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PubMed1990

Preparative separation of sphingolipids and of individual molecular species by high-performance liquid chromatography and their identification by gas chromatography-mass spectrometry.

Six fractions containing tri- to pentaglycosylceramides were isolated from the green, fresh water alga Chlorella kessleri, grown heterotrophically, by using preparative high-performance liquid chromatography (HPLC). Up to twelve fractions were obtained by further reversed-phase HPLC of each glycosylceramide. The use of a polar capillary column with Supelcowax 10 as the stationary phase allowed an excellent separation of the individual molecular species of ceramides, even though the separation did not occur when the ceramides differed only in the position of the amide bond. The individual molecular species (even if present in mixtures) were identified by gas chromatography-chemical ionization mass spectrometry. The evidence for a complete structure was obtained by enzyme splitting with alpha- and beta-galactosidases (the sequence of monosaccharides) and by negative ionization fast atom bombardment mass spectrometry. More than 400 molecular species of glycosylceramides were identified.

Ceramides↗
PubMed2000

Activation of sphingolipid turnover and chronic generation of ceramide and sphingosine in liver during aging.

Aging leads to a decreased ability of liver to metabolize drugs and increased expression and secretion of acute phase proteins, such as serum amyloid A (SAA), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). This phenomenon resembles some aspects of the acute phase response of host to inflammation; however, the molecular basis for the similarity is unclear. Ceramide and sphingosine are second messenger mediators of cellular responses to stress and inflammation. In liver, they play important role in mediating acute phase responses to IL1-beta. In this study, we use HPLC and thin layer chromatography to evaluate the effects of aging on steady-state levels of ceramide and sphingosine. We report that both lipids are elevated in liver of old (24 months) as compared to young (5 months) male Fisher 344 rats. To elucidate the mechanism(s) for ceramide elevation, we test the acidic (ASMase) and neutral sphingomyelinase (NSMase) in vitro using NBD-sphingomyelin as an exogenous substrate. SM synthase is also analyzed in vitro using NBD-ceramide and [3H]-dipalmitoylphosphatidylcholine (DPPC) as exogenous substrates. In accordance with the increases in the mass of ceramide, the activity of acid and neutral SMase is elevated in old animals. Michaelis-Menten analysis of NSMase implies that the apparent activation of this enzyme is caused by an increase in the Vmax of the enzyme. In contrast, SM synthase activity is lower in old animals as compared to young ones. These results show that aging is accompanied by an elevation in SM turnover and a decrease in its synthesis, resulting in accumulation of pro-inflammatory and growth inhibitory second messenger ceramide. Ceramidase, the only enzyme leading to sphingosine generation, is also measured in vitro using NBD-ceramide as a substrate and liver homogenate as an enzyme source. Its activity is higher in the old rats, as compared to young ones. The acid and neutral forms of the enzyme are affected the most, while the changes in the alkaline enzyme are not significant. The increases in the basal levels of ceramide and sphingosine in old animals may contribute to the onset of an inflammatory like state in liver during aging, exemplified by decreased P4502C11 mRNA expression and chronic induction of acute phase protein expression.

Aging↗