Clinical application of elevated platelet-activating factor acetylhydrolase in patients with hepatitis B

Background The aim of this study was to investigate the variation of platelet-activating factor acetylhydrolase (PAF-AH) in patients with various stages of hepatitis B infection and evaluate the association between PAF-AH activity and chronic severe hepatitis B (CSHB) and mortality in patients with hepatitis B. Methods Serum PAF-AH activity was measured in 216 patients with hepatitis B and in 152 healthy controls using an automatic biochemical analysis system. Spearman correlation was used to investigate the correlation between PAF-AH activity and other biochemical indicators. The receiver operating characteristic (ROC) curve and multivariable logistic regression analysis were used to evaluate the ability of PAF-AH activity to predict CSHB and mortality in patients with hepatitis B. Results The PAF-AH activities in patients with CSHB (1320 ± 481 U/L) were significantly higher than those in healthy controls and in other hepatitis B groups (all P < 0.01). In patients with hepatitis B, PAF-AH activity correlated with total bilirubin (r = 0.633), total bile acid (r = 0.559), aspartate aminotransferase (r = 0.332), apolipoprotein B (r = 0.348), high-density lipoprotein cholesterol (r = −0.493), and apolipoprotein AI (r = −0.530). The areas under the ROC curves for the ability of PAF-AH activity to predict CSHB and mortality in patients with hepatitis B were 0.881 (95% confidence interval (CI): 0.824–0.937, P < 0.001) and 0.757 (95% CI: 0.677–0.837, P < 0.001), respectively. Multivariate logistic regression analysis showed PAF-AH activity to be an independent factor predicting CSHB with an odds ratio of 1.003 (95% CI: 1.002–1.005, P < 0.001). Conclusion Elevated PAF-AH in patients with hepatitis B was significantly associated with liver damage. Thus, serum PAF-AH could be used as a novel indicator for predicting CSHB and mortality in patients with hepatitis B. Further, PAF-AH activity was an independent factor predicting CSHB.


Introduction
Platelet-activating factor acetylhydrolase (PAF-AH) is a Ca 2+ -independent catalyst of serine-dependent phospholipid hydrolysis and belongs to the superfamily of phospholipase A2 [1]. Also known as lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), the plasma PAF-AH is a single 45-kilodalton polypeptide composed of 441 amino acids encoded by the PLA2G7 gene [2]. Because PAF-AH hydrolyzes plateletactivating factor (PAF) and oxidizes phospholipids with a modified short fatty acyl chain esterified at the sn-2 position [3], PAF-AH plays an important role in human diseases such as severe anaphylaxis [4], rheumatic diseases [5], acute respiratory distress syndrome [6], necrotizing enterocolitis [7], and atherosclerosis [8]. Previous epidemiologic studies demonstrated that increased PAF-AH activity had a prognostic value and was associated with a high risk of future coronary and cerebrovascular events [9,10]. Additionally, Kamisako et al. reported increased PAF-AH activity in patients with hyperbilirubinemic hepatobiliary disease [11]. However, to our knowledge, the role of serum PAF-AH in hepatitis B has not yet been well defined. More importantly, whether serum PAF-AH activities are associated with different disease states of hepatitis B virus (HBV) infection such as acute hepatitis B (AHB), chronic hepatitis B (CHB), and chronic severe hepatitis B (CSHB) remains unknown. Thus, in the present study, we aim to determine the activity of serum PAF-AH in patients with various stages of hepatitis B and to evaluate the association of PAF-AH with different hepatitis B disease groups and with mortality in patients with hepatitis B.

Subjects
A total of 216 hepatitis B patients, including 155 male and 61 female patients aged 13-82 (45.1 ± 13.4) years from the Department of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, China, were enrolled in our prospective study. Of these patients, 23 were diagnosed with acute hepatitis B (AHB), 67 with chronic hepatitis B (CHB), 49 with chronic severe hepatitis B (CSHB), and 77 with liver cirrhosis (LC). All patients were diagnosed according to the criteria of the 2000 Xi'an viral hepatitis management scheme [12]. The standardized diagnosis of AHB, CHB, and CSHB has been previously described in detail [13][14][15]. The model for end-stage liver disease (MELD) score, calculated from the patient's serum total bilirubin (TBIL), creatinine level, and international normalized ratio (INR) of prothrombin time, was used to quantify the severity of liver disease [16]. A total of 152 healthy control patients with HBsAg negativity and normal liver and renal function and blood lipid levels at their annual health examination at the healthcare center of The First Affiliated Hospital of Zhejiang University were also recruited. The control group comprised 102 male and 50 female patients aged 17-78 (46.0 ± 12.9) years. Patients with a concurrent infection of hepatitis C virus (HCV), hepatitis D virus, hepatitis G virus, and/or human immunodeficiency virus and any autoimmune liver disease were excluded. There were no statistically significant differences in gender and age distribution between the case and control groups (both P > 0.05).

Ethics statement
This study was approved by the Ethics Committee of the First Affiliated Hospital of College of Medicine at Zhejiang University in China and was performed in accordance with the Helsinki Declaration. All participants provided written informed consent. For participants under 18 years of age, oral informed consent was obtained from the participants, and written informed consent was signed by their parents.

Specimen collection
All specimens for blood indicators and PAF-AH activity measurements were collected by venipuncture into 5-mL drying Vacuette vacutainers (Greiner Bio-One GmbH, Kremsmunster, Austria) in the morning after a 12 h fast on the second day after admission. The samples were sent to the laboratory, and serum was isolated by centrifugation (10 min, 3000 × g) and preserved at −80°C.

Statistical analysis
Statistical analysis was performed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD, and categorical data as percentages. For continuous variables, the differences between two groups were assessed with the independent samples t-test or the Mann-Whitney U test as appropriate. Multiple comparisons were performed by one-way analysis of variance (ANOVA) or Kruskal-Wallis H tests. Categorical variables were analyzed using the chi-square test. Spearman's rank correlation test was used in correlation analysis. For the univariate and multivariate analyses to identify independent predictors, the measurements for PAF-AH activity, biochemical parameters, and hematological parameters were presented as quartile ranks, with the lowest quartile being used as the reference category. A receiver operating characteristic (ROC) curve was generated and the area under the curve (AUC) was calculated to identify the best PAF-AH activity and/or MELD score for predicting CSHB and mortality in patients with HBV infection. Stepwise regression was performed to determine factors associated with the incidence of CSHB. A multivariable logistic regression analysis was used to evaluate PAF-AH activity and the MELD score as predictors of CSHB by adjusting for gender, LDL-c, and TBIL in the model. Statistical significance was defined at P < 0.05.

Detection of PAF-AH in patients with various clinical stages of hepatitis B
In this study, 216 patients with hepatitis B and 152 healthy control patients were enrolled as study participants. The clinical characteristics of the patients are listed in Table 1. The levels of ALT, AST, TBIL, CHE, TG, Tch, LDL-c, HDL-c, apoA I, apoB, and TBA were statistically different between each of the AHB, CHB, LC, and CSHB groups and the healthy control group (all P < 0.05). The MELD score and mortality were statistically different among the four hepatitis B groups (all P < 0.05). The PAF-AH activity was 820 ± 446 U/L in the 216 patients with hepatitis B, significantly higher than that measured in healthy controls, 450 ± 125 U/L (P < 0.01). The PAF-AH activities in the AHB, CHB, LC, and CSHB groups were all significantly higher than in control patients (all P < 0.05, Table 1). Moreover, the PAF-AH activities in patients with CSHB were significantly higher than those in patients with AHB, CHB, and LC by the Mann-Whitney U test (all P < 0.001).

Association of PAF-AH with CSHB and 3-month mortality in HBV-infected patients
To explore the association of PAF-AH with CSHB and with mortality, the 216 patients with hepatitis B were divided into four groups according to their PAF-AH activity percentiles (group 1: PAF-AH >1053 U/L; group 2: 709-1053 U/L; group 3: 477-708 U/L; group 4: <477 U/L). The prevalence of CSHB was calculated by dividing the number of patients with CSHB by the total numbers of patients in each PAF-AH percentile group. The clinical characteristics and differences in measurements for variables among the four groups are listed in Table 2. Patients with the highest values of PAF-AH (group 1) had higher MELD scores, incidence of CSHB, and mortality than patients with the lowest values of PAF-AH (group 4) (20.7 ± 9.9 vs. 7.0 ± 5.5, 35/19 vs. 1/53, and 17/37 vs. 1/53, respectively; all P < 0.05). ROC curve analysis was applied to estimate the ability of PAF-AH activity and the MELD score to predict CSHB in patients with hepatitis B (Figure 2, Table 3). The AUCs of PAF-AH activity and the MELD score were 0.881 (95% confidence interval (CI): 0.824-0.937, P < 0.001) and 0.921 (95% CI: 0.879-0.962, P < 0.001), respectively. When PAF-AH and the MELD score were combined, the AUC was 0.951 (95% CI: 0.919-0.982, P < 0.001).

Discussion
PAF is a potent inflammatory lipid mediator that, by binding to a high-affinity G-protein-linked receptor, can be activated to exert diverse actions including stimulating secretion and aggregation of platelets, initiating neutrophil and macrophage chemotaxis, and inducing release of   cytokines such as interleukins, tumor necrosis factor, and proteolytic enzymes, and thus may be involved in the development of circulation disorders and inflammation [18]. PAF accumulation has been implicated in pathological processes and diseases including inflammation, endotoxin shock, acute pancreatitis, and cardiovascular disease. PAF is also involved in acute liver damage, cirrhosis, severe hepatitis, and ischemia-reperfusion-induced liver injury [19,20]. As a major mediator of PAF inactivation, PAF-AH plays a crucial role in the regulation of serum PAF levels and in reducing PAF-induced damage [8,[21][22][23]. In experimental models of acetaminophen-induced liver injury in rats, PAF activities increased significantly between 24 and 32 h after acetaminophen administration, along with increases in other biochemical indexes (ALT, AST). The PAF-AH activity peaked between 72 and 96 h after acetaminophen treatment [24], indicating that PAF plays an important role in acetaminophen-induced liver injury and subsequent liver tissue repair, while PAF-AH can increase liver recovery and reduce liver damage [25,26]. The pathological mechanism of hepatitis B is complex, and patients' symptoms are often complicated by intestinal endotoxemia, the incidence of which can reach 80%-100% in severe hepatitis cases [27]. Lipopolysaccharide (LPS) is the major chemical endotoxin capable of stimulating PAF and PAF-AH secretion from monocytes and macrophages. Kupffer cells, specialized macrophages located in the liver, account for 80%-90% of the total monocytes and macrophages in the body. Kupffer cells are the primary mediators of endotoxin clearance and detoxification in the liver. By injecting bacterial LPS into the rat mesenteric vein, Howard et al. [28] observed a 20-fold increase in the PAF-AH mRNA level in Kupffer cells and a 2-fold increase in serum PAF-AH activity after 24 h. Svetlov et al. [29] reported that cultured primary Kupffer cells could express more PAF-AH mRNA than hepatocytes and had a 20-25-fold higher PAF-AH secretion rate than hepatocytes, indicating that Kupffer cells may be the main source of PAF-AH during liver damage.
We found that the activity of circulating PAF-AH in patients diagnosed with hepatitis B was positively correlated with TBIL, TBA, ALT, AST, TG, and apoB, negatively correlated with ChE, HDL-c, and apoAI, and not correlated with Glu, BMI, Tch, and LDL-c. However, the PAF-AH activity in healthy controls was positively correlated with TBIL, ALT, TG, Tch, LDL-c, and apoB, negatively correlated with HDL-c, and not correlated with Glu, BMI, TBA, AST, and apoAI. The differences in correlations between hepatitis patients and controls, especially in the correlation of PAF-AH with lipids and bile acids, could be explained by a change in the source of PAF-AH during the development of hepatitis. PAF-AH can be classified into intracellular types I and II and the plasma type [3]. Plasma PAF-AH exists in the blood and is predominantly produced by monocytes, macrophages, T lymphocytes, mast cells, and hepatocytes [30][31][32]. Under normal conditions, the main source of circulating PAF-AH is hematopoietic cells [31], and the main source of PAF-AH in bile juice is hepatocytes [29]. However, during hepatitis bile excretion disorder, the retention of bile components such as bile acid can cause the retention of hepatocyte-secreted PAF-AH [33,34]. This relationship could explain the association between serum PAF-AH activities and the TBIL and TBA levels in hepatitis. Another potential factor is the impact of liver damage on Kupffer cell PAF-AH secretion, leading to an increase in PAF-AH during hepatitis. Circulating PAF-AH mainly exists in complexes with lipoprotein particles [35]. During hepatitis, however, liver damage causes dysfunction in the synthesis of cholesterol and other lipids [36], resulting in the altered correlation between PAF-AH and blood lipids we observed. These findings indicate that serum PAF-AH may be involved in oxidative stress and inflammation of the liver.
We also found that serum PAF-AH activities in patients with various stages of hepatitis B were significantly higher than those in healthy controls, and serum PAF-AH activity was significantly positively correlated with TBIL. The greatest elevation in serum PAF-AH was observed in patients diagnosed with CSHB, suggesting that the PAF-AH activity is involved in pathological liver damage, and that the detection of PAF-AH may serve as a surrogate marker for hepatic inflammatory activity, allowing disease progress and prognosis to be monitored. Similar to our study, the study of Ma et al. found that among CSHB patients, serum PAF activities were significantly higher in the death group than