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Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults
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Clinical Nutrition xxx (2017) 1e7
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Original article
Q7 Q6
Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults Ming-Feng Xia a, b, 1, Hua Bian a, b, 1, Xiao-Peng Zhu a, b, Hong-Mei Yan a, b, Xin-Xia Chang a, b, Lin-Shan Zhang a, b, Huan-Dong Lin a, b, Xi-Qi Hu c, Xin Gao a, b, * a b c
Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China Fudan Institute for Metabolic Diseases, Shanghai, China Department of Pathology, Medical College, Fudan University, Shanghai, China
a r t i c l e i n f o
s u m m a r y
Article history: Received 17 January 2017 Accepted 21 June 2017
Background & aims: Non-alcoholic fatty liver disease (NAFLD) is a common and strong risk factor for cardiovascular disease and hepatocellular carcinoma. The rapid acceleration of the increase in NAFLD prevalence has exceeded the trends observed for obesity, and has been driven by multiple factors. The aim of this study was to investigate the correlation between the serum levels of folic acid, the endogenous source of methyl groups for DNA methylation, and NAFLD in Chinese adults. Methods: The correlations between the serum folic acid levels and NAFLD were investigated in two independent cohorts of 70 subjects who underwent a liver biopsy and 130 subjects with varying liver fat contents, as measured using proton magnetic resonance spectroscopy (1H-MRS). Independent correlations between serum folic acid levels and liver steatosis grades were detected using a multivariate ordinal regression analysis. The diagnostic performances of serum folic acid levels alone and in combination with existing NAFLD prediction scores were compared with those of traditional NAFLD prediction parameters using receiver operating characteristic (ROC) curve analyses. Results: Serum folic acid concentrations were inversely correlated with liver histological steatosis grades (r ¼ 0.371, P < 0.001) and the 1H-MRS-measured liver fat content (r ¼ 0.199, P ¼ 0.038). According to the multivariate ordinal regression analysis, serum folic acid levels were inversely correlated with liver steatosis grades (OR 0.739 [0.594e0.918], P ¼ 0.006) independent of age, gender, BMI, components of metabolic syndrome and the serum TC, LDL-c and HOMA-IR levels. The AUROC of serum folic acid for the diagnosis of NAFLD was 0.75 (0.65e0.83), and the addition of serum folic acid to NAFLD prediction scores significantly improved the diagnostic prediction of NAFLD (AUROC ¼ 0.88 [0.81e0.94]). Conclusion: Low serum folic acid levels were identified as an independent risk factor for NAFLD in the Chinese population. The addition of the serum folic acid levels to the current existing NAFLD prediction scores significantly improved the prediction of NAFLD. © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Folic acid NAFLD Liver biopsy Proton magnetic resonance spectroscopy
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease worldwide, with an estimated prevalence of 20e30% [1]. As the hepatic manifestation of metabolic syndrome [2], NAFLD increases the risks of cardiovascular disease and type 2 diabetes [3,4] and can progress to
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* Corresponding author. Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China. Fax: þ86 21 64037269. E-mail address: [email protected] (X. Gao). 1 These authors contributed equally to this work.
advanced liver disease, cirrhosis and hepatocellular carcinoma if it is accompanied by different degrees of liver inflammation and fibrosis [5,6]. Strikingly, the prevalence of NAFLD is still currently increasing, particularly in adolescents [7], despite the relative stability of the obesity epidemic from 2003e2004 to 2013e2014 [8,9]. The accelerated growth of the NAFLD prevalence over time has been driven by obesity and multiple factors related to NAFLD [10]. Based on emerging evidence, epigenetic mechanisms influence the development of NAFLD [11], and DNA methylation is the most intensively studied epigenetic mechanism that modulates susceptibility to NAFLD [12]. Methylome studies have observed pronounced global DNA hypomethylation and aberrant DNA
http://dx.doi.org/10.1016/j.clnu.2017.06.021 0261-5614/© 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Abbreviations NAFLD non-alcoholic fatty liver disease 1 H-MRS proton magnetic resonance spectroscopy NAS non-alcoholic fatty liver disease activity score BMI body mass index TC total cholesterol HDL high-density lipoprotein TG triglycerides ALT alanine amino transaminase AST aspartate amino transaminase LDL low-density lipoprotein HOMA-IR homeostatic model assessment of insulin resistance ROC receiver operating characteristic AdoMet S-adenosylmethionine AdoHcy S-adenosylmethionine PC phophatidylcholine VLDL very low-density lipoprotein AMPK 50 AMP-activated protein kinase
methylation at specific gene promoter regions in subjects with steatosis and NASH [13]. Therefore, the biological methylation function in the liver is important for the maintenance of hepatic lipid metabolism [14]. Folic acid, also called vitamin B9, is the endogenous source of methyl groups for hepatic DNA methylation, and may play an important role in the liver lipid metabolism [15]. Animal studies have shown that folate deficiency affects hepatic lipid storage and metabolism resulting in the development of NAFLD [16,17] and liver injury [18], and dietary supplementation with folic acid increases the DNA methylation status and reduces liver fat content in a mouse model [19,20]. In humans, the reported associations between serum folic acid levels and NAFLD are inconsistent and appear to depend on the potential influences of confounding factors, such as gender, age, ethnicity, and body weight [21e24]. As the largest ethnic group in the world, the Han Chinese are more prone to fat accumulation in the liver [25]. Despite an unparalleled 50% increase in the overall prevalence of obesity in China over the past decade [26], the prevalence of NAFLD doubled during that same period [27]. As the serum folic acid levels are deficient in approximately 20% of the Chinese adults [28], the serum folic acid level and the mechanism of DNA methylation should play an indispensable role in the pathogenesis of NAFLD in Chinese adults. However, the association between serum folic acid levels and the severity of NAFLD in the Chinese population has not previously been investigated. In the present study, we (1) analysed the relationship between serum folic acid levels and the severity of hepatic steatosis in 70 hospitalized subjects who were suspected to have NAFLD and 130 diabetic patients with varying liver fat content, as measured using proton magnetic resonance spectroscopy (1H-MRS), and (2) assessed the diagnostic performance of serum folic acid levels in predicting NAFLD and its clinical use in combination with other known NAFLD risk factors. 2. Materials and methods 2.1. Subjects We conducted this cross-sectional study with two cohorts from the Inpatient Department of Endocrinology and Metabolism,
Zhongshan Hospital (Shanghai, China). Seventy-one patients (34 men and 37 women) who initially enrolled in the study underwent a liver biopsy examination. All patients were diagnosed with fatty liver by ultrasonography, were clinically considered to have NAFLD, and had risks for steatohepatitis. Liver biopsies were performed in patients with metabolic syndrome (n ¼ 63) or chronic elevation of transaminase levels (n ¼ 8) [29]. After excluding one patient with pathological autoimmune hepatitis, 70 patients (33 men and 37 women) were included in the analysis. Another group of 130 eligible Chinese diabetic subjects with varying degrees of NAFLD, as determined by 1H-MRS were also enrolled. The patients included in the study were selected based on the following criteria: (1) no known acute or chronic disease with the exception of obesity or T2DM, based on a medical history, physical examination and laboratory tests conducted during hospitalization (including blood cell counts, serum creatinine, thyroid hormone, and concentrations of electrolytes, hepatitis B and C antibody and autoimmune hepatitis antibody levels); (2) no excessive alcohol consumption (140 g per week for men and 70 g per week for women) [30]; and (3) no use of hepatic protectants, hepatotoxic agents, folic acid or vitamin B12 supplements in recent years. The clinical characteristics of the two groups of participants are shown in Table 1. The study was approved by the Research Ethics Committee of Zhongshan Hospital, Fudan University, and all participants provided informed written consent prior to participating in the study. 2.2. Histological examination of the liver tissue A liver biopsy was performed by trained operators using an ultrasound-guided 1.6-mm-diameter needle. All liver tissue samples were examined by an experienced pathologist (Hu XQ) who was blinded to the study design. The NAS score (non-alcoholic fatty liver disease activity score) was used to assess hepatic histology based on a previously described standardized grading system [31], which included steatosis (scale of 0e3), lobular inflammation (scale of 0e3), hepatocellular ballooning (scale of 0e2) and fibrosis (scale of 0e4). Steatosis was graded as S0 (0e5%), S1 (6e33%), S2 (34e66%), or S3 (67e100%).
Table 1 Baseline characteristics of the study population. Diagnostic method for NAFLD
Number Age, year Gender (M/F) BMI, kg/m2 Waist, cm SBP, mmHg DBP, mmHg FBG, mmol/L TG, mmol/L TC, mmol/L HDL-c, mmol/L LDL-c, mmol/L ALT, U/L AST, U/L HOMA-IR Folic acid, ng/ml NAFLD n(%)
Liver biopsy
1
70 46.4 ± 14.6 33/37 28.4 ± 5.0 95.3 ± 11.9 128.5 ± 13.5 79.7 ± 9.2 6.1 ± 1.8 1.8(1.3e2.4) 4.6 ± 1.1 1.1 ± 0.3 2.6(2.0e3.1) 70(38e96) 38(25e56) 3.4(2.2e5.7) 10.2 ± 3.6 62(88.6%)
130 51.6 ± 14.8 69/61 26.7 ± 4.4 94.0 ± 10.5 131.3 ± 16.7 80.8 ± 9.5 7.0 ± 2.5 1.8(1.2e2.4) 4.5 ± 1.1 1.0 ± 0.2 2.6(2.0e3.1) 36(16e77) 26(17e42) 3.6(2.2e6.3) 10.3 ± 3.9 113(86.9%)
H-MRS
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TG, triglycerides; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine amino transaminase; AST, aspartate amino transaminase; HOMA-IR, Homeostatic model assessment of insulin resistance.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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2.3.
1
H-MRS measurements
1 H-MRS measurements were performed with a 1.5-T magnetic resonance scanner manufactured by Siemens (Erlangen, Germany). Prior to 1H-MRS measurements, T1-weighted sagittal, coronal, and axial images covering the entire liver were acquired for the localization of the spectroscopy acquisition voxel. A single 8-cm [3] (2 2 2-cm) voxel was placed within the right lobe avoiding large vessels, bile ducts, and the gall bladder. The proton spectrum was acquired after shimming over the volume of interest through a point-resolved spectroscopy (PRESS) sequence with the following parameters: repetition time ¼ 1500 ms and echo time ¼ 135 ms. The intensities of the peaks resonating from the protons of water at 4.8 ppm (Sw) and protons of methylene groups in the fatty acid chains at 1.4 ppm (Sf) were measured, and the hepatic fat percentage was calculated as a ratio of the signal from the methylene group to the total signal of methylene and water [32]. NAFLD was defined as a liver fat content of 5.56% [33].
2.4. Other measurements The body height and weight were measured when subjects were standing barefoot and wearing light indoor clothing, and these values were used for calculating the body mass index (BMI). The waist circumference was measured at the midpoint between the lower rib margin and the iliac crest while the subjects were in the standing position. Resting blood pressure was measured three times, and the mean value was used for the analysis. Blood samples were obtained after an at least 10-h fast. The total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and triglyceride (TG) concentrations were measured using the oxidase method, and the liver enzymes (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were measured using the UV lactate and malate dehydrogenase methods with an automated bio-analyser (Model 7600, Hitachi, Tokyo, Japan). The diagnostic kits used for the ALT and AST measurements were purchased from Donlim KELONG (Shanghai, China), the kits used for TG and TC measurements were obtained from Roche, and the kit used for HDLcholesterol measurement was purchased from Kyowa Medex. Low-density lipoprotein (LDL) cholesterol levels were calculated using the Friedewald equation, and the fasting serum glucose concentration was measured using the glucose oxidase method. The fasting insulin, folate, vitamin B12 and homocysteine concentrations were determined using an electrochemiluminescence immunoassay. The coefficients of variation for the folate, vitamin B12 and homocysteine concentrations were 4.8%, 4.0% and 5.0%, respectively. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated by dividing the product of the fasting blood glucose concentration (mmol/L) and fasting insulin concentration (mU/L) by 22.5.
3
hyperglycaemia treatment, or a previous diagnosis of T2DM. Dyslipidaemia was defined as the use of an antihyperlipidaemic drug treatment, triglycerides levels 1.7 mmol/L, HDL cholesterol (HDLc) levels <1.03 mmol/L, cholesterol levels 5.18 mmol/L or LDL cholesterol (LDL-c) levels 3.37 mmol/L [35]. 2.6. Statistical analysis All statistical analyses were performed using SPSS software version 15.0 (SPSS, Chicago, IL, USA). The data are shown as the means ± standard deviations (SDs), except for the skewed variables, which are presented as the medians with the interquartile ranges (25e75%) in parentheses. All subjects with liver histological examinations were divided into three subgroups according to serum folic acid tertiles. The continuous data were compared among groups using one-way analysis of variance or the ManneWhitney U-test, whereas the categorical variables were compared using either Chi-squared or Fisher's exact tests where appropriate. The univariate correlations between serum folic acid levels and grades of liver histological steatosis, inflammation, ballooning and fibrosis were analysed using Spearman's correlation analysis, and multivariate ordinal regression models were used to examine the independent correlations between serum folic acid levels and liver steatosis grades, after gradually adjusting for age, gender, BMI, waist circumference, SBP, TG levels, HDL-c levels, FBG levels and HOMA-IR. Receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic performance of serum folic acid levels in the diagnosing of NAFLD in the Chinese population and to compare its accuracy with the BMI, waist circumference, NAFLD liver fat score [36], and the Chinese NAFLD score [25]. A twotailed P-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Baseline characteristics of the study population Seventy subjects (33 males and 37 females) aged 18e70 years with a BMI of 19.0e40.3 kg/m2 were enrolled at the Department of Endocrinology, Zhongshan Hospital. Liver histological examinations showed liver steatosis grades of S0, S1, S2, and S3 in 8, 19, 33 and 10 of the subjects, respectively, according to the NAS score. The baseline characteristics of these subjects are shown in Table 2. Lower folic acid level was more commonly observed in younger males, and was accompanied with significantly higher values for BMI, waist circumference, and serum TG levels and lower vitamin B12 levels than subjects with higher folic acid levels after adjusting for age and gender (Table 2). The second group of 130 patients with diabetes and varying degrees of NAFLD as measured by 1H-MRS had an average BMI of 26.7 kg/m2 and a serum ALT concentration of 36U/L (Table 1).
2.5. Diagnosis of metabolic syndrome Metabolic syndrome was diagnosed using the Asian Guidelines of National Cholesterol Education Program Adult Treatment Panel III (ATPIII) (2005) for diagnosing metabolic syndrome [34]. Patients were diagnosed with metabolic syndrome, when they presented with at least three of the following characteristics: (1) waist circumference (WC) 90 cm (men) or 80 cm (women); (2) serum TG levels >1.7 mmol/L or receiving treatment for serum triglycerides; (3) serum HDL cholesterol levels <1.04 mmol/L (men) or <1.29 mmol/L (women); (4) systolic blood pressure (SBP)/diastolic blood pressure (DBP) 130/85 mmHg, use of antihypertensive treatments, or a previous diagnosis of hypertension; and (5) fasting blood glucose (FBG) levels 5.6 mmol/L, use of
3.2. Correlation between serum folic acid levels and histological changes in the liver A univariate correlation analysis showed that serum folic acid levels were significantly negatively associated with changes in liver histological steatosis (r ¼ 0.371, P < 0.001) and ballooning (r ¼ 0.304, P ¼ 0.010), but not associated with liver inflammation or fibrosis grades (Fig. 1). Subjects with lower serum folic acid levels had more severe liver histological steatosis and hepatocellular ballooning than subjects with higher folic acid levels (all P < 0.05, Table 3), but no significant differences were found in the proportion of NASH or severity of liver lobular inflammation or fibrosis among the subjects with varying serum folic acid levels (Table 3).
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Table 2 Baseline characteristics of the subjects who underwent liver histological examination.
Q1
Folic acid (ng/ml)
Number Age, year Gender (M/F) BMI, kg/m2 Waist circumference, cm SBP, mmHg DBP, mmHg FBG, mmol/L TG, mmol/L TC, mmol/L HDL-c, mmol/L LDL-c, mmol/L ALT, U/L AST, U/L HOMA-IR Hcy, umol/L VitB12, pmol/L
<8.2
8.2e11.8
11.8
22 37.5 ± 12.0 16/6 28.8 ± 5.5 98.6 ± 12.6 129.0 ± 13.9 81.4 ± 11.2 5.7 ± 1.2 2.2(1.6e2.9) 4.7 ± 1.2 1.0 ± 0.3 2.5 ± 0.8 83(47e95) 38(27e59) 3.5(2.8e5.8) 11.7(10.2e14.1) 540 ± 186
23 47.6 ± 13.5 10/13 28.8 ± 5.0 97.3 ± 9.8 129.4 ± 14.8 80.0 ± 9.1 6.7 ± 2.2 1.8(1.3e2.1)* 4.6 ± 1.1 1.0 ± 0.2 2.7 ± 1.1 63(30e84) 37(20e54) 3.4(2.2e5.7) 11.3(9.2e13.7) 546 ± 234
25 53.0 ± 14.2 7/18 25.9 ± 4.8* 89.6 ± 11.7* 127.3 ± 12.5 77.9 ± 7.3 6.0 ± 1.8 1.6(1.0e2.0)* 4.6 ± 1.1 1.1 ± 0.3 2.8 ± 1.0 65(30e94) 38(24e57) 3.0(1.8e5.2) 9.6(7.9e12.2) 684 ± 219*
Unadjusted p value
Adjusted p valuea
0.001 0.008 0.088 0.031 0.851 0.431 0.151 0.032 0.910 0.231 0.609 0.612 0.889 0.672 0.032 0.041
e e 0.091 0.071 0.566 0.333 0.214 0.013 0.544 0.402 0.894 0.303 0.516 0.893 0.258 0.085
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TG, triglycerides; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine amino transaminase; AST, aspartate amino transaminase; HOMA-IR, Homeostatic model assessment of insulin resistance. *P < 0.05, compared with subjects with folic acid<8.2 ng/ml after adjustment for age and gender. a Adjusted for age and gender.
Additionally, serum vitamin B12 and homocysteine levels were not associated with histological steatosis in liver (Supplementary Fig. 1). Multiple regression analysis indicated that serum folic acid levels were significantly correlated with liver steatosis grades after adjusting for age, gender and BMI (odds ratio (OR) 0.844 [95% confidence interval (CI): 0.720e0.990], P ¼ 0.037). Parameter
estimates of the correlation between serum folic acid levels and liver steatosis grades remained significant after successively adjusting for components of metabolic syndrome, including waist circumference, SBP, TG, TC, HDL-c, LDL-c and FBG levels, as well as after the addition of HOMA-IR (OR 0.739 [95% CI: 0.594e0.918], P ¼ 0.006) (Table 4).
Fig. 1. Relationships between serum folic acid levels and histological liver steatosis (upper left panel), ballooning (upper right panel), inflammation (lower left panel) and fibrosis grades (lower right panel). Serum folic acid levels are inversely correlated with liver steatosis (r ¼ 0.371, P < 0.001) and ballooning grades (r ¼ 0.304, P ¼ 0.010), but are not correlated with liver inflammation or fibrosis.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Table 3 Histologic features of the subjects with different serum folic acid levels.
Q5
Folic acid (ng/ml)
Steatosis grade [n(%)] <5% 5e33% 34e66% >66% Lobular inflammation [n(%)] No inflammatory foci <2 under 20 2e4 under 20 >4 under 20 Hepatocellular ballooning [n(%)] None Mild More than mild Fibrosis stagea [n(%)] None Mild to moderate, zone 3, perisinusoidal, or portal/periportal only Perisinusoidal and portal/periportal fibrosis Bridging fibrosis Cirrhosis NASH diagnosis [n(%)] No NASH Suspicious/borderline Definite
p value
<8.2 (N ¼ 22)
8.2e11.8 (N ¼ 23)
11.8 (N ¼ 25)
2(9.1%) 5(13.6%) 13(59.1%) 4(18.2%)
1(4.3%) 3(21.7%) 12(52.2%) 5(21.7%)
5(20.0%) 11(44.0%) 8(32.0%) 1(4.0%)
2(9.1%) 17(77.3%) 2(9.1%) 1(4.5%)
2(8.7%) 14(60.9%) 6(26.1%) 1(4.3%)
6(24.0%) 9(36.0%) 6(24.0%) 4(16.0%)
2(9.1%) 1(4.5%) 19(86.4%)
1(4.3%) 6(26.1%) 16(69.6%)
6(24.0%) 7(28.0%) 12(48.0%)
4(18.2%) 7(31.8%)
6(26.1%) 7(30.4%)
7(29.2%) 5(20.8%)
10(45.5%) 1(4.5%) 0(0.0%)
6(26.1%) 2(8.7%) 2(8.7%)
8(33.3%) 2(8.3%) 2(8.3%)
2(9.1%) 2(9.1%) 18(81.8%)
1(4.3%) 6(26.1%) 16(69.6%)
7(28.0%) 5(20.0%) 13(52.0%)
0.043
0.057
0.029
0.857
0.114
*P values from Fisher's exact test. a 1 subject was not scored for fibrosis. Table 4 Multivariate-adjusted associations between serum folic acid and liver steatosis grades by ordinal regression analyses. Parameters adjusted in the model
Folic acid Folic acid, gender and age Folic acid, gender, age, and BMI Folic acid, gender, age, BMI, and WC Folic acid, gender, age, BMI, WC, and SBP Folic acid, gender, age, BMI, WC, SBP, TG, and HDL-c Folic acid, gender, age, BMI, WC, SBP, TG, HDL-c, FBG, TC, LDL-c, and HOMA-IR
Liver steatosis grades OR (95% CI)
p-value
0.804(0.705e0.918) 0.809(0.692e0.946) 0.844(0.720e0.990) 0.816(0.680e0.979) 0.807(0.670e0.972) 0.812(0.670e0.984)
0.001 0.008 0.037 0.029 0.024 0.034
0.739(0.594e0.918)
0.006
two independent cohorts of Chinese subjects with high risks of NAFLD. The effect of serum folic acid levels on liver steatosis was independent of the traditional risk factors for NAFLD, including BMI, waist circumference and components of metabolic syndrome, and the addition of the serum folic acid concentrations to the existing NAFLD prediction scores significantly improved the clinical prediction of NAFLD in a Chinese population. Serum folic acid serves as a catalytic substrate for the transfer of one-carbon units and is responsible for the maintenance of the cellular S-adenosylmethionine (AdoMet) and S-adenosylmethionine (AdoHcy) concentrations, which are required for posttranslational methylation of DNA [37,38]. According to previous
3.3. The relationship between serum folic acid levels and the liver fat content measured using 1H-MRS The liver fat content of subjects in the second group was measured using 1H-MRS, and was found to be negatively correlated with the serum folic acid level (r ¼ 0.199, P ¼ 0.038, Fig. 2). The area under the ROC curve (AUROC) of serum folic acid was 0.75 (0.65e0.83) for the diagnosis of NAFLD, similar to the AUROC values observed for BMI, waist circumference and the NAFLD liver fat score (Fig. 3). Among all clinical parameters and NAFLD prediction scores, the Chinese NAFLD score presented the best accuracy for the diagnosis of NAFLD (AUROC ¼ 0.81 [0.72e0.88]). The addition of the information regarding serum folic acid levels to the Chinese NAFLD score increased the AUROC to 0.88 (0.81e0.94), revealing a significant improvement in the diagnostic prediction of NAFLD (P ¼ 0.028, compared with the Chinese NAFLD score). 4. Discussion The present study revealed inverse correlations between serum folic acid levels and histological and imaginal measures of NAFLD in
Fig. 2. Relationship between serum folic acid levels and liver fat content measured using 1H-MRS in a validation group of 130 Chinese subjects (r ¼ 0.199, P ¼ 0.038).
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Fig. 3. ROC analysis of the ability of the BMI, waist circumference, existing NAFLD prediction scores (NAFLD liver fat score and Chinese NAFLD score), and serum folic acid levels to predict of NAFLD. The AUROCs of serum folic acid levels, NAFLD liver fat score and Chinese NAFLD score for the diagnosis of NAFLD were 0.75 (0.65e0.82), 0.75 (0.65e0.82) and 0.81 (0.72e0.88), respectively. The combination of serum folic acid levels with the Chinese NAFLD score further improved the diagnostic performance (AUROC 0.88 [0.81e0.94]).
human epidemiological studies, folate deficiency is correlated with a higher body fat mass [39,40], an impaired lipid metabolism [41] and strong increases in the risk and incidence of cardiovascular disease [42e44]. This study also provides the first evidence and validation of a strong relationship between low serum folic acid levels and liver steatosis in a Chinese population, consistent with results previously obtained in European [21] and Mexican populations [22]. Numerous experimental studies have reported a connection between folic acid and liver steatosis. Administration of a folatedeficient diet alone induces hepatic steatosis in mice [45]. A folate-deficient diet can reduce the methylation capacity of the liver and induces liver steatosis through the following three possible mechanisms [15]: (1) a reduction in de novo phophatidylcholine (PC) synthesis, which induces an increase in liver TG synthesis and a decrease in very low-density lipoprotein (VLDL) secretion [46,47], (2) the inhibition of carnitine synthesis, which affects the transport of long-chain fatty acids into the mitochondria for b-oxidation [48,49]; and (3) the induction of the expression of genes involved in hepatic lipid synthesis, as detected using a microarray analysis [50]. Moreover, folic acid supplementation during the administration of a high-fat diet was recently shown to attenuate liver steatosis through the transcriptional regulation of hepatic oxidative stress-related genes [51] and 50 AMP-activated protein kinase (AMPK) activation [52]. A simple prediction score consisting of major risk factors for NAFLD may help identify patients with an increased risk of developing NAFLD in a clinical setting. Our previous study established and validated a Chinese NAFLD score based on the BMI, serum insulin, ALT, and AST levels and the presence of metabolic syndrome and T2DM [25], revealing that it presents good accuracy in
predicting NAFLD (Fig. 3). The strong association between serum folic acid levels and NAFLD obtained in our current study supports the role of serum folic acid levels as an important but neglected component of the NAFLD prediction score, and the addition of the serum folic acid level to the NAFLD prediction score improved the accuracy of NAFLD diagnosis by nearly 8% (AUROC 0.884 [0.807e0.938]). Thus, the use of serum folic acid levels will significantly improve the diagnosis of NAFLD if combined with the existing prediction scores. One major limitation of the current study is that the association between serum folic acid levels and hepatic steatosis was investigated in Chinese adults with high risks of NAFLD, therefore, further investigations of the relationship between serum folic acid levels and liver histological steatosis grades in the general Chinese population are still needed. Another limitation of our study is the lack of information regarding dietary folate intake. Serum folate levels are determined by its intake, absorption, metabolism and excretion and reflects the folate status of the body. Therefore, the relation between dietary folate intake and NAFLD still needs clarification. We concluded that serum folic acid levels are inversely correlated with liver steatosis grades in the Chinese population. The addition of the serum folic acid level to NAFLD prediction scores significantly improved the diagnostic performances of the scores. Dietary folate supplementation might prevent or attenuate liver steatosis in patients with NAFLD, but evidence from additional intervention studies is required to confirm this hypothesis. Authors' contributions XG and HB designed the study, MX, HB, XZ, HY, XC, LZ, HL performed the research, MX, HB, XZ collected and analysed the data, XH conducted the liver histology examination, MX drafted the article, and XG revised the manuscript. All authors approved the submitted manuscript. Conflict of interest None of the authors have conflicts of interest to declare. Acknowledgement This work was supported by the National Key Research Program of China [grant number 2012CB524906 to X. Gao], the Municipal Science and Technology Committee [grant numbers 16411954800 to X. Gao and 13441900303 to H. Bian], the Shanghai Health and Family Planning Commission Foundation [grant numbers 15GWZK0801 to X. Gao and H.D Lin, and 20164Y0029 to MF. Xia], the National Natural Science Foundation of China [grant numbers 81471073 to H. Bian and 81300682 to MF. Xia], and the Foundation for Distinguished Scholars from Zhongshan Hospital, Fudan University [grant numbers 2015ZSYXGG15 to H. Bian and 2015ZSYXQN20 to MF. Xia]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.clnu.2017.06.021. References [1] Younossi ZM, Stepanova M, Afendy M, et al. Changes in the prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 2008. Clin Gastroenterol Hepatol 2011;9:524e30. [2] de Alwis NM, Day CP. Non-alcoholic fatty liver disease: the mist gradually clears. J Hepatol 2008;48:S104e12.
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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Original article
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Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults Ming-Feng Xia a, b, 1, Hua Bian a, b, 1, Xiao-Peng Zhu a, b, Hong-Mei Yan a, b, Xin-Xia Chang a, b, Lin-Shan Zhang a, b, Huan-Dong Lin a, b, Xi-Qi Hu c, Xin Gao a, b, * a b c
Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China Fudan Institute for Metabolic Diseases, Shanghai, China Department of Pathology, Medical College, Fudan University, Shanghai, China
a r t i c l e i n f o
s u m m a r y
Article history: Received 17 January 2017 Accepted 21 June 2017
Background & aims: Non-alcoholic fatty liver disease (NAFLD) is a common and strong risk factor for cardiovascular disease and hepatocellular carcinoma. The rapid acceleration of the increase in NAFLD prevalence has exceeded the trends observed for obesity, and has been driven by multiple factors. The aim of this study was to investigate the correlation between the serum levels of folic acid, the endogenous source of methyl groups for DNA methylation, and NAFLD in Chinese adults. Methods: The correlations between the serum folic acid levels and NAFLD were investigated in two independent cohorts of 70 subjects who underwent a liver biopsy and 130 subjects with varying liver fat contents, as measured using proton magnetic resonance spectroscopy (1H-MRS). Independent correlations between serum folic acid levels and liver steatosis grades were detected using a multivariate ordinal regression analysis. The diagnostic performances of serum folic acid levels alone and in combination with existing NAFLD prediction scores were compared with those of traditional NAFLD prediction parameters using receiver operating characteristic (ROC) curve analyses. Results: Serum folic acid concentrations were inversely correlated with liver histological steatosis grades (r ¼ 0.371, P < 0.001) and the 1H-MRS-measured liver fat content (r ¼ 0.199, P ¼ 0.038). According to the multivariate ordinal regression analysis, serum folic acid levels were inversely correlated with liver steatosis grades (OR 0.739 [0.594e0.918], P ¼ 0.006) independent of age, gender, BMI, components of metabolic syndrome and the serum TC, LDL-c and HOMA-IR levels. The AUROC of serum folic acid for the diagnosis of NAFLD was 0.75 (0.65e0.83), and the addition of serum folic acid to NAFLD prediction scores significantly improved the diagnostic prediction of NAFLD (AUROC ¼ 0.88 [0.81e0.94]). Conclusion: Low serum folic acid levels were identified as an independent risk factor for NAFLD in the Chinese population. The addition of the serum folic acid levels to the current existing NAFLD prediction scores significantly improved the prediction of NAFLD. © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Folic acid NAFLD Liver biopsy Proton magnetic resonance spectroscopy
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease worldwide, with an estimated prevalence of 20e30% [1]. As the hepatic manifestation of metabolic syndrome [2], NAFLD increases the risks of cardiovascular disease and type 2 diabetes [3,4] and can progress to
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* Corresponding author. Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China. Fax: þ86 21 64037269. E-mail address: [email protected] (X. Gao). 1 These authors contributed equally to this work.
advanced liver disease, cirrhosis and hepatocellular carcinoma if it is accompanied by different degrees of liver inflammation and fibrosis [5,6]. Strikingly, the prevalence of NAFLD is still currently increasing, particularly in adolescents [7], despite the relative stability of the obesity epidemic from 2003e2004 to 2013e2014 [8,9]. The accelerated growth of the NAFLD prevalence over time has been driven by obesity and multiple factors related to NAFLD [10]. Based on emerging evidence, epigenetic mechanisms influence the development of NAFLD [11], and DNA methylation is the most intensively studied epigenetic mechanism that modulates susceptibility to NAFLD [12]. Methylome studies have observed pronounced global DNA hypomethylation and aberrant DNA
http://dx.doi.org/10.1016/j.clnu.2017.06.021 0261-5614/© 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
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Abbreviations NAFLD non-alcoholic fatty liver disease 1 H-MRS proton magnetic resonance spectroscopy NAS non-alcoholic fatty liver disease activity score BMI body mass index TC total cholesterol HDL high-density lipoprotein TG triglycerides ALT alanine amino transaminase AST aspartate amino transaminase LDL low-density lipoprotein HOMA-IR homeostatic model assessment of insulin resistance ROC receiver operating characteristic AdoMet S-adenosylmethionine AdoHcy S-adenosylmethionine PC phophatidylcholine VLDL very low-density lipoprotein AMPK 50 AMP-activated protein kinase
methylation at specific gene promoter regions in subjects with steatosis and NASH [13]. Therefore, the biological methylation function in the liver is important for the maintenance of hepatic lipid metabolism [14]. Folic acid, also called vitamin B9, is the endogenous source of methyl groups for hepatic DNA methylation, and may play an important role in the liver lipid metabolism [15]. Animal studies have shown that folate deficiency affects hepatic lipid storage and metabolism resulting in the development of NAFLD [16,17] and liver injury [18], and dietary supplementation with folic acid increases the DNA methylation status and reduces liver fat content in a mouse model [19,20]. In humans, the reported associations between serum folic acid levels and NAFLD are inconsistent and appear to depend on the potential influences of confounding factors, such as gender, age, ethnicity, and body weight [21e24]. As the largest ethnic group in the world, the Han Chinese are more prone to fat accumulation in the liver [25]. Despite an unparalleled 50% increase in the overall prevalence of obesity in China over the past decade [26], the prevalence of NAFLD doubled during that same period [27]. As the serum folic acid levels are deficient in approximately 20% of the Chinese adults [28], the serum folic acid level and the mechanism of DNA methylation should play an indispensable role in the pathogenesis of NAFLD in Chinese adults. However, the association between serum folic acid levels and the severity of NAFLD in the Chinese population has not previously been investigated. In the present study, we (1) analysed the relationship between serum folic acid levels and the severity of hepatic steatosis in 70 hospitalized subjects who were suspected to have NAFLD and 130 diabetic patients with varying liver fat content, as measured using proton magnetic resonance spectroscopy (1H-MRS), and (2) assessed the diagnostic performance of serum folic acid levels in predicting NAFLD and its clinical use in combination with other known NAFLD risk factors. 2. Materials and methods 2.1. Subjects We conducted this cross-sectional study with two cohorts from the Inpatient Department of Endocrinology and Metabolism,
Zhongshan Hospital (Shanghai, China). Seventy-one patients (34 men and 37 women) who initially enrolled in the study underwent a liver biopsy examination. All patients were diagnosed with fatty liver by ultrasonography, were clinically considered to have NAFLD, and had risks for steatohepatitis. Liver biopsies were performed in patients with metabolic syndrome (n ¼ 63) or chronic elevation of transaminase levels (n ¼ 8) [29]. After excluding one patient with pathological autoimmune hepatitis, 70 patients (33 men and 37 women) were included in the analysis. Another group of 130 eligible Chinese diabetic subjects with varying degrees of NAFLD, as determined by 1H-MRS were also enrolled. The patients included in the study were selected based on the following criteria: (1) no known acute or chronic disease with the exception of obesity or T2DM, based on a medical history, physical examination and laboratory tests conducted during hospitalization (including blood cell counts, serum creatinine, thyroid hormone, and concentrations of electrolytes, hepatitis B and C antibody and autoimmune hepatitis antibody levels); (2) no excessive alcohol consumption (140 g per week for men and 70 g per week for women) [30]; and (3) no use of hepatic protectants, hepatotoxic agents, folic acid or vitamin B12 supplements in recent years. The clinical characteristics of the two groups of participants are shown in Table 1. The study was approved by the Research Ethics Committee of Zhongshan Hospital, Fudan University, and all participants provided informed written consent prior to participating in the study. 2.2. Histological examination of the liver tissue A liver biopsy was performed by trained operators using an ultrasound-guided 1.6-mm-diameter needle. All liver tissue samples were examined by an experienced pathologist (Hu XQ) who was blinded to the study design. The NAS score (non-alcoholic fatty liver disease activity score) was used to assess hepatic histology based on a previously described standardized grading system [31], which included steatosis (scale of 0e3), lobular inflammation (scale of 0e3), hepatocellular ballooning (scale of 0e2) and fibrosis (scale of 0e4). Steatosis was graded as S0 (0e5%), S1 (6e33%), S2 (34e66%), or S3 (67e100%).
Table 1 Baseline characteristics of the study population. Diagnostic method for NAFLD
Number Age, year Gender (M/F) BMI, kg/m2 Waist, cm SBP, mmHg DBP, mmHg FBG, mmol/L TG, mmol/L TC, mmol/L HDL-c, mmol/L LDL-c, mmol/L ALT, U/L AST, U/L HOMA-IR Folic acid, ng/ml NAFLD n(%)
Liver biopsy
1
70 46.4 ± 14.6 33/37 28.4 ± 5.0 95.3 ± 11.9 128.5 ± 13.5 79.7 ± 9.2 6.1 ± 1.8 1.8(1.3e2.4) 4.6 ± 1.1 1.1 ± 0.3 2.6(2.0e3.1) 70(38e96) 38(25e56) 3.4(2.2e5.7) 10.2 ± 3.6 62(88.6%)
130 51.6 ± 14.8 69/61 26.7 ± 4.4 94.0 ± 10.5 131.3 ± 16.7 80.8 ± 9.5 7.0 ± 2.5 1.8(1.2e2.4) 4.5 ± 1.1 1.0 ± 0.2 2.6(2.0e3.1) 36(16e77) 26(17e42) 3.6(2.2e6.3) 10.3 ± 3.9 113(86.9%)
H-MRS
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TG, triglycerides; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine amino transaminase; AST, aspartate amino transaminase; HOMA-IR, Homeostatic model assessment of insulin resistance.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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2.3.
1
H-MRS measurements
1 H-MRS measurements were performed with a 1.5-T magnetic resonance scanner manufactured by Siemens (Erlangen, Germany). Prior to 1H-MRS measurements, T1-weighted sagittal, coronal, and axial images covering the entire liver were acquired for the localization of the spectroscopy acquisition voxel. A single 8-cm [3] (2 2 2-cm) voxel was placed within the right lobe avoiding large vessels, bile ducts, and the gall bladder. The proton spectrum was acquired after shimming over the volume of interest through a point-resolved spectroscopy (PRESS) sequence with the following parameters: repetition time ¼ 1500 ms and echo time ¼ 135 ms. The intensities of the peaks resonating from the protons of water at 4.8 ppm (Sw) and protons of methylene groups in the fatty acid chains at 1.4 ppm (Sf) were measured, and the hepatic fat percentage was calculated as a ratio of the signal from the methylene group to the total signal of methylene and water [32]. NAFLD was defined as a liver fat content of 5.56% [33].
2.4. Other measurements The body height and weight were measured when subjects were standing barefoot and wearing light indoor clothing, and these values were used for calculating the body mass index (BMI). The waist circumference was measured at the midpoint between the lower rib margin and the iliac crest while the subjects were in the standing position. Resting blood pressure was measured three times, and the mean value was used for the analysis. Blood samples were obtained after an at least 10-h fast. The total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and triglyceride (TG) concentrations were measured using the oxidase method, and the liver enzymes (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were measured using the UV lactate and malate dehydrogenase methods with an automated bio-analyser (Model 7600, Hitachi, Tokyo, Japan). The diagnostic kits used for the ALT and AST measurements were purchased from Donlim KELONG (Shanghai, China), the kits used for TG and TC measurements were obtained from Roche, and the kit used for HDLcholesterol measurement was purchased from Kyowa Medex. Low-density lipoprotein (LDL) cholesterol levels were calculated using the Friedewald equation, and the fasting serum glucose concentration was measured using the glucose oxidase method. The fasting insulin, folate, vitamin B12 and homocysteine concentrations were determined using an electrochemiluminescence immunoassay. The coefficients of variation for the folate, vitamin B12 and homocysteine concentrations were 4.8%, 4.0% and 5.0%, respectively. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated by dividing the product of the fasting blood glucose concentration (mmol/L) and fasting insulin concentration (mU/L) by 22.5.
3
hyperglycaemia treatment, or a previous diagnosis of T2DM. Dyslipidaemia was defined as the use of an antihyperlipidaemic drug treatment, triglycerides levels 1.7 mmol/L, HDL cholesterol (HDLc) levels <1.03 mmol/L, cholesterol levels 5.18 mmol/L or LDL cholesterol (LDL-c) levels 3.37 mmol/L [35]. 2.6. Statistical analysis All statistical analyses were performed using SPSS software version 15.0 (SPSS, Chicago, IL, USA). The data are shown as the means ± standard deviations (SDs), except for the skewed variables, which are presented as the medians with the interquartile ranges (25e75%) in parentheses. All subjects with liver histological examinations were divided into three subgroups according to serum folic acid tertiles. The continuous data were compared among groups using one-way analysis of variance or the ManneWhitney U-test, whereas the categorical variables were compared using either Chi-squared or Fisher's exact tests where appropriate. The univariate correlations between serum folic acid levels and grades of liver histological steatosis, inflammation, ballooning and fibrosis were analysed using Spearman's correlation analysis, and multivariate ordinal regression models were used to examine the independent correlations between serum folic acid levels and liver steatosis grades, after gradually adjusting for age, gender, BMI, waist circumference, SBP, TG levels, HDL-c levels, FBG levels and HOMA-IR. Receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic performance of serum folic acid levels in the diagnosing of NAFLD in the Chinese population and to compare its accuracy with the BMI, waist circumference, NAFLD liver fat score [36], and the Chinese NAFLD score [25]. A twotailed P-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Baseline characteristics of the study population Seventy subjects (33 males and 37 females) aged 18e70 years with a BMI of 19.0e40.3 kg/m2 were enrolled at the Department of Endocrinology, Zhongshan Hospital. Liver histological examinations showed liver steatosis grades of S0, S1, S2, and S3 in 8, 19, 33 and 10 of the subjects, respectively, according to the NAS score. The baseline characteristics of these subjects are shown in Table 2. Lower folic acid level was more commonly observed in younger males, and was accompanied with significantly higher values for BMI, waist circumference, and serum TG levels and lower vitamin B12 levels than subjects with higher folic acid levels after adjusting for age and gender (Table 2). The second group of 130 patients with diabetes and varying degrees of NAFLD as measured by 1H-MRS had an average BMI of 26.7 kg/m2 and a serum ALT concentration of 36U/L (Table 1).
2.5. Diagnosis of metabolic syndrome Metabolic syndrome was diagnosed using the Asian Guidelines of National Cholesterol Education Program Adult Treatment Panel III (ATPIII) (2005) for diagnosing metabolic syndrome [34]. Patients were diagnosed with metabolic syndrome, when they presented with at least three of the following characteristics: (1) waist circumference (WC) 90 cm (men) or 80 cm (women); (2) serum TG levels >1.7 mmol/L or receiving treatment for serum triglycerides; (3) serum HDL cholesterol levels <1.04 mmol/L (men) or <1.29 mmol/L (women); (4) systolic blood pressure (SBP)/diastolic blood pressure (DBP) 130/85 mmHg, use of antihypertensive treatments, or a previous diagnosis of hypertension; and (5) fasting blood glucose (FBG) levels 5.6 mmol/L, use of
3.2. Correlation between serum folic acid levels and histological changes in the liver A univariate correlation analysis showed that serum folic acid levels were significantly negatively associated with changes in liver histological steatosis (r ¼ 0.371, P < 0.001) and ballooning (r ¼ 0.304, P ¼ 0.010), but not associated with liver inflammation or fibrosis grades (Fig. 1). Subjects with lower serum folic acid levels had more severe liver histological steatosis and hepatocellular ballooning than subjects with higher folic acid levels (all P < 0.05, Table 3), but no significant differences were found in the proportion of NASH or severity of liver lobular inflammation or fibrosis among the subjects with varying serum folic acid levels (Table 3).
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Table 2 Baseline characteristics of the subjects who underwent liver histological examination.
Q1
Folic acid (ng/ml)
Number Age, year Gender (M/F) BMI, kg/m2 Waist circumference, cm SBP, mmHg DBP, mmHg FBG, mmol/L TG, mmol/L TC, mmol/L HDL-c, mmol/L LDL-c, mmol/L ALT, U/L AST, U/L HOMA-IR Hcy, umol/L VitB12, pmol/L
<8.2
8.2e11.8
11.8
22 37.5 ± 12.0 16/6 28.8 ± 5.5 98.6 ± 12.6 129.0 ± 13.9 81.4 ± 11.2 5.7 ± 1.2 2.2(1.6e2.9) 4.7 ± 1.2 1.0 ± 0.3 2.5 ± 0.8 83(47e95) 38(27e59) 3.5(2.8e5.8) 11.7(10.2e14.1) 540 ± 186
23 47.6 ± 13.5 10/13 28.8 ± 5.0 97.3 ± 9.8 129.4 ± 14.8 80.0 ± 9.1 6.7 ± 2.2 1.8(1.3e2.1)* 4.6 ± 1.1 1.0 ± 0.2 2.7 ± 1.1 63(30e84) 37(20e54) 3.4(2.2e5.7) 11.3(9.2e13.7) 546 ± 234
25 53.0 ± 14.2 7/18 25.9 ± 4.8* 89.6 ± 11.7* 127.3 ± 12.5 77.9 ± 7.3 6.0 ± 1.8 1.6(1.0e2.0)* 4.6 ± 1.1 1.1 ± 0.3 2.8 ± 1.0 65(30e94) 38(24e57) 3.0(1.8e5.2) 9.6(7.9e12.2) 684 ± 219*
Unadjusted p value
Adjusted p valuea
0.001 0.008 0.088 0.031 0.851 0.431 0.151 0.032 0.910 0.231 0.609 0.612 0.889 0.672 0.032 0.041
e e 0.091 0.071 0.566 0.333 0.214 0.013 0.544 0.402 0.894 0.303 0.516 0.893 0.258 0.085
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TG, triglycerides; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine amino transaminase; AST, aspartate amino transaminase; HOMA-IR, Homeostatic model assessment of insulin resistance. *P < 0.05, compared with subjects with folic acid<8.2 ng/ml after adjustment for age and gender. a Adjusted for age and gender.
Additionally, serum vitamin B12 and homocysteine levels were not associated with histological steatosis in liver (Supplementary Fig. 1). Multiple regression analysis indicated that serum folic acid levels were significantly correlated with liver steatosis grades after adjusting for age, gender and BMI (odds ratio (OR) 0.844 [95% confidence interval (CI): 0.720e0.990], P ¼ 0.037). Parameter
estimates of the correlation between serum folic acid levels and liver steatosis grades remained significant after successively adjusting for components of metabolic syndrome, including waist circumference, SBP, TG, TC, HDL-c, LDL-c and FBG levels, as well as after the addition of HOMA-IR (OR 0.739 [95% CI: 0.594e0.918], P ¼ 0.006) (Table 4).
Fig. 1. Relationships between serum folic acid levels and histological liver steatosis (upper left panel), ballooning (upper right panel), inflammation (lower left panel) and fibrosis grades (lower right panel). Serum folic acid levels are inversely correlated with liver steatosis (r ¼ 0.371, P < 0.001) and ballooning grades (r ¼ 0.304, P ¼ 0.010), but are not correlated with liver inflammation or fibrosis.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Table 3 Histologic features of the subjects with different serum folic acid levels.
Q5
Folic acid (ng/ml)
Steatosis grade [n(%)] <5% 5e33% 34e66% >66% Lobular inflammation [n(%)] No inflammatory foci <2 under 20 2e4 under 20 >4 under 20 Hepatocellular ballooning [n(%)] None Mild More than mild Fibrosis stagea [n(%)] None Mild to moderate, zone 3, perisinusoidal, or portal/periportal only Perisinusoidal and portal/periportal fibrosis Bridging fibrosis Cirrhosis NASH diagnosis [n(%)] No NASH Suspicious/borderline Definite
p value
<8.2 (N ¼ 22)
8.2e11.8 (N ¼ 23)
11.8 (N ¼ 25)
2(9.1%) 5(13.6%) 13(59.1%) 4(18.2%)
1(4.3%) 3(21.7%) 12(52.2%) 5(21.7%)
5(20.0%) 11(44.0%) 8(32.0%) 1(4.0%)
2(9.1%) 17(77.3%) 2(9.1%) 1(4.5%)
2(8.7%) 14(60.9%) 6(26.1%) 1(4.3%)
6(24.0%) 9(36.0%) 6(24.0%) 4(16.0%)
2(9.1%) 1(4.5%) 19(86.4%)
1(4.3%) 6(26.1%) 16(69.6%)
6(24.0%) 7(28.0%) 12(48.0%)
4(18.2%) 7(31.8%)
6(26.1%) 7(30.4%)
7(29.2%) 5(20.8%)
10(45.5%) 1(4.5%) 0(0.0%)
6(26.1%) 2(8.7%) 2(8.7%)
8(33.3%) 2(8.3%) 2(8.3%)
2(9.1%) 2(9.1%) 18(81.8%)
1(4.3%) 6(26.1%) 16(69.6%)
7(28.0%) 5(20.0%) 13(52.0%)
0.043
0.057
0.029
0.857
0.114
*P values from Fisher's exact test. a 1 subject was not scored for fibrosis. Table 4 Multivariate-adjusted associations between serum folic acid and liver steatosis grades by ordinal regression analyses. Parameters adjusted in the model
Folic acid Folic acid, gender and age Folic acid, gender, age, and BMI Folic acid, gender, age, BMI, and WC Folic acid, gender, age, BMI, WC, and SBP Folic acid, gender, age, BMI, WC, SBP, TG, and HDL-c Folic acid, gender, age, BMI, WC, SBP, TG, HDL-c, FBG, TC, LDL-c, and HOMA-IR
Liver steatosis grades OR (95% CI)
p-value
0.804(0.705e0.918) 0.809(0.692e0.946) 0.844(0.720e0.990) 0.816(0.680e0.979) 0.807(0.670e0.972) 0.812(0.670e0.984)
0.001 0.008 0.037 0.029 0.024 0.034
0.739(0.594e0.918)
0.006
two independent cohorts of Chinese subjects with high risks of NAFLD. The effect of serum folic acid levels on liver steatosis was independent of the traditional risk factors for NAFLD, including BMI, waist circumference and components of metabolic syndrome, and the addition of the serum folic acid concentrations to the existing NAFLD prediction scores significantly improved the clinical prediction of NAFLD in a Chinese population. Serum folic acid serves as a catalytic substrate for the transfer of one-carbon units and is responsible for the maintenance of the cellular S-adenosylmethionine (AdoMet) and S-adenosylmethionine (AdoHcy) concentrations, which are required for posttranslational methylation of DNA [37,38]. According to previous
3.3. The relationship between serum folic acid levels and the liver fat content measured using 1H-MRS The liver fat content of subjects in the second group was measured using 1H-MRS, and was found to be negatively correlated with the serum folic acid level (r ¼ 0.199, P ¼ 0.038, Fig. 2). The area under the ROC curve (AUROC) of serum folic acid was 0.75 (0.65e0.83) for the diagnosis of NAFLD, similar to the AUROC values observed for BMI, waist circumference and the NAFLD liver fat score (Fig. 3). Among all clinical parameters and NAFLD prediction scores, the Chinese NAFLD score presented the best accuracy for the diagnosis of NAFLD (AUROC ¼ 0.81 [0.72e0.88]). The addition of the information regarding serum folic acid levels to the Chinese NAFLD score increased the AUROC to 0.88 (0.81e0.94), revealing a significant improvement in the diagnostic prediction of NAFLD (P ¼ 0.028, compared with the Chinese NAFLD score). 4. Discussion The present study revealed inverse correlations between serum folic acid levels and histological and imaginal measures of NAFLD in
Fig. 2. Relationship between serum folic acid levels and liver fat content measured using 1H-MRS in a validation group of 130 Chinese subjects (r ¼ 0.199, P ¼ 0.038).
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Fig. 3. ROC analysis of the ability of the BMI, waist circumference, existing NAFLD prediction scores (NAFLD liver fat score and Chinese NAFLD score), and serum folic acid levels to predict of NAFLD. The AUROCs of serum folic acid levels, NAFLD liver fat score and Chinese NAFLD score for the diagnosis of NAFLD were 0.75 (0.65e0.82), 0.75 (0.65e0.82) and 0.81 (0.72e0.88), respectively. The combination of serum folic acid levels with the Chinese NAFLD score further improved the diagnostic performance (AUROC 0.88 [0.81e0.94]).
human epidemiological studies, folate deficiency is correlated with a higher body fat mass [39,40], an impaired lipid metabolism [41] and strong increases in the risk and incidence of cardiovascular disease [42e44]. This study also provides the first evidence and validation of a strong relationship between low serum folic acid levels and liver steatosis in a Chinese population, consistent with results previously obtained in European [21] and Mexican populations [22]. Numerous experimental studies have reported a connection between folic acid and liver steatosis. Administration of a folatedeficient diet alone induces hepatic steatosis in mice [45]. A folate-deficient diet can reduce the methylation capacity of the liver and induces liver steatosis through the following three possible mechanisms [15]: (1) a reduction in de novo phophatidylcholine (PC) synthesis, which induces an increase in liver TG synthesis and a decrease in very low-density lipoprotein (VLDL) secretion [46,47], (2) the inhibition of carnitine synthesis, which affects the transport of long-chain fatty acids into the mitochondria for b-oxidation [48,49]; and (3) the induction of the expression of genes involved in hepatic lipid synthesis, as detected using a microarray analysis [50]. Moreover, folic acid supplementation during the administration of a high-fat diet was recently shown to attenuate liver steatosis through the transcriptional regulation of hepatic oxidative stress-related genes [51] and 50 AMP-activated protein kinase (AMPK) activation [52]. A simple prediction score consisting of major risk factors for NAFLD may help identify patients with an increased risk of developing NAFLD in a clinical setting. Our previous study established and validated a Chinese NAFLD score based on the BMI, serum insulin, ALT, and AST levels and the presence of metabolic syndrome and T2DM [25], revealing that it presents good accuracy in
predicting NAFLD (Fig. 3). The strong association between serum folic acid levels and NAFLD obtained in our current study supports the role of serum folic acid levels as an important but neglected component of the NAFLD prediction score, and the addition of the serum folic acid level to the NAFLD prediction score improved the accuracy of NAFLD diagnosis by nearly 8% (AUROC 0.884 [0.807e0.938]). Thus, the use of serum folic acid levels will significantly improve the diagnosis of NAFLD if combined with the existing prediction scores. One major limitation of the current study is that the association between serum folic acid levels and hepatic steatosis was investigated in Chinese adults with high risks of NAFLD, therefore, further investigations of the relationship between serum folic acid levels and liver histological steatosis grades in the general Chinese population are still needed. Another limitation of our study is the lack of information regarding dietary folate intake. Serum folate levels are determined by its intake, absorption, metabolism and excretion and reflects the folate status of the body. Therefore, the relation between dietary folate intake and NAFLD still needs clarification. We concluded that serum folic acid levels are inversely correlated with liver steatosis grades in the Chinese population. The addition of the serum folic acid level to NAFLD prediction scores significantly improved the diagnostic performances of the scores. Dietary folate supplementation might prevent or attenuate liver steatosis in patients with NAFLD, but evidence from additional intervention studies is required to confirm this hypothesis. Authors' contributions XG and HB designed the study, MX, HB, XZ, HY, XC, LZ, HL performed the research, MX, HB, XZ collected and analysed the data, XH conducted the liver histology examination, MX drafted the article, and XG revised the manuscript. All authors approved the submitted manuscript. Conflict of interest None of the authors have conflicts of interest to declare. Acknowledgement This work was supported by the National Key Research Program of China [grant number 2012CB524906 to X. Gao], the Municipal Science and Technology Committee [grant numbers 16411954800 to X. Gao and 13441900303 to H. Bian], the Shanghai Health and Family Planning Commission Foundation [grant numbers 15GWZK0801 to X. Gao and H.D Lin, and 20164Y0029 to MF. Xia], the National Natural Science Foundation of China [grant numbers 81471073 to H. Bian and 81300682 to MF. Xia], and the Foundation for Distinguished Scholars from Zhongshan Hospital, Fudan University [grant numbers 2015ZSYXGG15 to H. Bian and 2015ZSYXQN20 to MF. Xia]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.clnu.2017.06.021. References [1] Younossi ZM, Stepanova M, Afendy M, et al. Changes in the prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 2008. Clin Gastroenterol Hepatol 2011;9:524e30. [2] de Alwis NM, Day CP. Non-alcoholic fatty liver disease: the mist gradually clears. J Hepatol 2008;48:S104e12.
Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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Please cite this article in press as: Xia M-F, et al., Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults, Clinical Nutrition (2017), http://dx.doi.org/10.1016/j.clnu.2017.06.021
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