A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat lysophosphatidic acid acyl transferase (LPAAT) produced in Escherichia coli was used. In the presence of [C]oleoyl CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmoles with a minimal detection of 0.2 pmoles. This method was used to quantify LPA in butanolextracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.

In order to understand the precise role of LPA in the development of particular pathologies such as cancer or obesity, and to clarify the metabolic origin of LPA produced by the cells, a precise and sensitive detection of LPA is necessary. In the present work, a recombinant lysophosphatidic acid acyltransferase (LPAAT) was used to develop a simple and highly sensitive radioenzymatic assay to quantify LPA in various biological fluids.

Preparation of recombinant LPAAT:
The rat 1-acyl-sn-glycerol-3-phosphate acyltransferase cDNA encoding for an enzyme behaving as a LPAAT was previously cloned in a bacterial expression vector pTrcHis (23).
Bacteria were collected by centrifugation at 3000 g for 10 min, and the pellet was disrupted by sonication in 0.2 M Tris-HCl (pH 7.4). After centrifugation at 10,000 x g for 20 min, the supernatant was recovered and further centrifuged at 100,000 x g (microsomes) for 90 min.
The yield of a typical preparation is 6 to 7 mg microsomal protein per liter of culture. The resulting pellet was resuspended in the same buffer to a final protein concentration of 1 µg/µl and was used for radioenzymatic detection of LPA. Kinetic analysis allowed to determined that the activity of transformation of LPA into PA of a typical microsome preparation varied from 0.8 to 1 nmol/min/mg protein. Lipid extraction from serum and plasma 1-oleoyl-LPA or lipids contained in serum or plasma were extracted with 1 volume of 1-butanol. After vigorous shaking and centrifugation (5 min at 3000 g), the upper butanol phase was collected and evaporated under nitrogen at 50°C. This procedure allowed 85 to 90% extraction of LPA.

Results
The ability of the LPAAT to acylate LPA into PA was tested after semi-purification of the enzyme from rat LPAAT cDNA-transformed E. coli. For that, 1-oleoyl-LPA or butanolextracted lipids from serum (known to contain significant amount of LPA (6-9)) were incubated in the presence of LPAAT-transformed E. coli microsomes and [ 14 C]oleoylCoA. In order to identify the products of the reaction, they were separated by two dimensional thin layer chromatography (Figure 1).   (Figure 3-B).
The radioenzymatic assay was finally used to quantify LPA in bovine sera from different origin. As shown in Table 1, the concentration of LPA measured in 20 µl of serum varied from 0.28 to 2.56 µM. Concentration of LPA in donor calf sera was significantly higher (2.4 to 4.7 fold) than the mean concentration (0.54 µM) of LPA found in fetal calf sera. Although present at much lower concentrations than in serum, significant concentrations of LPA (from 51 to 283 nM) were also found in human and mouse platelet -poor plasma (Table 2).

Discussion
Several procedures have previously been used to detect LPA. Gas chromatography (GC) allows quantification of methylated fatty acids derived from TLC-purified LPA (15). This is an indirect assay allowing determination of the relative fatty acid composition of LPA.
However, this method is not very sensitive and requires relatively high amounts of purified LPA. In addition, classical GC does not allow to determine the presence alkyl-LPA. For that it is necessary to employ both mild alkaline hydrolysis and gas chromatography coupled to mass spectrometry (26).
The presence of LPA in a biological fluid can also be demonstrated by using bioassays.
This type of assay consists in measuring the influence of a LPA-containing fluid on a cellular response known to be controlled by LPA, such as platelet aggregation (17), calcium mobilization in xenope oocytes (10), or cell spreading (11). Since bioactive phospholipids other than LPA are also able to generate those biological responses, bioassays cannot be considered specific enough for LPA quantification. LPA can also be detected following labeling cellular phospholipid with [ 32 P]ortho-phosphate, and separation of [ 32 P]LPA by TLC (7,11,18). Although very sensitive, this method is only applicable on cultured cells, and is not quantitative.
The LPAAT, also known as 1-acyl-sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.51) catalyses LPA conversion into phosphatidic acid (PA), a key step in glycerolipid synthesis. LPAAT has been demonstrated to be highly specific for LPA and not influenced by LPA fatty acid composition (27). Therefore the high specificity of LPAAT made this enzyme an interesting partner to develop a new assay for LPA quantification.
By using a rat recombinant LPAAT produced in E. coli (23) we have developed a simple, rapid, and highly sensitive radioenzymatic assay to quantify LPA in various biological fluids. By using this assay it is possible to quantify LPA in crude butanol-lipid extracts without prior time consuming and poorly efficient TLC-purification of LPA. It is also important to notice that, this assay allows to quantify both acyl-and alkyl-LPA. Although present to a much lower concentration than acyl-LPA, alkyl-LPA has been reported to be much more active as acyl-LPA on platelet aggregation( 28). Although our assay does not discriminate between acyl-LPA and alkyl-LPA, the amount of alkyl-LPA present in a biological fluid can nevertheless be determined. For that the biological fluid can be treated with a lysophospholipase, such as the phospholipase B, known to specifically hydrolyze acyl-LPA without altering alkyl-LPA. Phospholipase B-resistant LPA will correspond to alkyl-LPA.
Our radioenzymatic assay is also highly sensitive. It is possible to detect less than 0. Finally, the method is reliable since the concentration values determined in bovine sera are in agreement with those previously published (6)(7)(8)(9). However, it is noticeable that donor calf sera contain a higher concentration of LPA than fetal calf sera. Although the precise reasons of this difference remain to be determined, this information needs to be taken into consideration to choose cell culture serum.
In conclusion, this radioenzymatic assay represents a new, and highly sensitive method for LPA quantification. Because of its simplicity and rapidity of execution, this method could easily be introduced in any research or hospital laboratory interested in fundamental or pathological issues concerning LPA.  Tables   Table 1 :