Secretory phospholipase A₂ activity is linked to hypercholesterolemia and gender in non-alcoholic fatty liver disease individuals

Introduction

Non-alcoholic fatty liver disease (NAFLD) is closely linked to accumulation of lipid droplets in hepatocytes [1]. A consequence of lipid accumulation in the liver is the autocatalytic process of lipid peroxidation, demonstrated by elevation of plasma lipid peroxidation markers – 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODEs) and isoprostane 8-epi-PGF_2α III. Both were si­gnificantly elevated in patients with nonalcoholic steatohepatitis (NASH) [2].

Phospholipases A₂ are enzymes releasing free fatty acids from cytoplasmic membranes and allowing their further transformation to HODEs, hydroxyeicosatetraenoic acids (HETEs) and eicosanoids. There are two main classes of phospholipases A₂, cytoplasmic (cPLA₂) and secretory phospholipase A₂ (sPLA₂), and both regulate physiological and pathological processes [3-5]. The sPLA₂ is expressed in the atherosclerotic arterial walls, highlighting the important role of that enzyme in the development of inflammation [3, 4, 6]. The role of sPLA₂ during progression of inflammation in NAFLD is practically unknown. However, lipoprotein-associated phospholipase A₂ was recently characterized as a novel in­flammatory biomarker that is correlated with several components constituting the metabolic syndrome [7].

Aim

In this paper we look for plasma sPLA₂ activity in NAFLD patients. We also investigated the intensity of lipid peroxidation through measurement of plasma concentration of non-enzymatic oxidative stress markers:

9- and 13-HODEs, and isoprostane 8-epi-PGF_2α III.

Material and methods

Patients



Ninety-two consecutive patients with NAFLD were enrolled in the study as well as 20 healthy individuals. All analyzed subjects were Caucasians. The exclusion criterion was consumption of more than 20 g/day of ethanol. The subgroup of patients with clinically diagnosed NAFLD and permanently normal liver enzymes consisted of 30 individuals. All the patients tested negative for HBV infection (with HBs antigen and anti-HBc total antibodies) and anti-HCV antibodies. Gamma-globulins, immunoglobulins, and auto-antibodies were tested and imaging diagnostics was performed to exclude autoimmune hepatitis as well as primary biliary cirrhosis and primary sclerosing cholangitis. In a subgroup of 60 patients with biopsy-proven NAFLD, 12 patients (20%) had severe fibrosis of the liver defined as bridging fibrosis and cirrhosis (F3 and F4) and 24 (40%) had no fibrosis (F0) according to the Kleiner and Brunt classification [8]. Patients were fully informed as to the study objectives and benefits, and provided written consent prior to enrollment. The control group consisted of healthy individuals from general practitioners’ registers. The study protocol complied with ethical standards laid down in the Declaration of Helsinki and was approved by the Committee on Human Research at the Pomeranian Medical University. Venous blood for lipid analyses was collected into tubes (with EDTA) after an overnight fast. After that, the plasma was obtained by centrifugation (1200 γ × 10 min), and then frozen at –80°C until analyses.



Measurement of secretory phospholipase A₂ content in plasma



Before analysis samples were thawed slowly in an ice bath, vigorously vortexed and then equal amounts of samples were taken. The procedure was performed according to instructions of the manufacturer (Cayman, USA). Analysis was performed with En Vision 2104 Multilabel Reader (Perkin Elmer, USA).



Measurement of arachidonic and linoleic acid derivatives from plasma



Plasma 9- and 13-HODE were assessed with the HPLC method. First plasma (0.5 ml) was suspended with 1 ml of 100% acetonitrile (cooled to 4°C), then it was vortexed vigorously for 3 min. After that PGB2 (1 µg/ml) was added as an internal standard, and all was vortexed for the next 1 min. The mixture was cooled (10 min/

–20°C), centrifuged (3200 γ for 10 min, 4°C) and supernatant was taken. After that, the pH value of the samples was adjusted to 3.0 (with 1 mM HCl). The lipid

fraction was extracted with solid-phase extraction SPE columns (Bakerbond Spe, RP-18, J. T Baker). Then derivatives were eluted by a mixture of methanol and ethyl acetate (1 : 1, v/v), carefully collected and evaporated to dryness in a nitrogen steam. Residue after evaporation was reconstituted in 200 µl methanol/water/acetic acid (60/40/0.1, v/v/v) and analyzed by HPLC. The Agilent 1200 chromatographic system consisted of a vacuum solvent degassing system, quaternary gradient pump, thermostated high-performance autosampler, thermostated column compartment and diode array detector (DAD). Chromatographic data were processed by Agilent Chemstation software as described in detail previously [9]. The total recovery for all sample extraction and processing steps was 46 ±8%.



Measurements of 8-epi-PGF_2α III concentration in plasma



Lipid fractions were extracted with a Folch mixture (chloroform : methanol 2 : 1, v/v) with butylated hydroxytoluene (0.05%) as an antioxidant; the mixture was vortexed and centrifuged (3800 γ × 10 min). The lower phase was then taken and evaporated under a stream of N2. The sediment was saponified and hydrolyzed with 15% KOH and methanol (1 : 1, v/v). The mixture was vortexed and incubated at 37°C for 30 min. Then 1 mM of HCl was added and the mixture was applied to an SPE column (Bakerbond Spe, RP-18, J. T Baker).

The 8-epi-PGF_2α III was eluted from the column by a mixture of ethyl acetate : methanol (1 : 1, v/v) and samples were evaporated under N2. After that solvent was resuspended in dilution buffer, and assayed as de­scribed by the Cayman protocol. Analysis was performed with En Vision 2104 Multilabel Reader (Perkin Elmer, USA).



Statistical analysis



Statistica 7.1 software was used for the statistical analysis and all results are expressed as mean ± standard deviation. As the distribution in most cases deviated from normal (Shapiro-Wilk test), non-parametric Mann-Whitney test was used for comparisons between groups. A multiple linear regression model was used to find the independent predictors of sPLA₂ plasma concentration in NAFLD patients. Variables with distribution different from normal were transformed logarithmically before the multivariate analysis. Value of p below < 0.05 was considered significant.

Results

Selected patients’ data are summarized in Table I. The difference in content of sPLA₂ (among NAFLD individuals) between the genders nearly reached statistical significance (p = 0.054) (Table II). Additionally, among NAFLD patients multivariate analysis (adjusted for patient age, body mass index and gender as well as some biochemical data) showed that independent factors associated with higher sPLA₂ activity were higher total cholesterol concentration in plasma and (with borderline significance) female gender (Table III).

We did not find higher HODE concentration in plasma of the NAFLD subjects in comparison to healthy controls (Table IV), whereas concentration of isoprostane 8-epi-PGF_2α III was higher in NAFLD patients compared to healthy individuals (p = 0.00028).

Discussion

In this paper we have presented the results of evaluation of the inflammatory aspect of NAFLD pathogenesis.

We found an interesting correlation between quantity of sPLA₂ and male gender, and total plasma cholesterol concentration. In our previous study we found that male gender was associated with a lower level of severe fibrosis in the Polish NAFLD group [10]. It seems to be more interesting, although it is not certain, whether the higher activity of sPLA₂ observed in women was the result of more advanced age and dyslipidemia. On the other hand, it cannot be excluded that intensification

of inflammatory processes (observed in women after menopause) may occur due to up-regulation of inflammatory enzymes, e.g. sPLA₂). The sPLA₂ is one of the main factors associated with progression of inflammatory disease (e.g. cardiovascular disease) and the serum level of lipoprotein-associated phospholipase A₂ was found to be elevated in NAFLD [7]. The enzyme plays a role by the activation of low-density lipoprotein (LDL) oxygenation and recruitment of macrophages to the atherosclerotic plaque [6]. Therefore a positive correlation between total cholesterol in plasma and activity of sPLA₂ is not surprising. The increase in cholesterol concentration is probably associated with the ongoing inflammatory process, in the development of which sPLA₂ is involved [6]. Based on the results of our study, we could support the thesis of Simonen et al. published recently [11]. Enhanced cholesterol synthesis leads in hepatocytes to excess free cholesterol, which is toxic to cells and could contribute to the pathogenesis of NAFLD and its progression to steatohepatitis [11]. Perhaps also lipids could be a bridge between inflammation in the liver and in the circulation. Fatty acids released by sPLA₂ are substrates for enzymes as well as free radical pro­cesses. Our study showed that the radical peroxidation product isoprostane 8-epi-PGF_2α III is increased in plasma of NAFLD patients.

Acknowledgments

This paper was supported by a grant from the State Committee for Scientific Research, 2006-2009, No. N 40209931/3037.

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