Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance...

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.