The measurement of lysosomal phospholipase A2 activity in plasma

Abstract

A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.

Fig. 1.

Effect of plasma or serum on LPLA2 activity. A: Transacylase activity of LPLA2. The reaction mixture contained 48 mM sodium citrate (pH 4.5), 10 µg/ml BSA, liposomes (130 µM phospholipid), 1.3% human serum, 1.3% human plasma, or 14.5 ng/ml of recombinant mouse LPLA2 in the presence or absence of 1.3% human serum or 1.3% human plasma in 500 µl of total volume. Liposomes consisting of DOPC/sulfatide/NAS (3:0.3:1, molar ratio) were incubated with the enzyme for 2 h or 15 min at 37°C as shown in A. The reaction products were extracted and separated by an HPTLC plate using a solvent system consisting of chloroform/acetic acid (9:1, v/v). The reaction product, 1-O-acyl-NAS, is produced by LPLA2. B: Esterase activity of LPLA2. The reaction mixture contained 200 µM p-nitro-phenylbutyrate, 48 mM sodium citrate (pH 4.5), 10 µg/ml BSA, and 14.5 ng/ml of recombinant mouse LPLA2 in the presence or absence of 1.3% serum in 500 µl of total volume. The reaction was initiated by adding the recombinant LPLA2 and kept for 10, 20, 30, and 40 min at 37°C. One hundred microliters of the reaction mixture was taken at each time point and mixed with 100 µl of cold 0.2 M NaHCO₃. The absorbance of the mixture at 400 nm was measured immediately.

Fig. 2.

Substrate specificity of LPLA2. A. The reaction mixture contained 48 mM sodium citrate (pH 4.5), 10 µg/ml BSA, liposomes (130 µM phospholipid) and recombinant mouse LPLA2 in 500 µl of total volume. Before starting the reaction, liposomes consisting of 1,2-di-(9Z-octadecenyl)-PC, DOPC or anionic glycerophospholipid and NAS (54:22:24, molar ratio) were preincubated for 5 min at 37°C. The reaction was initiated by the addition of recombinant LPLA2 and kept for 1.5 to 10 min at 37°C. In the reaction, 4.83 ng/ml and 58 ng/ml of recombinant mouse LPLA2 were used for DOPC and anionic phospholipid liposomes, respectively. The reaction products were extracted and separated with an HPTLC plate using a solvent system consisting of chloroform/acetic acid (9:1, v/v). The reaction product, 1-O-oleoyl-NAS, was quantified by scanning the plate. Error bars indicate S.D. (n = 3). PC, PI, CL, PA, PS, PG and PEt indicate 1,2-dioleoyl phosphatidylcholine, 1,2-dioleoyl phosphatidylinositol, 1,1′, 2,2′-tetraoleoyl cardiolipin, 1,2-dioleoyl phosphatidic acid, 1,2-dioleoyl phosphatidylserine, 1,2-dioleoyl phosphatidylglycerol and 1,2-dioleoyl phosphatidylethanol, respectively. B: Different concentrations of liposomes consisting of DOPC/sulfatide/NAS (3:0.3:1, molar ratio) or DOPG/NAS (3:1, molar ratio) were preincubated for 5 min at 37°C and then incubated with the recombinant LPLA2. The reaction was performed as described in A.

Fig. 3.

Transacylase activity in human plasma. A: The reaction mixture contained 48 mM sodium citrate (pH 4.5), liposomes (130 µM phospholipid), and 1.6% of human plasma in 500 µl of total volume. The liposomes consisted of DOPG/NAS (3:1, molar ratio). The reaction was initiated by adding 8 µl of human plasma and kept for 30, 60, and 90 min at 37°C. The reaction product was determined and quantified as described in Fig. 2 and plotted against incubation time (B). C: Different concentrations of plasma were incubated with DOPG/NAS liposomes for 90 min at 37°C in 500 µl of 48 mM Na-citrate (pH 4.5). D: The 1-O-acyl-NAS formed after 90 min incubation was plotted against the plasma concentration in the reaction mixture.

Fig. 4.

The absence of transacylase activity in LPLA2-knockout mouse plasma. The reaction mixture contained 48 mM sodium citrate (pH 4.5), 10 µg/ml BSA, liposomes (130 µM phospholipid), 4% mouse plasma, or 4% LPLA2-knockout mouse plasma in 500 µl of total volume. Liposomes consisting of DOPG/NAS (3:1, molar ratio) were incubated with the mouse plasma for 90 min at 37°C. The reaction products were extracted and separated by an HPTLC plate using a solvent system consisting of chloroform/acetic acid (9:1, v/v).

Fig. 5.

Effect of MAFP on LPLA2. A: Recombinant mouse LPLA2 was treated with 0, 10, or 100 µM MAFP for 1 h on ice. LPLA2 was incubated with DOPG/NAS (3:1, molar ratio) liposomes. The reaction mixture contained 130 µM phospholipid, 48 mM sodium citrate (pH 4.5), 10 µg/ml BSA, and 14.5 ng/ml of the enzyme in 500 µl of total volume and was incubated for 1.5–4.5 min at 37°C. The reaction products were extracted, separated, and quantified as described in Fig. 2. Error bars indicate SD (n = 3). B: Esterase activity of MAFP-treated LPLA2 was performed as described in Fig. 1. The reaction was initiated by adding vehicle or the enzyme and kept for 5, 15, 25, and 35 min at 37°C. C: Recombinant mouse LPLA2 (4 µg) pretreated with or without 100 µM MAFP as described in A was incubated with DOPC/ sulfatide-liposomes or DOPG-liposomes for 30 min on ice and centrifuged at 150,000 g for 1 h at 4°C. The resultant pellet was applied on SDS-PAGE as described in “Materials and Methods.” D and E: Human plasma (D) and mouse WT and LPLA2-knockout plasma (E) were pretreated with or without 100 µM MAFP for 1 h on ice. The treated plasma samples (2% for human and 4% for mice) were incubated with DOPG/NAS liposomes for 90 min at 37°C. The reaction products were analyzed by TLC as described in Fig. 1.