Conjugated linoleic acid: Techniques for analysis and effects on PUFA formation in rat hepatocytes
This study focused on developing techniques for the analysis of conjugated linoleic acid (CLA) and determining the effects of CLA on PUFA formation in suspension cultures of rat hepatocytes. First, two methylation procedures were established for analyzing neutral or polar lipids containing CLA, regardless of the free fatty acid level. Both methods showed less alteration of CLA isomeric distribution compared to conventional methods of methylation. Second, the positional isomers of CLA in commercial preparations and the neutral lipids isolated from rat hepatocyte cultures were determined by GC-MS analysis. All 18:2[7,9], 18:2[8,10], 18:2[9,11], 18:2[10,12], and 18:2[11,13] isomers were detected in the commercial CLA mixture and in the neutral lipid fraction isolated from hepatocytes. Third, the effects of CLA on rat hepatocyte fatty acid composition, and cell physiology were investigated. CLA enrichment did not affect LDH activity or lactate oxidation to produce CO2. Greater amounts of CLA isomers were detected in neutral lipids compared to that found in polar lipids of the hepatocytes. Individual CLA isomers tended to be incorporated into different lipid classes. Enrichment of rat hepatocytes with 200 μM CLA resulted in a greater CLA incorporation compared to 100 μM CLA enrichment. Greater amounts of CLA isomers were detected in the hepatocyte cultures at 120 minutes of incubation. In addition, no c,c-CLA was detected in hepatocyte polar lipids. The formation of arachidonic acid (AA) from 14C-linoleic acid was reduced by treating the hepatocytes with 100 μM of CLA. Compared to the control, a maximal 28% suppression of AA formation was observed with 100 μM CLA enrichment for 120 min. Therefore, CLA may directly affect membrane structure and function by altering membrane lipid composition, and indirectly influence cell physiology by down-regulating arachidonic acid production.
Watkins, Purdue University.
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