Analysis of micellar and vesicular lecithin and cholesterol in model bile using 1H- and 31P-NMR

The distribution of phospholipid and cholesterol between the vesicular and micellar phases in bile plays an important role in the formation of cholesterol gallstones. Conventional analytical procedures to determine the distribution are potentially unreliable because they disturb the distribution of these compounds between the two phases. In this work, we circumvent this problem by using NMR. 31P-NMR is used to quantify directly the micellar and the vesicular amount of lecithin. The previously described 1H-NMR method to determine directly the micellar lecithin (Groen et al., J Lipid Res 31: 1315-1321) has been optimized by the implementation of a spectral quantification method. The agreement between the 31P and 1H methods was excellent. In our hands, the method of Ellul et al. (FEBS Lett 300: 30-32) did not allow quantification of micellar cholesterol, although our fitting procedure offered the possibility of quantifying overlapping spectral peaks by the use of prior knowledge about all the parameters of the compounds visible in the spectrum. The micellar cholesterol concentration was so low compared to the overlapping lecithin peaks that no reliable quantification was possible. The same problem was encountered when using other characteristic cholesterol resonances for quantification. Our data suggest that cholesterol present in the vesicular phases is too immobile to give rise to high-resolution peaks and the amount of cholesterol present in the micellar phase is too low to allow quantitation by NMR. We conclude that 1H-NMR can be used to determine micellar lecithin, and 31P-NMR to determine micellar as well as vesicular lecithin in model bile.