A rapid procedure for the preparation of delipidized serum protein is described. The delipidized protein can be used for the maintenance and growth of tissue culture cells in a lipid-free environment. The extraction procedure greatly reduces all serum lipid classes and the delipidized protein supports the growth of a variety of cells in culture.

The influence of lipoprotein composition on free cholesterol (FC) esterification and accumulation of esterified cholesterol (EC) was studied in cells of Fu5AH rat hepatoma exposed to culture media supplemented with FC-lecithin dispersions having mole ratios of FC to phospholipid (FC/P) ranging from 0.6 to 2.8. In the presence of normal human serum, FC-lecithin dispersions with a FC/P greater than one stimulated both the We sought to determine if variations in the exogenous FC/P composition could influence the accumulation of EC by tissue culture cells. The Fu5AH rat hepatoma cell line was selected because these cells are capable of accumulating large quantities of EC when grown in the presence of hyperlipemic animal sera (11), and because the accumulation of EC correlates closely with the extent of esterification of FC (12). Thus, this cell, with its high capacity for cholesterol esterification and EC accumulation, can serve as a sensitive experimental tool for the quantitation of cholesterol flux between the cells and the culture medium. MATERIALS AND METHODSCholesterol/Lecithin Dispersions. Cholesterol/lecithin dispersions of various molar ratios were prepared as previously described (5, 6). L-a-Dipalmitoyl lecithin (General Biochemicals, Div. Mogul Corp., Chagrin Falls, Ohio) and FC (Sigma Chemical Co., St. Louis, Mo.) were added to 10 ml of physiological saline and subjected to 70 W with a Branson sonifier for 60 min at 450 under N2. Cholesterol dispersions prepared with 40 mg of lecithin and 23 mg of cholesterol resulted in a free cholesterol-to-phospholipid mole ratio (FC/P) of approximately one. Cholesterol rich dispersions (40 mg of lecithin, plus 30-80 mg of cholesterol) had a FC/P of greater than one. Cholesterol poor dispersions (FC/P less than one) were prepared with 40 mg of lecithin and less than 20 mg of cholesterol. Albumin (5 mg/ml) was added to the sonicated dispersions and the mixture was centrifuged at 26,000 X g for 30 min to sediment the undispersed lipid. Dispersions were sterilized by filtration through a 0.45 ,um Millipore filter and mixed at various concentrations, with tissue culture medium supplemented with either albumin (bovine fraction V powder, fatty acid-free, Miles Laboratories, Inc., Kankakee, Ill.), normal human serum, or human serum lipoproteins.Cells and Incubation Conditions. The Fu5AH rat hepatoma cell line has been described previously (11,12

Previous investigations had demonstrated that Fu5AH rat hepatoma cells accumulated large quantities of esterified cholesterol when grown in hyperlipemic rabbit serum. The present investigation has determined the sources of the cellular esterified cholesterol when the cells were grown in hyperlipemic serum. Cellular esterification of endogenous and exogenous free cholesterol contributed 10% and 30%, respectively. The remaining 60% of the accumulated cellular esterfied cholesterol was derived from exogenous (serum) cholesteryl esters. Evidence for the hydrolysis of a portion of the incorporated esterified cholesterol is presented. A stimulation of free cholesterol incorporation and cellular esterification is elicited by hyperlipemic serum and serum lipoproteins when compared to normolipemic serum present at equivalent exogenous cholesterol concentrations. The effect of hyperlipemic serum is reduced by Tween-80 and Triton WR-1339. Comparative data on esterified cholesterol accumulation, free cholesterol incorporation, and cellular cholesterol esterification in Fu5-5 rat hepatoma cells, L-cells, and rabbit aortic medial cells are presented.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50,ug/ml)

Past research has associated ABO blood type and mental stress with cardiovascular risk. We studied the effects of blood type (A vs. O) coupled with a mirror drawing stressor on very low density lipoprotein toxicity-preventing activity (TxPA) and plasma cortisol levels. Exposure to the stressor significantly decreased TxPA and increased cortisol for the total group of 25 older adult males. However, the stress response patterns of the 15 blood type A males were different from those of the 10 type O subjects. The blood type A group had higher initial levels of TxPA and cortisol as well as quicker stress recovery rates than the type O group. ABO blood type may be an important behavioral hematologic variable to assess in studies concerning biochemical stress response or cardiovascular risk.