Association of Meat Consumption With NAFLD Risk and Liver-related Biomarkers in Older Chinese: A Cross-sectional Study

Background: High meats intake contributes to unhealthy status. The present study aims to evaluate the association of meat consumption with non-alcohol fatty liver disease (NAFLD) risk and liver-related biomarkers in middle-aged and elderly Chinese. Methods: A cross-sectional study was conducted in individuals who were 45 years or older and underwent physical examination from April 2015 to August 2017 in Southeast China. NAFLD was evaluated by abdominal ultrasonography. Results: High consumptions of red meat (28.44-49.74 and >71.00 g/day) was positively associated with NAFLD risk on inverse probability of treatment weighting analysis, adjusting for smoking, tea intake, weekly hours of physical activity and presence of hypertension, dyslipidemia and diabetes (OR adjusted =1.948 and 1.1.716, respectively). Exposure-response relationship analysis presented that red meat intake was positively associated with NAFLD risk. Signicant associations of red meat intakes with serum levels of γ-glutamyl transferase, alanine transaminase, aspartate aminotransferase, total triglyceride and high-density lipoprotein cholesterol were found (r s =0.176, 0.128, 0.060, 0.085 and -0.074, respectively). Conclusions: These ndings suggest that reduction of meat consumption may decrease NAFLD risk and should warrant further investigations. Meat consumptions were measured by a semi-quantitative food frequency questionnaire. of meat subtypes intakes with the concentrations of serum GGT, ALT, AST, fasting plasma glucose, total cholesterol, TG, low-density lipoprotein cholesterol and HDL-C, spearman’s rank correlation was performed. For the statistical analyses, SPSS, SPSS, IL, and (R Project for Statistical Computing) were performed. All P values were two-tailed and results were considered to be statistically signicant at P values < 0.05.

than 25 food-related items on the questionnaire. (E) Individuals who did not provide information on smoking and tea consumption. All subjects provided their informed consent prior to participating in this study.
The current study was carried out in compliance with the Declaration of Helsinki, and the Ethics Committee of Fujian Medical University approved the study protocol (ethics number 2014096).

Data Collection
NAFLD ascertainment NAFLD was diagnosed by abdominal ultrasonography using established criteria [23]. Abdominal ultrasonography examination was done by experienced radiologists who were unaware of the laboratory and clinical data.

Meat intakes assessment
Semi-quantitative food frequency questionnaire included meat consumption were obtained from participants who were interviewed face to face by trained investigators. For each food item, participants used the following response options to indicate how often they ate the selected food on average: 3 times/day, twice/day, once/day, 5-6 times/week, 3-4 times/week, 1-2 times/week, 1-3 times/month, <once/month and rarely. Red meat consisted of pork, beef and lamb. Poultry was composed of chicken and duck. Processed meat contained fried and smoked meat. Fish included: sh, shell sh and crab.

Covariate assessment
The following variables were self-reported: age, sex, marital status, income, educational level, smoking status, tea intake status, physical activity, medication use and medical conditions. All subjects underwent physical examinations (height, weight, waistline, hipline and blood pressure) and blood tests (fasting plasma-glucose, low-density lipoprotein cholesterol, total cholesterol, TG, HDL-C, AST, ALT and GGT) performed by trained physicians. BMI was calculated as weight/ (height) 2 . Participants with a systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg were de ned as having hypertension. And subjects who had one or more of the following abnormalities were diagnosed as dyslipidemia: total cholesterol ≥6.2 mmol/L, TG > 2.25 mmol/L, low-density lipoprotein cholesterol > 4.13 mmol/L or HDL-C < 1.03 mmol/L [24]. Diabetes was diagnosed as follows: fasting plasma glucose of 7.0 mmol/L or greater or 2-hour postprandial glucose greater than or equal to 11.1 mmol/L.

Statistical Analysis
Nonparametric Kruskal-Wallis test and Chi-Square test were performed for comparisons between groups. Continuous variables were expressed as median (interquartile range, IQR). Propensity scores were used to explain potential confounders by observed characteristics at baseline [25]. Age, gender and BMI were used to calculate propensity score. Inverse probability of treatment weighting was performed to evaluate the associations of red meat, processed meat, poultry and sh intakes with NAFLD, adjusting for smoking status, tea intake status, weekly hours of physical activity, and presence of hypertension, dyslipidemia and diabetes. The lowest quartile (Q 1 ) of each type of meat intake was served as the reference group and P for trend was calculated by set the meat intake quartiles as continuous variable. To evaluate the dose-response relationships between continuous exposure variables (red meat, processed meat, poultry and sh intakes) and NAFLD, a logistic model with restricted cubic spline using ve knots at 0.05, 0.275, 0.5, 0.725 and 0.95 was built, adjusting for age, sex, BMI, smoking status, tea intake status, weekly hours of physical activity, and presence of hypertension, dyslipidemia and diabetes.
We also performed subgroup analysis to examine the relationships of red meat, processed meat, poultry and sh with NAFLD by the following subgroups: age (< 60 years or ≥60 years), gender (men or women), BMI (< 24 kg/m 2 or ≥24 kg/m 2 ), smoking status (never, former or current), tea consumption status (yes or no), hypertension (yes or no), dyslipidemia (yes or no), diabetes (yes or no), and weekly hours of physical activity (< 9 hours/week or ≥9 hours/week). P value for interaction was calculated. Two sensitivity analyses were conducted: (1) logistic regression analysis without IPTW; and (2) propensity score-matching logistic regressions. Furthermore, to investigate the associations of meat subtypes intakes with the concentrations of serum GGT, ALT, AST, fasting plasma glucose, total cholesterol, TG, lowdensity lipoprotein cholesterol and HDL-C, spearman's rank correlation was performed.
For the statistical analyses, SPSS, version 19.0.0.1(IBM SPSS, 2010, Chicago, IL, USA) and R, version 4.0.0 (R Project for Statistical Computing) were performed. All P values were two-tailed and results were considered to be statistically signi cant at P values < 0.05.

Baseline characteristics
A total of 1594 individuals were included in this analysis. In the entire study sample, 53.5% were men, average age was 53.54 ± 6.90 years and average body mass index (BMI) was 23.77 ± 2.99 kg/m 2 . As shown in Table 1,compared with participants with lower total intake of red, processed meat, poultry and sh, participants with higher total intake (1) were younger and more likely to be married persons, male, smokers and tea drinkers; (2) had higher educational level, higher income, higher BMI, higher waist and hip circumference; (3) had higher levels of plasma γ-glutamyl transferase and alanine aminotransferase; and (4) had higher prevalence rate of NAFLD (each P < 0.05).

Meat subtypes consumptions and NAFLD
As presented in Table 2, after propensity score weighting, red meat intake was positively related with the risk of NAFLD (OR per 50 g/day of red meat: 1.128, 95%CI: 1.005-1.266). After further adjustment for smoking status, tea intake status, weekly hours of physical activity and the presence of hypertension, dyslipidemia and diabetes, the association with NAFLD remained signi cant (OR adjusted =1.143). High consumptions of red (Q 2 and Q 4 ) was signi cantly relevant to higher odds for NAFLD (OR adjusted = 1.948 and 1.716, respectively), adjusting for those potential confounders. The restricted cubic splines analyses were applied to explain the exposure-response association between meat subtypes intakes and the risk of NAFLD (Fig. 1). The ORs of NAFLD increased with red meat and poultry consumptions, but decreased with sh intake (Fig. 1A, 1C and  1D). Moreover, the trend for OR of processed meat intake was not found (Fig. 1B).

Strati ed analyses
The positive association between meat intake and NAFLD was consistent across strata of age, sex, smoking status, tea intake status, BMI, weekly hours of physical activity, and the presence of hypertension, dyslipidemia and diabetes. Signi cant associations between red meat intake and the risk of NAFLD were not only found in males, but also existed in people whose BMI ≥24 kg/m 2 , tea-drinkers, people with hypertension and people without dyslipidemia. Adjusted ORs were estimated to be 1.177, 1.195, 1.229, 1.304 and 1.203, respectively. Moreover, sh intake was found positively relevant to NAFLD in people with diabetes (OR adjusted =1.447). Nonetheless, no signi cant interactions of meat subtypes intakes and potential confounders were identi ed (Table 3).

Sensitivity analyses
Results from unweighted analysis were similar to those from propensity score-weighted analysis. Compared with the lowest quartile of meat intake, high intakes of red meat (Q 2 and Q 4 ) were signi cantly associated with NAFLD risk without inverse probability of treatment weighting (OR adjusted =1.484 and 1.558, respectively) (see Additional le 1). Nonetheless, positive associations of red meat (Q 4 ) and processed meat (Q 3 ) consumptions with NAFLD risk were observed on propensity score matching analysis, with adjusted OR of 1.673 and 1.800, respectively (see Additional le 2).

Meat subtypes intakes and liver-related biochemical indexes
Associations between meat subtypes intakes and liver-related biochemical indexes were shown in Fig and − 0.066, respectively). In addition, GGT was also found to be signi cantly related to the consumption of sh (Spearman test correlation coe cient = 0.063).

Discussion
In this cross-sectional study, we observed that NAFLD was associated with higher intake levels of red meat. Signi cant associations of serum levels of GGT, ALT, AST, TG and HDL-C with meat subtypes intakes were found as well. Additionally, no signi cant interactions between meat consumptions and potential confounders for NAFLD were detected.
Signi cant associations between high meat consumptions and NAFLD were demonstrated in a few studies [7,9,10,26]. Our results are in accordance with the previous studies, indicating a positive association between high red meat intake and NAFLD. Two cross-sectional studies have presented that red meat was signi cantly correlated with NAFLD [7,10]. In another cross-sectional study, high intakes of total meat, especially red meat and/or processed meat were positively linked to NAFLD and insulin resistance, while processed meat alone was only relevant to insulin resistance. This is mainly due to a relatively low level of processed meat consumption in their research set [9]. In addition, a nested case-control study also showed that high consumptions of red meat, processed red meat and poultry were positively associated with NAFLD [26]. Due to the better accuracy of the continuous ORs [27], the dose-response analysis we have employed can better measure the overall trends of the ORs for meat intakes. The 95% CI for red meat intake beyond 200 g/day was slightly wider, because that less than 2% of participants had red meat intake > 200 g/day, that was, the tendency of red meat intake within the range 0 ~ 200 g/day was relatively reliable and stable.
There are several plausible mechanisms by which meat intake is related to NAFLD. NAFLD was reported to be closely linked to hepatic insulin resistance, which had strong correlation with liver biomarkers such as ALT, AST and GGT [28]. GGT and ALT had been considered as biomarkers of hepatic fat accumulation, which can lead to hepatic insulin resistance and increase the contribution of gluconeogenesis to total endogenous glucose production [29]. A cross-sectional study of 2198 European reported a signi cant positive association between red meat and GGT. As GGT is also a potential nonspeci c marker of oxidative stress, the author suggested that oxidative stress may plays a vital role underlying the development of chronic diseases with red meat intake [30]. Another cross-sectional study indicated that TG/HDL-C was independently relevant to the risk of NAFLD. The author attributed this result to insulin resistance [31]. Positive associations of serum levels of GGT, ALT and TG with red meat intakes were found in this study. Inversely, serum HDL-C concentration was negatively relevant to red meat and processed meat intakes. Hence, it's plausible that increased hepatic lipid accumulation and insulin resistance play a substantial role in the relationship of meat intakes with the development of NAFLD. In addition, a study by Avila et al. found that red meat was positively relevant to serum ferritin [32], which can increase the risk of NAFLD [33,34]. Fried food will produce some hazardous chemicals, such as advanced glycation end products and trans fatty acids [35,36], which were reported to play a critical role in NAFLD [37,38].
Signi cant associations were found in several subgroups. A positive association was observed between red meat and NAFLD in males, perhaps it is because males had a higher meat intakes and a higher prevalence of fatty liver than females [39]. Moreover, several studies had found signi cant associations of high meat intakes with obesity, type 2 diabetes and hypertension [14,17,40], which were considered as risk factors in the development of NAFLD [19,41,42]. In our study, positive association of red meat intake with NAFLD was found in people with BMI≥24 kg/m 2 and people with hypertension.
This study, however, had several limitations. Firstly, because of the cross-sectional study design, casual inference was not allowed.
Secondly, measurement error was unavoidable for self-reported diet and other data. Nevertheless, since all participants and researchers in this study were blinded to the results of abdominal ultrasonography and blood test, a reporting bias without differences is likely only to attenuate our observed association. Thirdly, since the study subjects were middle-aged and elders, it should be cautious in generalizing our ndings to the wider population. Lastly, although a comprehensive set of confounders were considered, as an observational study, the presence of unmeasured confounders is possible.

Conclusions
In conclusion, positive relationship between high consumptions of red meat and the risk of NAFLD was observed. In addition, serum levels of liver-related biomarkers were signi cantly relevant to red meat intake. Our ndings suggested that reduction of meat consumption may decrease the risk of NAFLD.

Availability of data and materials
The datasets used can be available from the corresponding author on reasonable request.