Serum folate associated with nonalcoholic fatty liver disease and advanced hepatic fibrosis

The role played by serum folate in the progression of nonalcoholic fatty liver disease (NAFLD) remains controversial. The purpose of this study was to investigate the association of serum folate with NAFLD and advanced liver fibrosis (AHF). We conducted a cross-sectional study with 5417 participants using 2011–2018 NHANES data. Multiple logistic regression analysis and propensity score matching analysis were used to investigate the association of serum folate with NAFLD and AHF. In the completely adjusted model, participants in the high serum folate group had a 27% (OR 0.73, 95% CI 0.62, 0.87, p = 0.0003) and 53% (OR 0.47, 95% CI 0.35, 0.63, p < 0.0001) lower odds of suffering from NAFLD and AHF, respectively, compared to the low serum folate group. The similar results in propensity score matching further validated the above association. Stratified analysis showed that the negative correlation of serum folate with NAFLD and AHF demonstrated a broad consistency across populations. The results of this study indicate that higher serum folate level was associated with lower odds of NAFLD and AHF among US adults. Further prospective studies are necessary due to the limitations of cross-sectional studies.


Definition of NAFLD and AHF.
The fatty liver index (FLI) was used to define NAFLD in this study.FLI is a widely used surrogate marker to predict the risk of NAFLD and is recommended by European guidelines for the management of NAFLD 15,16 .Participants with an FLI score greater than or equal to 60 were considered to have NAFLD 17 .The NAFLD fibrosis score (NFS) is a nondiffusion system for identifying nonalcoholic fatty liver fibrosis, and participants in this study with NFS > 0.676 were considered to have AHF 18 .It is important to note that the definitions of both NAFLD and AHF are based on non-invasive scores.The equations for FLI and NFS are shown below 17,18 .NFS = − 1.675 + 0.037 × age + 0.094 × BMI + 1.13 × impaired fasting glycemia or diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio − 0.013 × platelet − 0.66 × albumin.TG, Triglycerides; GGT, gamma-glutamyl transferase; WC, waist circumference.

Covariates.
Since the results of the study may be influenced by multiple factors, we included age, sex, race, education level, poverty income ratio (PIR), BMI, smoking status, work activity status, recreational activity status, dietary energy, protein, alcohol, folate intake, hypertension status, diabetes status and biochemical indicators, including total cholesterol and HDL cholesterol, as covariates of the study.Five racial classifications, including Mexican American, Other Hispanic, Non-Hispanic Black, Non-Hispanic White, and Other races, were used to define the race variable.Education level is classified as < high school, high school, and > high school.The poverty income ratio was categorized as < 1, 1-3, and > 3. BMI was classified as < 18.5 (underweight), 18.5-24.9(healthy weight), or > 25 (overweight or obesity) 19 .Smoking status was categorized as never, former, and now.Four scales, including no, vigorous, moderate and both, were used to evaluate the work or recreational activities status of participants.The dietary intake status used the sum of the dietary intake of the first and second day.Participants using hypertensive medications or with past/current diagnosis of hypertension were diagnosed with hypertension.Diabetes status was grouped as yes, no, impaired fasting glucose, impaired glucose tolerance based on hypoglycemic medication status, diabetes diagnosis status, glycated hemoglobin, and fasting glucose.
All covariate data in this study were obtained from the NHANES website (https:// www.cdc.gov/ nchs/ nhanes/ index.htm).
Multiple logistic regression analysis was used to evaluate the association of serum folate with NAFLD or AHF, and adjusted models were constructed based on the included covariates.No variables were adjusted in the crude model.Model 1 was adjusted by age, sex, race, education level, and PIR.Model 2 further adjusted for total cholesterol, HDL cholesterol, hypertension status and diabetes status.Model 3 is a fully adjusted model, with the addition of adjusted smoking status, work activities status, recreational activities status, dietary energy intake, dietary protein intake, dietary folate intake and dietary alcohol intake.In multiple logistic regression, serum folate was trisected into low (1.8-12.6 ng/mL, n = 1806), medium (12.7-20.5 ng/mL, n = 1789) and high (20.6-49.9ng/mL, n = 1822) groups, and the low serum folate group was used as the reference group.We calculated the z score of serum folate and reported the odds ratios (OR) of NAFLD and AHF with each standard deviation (SD) increase in serum folate.Subsequently, we visualized the association by plotting a smoothed fit curve based on adjusted model 3 (ln-transformed data).
Propensity score matching (PSM) has been widely used to control for selection bias in observational studies.In this study, based on a 1:1 nearest neighbor matching algorithm, we used PSM to match participants with NAFLD or AHF to controls.Confounding factors, including age, sex, race, education level, poverty income ratio (PIR), smoking status, work activity status, recreational activity status, dietary energy, protein, alcohol, folate intake, hypertension status, diabetes status, total cholesterol, and HDL cholesterol, were chosen for matching.In addition, stratified analyses were constructed based on age, sex, race, education, and PIR to examine the stability of the association of serum folate (per SD increment) with NAFLD or AHF.For all analyses, the level of statistical significance was determined to be 2-sided p < 0.05, and 95% confidence intervals were calculated in this study.Using appropriate strata, clusters, and weights in the statistical analysis process to illustrate the complex multistage stratified sampling design of NHANES.The researchers used the statistical packages R (The  For AHF, no association was observed between serum folate and AHF in the crude model.In adjusted model 3, participants in the high serum folate group exhibited 53% lower odds of AHF than those in the low serum folate group (OR 0.47, 95% CI 0.35, 0.63, p < 0.0001).For each standard deviation increase in serum folate, the odds of AHF decreased by 23% in participants (OR 0.77, 95% CI 0.69, 0.86, p < 0.0001).
In addition, age, sex, race, education, PIR, smoking status, physical activity status, hypertension status, diabetes status, total cholesterol, HDL cholesterol, dietary protein intake, and dietary folate intake were significantly associated with NAFLD status in adjusted model 3. Age, sex, race, education, PIR, smoking status, physical activity status, hypertension status, diabetes status, total cholesterol, HDL cholesterol, and dietary folate intake were significantly associated with AHF status (Appendix Table 1).
Figure 2 demonstrates a smoothed curve fit plot of the association, with serum folate showing a linear negative trend with both NAFLD and AHF.

Propensity score matching.
A comparable control group constructed based on nearest neighbor propensity score matching (1:1) was used to further explore the association of serum folate with NAFLD and AHF.For NAFLD, 1640 participants were included in both the NAFLD and control groups after propensity score matching.Figure 3 shows the results of the multivariate analysis before and after matching.After matching, participants in middle and high serum folate group exhibited 16% (p = 0.0475) and 21% (p = 0.0053) lower odds of NAFLD in comparison to the low serum folate group, respectively.For each standard deviation increase in serum folate, the odds of NAFLD decreased by 11% in participants (OR 0.89, 95% CI 0.83, 0.95, p = 0.0007).
For AHF, 519 participants were included in both the AHF and control groups after propensity score matching.Figure 4 shows the results of the multivariate analysis before and after matching.After matching, participants in middle and high serum folate group exhibited 28% (p = 0.0303) and 40% (p = 0.001) lower odds of AHF in comparison to the low serum folate group, respectively.For each standard deviation increase in serum folate, the odds of AHF decreased by 16% in participants (OR 0.84, 95% CI 0.74, 0.95, p = 0.0054).

Stratified analysis.
We constructed stratified analyses based on age, sex, race, education level, PIR, BMI, smoking status, work activities status, recreational activities status, dietary energy intake, dietary protein intake, dietary folate intake, hypertension status, diabetes status, total cholesterol, and HDL cholesterol.The results of the stratified analysis are shown in Fig. 5

Discussion
This study analyzed NHANES data from 2011 to 2018 and elucidated the association between serum folate and NAFLD and AHF in US adults based on epidemiological studies for the first time.We found that higher serum folate level was associated with lower odds of NAFLD and AHF after controlling for confounding factors.Subsequently, a stratified analysis was conducted to explore the stability of the association across populations.The results of stratified analysis indicated that the association between serum folate and NAFLD and AHF exhibited excellent stability, with similar associations observed in almost all subgroups.Although results contradicting the findings were observed in a very small number of subgroups, none were statistically significant.There were no interactions for any of the covariates included in this study.
The association between folate and NAFLD is not the first time that attention has been drawn to it, so some of the previous relevant studies should not be overlooked.A randomized controlled trial of dietary intervention in Israel observed greater reductions in intrahepatic fat (IHF) in subjects with the most significant elevations in serum folate, suggesting that serum folate is effective in reducing the risk of developing NAFLD 20 .Mahamid et al. found that low folate levels were significantly associated with the severity of fibrosis 21 .The risk of NAFLD was negatively associated with serum folate in a recent meta-analysis 12 .The above findings were consistent with our study's conclusions.Nevertheless, two past studies based on US populations reached conclusions that contradict this study.Li Li et al. studied the association of vitamin B12 markers with NAFLD with data from NHANES 1999-2004 and claimed that serum folate was not associated with NAFLD 14 .Sources of inconsistency in the conclusions are the differences in year and adjustment models, and we added variables adjusting for physical activity, smoking, and dietary intake of the participants.Xiaohui Liu et al. researched the association between vitamins and NAFLD, but no association was found between dietary intake of folic acid and NAFLD 13 .We considered that differences in dietary intake of folic acid and serum folate level were the main reason for the different conclusions.Overall, we have strong confidence in the findings of this study due to the well-adjusted model, detailed stratification study and large sample size.
The current research on the possible mechanisms by which folic acid reduced the risk of NAFLD and AHF focused on improving abnormalities in lipid metabolism.Cellular AdoMet-dependent methylation reactions are required for the synthesis of phosphatidylcholine (PC), which is normally converted to triglycerides (TG) 22 .High levels of serum folate help to control or reduce AdoMet concentrations so that PC synthesis is inhibited to reduce the accumulation of triglycerides in the liver.Moreover, it has been shown that phosphatidylethanolamine (PE) is mediated by AdoMet via N-methyltransferase (PEMT) to accelerate PC synthesis, followed by the induction of hepatic steatosis 22 .In contrast, high serum folate levels can reduce AdoMet concentrations and in turn inhibit the above PC synthesis pathway, ultimately improving abnormalities in hepatic lipid metabolism 23 .The protective effect of folic acid against oxidative stress in hepatocytes may also be a potential mechanism 24 .Serum folate promoted mitochondrial beta oxidation, reduced oxidative stress in vivo and inhibited peroxisome proliferator-activated receptor γ (PPARγ).PPARγ is the key factor in regulating adipogenesis and decreasing TG accumulation in the liver, thus reducing hepatic steatosis 25 .In addition, the severity of NAFLD and the progression of AHF were associated with higher systemic levels of some cytokines, such as IL-6 and TNF-α 26 .High serum folate levels can help to reduce the expression of proinflammatory cytokines and inhibit the recruitment and activation of Kupffer cells, thereby lowering the risk of NAFLD and AHF 27,28 .
In this study, age, sex, race, education, PIR, smoking status, physical activity status, hypertension status, diabetes status, total cholesterol, HDL cholesterol, dietary protein intake and dietary folate intake were significantly associated with NAFLD status.Previous studies have shown that hypertension and diabetes are important risk factors for NAFLD 29 .Smoking is positively associated with NAFLD and the underlying mechanisms have been initially elucidated 30,31 .The association between physical activity status and NAFLD has also been previously reported, with exercise helping to reduce functional adaptations in patients with NAFLD 32,33 .In addition, race, education, and PIR are important social determinants of NAFLD [34][35][36] .Of note, dietary folate intake was significantly associated with NAFLD and AHF.Participants with high dietary folate intake had 20% lower odds of suffering from NAFLD (OR 0.80, 95% CI 0.66, 0.98, p = 0.031) and 36% lower odds of suffering from AHF (OR 0.64, 95% CI 0.46, 0.90, p = 0.0109) compared to participants with low dietary folate intake.The intake of Notably, this study observed significant sex differences in the correlation between serum folate and NAFLD, which was lacking in previous studies.One possible explanation may be that higher levels of estrogen in women exerted a protective effect.A study by Nemoto et al. found that estrogen supplementation prevented the progression of hepatic steatosis adenopathy in estrogen-deficient mice, suggesting that estrogen receptor-mediated signaling pathways may play a key role in lipid metabolism in the liver 37,38 .Additionally, Kupffer cells in men expressed higher levels of TLR4 than those in women to the extent that they produced more proinflammatory cytokines, further activating liver inflammation and fibrosis 39 .Unlike men, Kupffer cells in women exhibited more anti-inflammatory and anti-fibrotic properties.
A highlight of this study is the larger and scientifically designed sample source, which enhanced the credibility and universality of the findings.In addition, the well-established adjustment model and stratified analysis make the conclusions more reliable.However, there are still some limitations of our study that cannot be ignored.First, due to the nature of cross-sectional studies, we cannot establish a causal relationship between serum folate and NAFLD and AHF, and further prospective cohort studies are necessary.Second, although we included as many covariates as possible to exclude bias from confounding factors, there may still be potential confounders that were not included in the analysis.Third, all participants in this study were from the United States, and the applicability of the results to populations in other countries needs to be carefully considered, given the differences in physical condition, dietary habits and environmental factors that exist between populations.In addition, although FLI showed a high diagnostic value, it is not a substitute for biopsy.The diagnosis of NAFLD in this study is not a clinical diagnosis, and further studies are still needed in the future.Overall, despite the strong statistical efficacy of this study, there is a requirement for greater modesty and caution in interpreting the results due to the limitations of cross-sectional studies as well as the diagnosis of NAFLD.

Conclusions
The results of this study indicate that higher serum folate level was associated with lower odds of NAFLD and AHF among US adults.Future prospective cohort studies are still necessary to validate our conclusions.

Figure 4 .Figure 5 .
Figure 4. Multivariate analysis before and after matching for AHF.

Table 1 .
Baseline characteristics of participants based on NAFLD stratification.NAFLD Nonalcoholic Fatty Liver Disease, PIR Poverty Income Ratio, IFG Impaired Fasting Glycemia, IGT Impaired Glucose Tolerance, BMI Body Mass Index, GGT Gamma glutamyl transferase, AST Aspartate Transaminase, ALT Alanine Aminotransferase.

Table 2 .
Baseline characteristics of participants based on serum folate stratification.NAFLD Nonalcoholic fatty liver disease, AHF advanced hepatic fibrosis, PIR poverty income ratio, IFG impaired fasting glycemia, IGT impaired glucose tolerance, BMI body mass index, GGT gamma glutamyl transferase, AST aspartate transaminase, ALT alanine aminotransferase.

Table 3 .
Association of serum folate with NAFLD and AHF.NAFLD Nonalcoholic fatty liver disease, AHF advanced hepatic fibrosis, PIR poverty income ratio.Crude model was not adjusted.Adjusted model 1 adjusted for age, sex, race, education level, PIR.Adjusted model 2 adjusted for model 1 + total cholesterol, HDL cholesterol, Hypertension status, Diabetes status.Adjusted model 3 adjusted for model 2 + smoking status, work activities status, recreational activities status, dietary energy intake, dietary protein intake, dietary alcohol intake, dietary folate intake.