Abstract
The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (dipalmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6%±1.0%, 104.4%±7.6%, and 109.4%±3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended wheneve possible. Validation experiments revealed an absolute quantification limit of 1.25 μg per assay, a good linearity in the range of 25 to 1000μg/mL PC (r2≥0.9990) and a quite high accuracy (99.8%–101.4% of theory) and precision (relative standard deviation ≤3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.
Keywords: quantification, phosphatidylcholine, liposome, microplate, enzymatic assay, colorimetric assay, phospholipase D
Full Text
The Full Text of this article is available as a PDF (417.1 KB).
References
- 1.Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234:466–468. [PubMed] [Google Scholar]
- 2.Eibl H, Lands WE. A new, sensitive determination of phosphate. Anal Biochem. 1969;30:51–57. doi: 10.1016/0003-2697(69)90372-8. [DOI] [PubMed] [Google Scholar]
- 3.Stewart JC. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem. 1980;104:10–14. doi: 10.1016/0003-2697(80)90269-9. [DOI] [PubMed] [Google Scholar]
- 4.Takayama M, Itoh S, Nagasaki T, Tanimizu I. A new enzymatic method for determination of serum choline-containing phospholipids. Clin Chim Acta. 1977;79:93–98. doi: 10.1016/0009-8981(77)90465-X. [DOI] [PubMed] [Google Scholar]
- 5.Gurantz D, Laker MF, Hofmanns AF. Enzymic measurement of choline-containing phospholipids in bile. J Lipid Res. 1981;22:373–376. [PubMed] [Google Scholar]
- 6.McGowan MW, Artiss JD, Zak B. A procedure for the determination of high-density lipoprotein choline-containing phospholipids. J Clin Chem Clin Biochem. 1982;20:807–812. doi: 10.1515/cclm.1982.20.11.807. [DOI] [PubMed] [Google Scholar]
- 7.Bradley CA, Salhany KE, Entman SS, Aleshire SL, Parl FF. Automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid. Clin Chem. 1987;33:81–86. [PubMed] [Google Scholar]
- 8.Nie Y, He JL, Hsia SL. A micro enzymic method for determination of choline-containing phospholipids in serum and high density lipoproteins. Lipids. 1993;28(10):949–951. doi: 10.1007/BF02537506. [DOI] [PubMed] [Google Scholar]
- 9.Campanella L, Pacifici F, Sammartino MP, Tomassetti M. Analysis of lecithin in pharmaceutical products and diet integrators using a new biosensor operating directly in non aqueous solvent. J Pharm Biomed Anal. 1998;18:597–604. doi: 10.1016/S0731-7085(98)00212-X. [DOI] [PubMed] [Google Scholar]
- 10.Yamamoto I, Nishii M, Tokuoka E, Handa T, Miyajima K. Productretardation and-activation of catalytic hydrolysis by phospholipase D in small unilamellar vesicles of egg yolk phosphatidylcholine. Colloid Polym Sci. 1997;275:627–633. doi: 10.1007/s003960050128. [DOI] [Google Scholar]