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United States Department of Agriculture

Agricultural Research Service

Title: A Rapid Method for Quantitating Free Sphingoid Bases and Complex Sphingolipids in Microgram Amounts of Cells Following Exposure to Fumonisinb1

item Yoo, Hwan-Soo - NIH, BETHESDA, MD
item Norred, William
item Riley, Ronald

Submitted to: Toxicology In Vitro
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 13, 1995
Publication Date: N/A

Interpretive Summary: Fumonisins are toxic chemicals produced by fungi that occur on corn. In animal and plant cells the fumonisins cause changes in the amount of a special type of fat called sphingolipids. The change in the amount of certain sphingolipids is used as an indicator of exposure of animals and plants to the fumonisins. The marker for exposure is the increase in a sphingolipid called sphinganine and a decrease in a group of sphingolipids called "complex sphingolipids". The use of the sphingolipid marker is currently patented by the USDA and Emory University. This paper describes a rapid method for measuring the changes in the marker in small amounts of tissues and cells. Development of rapid methods is a step towards developing commercially useful methods for detection of the presence of the fumonisins in foods and animals based on the patented marker.

Technical Abstract: Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyltranferase, key enzymes in sphingolipid metabolism. The purpose of this study was to develop and validate rapid methods for determination of free sphingoid bases and total sphingolipids in small quantities of cells exposed to pure fumonisin B1 (FB1). The developed rapid methods were a modification of an earlier "original" method and used a single CHCl3 extraction following base or acid hydrolysis of cell suspensions. Average recovery of the C20-sphinganine internal standard using the rapid extracation method for free sphingoid bases was 48% and 84% for control and FB1-treated LLC-PK1 cells (appr. 100 ug protein), respectively, while the recoery using the original extraction methods was <1%. The average total sphingolipid concentrations (free sphingoid bases plus complex sphingolipids) determined by the rapid and original method were similar. The rapid extraction method provided simpler and shorter extractions with improved recovery, and was more economical by allowing experimental designs using smaller quantities of cells (10-100 ug protein) and fumonisins. In conclusion, the rapid method is useful for study of fumonisin-induced disruption of sphingolipid metabolism in cultured cells where free sphingoid base concentratiaon increases and complex sphingolipds decreased markedly.

Last Modified: 4/24/2014