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Journal of Lipid Research, Vol. 42, 1706-1713, October 2001
Copyright © 2001 by Lipid Research, Inc.

Methods Article

A simple assay for a human serum phospholipase A₂ that is associated with high-density lipoproteins

Correspondence to: Philip J. Thompson, To whom correspondence should be addressed., pjthomps{at}cyllene.uwa.edu.au (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Phospholipase A₂ (PLA₂) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. We have designed a simple microplate assay for human serum PLA₂ using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid. Using this substrate, serum PLA₂ activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthio-glycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA₂ activity in serum from healthy volunteers (n = 30) measured by this assay was 10.4 ± 1.6 µmol · h^-1 · ml^-1. The assay is reproducible and is suitable for the analysis of large numbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA₂ activity in normal human serum is associated with high-density lipoproteins and that serum PLA₂ activity is positively correlated with the lipoprotein parameters total triglyceride (P < 0.0001), total cholesterol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA₂ activity was calcium dependent and was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin (EC₅₀ = 0.4 mM).

The PLA₂ activity characterized here is unlikely to be due to plasma platelet-activating factor acetylhydrolase or low molecular weight His-Asp sPLA₂, and may represent a new sPLA₂ type. — Petrovic, N., C. Grove, P. E. Langton, N. L. A. Misso, and P. J. Thompson. A simple assay for a human serum phospholipase A₂ that is associated with high-density lipoproteins. J. Lipid Res. 2001. 42: 1706–1713.

Supplementary key words: enzyme, substrate, cholesterol, atherogenic

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Phospholipase A₂ (PLA₂) plays a crucial role in a number of diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction, and they also provide precursors for eicosanoid generation (1). PLA₂ comprises a large group of enzymes that share the capacity to hydrolyze fatty acids from the sn-2 position of glycerophospholipids. PLA₂ provides precursors for the generation of eicosanoids when the cleaved fatty acid is arachidonic acid and precursors for platelet-activating factor when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid or lysophospha-tidylcholine. Mammalian cells generally contain more than one PLA₂, each of which is regulated independently and exerts a distinct effect (2). PLA₂ enzymes are found in all mammalian tissues including plasma and serum.

PLA₂ enzymes are generally classified into two large groups on the basis of their localization, namely, extracellular or secreted (sPLA₂ ) (3) and intracellular or cytosolic (cPLA₂) (1). In mammalian extracellular fluids, several types of small (14 kDa), calcium-dependent sPLA₂ enzymes have been characterized (4). Although plasma sPLA₂ are physiologically important, they have not been well characterized. It has not been determined how many different PLA₂ enzymes exist in plasma and what are their respective roles in different physiological states. The studies performed to date on plasma PLA₂ vary in their conclusions partly because plasma was taken from a variety of organisms (including humans) and in different physiological states. These include samples from patients with severe inflammatory diseases (5), sepsis, rheumatoid arthritis (6), and acute pancreatitis (7) or from rats treated intravenously with heparin (8). In these reports, the presence of several different PLA₂ enzymes in plasma has been suggested. Furthermore, it was also observed that these enzymes exist in plasma as high molecular weight complexes with unidentified molecular species. Gijon et al. (9) have suggested that in patients with septic shock, plasma sPLA₂ could be physically associated with HDL.

Other recent studies also suggest that plasma lipoproteins could be the major source of PLA₂ in plasma or serum. The best-characterized lipoprotein-associated PLA₂ is the type VII enzyme found in plasma under normal physiological conditions. This PLA₂ was initially characterized as platelet-activating factor (PAF) acetylhydrolase (PAF-AH) and is also known as human plasma LDL-PLA₂ because it is associated with the LDL class of lipoproteins (10) (11). This enzyme is a single polypeptide containing a lipase consensus motif and is expressed in inflammatory cells, in particular macrophages, or in tissues infiltrated with large numbers of inflammatory cells. In addition to its PAF-AH activity, this enzyme also hydrolyzes phosphatidylcholines with either oxidized or short chain fatty acids at the sn-2 position (11). An important characteristic of this enzyme is that it has no requirement for calcium.

There may be other sPLA₂ enzymes, besides LDL-PLA₂, that are associated with lipoprotein particles. Thus, apolipoprotein B-100, an integral component of some lipoproteins (VLDL, IDL, and LDL) may itself possess PLA₂ activity (12) (13). An intrinsic PLA₂-like activity in LDL, which might be LDL-PLA₂ or a completely separate enzyme, has also been proposed (14) (15). Interestingly, human acute phase HDL and LDL are the first reported biological substrates for human group IIA PLA₂, with this activity resulting in the formation of numerous biologically active lipid products (16).

PLA₂ activity is usually assayed with expensive radioactive or chromogenic substrates that are unsuitable for assaying large numbers of patient samples in a clinical setting. The aim of this work was to design and validate a simple microplate spectrophotometric assay for serum PLA₂ using the chromogenic lipid substrate 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) and examine its relationship to serum lipoprotein parameters. The use of this substrate was originally reported by Cho and Kezdy (17), and it has been subsequently used to measure PLA₂ activity in snake venoms (18) (19) (20) and purified immobilized PLA₂ activity (21). 4N3OBA was originally used in a kinetic assay protocol to measure PLA₂ enzyme activity purified from snake venom and porcine pancreas (17). However, the assay as originally described may not be directly applicable to high-throughput screening of enzyme activity in a complex medium such as serum. Therefore, we have optimized the assay with 4N3OBA as substrate to measure PLA₂ activity in serum from healthy volunteers and have demonstrated that this PLA₂ activity is exclusively associated with HDL and is strongly correlated with serum lipoprotein parameters.

	METHODS

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Human serum collection
Blood samples were collected by forearm venepuncture from healthy volunteers who had fasted overnight. Serum was separated and stored at -70°C until assayed for PLA₂ activity. The study was approved by the Human Research Ethics Committee of the University of Western Australia, and all subjects gave written informed consent.

PLA₂ assays
4N3OBA as substrate 4N3OBA (Sigma, Castle Hill, New South Wales, Australia) was dissolved in chloroform at a concentration of 50 mg/ml. Aliquots (80 µl, 4 mg) were distributed into Eppendorf tubes and evaporated under vacuum. The dry 4N3OBA residue (4 mg per tube) was stored at -20°C. Immediately before the assay, substrate (4 mg) was resuspended in 1 ml of PLA₂ assay buffer (150 mM KCl, 10 mM CaCl₂, 50 mM Tris-HCl, pH 7.5). The suspension was vortexed vigorously for 1 min and centrifuged (2,000 g, 2 min) at room temperature, and the supernatant was used as a substrate solution (190 µl) in enzyme assays. There was no obvious micelle formation during substrate solubilization, indicating that 4N3OBA was completely soluble at the final concentration of 2 mM used in the standard assay. Dilutions of serum in a final volume of 10 µl were added to 190 µl substrate solution, and samples were incubated in microtiter plate wells at room temperature for 1 h. The absorbances at 425 and 600 nm (to correct for any turbidity in the sample) were determined on a microplate reader (Molecular Devices, Sunnyvale, CA). In experiments in which PLA₂ activity was expressed as units, one unit was defined by the following equation:

PLA₂ activity [µmol · h^-1 · ml^-1] = (A_425nm - A_600nm) [OD_425nm/h] x 0.07862 [µmol/OD_425nm] x (1/serum volume [1/ml])

The correction factor (0.07862) represents the concentration of product (µmoles in 200 µl) producing an absorbance of 1.0 at 425 nm.

1,2-Bis-heptanoylthio-glycerophosphocholine as substrate Aliquots (250 µg) of 1,2-bis-heptanoylthio-glycerophosphocholine (1,2bHGPC; Cayman Chemical, Ann Arbor, MI) supplied as a solution in methyl acetate were distributed into Eppendorf tubes and evaporated under vacuum, and the dry substrate was stored at -20°C. Immediately before the assay, substrate (250 µg) was resuspended in 190 µl of PLA₂ assay buffer. 5,5'-Dithiobis-2-nitro-benzoic acid (DTNB, Sigma) in PLA₂ assay buffer (1 mM, 40 µl) was added, and the resulting mixture was vortexed vigorously for 1 min and used as a substrate solution (190 µl) in enzyme assays (final substrate concentration 2 mM). In separate experiments we determined that DTNB did not inhibit human serum PLA₂ activity, and it was therefore included in the substrate solution for simplicity. After addition of serum dilutions, samples were incubated in microtiter plate wells at room temperature for 1 h. The absorbances at 415 and 600 nm were measured.

Effects of inhibitors on PLA₂ activity The protease inhibitor 3,4-dichloroisocoumarin (DCIC; Sigma) was dissolved in DMSO (Sigma) and stored at -20°C. In PLA₂ assays, the effects of DCIC were corrected for the effects of DMSO. A second protease inhibitor, 4-[2-aminoethyl]-benzenesulfonyl fluoride (Pefabloc; Roche Diagnostics, Castle Hill, New South Wales, Australia) was dissolved in PLA₂ assay buffer and stored at -20°C. The effects of these inhibitors on PLA₂ activity were assessed by adding 5 µl of inhibitor solution to the serum/substrate mixture (5 µl/190 µl) followed by incubation at room temperature for 1 h before measurement of the absorbances at 425 and 600 nm.

Isolation of lipoproteins
Lipoprotein classes (HDL, LDL, and VLDL) were isolated by ultracentrifugation of serum through NaBr/NaCl gradients, as previously described (22) (23). Briefly, the serum density was adjusted to 1.26 g/ml by addition of NaBr. The serum was then overlayed on several layers of NaCl and NaBr solutions of decreasing density to produce a discontinuous density gradient that by diffusion becomes a continuous gradient. Lipoprotein fractions isolated from the gradients were desalted on gel permeation PD-10 columns (Sigma) before assay for PLA₂ activity. Lipoproteins were collected at the following gradient densities: VLDL (<1.006 g/ml), LDL (1.006–1.063 g/ml), HDL (1.063–1.210 g/ml).

Fractionation of human serum by size-exclusion Superose 6 chromatography
Serum samples (0.5 ml) were applied to a column (10 ml, 7 x 1.5 cm) packed with Superose 6 gel (Amersham Pharmacia Biotech, Castle Hill, New South Wales, Australia) according to the manufacturer's instructions. The column was equilibrated and run in PLA₂ assay buffer, and chromatography was performed at a flow rate of 0.2 ml/min at room temperature with collection of 0.5-ml fractions.

Measurement of lipoprotein parameters
Total cholesterol, HDL cholesterol, and total triglyceride concentrations in human serum samples were determined at the Department of Clinical Biochemistry (PathCentre, Perth, Western Australia). The concentration of LDL cholesterol was calculated according to the following equation:

LDL cholesterol = total cholesterol - HDL cholesterol - total triglyceride/2.2

Statistical analyses
Results are expressed as means of three independent experiments, each done in triplicate. In independent measurements of PLA₂ activity using the same serum sample, the coefficient of variation did not exceed 10%. In enzyme activity measurements using different serum samples, results were expressed as mean ± standard deviation. Correlation between parameters was evaluated by linear regression. Statistical significance was set at P < 0.05.

	RESULTS

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A simple assay for the determination of serum PLA₂ activity
To determine the correlation between absorbance at 425 nm and chromophore concentration, known amounts of 4N3OBA were subjected to NaOH hydrolysis of the ester bond ( Scheme 1), causing complete conversion to the chromophore, 4-nitro-3-hydroxy-benzoic acid. The resulting absorbance at 425 nm was measured in a total volume of 0.2 ml using a microplate reader. The relationship between chromophore concentration and absorbance at 425 nm was linear ( Fig 1A). A chromo-phore concentration of 1.0 mM produced an absorbance at 425 nm of 2.54 ± 0.04 OD. This correlation factor was used to calculate the concentration of chromophore produced as a result of PLA₂ activity and thereby the unit of PLA₂ activity.

View larger version (17K): [in this window] [in a new window]   Figure 1. Optimization of the human serum PLA2 assay. A: Relationship between absorbance at 425 nm and chromophore concentration. Known concentrations of 4N3OBA were subjected to quantitative NaOH hydrolysis. B: Substrate concentration dependence of the serum PLA2 activity assay. A range of 4N3OBA concentrations were incubated with 2 µl serum for 60 min. C: Time dependence of the serum PLA2 activity assay. Different amounts of serum [0.625 µl (open circle), 1.5 µl (solid square), 2.5 µl (open triangle), 5 µl (solid triangle) and 10 µl (solid circle) were incubated with 2 mM 4N3OBA for a range of times (5–120 min). D: Serum concentration dependence of the PLA2 activity assay. A range of serum volumes (0.1–10 µl) were incubated with 2 mM 4N3OBA for 60 min. All experiments were performed in a final volume of 0.2 ml in microtiter plates, and the absorbance at 425 nm was measured on a microplate reader. Each point is the mean of three independent experiments, each performed in triplicate.

The solubility of 4N3OBA in aqueous solutions is limited. To determine its maximum solubility, known amounts of 4N3OBA were resuspended in PLA₂ reaction buffer. The suspension was centrifuged (2000 g, 2 min) to remove undissolved substrate, and the concentration of dissolved 4N3OBA was determined by quantitative hydrolysis with NaOH. The maximum solubility of 4N3OBA in PLA₂ assay buffer was 3.14 ± 0.15 mM. In subsequent assays for PLA₂ activity, 2 mM 4N3OBA was chosen as the standard substrate concentration. In a separate experiment, the reproducibility of substrate solubilization was determined by repeated resuspension of 4 mg of dry 4N3OBA in 1 ml of assay buffer, centrifugation to remove insoluble material, and measurement of the soluble substrate concentration by quantitative hydrolysis with NaOH. The mean 4N3OBA concentration was 1.93 mM (SD 0.097, coefficient of variation 5%, n = 9), indicating that interassay variation in the final substrate concentration obtained by this method was not significant. Furthermore, as shown in Fig 1B, a substrate concentration of 2 mM was sufficient to saturate PLA₂ enzyme activity under the assay conditions used.

To determine the optimum incubation time, different amounts of serum were assayed for PLA₂ activity with incubation times ranging from 5 to 120 min (Fig 1C). The PLA₂ enzyme reaction showed biphasic kinetics for all serum concentrations. An initial fast phase during the first 40 min was followed by a slower linear reaction rate for incubation times >40 min. For subsequent assays of PLA₂ activity, 60 min was chosen as the standard incubation time. One unit of serum PLA₂ activity was defined as the formation of 1 µmol of product by 1 ml of serum during 1 h of incubation at room temperature in the standard microplate assay.

To determine the optimum amount of serum in the assay, different amounts of serum were incubated for 60 min in the standard PLA₂ assay (Fig 1D). A linear relationship between serum concentration and product formation was observed when the volume of serum did not exceed 2.5 µl per 200 µl of reaction mixture and the final absorbance did not exceed 0.325 OD (Fig 1D). Therefore, in subsequent PLA₂ assays, values were accepted only if the absorbance at 425 nm did not exceed 0.325 OD. If a higher absorbance was observed, then the serum was diluted and the assay repeated.

Identification of HDL as a major source of serum PLA₂ activity
To identify the molecular characteristics of the human serum enzyme(s) exhibiting PLA₂ activity, initial gel permeation chromatography experiments were performed using Sephadex G-100. In these experiments, PLA₂ activity eluted in the column void volume without any PLA₂ activity being detected in the lower molecular weight fractions (data not shown). To further characterize the higher molecular weight species with PLA₂ activity in serum, we performed gel permeation chromatography on Superose-6 (5–5000 kDa separation range). To eliminate potential differences between individual serum samples, we used a pooled sample from 30 healthy volunteers. In these experiments, a single peak of PLA₂ activity was detected using both the artificial substrate 4N3OBA and the structurally more appropriate phospholipid substrate 1,2bHGPC ( Fig 2A). However, 4N3OBA was a more sensitive substrate for the measurement of serum PLA₂ activity than was 1,2bHGPC (Fig 2A). In separate experiments, activity assays optimized for each substrate showed that 4N3OBA was five times more sensitive for assaying PLA₂ activity in human serum samples compared with 1,2bHGPC (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Identification of HDL as the major source of human serum PLA2 activity. A: Superose-6 gel permeation chromatography of human serum was performed using pooled serum from 30 healthy volunteers. PLA2 activity in the eluted fractions was determined using 4N3OBA (solid circle) and 1,2-bis-heptanoylthio-glycerophosphocholine (1,2bHGPC) (open circle) as substrates (both at a concentration of 2 mM). Absorbance was measured at 425 nm for 4N3OBA and at 415 nm for 1,2bHGPC. The elution positions of the three lipoprotein fractions (HDL, LDL, and VLDL) are indicated (arrows). The elution positions for Blue Dextran (BD), indicating column void volume, and for bromphenol blue (BPB), indicating the low-molecular-weight end of the separation range, are also shown (arrows). Each point is the mean of three independent experiments. B: PLA2 activity in purified lipoprotein fractions. PLA2 activity was determined in purified HDL, LDL, and VLDL fractions from healthy volunteers (n = 3) and compared with total serum PLA2 activity from healthy volunteers (n = 30). The unit of PLA2 activity in lipoprotein fractions is corrected for the initial volume of serum from which these lipoproteins were isolated. Bars represent the mean (± standard deviation) of three independent experiments, each performed in triplicate.

To test the hypothesis that serum lipoproteins might be the carriers of PLA₂ activity, HDL, LDL, and VLDL were separated from normal human serum, and Superose 6 chromatography was performed on each of these fractions to determine the elution profiles of the three lipoprotein types. Only HDL eluted in a similar molecular weight range to serum PLA₂ activity, whereas LDL and VLDL eluted in the higher molecular weight range (Fig 2A).

To further substantiate the findings that PLA₂ activity might be linked with serum HDL, we determined PLA₂ activity in purified HDL, LDL, and VLDL samples from healthy volunteers (n = 3) (Fig 2B). Most of the total lipoprotein PLA₂ activity (HDL, LDL, and VLDL combined) was detected within the HDL fraction (9.848 ± 1.497 units or 94%) (Fig 2B). LDL and VLDL contained 4.9% (0.516 ± 0.128 units) and 1.1% (0.114 ± 0.042 units) of the total PLA₂ activity, respectively. The total PLA₂ activity in the HDL fractions was similar to the total serum PLA₂ activity measured in a pooled sample from 30 healthy volunteers (10.40 ± 1.56 units). These findings, taken together, suggest that most of the PLA₂ activity in normal human serum is associated with HDL.

Inhibitors of serum PLA₂ activity
To further characterize human serum PLA₂, we determined the effects of several known PLA₂ inhibitors ( Fig 3). Serum PLA₂ activity was not inhibited by manoalide or 4-bromo-phenacyl bromide, which have previously been reported to inhibit the known calcium-dependent small molecular weight PLA₂ (24) (25) (data not shown). However, the PLA₂ activity detected in our assay appears to be calcium dependent, because EDTA and EGTA inhibited this activity (Fig 3A). EGTA and EDTA inhibited PLA₂ activity in the presence of 10 mM calcium with EC₅₀ values of 5 and 11 mM, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 3. Inhibition of serum PLA2 activity. A: The effects of increasing concentrations of EDTA (solid circle) and EGTA (open circle) in assay buffer (150 mM KCl, 10 mM CaCl2, 50 mM Tris-HCl, pH 7.5) on PLA2 activity in pooled serum from healthy volunteers (n = 30). PLA2 activity is expressed as a percentage of the activity in a control sample without EDTA or EGTA, and each point is the mean of three independent experiments, each performed in triplicate. B: The effects of increasing concentrations of 3,4-dichloroisocoumarin (DCIC) (open triangle) and Pefabloc (solid triangle) on PLA2 activity in pooled serum from healthy volunteers (n = 30). PLA2 activity is expressed as a percentage of the activity in a control sample without inhibitor, and each point is the mean of three independent experiments, each performed in triplicate.

The effect of DCIC, which has been reported to inhibit lipoprotein-linked serine-dependent PLA₂ activity (11), was also determined. DCIC inhibited serum PLA₂ activity with an EC₅₀ of 0.4 mM (Fig 3B). The irreversible serine protease inhibitor Pefabloc has been reported to efficiently inhibit both human and rat PAF-AH and to totally inactivate human plasma - , VLDL-, IDL-, LDL-, and HDL-associated PAF-AH (LDL-PLA₂) (26) (27). In contrast, Pefabloc, at concentrations up to 2 mM, did not inhibit the serum PLA₂ activity measured in our assay (Fig 3B).

The possibility that the PLA₂ enzyme activity assayed in our experiments is due to a known secretory PLA₂ (type II or type V) was investigated by determining the effects of blocking polyclonal antibodies to PLA₂ IIA and PLA₂ V on serum PLA₂ activity. Neither of these antibodies had any inhibitory effect on the PLA₂ activity (data not shown).

It is unlikely that the PLA₂ activity observed in our study is due to lipoprotein lipase because 1M NaCl did not inhibit serum PLA₂ activity, whereas it has been shown to inhibit lipoprotein lipase activity (28). Furthermore, serum PLA₂ activity was not inhibited by tetrahydrolipstatin, a potent inhibitor of serine lipases (29).

Correlation between serum lipoprotein parameters and PLA₂ activity
To further assess the relationship between serum lipoproteins and PLA₂ activity, we collected fasting serum samples from 30 healthy volunteers. PLA₂ activity as well as serum lipoprotein-related parameters (total cholesterol, LDL cholesterol, HDL cholesterol, total triglyceride concentration, and atherogenic index) were determined in these samples. Atherogenic index is defined as the ratio of total cholesterol concentration to HDL concentration. Linear regression analysis showed that serum PLA₂ activity was positively correlated with total triglyceride (r² = 0.6244, P < 0.0001), total cholesterol (r² = 0.4651, P < 0.0001), and LDL (r² = 0.3213, P = 0.0011) concentrations ( Fig 4A) and with atherogenic index (r² = 0.225, P = 0.0081) (Fig 4B). Interestingly, serum PLA₂ activity did not correlate significantly with HDL cholesterol concentration (P = 0.4236).

View larger version (16K): [in this window] [in a new window]   Figure 4. Correlation between serum PLA2 activity and serum lipoprotein-related parameters. PLA2 activity, total cholesterol (solid triangle), LDL cholesterol (open circle), total triglyceride concentrations (solid circle) (A) and atherogenic index (total cholesterol/HDL) (B) were determined in serum samples from healthy volunteers (n = 30). PLA2 activity was correlated with triglyceride (r2 = 0.6244, P < 0.0001), total cholesterol (r2 = 0.4651, P < 0.0001), and LDL (r2 = 0.3213, P = 0.0011) concentrations and with atherogenic index (r2 = 0.225, P = 0.0081). Each point is the mean of three independent experiments, each performed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our findings suggest that the chromogenic substrate 4N3OBA can be successfully used to measure PLA₂ activity in human serum. Cleavage by PLA₂ of the ester bond between the octanoyl group and the chromophore 4-nitro-3-hydroxy-benzoic acid results in absorbance at 425 nm (17).

Here we describe a simple PLA₂ assay that employs 4N3OBA and microplate spectrophotometric measurement of PLA₂ activity. The assay does not require radioactive or expensive substrates and is suitable for the analysis of large numbers of patient samples in a clinical setting. PLA₂ assays performed on human serum fractions separated by Superose-6 chromatography gave similar results using 4N3OBA compared with the previously characterized chromogenic phospholipid PLA₂ substrate 1,2bHGPC (30), although 4N3OBA appears to be a more sensitive substrate.

Biochemical characterization of the molecular species exhibiting PLA₂ activity in normal human serum indicates that the majority of serum PLA₂ appears to be physically associated with HDL. Several previous reports support the conclusion that serum HDL could be the major source of sPLA₂ in normal serum. The sPLA₂ activity has been previously shown to be associated with HDL in patients with septic shock (9). Furthermore, acute phase HDL has been shown to be the primary natural substrate for type IIA sPLA₂ (16), and transgene overexpression of sPLA₂ IIA specifically increases HDL catabolism in acute and chronic inflammatory conditions (31) (32).

In spite of our findings that serum PLA₂ is found almost exclusively on HDL particles, serum PLA₂ activity did not show a significant correlation with HDL cholesterol, whereas significant correlations were observed with total cholesterol, total triglyceride, and LDL cholesterol concentrations. The observed correlation with LDL cholesterol could be a consequence of the correlation with total cholesterol. The absence of a correlation with HDL cholesterol suggests that the PLA₂ activity of HDL is not related to total HDL concentration but rather to an as yet undetermined functional or structural property of HDL particles, possibly associated with total cholesterol or triglyceride levels. Structurally and functionally, HDL are a heterogeneous population of lipoproteins, particularly during inflammation and acute phase responses. Thus, the acute phase protein serum amyloid A is also expressed during inflammation and associates with HDL, altering its physicochemical composition. Acute phase serum amyloid A, but not the constitutive form, markedly enhances sPLA₂ activity (33).

Considering the established cardioprotective role of HDL, an associated proinflammatory enzyme such as sPLA₂ could be involved in catabolism of HDL and the consequent loss of its anti-inflammatory properties. In the acute phase response and in diseases with prolonged inflammation, normal HDL is converted into acute phase HDL, which is proinflammatory and unable to protect LDL against oxidative modification (34). These changes in HDL metabolism could be responsible for the depressed HDL levels in chronic inflammatory diseases. In support of such a hypothesis, it has been reported that HDL from transgenic mice and human HDL treated with recombinant sPLA₂ failed to protect against the formation of biologically active phospholipids in LDL (35).

In addition, biologically active lipids formed as a result of PLA₂ action on serum lipoproteins may also affect cellular function. Thus, lysophosphatidylcholine, which is generated as a consequence of PLA₂ activity during LDL oxidation, is a potent chemotactic factor for monocytes (36). Enzymatic generation of lysophosphatidylcholine by sPLA₂ using plasma lipoprotein phospholipids as a substrate is essential for the sPLA₂-mediated inhibition of platelet activation (37). Incubation of LDL with purified soybean lipoxygenase, in the presence of pure PLA₂, can mimic endothelial cell - induced oxidative modification leading to enhanced recognition of LDL by macrophages (38). Our finding that serum PLA₂ activity correlated positively with atherogenic index supports the hypothesis that the HDL-associated PLA₂ characterized in this work has a proatherogenic role.

Although there is some evidence to suggest that sPLA₂ IIA may be the PLA₂ associated with HDL, the PLA₂ activity characterized in this work does not appear to belong to this type. Thus, serum PLA₂ activity was not inhibited by manoalide or 4-bromo-phenacyl bromide, which have previously been reported to inhibit the known calcium-dependent small molecular weight PLA₂ (24) (25). However, the PLA₂ detected in our assay appears to be calcium dependent, and this is one of the main biochemical characteristics of PLA₂ IIA. The previously characterized calcium-dependent sPLA₂ enzymes that have histidine- and asparagine-dependent active sites have not been reported to be inhibited by DCIC, an inhibitor of serine-dependent PLA₂ activity. However, DCIC did inhibit the serum PLA₂ activity identified in our experiments. Furthermore, blocking polyclonal antibodies to PLA₂ IIA and PLA₂ V did not inhibit the PLA₂ enzyme activity that we measured in serum. Experiments using antibodies to block the activity of an enzyme bound to a lipoprotein particle should be interpreted with caution. Lack of specificity of the antibody for the active site, or steric hindrance and inaccessibility of the active site to the antibody, may limit the ability of the antibody to block enzyme activity. Nevertheless, taken together with the data obtained with low molecular weight PLA₂ inhibitors, our findings strongly suggest that this enzyme is not a type IIA or type V PLA₂. The possibility that HDL-associated PLA₂ activity is the result of several distinct PLA₂ types within the lipoprotein particle cannot be excluded. Further studies involving purification of HDL-associated PLA₂ will be needed to specifically address that issue.

The PLA₂ activity characterized in this work is unlikely to be due to the previously characterized plasma PAF-AH or LDL-PLA₂ that were found to be calcium-independent enzymes, a characteristic not observed in our PLA₂ assays. Furthermore, the new irreversible serine protease inhibitor, Pefabloc, which efficiently inhibits both human and rat PAF-AH and totally inactivates human plasma - , VLDL-, LDL-, and HDL-associated PAF-AH (LDL-PLA₂) (26) (27) did not inhibit serum PLA₂ activity.

We also tested the possibility that the observed PLA₂ activity might be due to the action of lipoprotein lipase, although in our experiments we did not use post-heparin plasma, which usually contains high levels of this enzyme. Serum PLA₂ activity was not inhibited by 1 M NaCl, which has been reported to inhibit lipoprotein lipase (28). Furthermore, PLA₂ activity was not inhibited by tetrahydrolipstatin, a potent inhibitor of serine lipases (29).

Enzymes within lipoproteins are just beginning to be characterized, and their physiological roles are still unclear. Circulating lipoproteins contain a number of associated enzymes other than PLA₂, including lecithin cholesterol acyl transferase, phospholipid transfer protein, cholesterol ester transfer protein, and paraoxonase 1. Together with lipoprotein lipase and hepatic lipase, these enzymes are involved in lipoprotein remodelling and modulation of lipoprotein structure and function in normal physiological conditions as well as in inflammation and the acute phase response (39).

Lipoproteins and PLA₂ play key roles in a number of important processes in mammalian organisms, and further studies are therefore necessary to characterize the structural and functional interactions between these molecules in a variety of physiological states.

methods

View larger version (15K): [in this window] [in a new window]   Scheme S1. Structures of the PLA2 substrate, 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA), and the chromophore, 4-nitro-3-hydroxy-benzoic acid, detected at 425 nm.

	ACKNOWLEDGMENTS

The authors wish to thank Ms. Katie Coles, B.Sc. (Hons), Department of Public Health, University of Western Australia for kindly providing purified preparations of human serum HDL, LDL, and VLDL. We also thank Roche Products for the supply of tetrahydrolipstatin (Xenical^®).

Manuscript received February 8, 2001; and in revised form May 31, 2001

Abbreviations: DCIC, 3,4-dichloroisocoumarin; 1,2bHGPC, 1,2-bis-heptanoylthio-glycerophosphocholine; PAF-AH, platelet-activating factor acetylhydrolase; Pefabloc, 4-[2-aminoethyl]-benzenesulfonyl fluoride; PLA_2, phospholipase A₂; 4N3OBA, 4-nitro-3-octanoyloxy-benzoic acid

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TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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