HPLC Quantification of Sphingolipids in Soybeans with Modified Palmitate Content

Abstract

Efficient separation and accurate quantification of sphingolipids (SL) are important for studying SL concentrations and biological functions. The objectives of this study were to develop effective methods for the separation and quantification of SL and to determine the relationship between palmitate and SL contents of mature soybean seeds. Methods using column chromatography and high-performance liquid chromatography−evaporative light scattering detector (HPLC-ELSD) were developed to separate and quantify glucosylceramide (GlcCer) and ceramide (Cer) in 15 soybeans lines in which palmitate content ranged from 3.7 to 40.7%. There were significant differences among the lines for GlcCer (83.4−397.6 nmol/g) and major Cer contents (8.4−20.7 nmol/g) on a dry weight basis. The correlations of palmitate content with GlcCer and Cer concentrations were not significant. The results indicated that the palmitate content of soybean seed did not affect their GlcCer and Cer contents. Genetic factors other than those that control palmitate content seemed to be responsible for the variation among soybean lines for GlcCer and Cer contents. Efficient separation and accurate quantification of sphingolipids (SL) are important for studying SL concentrations and biological functions. The objectives of this study were to develop effective methods for the separation and quantification of SL and to determine the relationship between palmitate and SL contents of mature soybean seeds. Methods using column chromatography and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) were developed to separate and quantify glucosylceramide (GlcCer) and ceramide (Cer) in 15 soybeans lines in which palmitate content ranged from 3.7 to 40.7%. There were significant differences among the lines for GlcCer (83.4-397.6 nmol/g) and major Cer contents (8.4-20.7 nmol/g) on a dry weight basis. The correlations of palmitate content with GlcCer and Cer concentrations were not significant. The results indicated that the palmitate content of soybean seed did not affect their GlcCer and Cer contents. Genetic factors other than those that control palmitate content seemed to be responsible for the variation among soybean lines for GlcCer and Cer contents.

Plant sphingolipids: decoding the enigma of the Sphinx

“…Vesper et al (1999) estimated that the ceramide content of plant tissues is c . 10–20% of the glycosylceramide content while Wang et al (2006) observed a level of ceramide in the range of 4–10 mol% of glycosylceramide content. In rice and Arabidopsis , ceramide content was estimated at 6 mol% in leafy stems of rice and between 2–7 mol% in Arabidopsis leaves (Ohnishi et al , 1985; Markham et al , 2006; Markham & Jaworski, 2007).…”

“…Due to the complexity of sphingolipids, and their diversity between plants and also between organs within the same plant, powerful analytical tools are required to directly identify individual molecules. Some of the techniques used include capillary gas chromatography, infra-red spectrometry, high-performance liquid chromatography and mass spectrometry (Cahoon & Lynch, 1991; Sullards et al , 2000; Ng et al , 2001; Ternes et al , 2002; Bielawski et al ., 2006; Wang et al , 2006; Markham et al , 2006; Markham & Jaworski, 2007; Shi et al , 2007). It is noteworthy that the success of LC/MS/MS approaches for characterizing sphingolipids is dependent on the development of efficient extraction protocols due to the diverse polarity associated with sphingolipids (Markham et al , 2006; Merrill et al , 2007, 2009).…”

Oxidative Stability of Egg and Soy Lecithin as Affected by Transition Metal Ions and pH in Emulsion

“…The PL class composition was determined by a HPLC method as described by Wang and others (13). Briefly, a Beckman-Coulter Gold HPLC system equipped with a model 508 autosampler and model 126 delivery pumps was connected with Alltech 2000 evaporative light scattering detector (ELSD).…”

Effect of Seed Development Stage on Sphingolipid and Phospholipid Contents in Soybean Seeds

“…Because the amount of total lipid extract from each replicate of immature seeds was too low to be used in the separation and quantification of the SL contents, the two replicates of the total lipid extract of each immature seed sample were combined. The total lipid was separated into the neutral lipid fraction, the intermediate polar lipid fraction, and the polar lipid fraction using the procedures described by Wang et al (11). The intermediate polar lipid fraction mainly contained SL, glycolipid, and some PL.…”

“…Because ESG and SG also are membrane lipid components, they were quantified in the same run for the GlcCer quantification with HPLC-ELSD. Only the major Cer, TA, was determined and quantified in this study using the HPLC method reported by Wang et al (11). Different concentrations of the GlcCer, ESG, and SG standard mixtures and the TA standard were used for establishing standard calibration equations ( Table 1).…”