Carbon stripping extracts serum free fatty acids: implications for media supplementation of cultured type II pneumocytes.
Carbon stripping is a process that is widely used to remove hormones from serum. Because addition of serum to culture media also provides exogenous fatty acids that influence lipid metabolism of cultured cells, we investigated the effects of carbon stripping on the composition of the phospholipid and free fatty acid fractions in fetal bovine serum and the effects of these changes on phosphatidylcholine synthesis by cultured adult alveolar type II cells. Carbon stripping resulted in quantitative and qualitative changes in serum free fatty acids. The process effectively extracted greater than or equal to 99% free fatty acids and, to a lesser extent, phospholipids. There were also qualitative changes in the relative composition of the remaining free fatty acids with a selective loss of oleic and linoleic free fatty acids. However, the relative composition of the serum phospholipid fatty acid fraction was unaffected. Type II cells isolated from adult male rat lung and cultured in Dulbecco's modified Eagle's medium supplemented with 10% carbon-stripped fetal bovine serum (FBS-CS) incorporated [3H]choline into phosphatidylcholine at a rate 36% less than the rate of control cells cultured with unstripped FBS. Addition of oleic acid to FBS-CS supplemented media increased total phosphatidylcholine synthesis by adult type II cells by 67-71%. In contrast, addition of palmitic acid inhibited PC synthesis 51-67%. The combination of oleic and palmitic acids resulted in a rate of [3H]choline incorporation into phosphatidylcholine similar to the rate for control cells cultured in FBS-CS-supplemented media alone. Although synthesis of disaturated phosphatidylcholine was unaffected by exogenous fatty acids, addition of fatty acids altered the proportion of disaturated phosphatidylcholine synthesis relative to total phosphatidylcholine synthesis. The presence or absence of the hormones, dexamethasone and triiodothyronine, did not explain the difference in rate of phospholipid synthesis by type II cells cultured in untreated versus carbon-stripped serum supplemented media. These results suggest that the removal of serum free fatty acids by carbon stripping can influence phospholipid metabolism of cultured type II cells. Because serum free fatty acids influence cellular lipid composition and potentially cell metabolic functions, carbon-stripped serum may not be the optimal choice for media supplementation of cultured cells.
Viscardi
R M
RM
Department of Pediatrics, University of Maryland, Baltimore School of Medicine.
Ullsperger
S
S
McKenna
M C
MC
D000818:Animals; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D002849:Chromatography, Gas; D003470:Culture Media / Q000737:chemistry; D005230:Fatty Acids, Nonesterified / Q000097:blood*; D006728:Hormones / Q000097:blood*; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008297:Male; D010713:Phosphatidylcholines / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains
D003470:Culture Media; D005230:Fatty Acids, Nonesterified; D006728:Hormones; D010713:Phosphatidylcholines; D002794:Choline