Branched Long-chain Bases in Ceramide 2-Aminoethylphosphonate from Ova of the Fresh-water Bivalve, Hyriopsis schlegelii

Abstract

In our previous communication the discovery of ceramide 2-aminoethylphosphonate (CAEP) in ova of a fresh-water bivalve, H. schlegelii was reported. The ova sphingolipid contained mostly 4-sphingenines which were composed of octadecasphingenine (predominant) and branched octadeca- and nonadecasphingenines. In this paper we wish to report on the characterization of the above branched compounds. A ceramide fraction derived by enzymatic degradation of CAEP was subjected to catalytic hydrogenation, hydrolyzed and separated into long-chain bases and fatty acids as described earlier. Thin-layer chromatogram of the hydrogenated long-chain bases revealed one major ninhydrin positive component with the same Rf value as that of authentic octadecasphinganine, with a very small amount of artifacts (O-methyl derivatives) (Fig. -1). The hydrogenated bases were oxidatively cleaved to aldehydes with lead tetra-acetate (this resulted in the loss of two carbon atoms from the parent carbon skeleton), and were analyzed by gas-liquid chromatography (GLC) using a 3m×3mm glass column containing 25% EGS on 6080 mesh Celite 545. Based on equivalent chain length data described by Morrison on straight and branched chain aldehydes, all of the aldehydes except Peak 3 in Fig. -2 were tentatively identified as iso-hexadecanal (Peak 1), n-hexadecanal (Peak 2), iso-heptadecanal (Peak 4) and anteiso-heptadecanal (Peak 5), respectively. For the further confirmation of the above branched chain compounds, the aldehydes were oxidized with silver oxide, and the resulting fatty acids after methylation were analyzed by GLC. Fatty acid methyl esters derived from the aldehydes of Peak 1, 2, 4 and 5 had identical retention times as iso-palmitic acid, palmitic acid, iso-margaric acid and anteiso-margaric acid (Fig. -4). However, Peak 3 in Fig. -4 did not correspond to any of the authentic saturated methyl esters available in the C14C17 range and no further characterization of this peak was made. From the gas chromatographic analyses of aldehydes and the corresponding fatty acid methyl esters, the composition of long-chain base mixtures in the ova CAEP was calculated and given as follows; iso-sphingosine (12%), sphingosine (60%), iso-nonadeca-4-sphingenine (2%), anteiso-nona-deca-4-sphingenine (22%) and unidentified long-chain base (4%). This is the first paper on the branched long-chain bases found in shellfish.