Association between serum free fatty acid levels and nonalcoholic fatty liver disease: a cross-sectional study

High serum free fatty acid (FFA) levels are associated with metabolic syndrome (MS). This study aimed to assess the association of fasting serum FFAs with nonalcoholic fatty liver disease (NAFLD) in a Chinese population. A total of 840 subjects fulfilled the diagnostic criteria of NAFLD and 331 healthy control participants were enrolled in this cross-sectional study. Fasting serum FFA levels and other clinical and laboratory parameters were measured. NAFLD patients had significantly higher serum FFA levels than controls (P < 0.001). Serum FFA levels were significantly and positively correlated with parameters of MS, inflammation indexes, and markers of hepatocellular damage. Elevated serum FFA levels were found in NAFLD subjects with individual components of MS (obesity, hypertriglyceridaemia, and hyperglycaemia). Stepwise regression showed that serum FFA levels were an independent factor predicting advanced fibrosis (FIB-4 ≥ 1.3) in NAFLD patients. Serum FFA levels correlated with NAFLD and could be used as an indicator for predicting advanced fibrosis in NAFLD patients.

analysis of the relationship between serum FFA levels and parameters of metabolic syndrome (body mass index, BMI; systolic blood pressure, SBP; triglyceride, TG; total cholesterol, TC; fasting plasma glucose, FPG), inflammatory indexes (sialic acid, SA; high-sensitivity C-reactive protein, hsCRP; white blood cells, WBC) and markers of hepatocellular damage (ALT, AST and GGT) may indirectly lead to a further understanding of serum FFA levels and NAFLD. This crosssectional study aimed to characterise the relationship between changes in serum FFA and NAFLD in a Chinese population.

Methods
Subjects. The study initially enrolled 920 patients diagnosed with fatty liver based on abdominal ultrasonography. Subjects who met the following criteria were excluded: (i) those with alcohol consumption . 140 g/week for men and . 70 g/week for women (n 5 20); (ii) those with a history of viral hepatitis (n 5 46), autoimmune hepatitis or other forms of chronic liver disease (n 5 14). The remaining 840 patients with NAFLD (mean age: 46.1 6 12.2 years; female: 239; male: 601) and 331 age-and gender-matched healthy subjects (mean age: 47.0 6 10.7 years; female: 96; male: 235) were used in the current analyses. Informed consent was obtained from all the subjects and the study was approved by the ethics committee of the first affiliated hospital of the medical college at Zhejiang university in China.
Clinical and anthropometric parameters. The clinical examinations were conducted in the morning after an overnight fast. Weight, height and blood pressure (SBP; DBP, diastolic blood pressure) were measured, and BMI was calculated as weight in kilograms divided by height in meters squared. FIB-4 index was calculated according to the following equation: Age (years) 3 AST (U/L)/PLT (10 9 /L) 3 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ALT(U=L) p 28 . Liver ultrasound examinations were performed by experienced radiologists who were unaware of the clinical and laboratory data, using a Toshiba Nemio 20 sonography machine with a 3.5-MHz probe (Toshiba, Tokyo, Japan).
Diagnostic criteria for NAFLD and MS. Hepatic steatosis was diagnosed according to the guidelines established for the diagnosis and treatment of NAFLD issued by the Fatty Liver Disease Study Group of the Chinese Liver Disease Association 29 . Specifically, hepatic steatosis was diagnosed according to characteristic echo patterns, such as diffuse hyperechogenicity of the liver relative to the kidneys, ultrasound beam attenuation, and poor visualisation of intrahepatic structures. The prevalence of MS differs widely in varying studies according to the definition criteria used and the population sample studied 30,31 . To adopt an ethnicity-specific value of waist circumference, the International Diabetes Federation (IDF) has proposed regional definitions of central obesity according to the characteristics of Chinese people. According to IDF guidelines, for a person to be defined as having MS they must have three or more of the following: central obesity (defined as a waist circumference . 90 cm for Chinese men and . 80 cm for Chinese women); BMI . 25 kg/m 2 ; raised circulating triglyceride levels (defined as triglycerides $ 1.7 mmol/L) or specific treatment for this lipid abnormality; reduced HDL-C levels (defined as HDL-C , 1.03 mmol/L in male patients and , 1.29 mmol/L in female patients); raised blood pressure (defined as SBP $ 130 mm Hg or DBP $ 85 mm Hg) or treatment for previously diagnosed hypertension; and raised FPG (defined as FPG $ 5.6 mmol/L) or previously diagnosed type 2 diabetes.
Data and statistical analysis. Statistical analyses were performed using SPSS, version 16 (SPSS, Chicago, IL, USA). Data are presented as the mean 6 standard deviation when data were found to be normally distributed or as the median if the distribution was skewed. Differences between groups were analysed using the Student's t-test or the Mann-Whitney U test. Spearman correlation analysis was used to examine the correlation between serum FFA levels and clinical and laboratory parameters. The parameters for multivariate logistic regression were selected by univariate regression analysis.
Ethics Statement. This study was approved by the Hospital Ethics Committee and was performed in accordance with the Helsinki Declaration.

Results
Characteristics of Subjects. Demographic and biochemical characteristics of study participants are summarised in Table 1. We found that patients with NAFLD had significant differences in terms of BMI, SBP, DBP, ALT, AST, GGT, CK, TG, TC, HDL-C, LDL-C, FPG, UA, HbA 1C , Hcy, Fe, SA, hsCRP, WBC, P-LIP, AMY, HGB, PLT and RDW compared with controls. Specifically, the levels of these parameters were less favourable in subjects with NAFLD compared with the controls. Consistent with our hypothesis, we found that serum FFA levels were markedly higher in patients with  . MS and inflammation are two major factors that are closely associated with NAFLD 23 . The present observation suggest that they may act as cofactors for the link between serum FFA levels and NAFLD. As shown in Figure 1, serum FFA levels were significantly higher in obese subjects (BMI . 25 kg/m 2 ) than in lean subjects (BMI # 25 kg/m 2 ). However, there was no significant difference when subjects were classified according to blood pressure. The group with higher FPG levels (FPG $ 5.6 mmol/L) showed increased serum FFA levels. Similarly, the FFA levels were increased in the higher TG group (TG $ 1.7 mmol/L) compared with the lower TG group (TG , 1.7 mmol/L).
Serum FFA levels in the group with FIB-4 $ 1.3 were significantly higher than those in the group with FIB-4 , 1.3 (median 0.72 mmol/ L vs. 0.58 mmol/L; P , 0.001). We used univariate regression to analyze the odds and p-values (shown in Table 3) with forward selection, and we subsequently considered FFA, age, gender, BMI, c-GT, ALT, hsCRP, and presence of diabetes (all p , 0.05) within the multivariate regression analysis. The results of unadjusted and adjusted multivariate logistic regression analysis models are shown in Table 4. Following adjustment for age, gender, BMI, c-GT, ALT, hsCRP, and presence of diabetes (all P , 0.05) selected by stepwise regression, showed that serum FFA levels were an independent factor predicting advanced fibrosis (FIB-4 $ 1.3) in NAFLD participants.

Discussion
This is one of the few studies performed to date addressing changes in blood biochemical parameters in a large cohort (n 5 840) of subjects of the same ethnicity (Chinese) with NAFLD. The results suggest that serum FFA levels are strongly association with NAFLD. First, NAFLD patients had increased serum FFA levels, and these levels were positively correlated with parameters of MS (BMI, TG, TC and FPG), indexes of inflammation (SA, hsCRP and WBC) and markers of hepatocellular damage (ALT, AST and GGT). Second, NAFLD subjects with diabetes had higher serum FFA levels. Third, to obtain a better understanding of the association between serum FFA levels and NAFLD, all subjects with NAFLD were classified according to components of MS. This subgroup analysis indicated elevated serum FFA levels were significantly associated with individual components of MS (obesity, hypertriglyceridaemia and hyperglycaemia). Furthermore, logistic regression analysis showed that serum FFA levels were an independent factor predicting advanced fibrosis (FIB-4 $ 1.3) in NAFLD subjects.
One of the possible explanations for the increased FFA levels in NAFLD patients is insulin resistance 32,33 . Insulin resistance is well known as a pathophysiological hallmark of NAFLD 34 . Fasting serum FFA primarily originate from lipolysis in adipose tissue. The human body mobilises stored fat by catalysis of hormone sensitive lipase, which hydrolyses the fat into glycerol and fatty acids in the fasted state 35 . Insulin induces the dephosphorylation of hormone-sensitive lipase by reducing the concentration of cyclic adenosine monophosphate (cAMP) and activating phosphoprotein phosphatase, thus inhibiting lipase activity and decreasing fat hydrolysis 36 . It has been extensively documented that insulin resistance results in an increased flux of FFA. The other possible mechanism linking serum FFA with NAFLD is the impaired ability of the liver to export or utilise FFA. Besides beta oxidation, the majority of FFA entering the hepatocyte are used for the synthesis of triglyceride, which then becomes a constituent of very low density lipoprotein (VLDL) particles that are exported into the blood 37 . It has been demonstrated that beta oxidation of FFA and export of VLDLs in the liver are impaired in NAFLD patients 38 . The elevated serum levels of FFA are therefore dependent on the imbalance between its origin and use in NAFLD.
The link between serum FFA levels and NAFLD suggested that FFA could play a pivotal role in the development of NAFLD 39 . Increased systemic oxidative stress in patients with NAFLD has long been recognised both in animal experiments and clinical studies 40,41 . FFA are responsible for development of hepatocellular apoptosis and injury, acting as a strong oxidant in several pathological states such as MS or type 2 diabetes 42,43 . High FFA levels also attenuate the insulin-mediated suppression of endogenous glucose production and gluconeogenesis, leading to the impairment of whole-body glucose tolerance 44 . High serum ALT, AST and GGT levels are associated with the more severe form of NAFLD, although low serum ALT, AST or GGT levels do not rule out the possibility of an advanced stage of NAFLD 45,46 . The correlation between FFA and markers of hepatocellular damage in the present study indirectly confirm the hypothesis that FFA significantly correlate with hepatocellular injury. Further studies are needed to clarify the detailed mechanism by which FFAs influence the progression of NAFLD.
NAFLD is a well-known risk factor for cardiovascular disease 47 . The association between FFA and NAFLD may provide a possible explanation for why NAFLD is closely related with cardiovascular disease. FFA can stimulate vascular smooth muscle proliferation and induce endothelial dysfunction, which may contribute to the cardiovascular events of NAFLD 48 . Furthermore, FFA have been implicated in the physiological mechanism of cardiovascular disease resulting from low-grade systemic inflammatory processes 49,50 . On the basis of our study, we can only propose a role of FFA in the aetiology of NAFLD and inflammation from a snapshot of the circulating FFA state. However, the question of whether or not elevated serum FFA are an unrelated phenomenon, a cause, or a consequence of NAFLD could not be determined from this cross-sectional study. Traditionally, NAFLD has been viewed as a spectrum that progresses from pure fatty liver through NASH to severe fibrosis, cirrhosis and eventually hepatocellular carcinoma 51 . According to recent views, uncomplicated steatosis and NASH tend to be considered as potentially unrelated disorders, not only based on histological definitions but also from pathophysiological evidence 52,53 . The identification of differences in serum FFA between simple steatosis and NASH in further studies may be important to clarify their precise interrelationship.
This study has some limitations. First, the diagnosis of NAFLD was based on ultrasonographic examination. Ultrasonography is not sensitive enough to detect mild steatosis, but overall it significantly correlates with both liver histology and metabolic derangements 54 . Given the acceptable sensitivity and specificity for detecting hepatic steatosis, ultrasonography is widely used in epidemiological studies of NAFLD 55 . Second, postprandial serum levels of FFA have not been examined in this study. Nevertheless, fasting serum FFA constitute the majority of fatty acids delivered to the liver and contribute to liver fat metabolism and accumulation in NAFLD. Third, we used the FIB-4 index for evaluating advanced fibrosis of NAFLD according the studies of Xun YH et al. 56 . FIB-4, a simple non-invasive index  composed of readily available routine laboratory tests, can accurately predict advanced fibrosis and cirrhosis in patients with NAFLD. However, it is not sensitive enough to identify patients with a mild degree of fibrosis who are at risk of progression 57,58 .
In conclusion, our results demonstrate a significant correlation between serum FFA levels and NAFLD. Further research on the involvement of FFA in NAFLD will not only enhance our understanding of the development of NAFLD, but also benefit in the eventual development of new prevention and treatment strategies for the disease.