The field of extracellular vesicles is an exponentially growing segment of biomedical
sciences. However, the problems of normalisation and quantification of extracellular
vesicle samples have not been completely solved yet. Currently, extracellular vesicle
samples are standardized on the basis of their protein content sometimes combined
with determination of the particle number. However, even this combined approach may
result in inaccuracy and overestimation of the extracellular vesicle concentration.
Lipid
bilayers are indispensable components of extracellular vesicles. Therefore, a lipidbased
quantification, in combination with the determination of particle count and/or
protein content, appears to be a straightforward and logical approach for the
extracellular vesicle field. In this study we set the goal to improve the previously
reported sulfo-phospho-vanillin total lipid assay. We introduced an aqueous phase
liposome standard (DOPC) to replace the purified lipid standards in organic solvents
(used commonly in previous studies). Furthermore, we optimized the concentration of
the vanillin reagent in the assay. We found that elimination of organic solvents from
the
reaction mixture could abolish the background colour that interfered with the assay.
Comparison of the optimised assay with a commercial lipid kit (based on the original
sulfo-phospho-vanillin lipid assay) showed an increase of sensitivity by approximately
one order of magnitude. Thus, here we report a quick, reliable and sensitive test
that
may fill an existing gap in extracellular vesicle standardisation. When using the
optimised total lipid assay reported here, extracellular vesicle lipid measurements
can
be as easy as measuring proteins with a simple BCA test.
