not surprising, and it is expectable that all food sources of nitrate are not necessarily identical with regards to their potential health impacts . Previous studies have reported that the inducing production of nitric oxide (NO), a gas produced by nitric oxide synthase (NOS) enzyme, slows NAFLD development , and also the reduction or deficiency of eNOS accelerate early-stage NAFLD progression via alteration of the fat distribution . The bioactivity of nitrate, nitrite, and NO metabolism is a complicated process that can be affected by the nature of the consumed foods and their nutrients. The higher intakes of pro-inflammatory nutrients, including saturated fat, sodium, and amine compounds from animal food sources such as dairy foods, meat, and processed meat may interfere with eNOS and decreased the NO production from nitrate , however, the higher intakes of anti-inflammatory compounds including polyphenols, fiber, vitamin C, and other antioxidants from vegetables and fruits can have enhancing role on NO production from nitrate [31,32,33]. Also, it has been reported that higher intakes of nitrate/nitrite from vegetables may contribute to improving cardiometabolic conditions via beneficial impact on insulin signaling pathway, insulin resistance, and inflammation and oxidative stress .
The current study had several strengths. This study is the first study that assesses the association of all dietary nitrate intakes and all NCVs with odds of NAFLD. We also analyzed the role of three categories of NCVs consumption based on nitrate content (low-, medium-, and high NCVs) with NAFLD. In addition, the relationship of nitrate intakes from various other food groups (fruits, grains, legumes, nuts, dairy products, red meats, and processed meats) with odds of NAFLD was examined to provide more explanation on the role of nitrate intake in the development of NAFLD based on different food sources. We have used a validated and reproducible FFQ to collect participants’ dietary data, which was completed by trained dietitians in a face-to-face interview; this questionnaire was not a self-reported questionnaire that led to minimizing measurement bias. Furthermore, we used a validated questionnaire to assess the physical activity levels of individuals.
Despite the above-listed strengths, some limitations deserve to mention. First, this study cannot discover the causality between exposures and outcomes because of the case-control design. Second, we used ultrasonography test to diagnose NAFLD in participants, but a biopsy of the liver and magnetic resonance imaging (MRI) technique is the gold standard and more accurate tests for diagnosis of NAFLD; it is worth mentioning that because of the limitations and complications of biopsy and high cost and low availability of MRI, using noninvasive methods such as ultrasonography is applicable and reliable to diagnosis NAFLD in clinical practice . Furthermore, in this case-control study, the matching process of the cases and controls was not performed based on main variables including age, sex, and BMI, because it could make it more difficult to select eligible individuals and lengthen the sampling time. Also, it could increase the possibility of over-matching; however, it should be noted based on baseline results, age and sex distribution did not differ in participants based on cases and controls groups and also the possible confounding role of these variables such as BMI were controlled in the multivariable-adjusted analysis. Moreover, some inherent limitations of case-control studies, including selection bias and information bias in determining exposure or outcome, should be considered in interpreting the results. Finally, despite the adjustment of various potential confounders, our study design cannot eliminate all possible confounders, and the effects of some residual confounders may have occurred.