Association of Lipoprotein-Associated Phospholipase A2 with the Prevalence of Nonalcoholic Fatty Liver Disease: A Result from the APAC Study

Nonalcoholic fatty liver disease (NAFLD) is a worldwide chronic liver disease. Few studies have investigated the association between NAFLD and Lipoprotein-associated phospholipase A2 (Lp-PLA2), a unique enzyme correlated with oxidative stress. The aim of this study was to assess the relationship between Lp-PLA2 and NAFLD in a Chinese community-based cohort. A total of 1587 adults aged ≥40 years were enrolled in the current study. Participants underwent a standardized evaluation. The serum Lp-PLA2 concentration was measured by ELISA and NAFLD was diagnosed by ultrasonography. Multivariable logistic regression was used to assess the association between Lp-PLA2 and NAFLD. Increased Lp-PLA2 levels were significantly associated with decreased NAFLD prevalence after adjusting for other potential confounders. The adjusted ORs of NAFLD in Q2, Q3 and Q4 compared with Q1 were 0.88 (0.64–1.21), 0.71 (0.51–0.98) and 0.67 (0.48–0.95), respectively (P < 0.05). Furthermore, the adjusted ORs of moderate and heavy NAFLD in Q2, Q3 and Q4 compared to Q1 were 0.64 (0.41–1.01), 0.48 (0.29–0.80) and 0.47 (0.28–0.79), respectively (P < 0.01). In conclusions, increased Lp-PLA2 levels were independently associated with decreased NAFLD prevalence.

Nonalcoholic fatty liver disease (NAFLD), one of the most common causes of chronic liver disease, is now regarded as a manifestation of metabolic syndrome with abnormal lipid deposition in liver 1,2 . Currently, the prevalence of NAFLD approaches 30% in the general population worldwide 3 . An increasing number of studies have indicated that NAFLD is linked to increased risk of cardiovascular disease (CVD) [4][5][6] . Meanwhile, some studies have demonstrated that lipoprotein-associated phospholipase A2 (Lp-PLA 2 ), which was named firstly as platelet-activating factor acetylhydrolase (PAF-AH), is significantly associated with CVD 7,8 .
Oxidative stress is regarded as a major leading cause of NAFLD 9,10 , and the anti-oxidative property of Lp-PLA 2 has been well addressed by several studies 11,12 . However, few studies were performed to explore the relationship between Lp-PLA 2 and NAFLD occurrence. Previous studies have mainly focused on small, foreign populations 13,14 , but no studies have investigated this association specifically in a Chinese population. Therefore, we hypothesized that an association between Lp-PLA 2 and NAFLD also exists in Chinese populations. In this study, we aimed to investigate the association between serum Lp-PLA 2 levels and NAFLD occurrence in a large Chinese community.
The association between Lp-PLA 2 concentration and different NAFLD levels in was similar to the above results in Fig. 2. In model 1, the ORs (95% CI) for moderate to heavy NAFLD in Q2, Q3 and Q4 compared with Q1 were 0.71 (0.46-1.07), 0.49 (0.31-0.77) and 0.41 (0.25-0.66), respectively (P < 0.01). This relationship also persisted in models 2 and 3 even after adjusting for other potential confounders (Table 2). Our results indicated that increased Lp-PLA 2 levels are associated with a decreased prevalence of NAFLD.

Discussion
In this present, large community-based study, we first demonstrated that higher Lp-PLA 2 concentrations are associated with decreased NAFLD prevalence, and multivariate analysis revealed a significant trend over the Lp-PLA 2 quartiles and a decreasing NAFLD OR gradient. In addition, a similar association was seen between the Lp-PLA 2 quartiles and different NAFLD levels.  Our finding is inconsistent with a small sample sized study. A case-control analysis of 95 Turkish participants age around 40 years conducted by Yasar Colak revealed that Lp-PLA 2 levels are higher in NAFLD patients than in healthy controls 13 . The study of Colak is a small sample size case-control study. Some unavoidable biases   such as recall bias may exist in this type of retrospective study. Therefore, association between Lp-PLA 2 and NAFLD was explored in our cohort study with a large sample size. Moreover, NAFLD is strongly associated with cardiovascular disease, hence we excluded participants suffering from cardiovascular diseases (stroke, coronary disease, transient ischemic attack). Therefore, the study population in the present study is a cardiovascular disease-free population, which might explain the inconsistency with Colak's study. In addition, our participants were middle-aged and elder people (mean ages 61.6 ± 11.8) but subjects in Colak study were relative young. Hence, the relationship between Lp-PLA 2 and NAFLD should be further explored. However, several studies have also similar findings with us. Nozomu Kono, et al. indicated that Lp-PLA 2 was protective against ox-LDL-induced hepatic injury, which is regarded as a leading cause of NAFLD 15,16 . In addition, Stanislav, et al. demonstrated that the serum Lp-PLA 2 secretion was increased in response to endotoxin-induced hepatic damage. Increased Lp-PLA 2 levels promoted the elimination of excess oxidized phospholipids to prevent further hepatic damage 17 . Meanwhile, Grypioti further determined that Lp-PLA 2 inhibits oxidative stress-induced liver damage and promotes liver recovery through animal experiments 18 . These studies suggest that Lp-PLA 2 has a positive effect on liver tissue protection. In addition, our results supported the idea that increased Lp-PLA 2 levels are associated with decreased NAFLD prevalence.
We proposed several explanations to analyze the results in our study. First, serum Lp-PLA 2 can hydrolyze oxidized phospholipids (oxPL), a part of ox-LDL, to decrease oxidative stress which has been demonstrated as a major leading cause of NAFLD 9,11,12,19-21 . Second, Lp-PLA 2 may hydrolyze oxPL to increase paraoxonase (PON) expression, which is a beneficial enzyme for NAFLD 22,23 . Third, serum Lp-PLA 2 may bind HDL to form HDL-Lp-PLA 2 ; then, HDL-Lp-PLA 2 exerts a positive regulatory effect on inflammation and dyslipidaemia to protect against NAFLD [24][25][26][27] . In addition, several studies have revealed the serum lipid regulatory effect of Lp-PLA 2 24,25,28 , so our results that hyperlipidaemia significantly decreased with the elevation of Lp-PLA 2 level in baseline further supported our finding. However, these proposed underlying mechanisms require further study.
To the best of our knowledge, previous studies have mainly focused on basic studies, not epidemiological studies, to show evidence that Lp-PLA 2 plays a protective effect in the liver. Our study is the first epidemiological study especially indicates that Lp-PLA 2 is beneficial for NAFLD in a large Chinese population. NAFLD is a worldwide health problem, particularly in subjects with middle or old age. Currently, diagnosis of NAFLD mainly depends on imaging examination and liver biopsy, therefore exploring a new effective serum biomarker is necessary for the diagnosis and prevention of NAFLD. Our finding that Lp-PLA 2 associates with the initiation and progression of NAFLD might suggests that detection of Lp-PLA 2 is helpful for the diagnosis and prevention of NAFLD.
Nevertheless, several limitations in our study must be noted. We were unable to measure the hepatic Lp-PLA 2 expression, which is a better marker to study. Liver biopsies had the more accuracy to diagnose NAFLD than abdominal ultrasonography did, but it was with invasive property. So, abdominal ultrasonography was used in the current study for diagnosis of NAFLD. Besides, we did not measure Lp-PLA 2 activity, which is more representative, but the Lp-PLA 2 concentration is closely correlated with activity; thus, we selected concentration in our study. In addition, the present study was a cross-sectional study. These results need to be further investigated in a longitudinal design for testing the causality. Since the study is ongoing, more data are needed to further confirm the correlation between Lp-PLA 2 and NAFLD in the future.
In conclusion, we have demonstrated that Lp-PLA 2 is significantly associated with decreased NAFLD risk in a large Chinese population, and increased Lp-PLA 2 levels may be useful as an independent protector against NAFLD.

Study population.
In the current study, we included 2012 participants ≥40 years old who had their serum Lp-PLA 2 levels measured in the APAC (Asymptomatic Polyvascular Abnormalities in Community) study 29 . We further excluded the participants who had (1) no or incomplete NAFLD information (n = 36), (2) a history of cancer (n = 27), (3) excessive alcohol consumption (male ≥ 20 g/d, female ≥ 10 g/d) and a positive HbsAg test (n = 362). In total, 1587 individuals were included in this analysis (Fig. 3).
All participants underwent a standardized questionnaire survey, physical examinations and laboratory assessments conducted by 11 hospitals in the Kailuan community. The study was performed according to the Declaration of Helsinki guidelines and was approved by the Ethics Committees of Beijing Tiantan Hospital and Kailuan General Hospital. All subjects provided informed consent for participation in the study.
Lp-PLA 2 Assessment. Blood samples were drawn from the antecubital vein after an overnight fast and were collected in ethylene diamine tetraacetic acid (EDTA) tubes. The samples were stored at −80 °C for subsequent analyses after a centrifugation at least 10 min at 3000 r/min. The serum Lp-PLA 2 concentration was measured by enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer's instructions 29 . The intra-and inter-assay coefficients of variation were <8% and <10%. To minimize the measurement error, the serum levels of Lp-PLA 2 were also assessed by professional technicians in Beijing Tiantan Hospital simultaneously. The participants were classified into 4 groups according to the quartile of Lp-PLA 2 concentration. NAFLD Assessment. NAFLD examination was performed via abdominal ultrasonography by two experienced radiologists who were blinded to laboratory participants. Fatty liver was diagnosed by a high-resolution B-mode topographic ultrasound system with a 3.5 MHz probe (ACUSON × 300, Siemens, Germany). The fatty liver diagnosis was based on the presence of at least two of the following three clinical findings: (1) diffusely increased liver near field ultrasound echo ('bright liver'); (2) liver echo greater than kidney; (3) vascular blurring and the gradual attenuation of far field ultrasound echo. NAFLD was also categorized into three types: Light (5% < lipid content ≤ 10%), Moderate (10% < lipid content ≤ 25%), Heavy (25% < lipid content) 30 Assessment of demographic variables and potential covariates. Participants reported basic information by answering questionnaires including age, sex, lifestyle behaviours and medical history. Smoking was categorized as none or current smoking on the basis of the self-report. Physical activity was evaluated from subject responses, and the subjects were categorized as inactive, moderate and active. Education level was classified as illiterate or primary, middle/high school or college and above.
All participants were measured by standing in light clothing without shoes and hats. Body weight was measured to the nearest 0.1 kg with the use of calibrated platform scales, and height was measured to the nearest 0.1 cm with the use of a tape ruler. Body mass index (BMI) was calculated as weight divided by the square of height.
Diseases history mainly included hypertension, diabetes and hyperlipidaemia. After a ≥5-min resting period, blood pressure was measured with a mercury sphygmomanometer twice at 5-min intervals on the left arm while the patient was in a seated position. The mean of 2 readings was used for analysis. A systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mm Hg or a history of hypertension was defined as hypertension. Diabetes was defined as a fasting blood glucose concentration >7.0 mmol/L or a medical history of diabetes. The total cholesterol (TC) level was measured with the use of an enzymatic colorimetric method, and high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) levels were measured by direct tests (Mind Bioengineering Co. Ltd). Triglyceride (TG) and alanine aminotransferase (ALT) levels were also measured. Hyperlipidaemia was defined as a history of hyperlipidaemia, TC ≥ 5.7 mmol/L, TG ≥ 1.7 mmol/L, or the current use of lipid-lowering drugs. An autoanalyser (Hitachi 747; Hitachi) was used to analyse all plasma samples at the central laboratory of Kailuan General Hospital.

Statistical analyses.
Continuous variables are shown as the means ± standard deviation (ME ± SD) and were compared using an analysis of variance (ANOVA) or a t-test. Categorical variables are presented as frequencies (percentage) and were compared using the Chi-squared test. Multivariate logistic regression was used to evaluate the relationship between Lp-PLA2 concentration and NAFLD by calculating the odds ratio (OR) and 95% confidence interval (CI). Moreover, the association between Lp-PLA 2 and different levels of NAFLD was analysed by logistic regression. Potential confounders such as age, sex, BMI, smoking, physical activity, education level, ALT, diabetes, hypertension and hyperlipidaemia were adjusted in statistical analyses. Statistical analysis was performed using SAS software, version 9.3 (SAS Institute Inc., Cary, NC, USA). P < 0.05 was regarded as significant for 2-sided tests.