Optimized Lipid Handling for LPA Assays

Various chemical forms of LPA were assayed: 1-oleoyl-LPA (18:1), 1-palmitoyl-LPA (16:1), 1-arachidonoyl-LPA (20:4), 1-linoleoyl-LPA (18:2), and 1-oleoyl-lysophosphatidylcholine (18:1 LPC) (Avanti Polar Lipids Inc.). Saturated or mono-unsaturated samples (16:0, 18:1 LPAs, and 18:1 LPC) were completely dissolved in ethanol:water (1:1 v/v) by sonicating for 3–5 min, aliquoted in glass vials layered with N₂, and stored under N₂ atmosphere at −20°C for several uses (up to 9 months). Unstable and unsaturated LPA samples (18:2 and 20:4; received in CHCl₃) were desiccated and then reconstituted in fresh ethanol:water (1:1 v/v) for immediate use in binding assays. Redissolving desiccated LPAs in aqueous BSA solutions for storage purposes was eliminated because it resulted in 95–97% loss of LPA during reconstitution (38 (link)). Stored or reconstituted LPAs in ethanol:water solution show a monodispersed distribution of LPA as measured by dynamic light scattering (DLS). Saturated LPAs are relatively stable under atmosphere, whereas unsaturated ones are highly unstable, extremely hygroscopic, and, therefore, cannot be stored for subsequent use in this assay.

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Ray M., Nagai K., Kihara Y., Kussrow A., Kammer M.N., Frantz A., Bornhop D.J, & Chun J. (2020). Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader. Journal of Lipid Research, 61(8), 1244-1251.

Publication 2020

1-oleoyl-LPA

1 oleoyl lysophosphatidylcholine Assay Atmosphere Chcl3 Ethanol Lipids

Corresponding Organization : Sanford Burnham Prebys Medical Discovery Institute

Other organizations : Vanderbilt University, University of California, San Diego

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Variable analysis

independent variables

Various chemical forms of LPA: 1-oleoyl-LPA (18:1), 1-palmitoyl-LPA (16:1), 1-arachidonoyl-LPA (20:4), 1-linoleoyl-LPA (18:2), and 1-oleoyl-lysophosphatidylcholine (18:1 LPC)

dependent variables

Binding assays

control variables

Saturated or mono-unsaturated samples (16:0, 18:1 LPAs, and 18:1 LPC) were completely dissolved in ethanol:water (1:1 v/v) by sonicating for 3–5 min, aliquoted in glass vials layered with N2, and stored under N2 atmosphere at −20°C for several uses (up to 9 months)
Unstable and unsaturated LPA samples (18:2 and 20:4; received in CHCl3) were desiccated and then reconstituted in fresh ethanol:water (1:1 v/v) for immediate use in binding assays
Redissolving desiccated LPAs in aqueous BSA solutions for storage purposes was eliminated because it resulted in 95–97% loss of LPA during reconstitution

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