Effect of Orotic Acid on the Distribution of Phosphatidylcholine [32P]

Abstract

Triglyceride accumulation in the liver can be brought about by a disease state in man, and by the administration of a large variety of agents (1, 2). A block in the release of hepatic beta-lipoprotein into blood stream appears to be responsible for the development of a number of experimentally induced fatty liver conditions (3).Our previous results demonstrated that a decreased release of beta-lipoprotein from liver slices in rats fed orotic acid was restored by the addition of phosphatidylcholine. It was further found that phospholipid has a curative effect on fatty liver induced by orotic acid or ethionine. These findings suggest that phospholipid is an important factor in the normal release of beta-lipoprotein. Accordingly, it is of interest to examine the fate of ingested phosphatidylcholine. The present experiments were done to examine the distribution of phosphatidylcholine [32P] in rats fed orotic acid.Female Wistar strain rats weighing 180 to 210 g were first fed a semisynthetic diet (4) for 2 days. Control rats were given a semisynthetic diet alone. Groups of animals then were put on a dietary supplement of I ;o orotic acid for 7 days after which 0.373 or 0.824 /iCi of phosphatidylcholine [32P] to which was added 14 mg of non-radioactive phosphatidyl choline as carrier in 0.5 nil of saline solution (1.91 or 4.23 /tCi/Kg body weight) was in jected intraperitoneally. At the indicated time after administration of the phospholipid, the rats were sacrificed by heart puncture under ether anesthesia. Tissues were removed, homogenized with ice-cold 5 trichloroacetic acid, and centrifuged. The precipitate was washed twice with ice-cold trichloroacetic acid. Lipids were then extracted once with 80% ethanol, once with 100% ethanol, twice with chloroform-ethanol (1 : 1, by vol.), and once with ether. Trichloroacetic acid was added to the serum, and centrifuged. Serum lipids were then extracted with chloroform-methanol (2: 1, by vol.). Radioactivity of acid-soluble and lipid fraction was determined in a liquid scintillation spectrometer using dioxan and toluene-phosphor solution, respectively.Phosphatidylcholine [32P] was prepared as follows: Liver slices were incubated with 1.25 mCi orthophosphate [32P] per ml of Krebs-Ringer bicarbonate buffer at 37' in 02 CO2 (95: 5, by vol.) for 5 hr. Lipids were extracted as described above. Phosphatidyl choline was separated by paper chromatography according to the method of Marinetti et al. (5). The specific radioactivity of the prepared phosphatidylcholine [32P] was 18.4 1eCi/