**4. Assays for Lp-PLA2 mass and activity determination**

**2. Lipoprotein-associated phospholipase A2: structure and biology**

distribution of Lp-PLA2 in various plasma lipoproteins affects its functions.

**3. Actions of lipoprotein-associated phospholipase A2**

(oxFFAs) and lysophosphatidylcholine.

114 Lipoproteins - From Bench to Bedside

Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), belongs to the phospholipase A2 superfamily [1,2]. This Ca2+ independent phospholipase is encoded by *PLA2G7* gene that consists of 12 exons and 11 introns located on chromosome 6p21.2 to 12 [3,4]. Lp-PLA2 is protein of 45,4 kDa that consists of 441 amino acid residues [5]. The major sources of Lp-PLA2 in plasma are T lymphocytes, mono‐ cytes/macrophages, activated bone marrow-derived mast cells, and liver cells [6-8]. The secreted Lp-PLA2, circulates in plasma in active form. It predominantly binds to LDLs, and in a much smaller extent to HDLs, Lp(a), lipoprotein remnants, and platelet-borne microparticles [6,9-12]. Indeed, Lp-PLA2 is highly associated with the smallest LDL and HDL subclasses [13] and with electronegative LDL, which overlaps with small dense LDL [14]. Lp-PLA2 bound to HDL has a much lower specific activity compared to when bound to LDL [10]. Different

Lp-PLA2 catalyzes hydrolysis of the acetyl group at sn-2 position of PAF to generate lyso-PAF and acetate [15]. On the other side, Lp-PLA2 cleaves oxidatively modified lipoproteins from the sn-2 position of the apoB100-containing lipoproteins into oxidized nonesterified fatty acids

We can think about Lp-PLA2 as a friend and foe at the same time. On the one hand, it functions as PAF-AH, which hydrolyzes inflammatory mediator PAF, inhibits foam cell formation,

On the other hand, Lp-PLA2 has a proatherogenic role. Lp-PLA2 relates to a number of different proatherogenic biological processes. Lp-PLA2 hydrolyzes oxidized phospholipids on modified LDL particles within the arterial intima, and thus contributes to the initiation and progression of atheroma. Lp-PLA2 is produced by macrophages and foam cells within atherosclerotic plaque. Its expression is mainly confined to plaque areas with massive lipid accumulation and leukocyte infiltration, cellular necrosis, and calcification, suggesting that Lp-PLA2 is a marker for rupture-prone plaque [16]. The amount of Lp-PLA2 and its by-product, lysophosphatidyl‐ choline in the coronary circulation is proportional to the extent of the atheroma and indirectly affects local endothelial function [17]. Proatherogenic activities of lysophosphatidylcholine are: expression of adhesion molecules; upregulation of cytokines and CD40 ligand by T-cells, cytotoxic at concentrations higher than 30–50 µM; stimulation of macrophage proliferation; release of arachidonic acid from endothelial cells; induction of MCP-1 and genes for growth factors; release of myeloperoxidase; migration of vascular smooth muscle cells; chemoattrac‐ tant for monocytes, macrophages, and T-cells; induction of apoptosis in smooth muscle cells and macrophages; involvement in the antigenicity of oxidized LDL; inhibition of endotheliumderived nitric oxide [18-20]. Furthermore, oxFFAs promote atherosclerosis by direct and

enhances cholesterol efflux in macrophages, and exerts its atheroprotective role [16].

It is well known that circulating Lp-PLA2 can be measured by different assays ascertaining either its mass or activity. There is no definite decision about the potential superiority of the tests.

According to the literature data, Lp-PLA2 levels were determined preferentially using PLAC® test. Lp-PLA2 mass was determined by diaDexus PLAC Test ELISA, sandwich enzyme immunoassay, followed by diaDexus PLAC® test based for Lp-PLA2 mass measurement on turbidimetric immunoassay technology. More recently, enzyme assay PLAC® Test for measurement of Lp-PLA2 activity has been developed and commercially available. The preferred sample type is EDTA plasma, and serum is also acceptable. It should be noted that methodological issues associated with Lp-PLA2 measurement make comparisons between studies difficult.

Recently published meta-analysis of 32 prospective studies in persons with stable vascular disease or recent acute ischemic event revealed a moderate correlation between mass and activity of Lp-PLA2 [24]. The PROVEIT-TIMI 22 study [25] has also found a moderate corre‐ lation between Lp-PLA2 activity and mass measured at baseline and at 30-day after ACS. There is still controversy about the method of estimating Lp-PLA2 level. While Koenig et al. [26] reported that Lp-PLA2 mass was the better risk predictor of future cardiovascular events than Lp-PLA2 activity, Persson et al. [27] has reached the opposite conclusion. Jenny et al. [28] showed no difference in Lp-PLA2 activity and mass with respect to risk prediction.

Currently, there is no consensus on the best method to estimate Lp-PLA2 level. A consensus panel recommendation for incorporating Lp-PLA2 testing into the cardiovascular disease risk assessment guidelines used Lp-PLA2 mass for stratifying patients [23].

Blood samples should be refrigerated after processing and should be kept frozen for long-term storage. There are no restrictions to the time of day that the sample should be drawn and no dietary restrictions. In contrast to other emerging risk markers, a very minimal biological variation in Lp-PLA2 concentrations has been demonstrated among individuals monitored serially over several weeks [29].

In addition, Lp-PLA2 levels are typically unaffected by conditions of systemic inflammation, such as osteoarthritis and chronic obstructive pulmonary disease, whereas markers of inflammatory response are often elevated by these conditions. The normal population medians for men and women are in the range of 230–250 ng/mL, and a value of >300 ng/mL may be considered elevated [30].

Gender differences in Lp-PLA2 levels were found; men had significantly higher levels than women. Also, a significant association between Lp-PLA2 levels and smoking was noticed. These finding has been observed in previous studies [31,32,33,34]. Lower Lp-PLA2 levels in women could be explained by estrogen-mediated down-regulation of Lp-PLA2 expression, due to lower concentrations of LDL cholesterol in women or estrogen-related decrease in platelet activating factor acetyl hydrolase activity [35,36]. Estrogen-replacement therapy can significantly reduce Lp-PLA2 activity in healthy postmenopausal women [37], while admin‐ istration of steroids with progesterone-like activity increases Lp-PLA2 activity [38]. Smoking may increase the carrier (LDL) and the substrate (oxidized LDL) for Lp-PLA2 [39].
