Donald A. Beeler

The entry of choline into lecithin, in vivo, by base exchange

Abstract 1. 1. After intravenous administration to bile-cannulated male rats of 32 Pi, [ Me - 3 H]choline and [ Me - 14 C]methionine simultaneously, the specific activities were determined of free choline, phosphorylcholine, CDP-choline, sphingomyelin and also of linoleoyl and arachidonoyl lecithins of both bile and liver at intervals up to 6 h. 2. 2. The ratio of 73 H/ 32 P of lecithins was much higher than that of phosphorylcholine indicating that free choline enters lecithins by base exchange. The rate of base exchange was estimated at about 4 times the rate at which phosphorylcholine is converted to lecithin. 3. 3. The low specific activity of CDP-choline in respect of choline observed by BJ0RNSTAD and Bremer is confirmed. A high ratio of 3 H/ 32 P and 14 C/ 3 H in CDPcholine compared to phosphorylcholine and a high specific activity ( 32 P) of the CMP portion of CDP-choline confirms their suggestion of a phosphorylcholine transferase catalyzed exchange, CDP-choline and diglyceride lecithin and CMP. 4. 4. Because of rapid base exchange, estimates of the rate of phospholipid turnover based upon radioactive lipid bases are probably too high.

The biosynthesis of phospholipids and their precursors in rat liver involving De novo methylation, and base-exchange pathways, in vivo☆

Abstract 1. 1. The livers of male rats were exposed to [Me-3H]choline, [1,2-3H]ethanol-amine, or [Me-3H]methionine by intraportal injection for periods varying from 15 s to 2 h. 2. 2. The incorporation of choline into hepatic phosphatidylcholines was predominantly by phosphorylation, utilized CDPcholine as an intermediate, and favored the more saturated subfractions of lecithin; whereas choline-exchange was negligible. The oxidation of choline to betaine was at least equal to the phosphorylation of choline. Betaine was rapidly demethylated and the methyl groups reutilized. 3. 3. The incorporation of ethanolamine into hepatic phosphatidylethanol-amines was predominantly by phosphorylation and utilized CDPethanolamine as an intermediate; whereas ethanolamine-exchange was negligible. 4. 4. After the injection of [1,2-3H]ethanolamine, radioactive maxima were observed in phosphorylcholine and CDPcholine prior to that in phosphatidylcholine, indicating that phosphorylethanolamine and/or CDPethanolamine were substrates for methylation. 5. 5. After the injection of methionine, radioactive maxima were observed in choline, betaine, and phosphorylcholine well before that in phosphatidylcholine, indicating their direct synthesis by the methylation of ethanolamine and phosphoryl-ethanolamine. 6. 6. Tritium from either [1,2-3H]ethanolamine or [Me-3H]methionine was preferentially incorporated into the more unsaturated subfractions of phosphatidylcholine.

Alterations in phosphatidylcholine species and their reversal in pulmonary surfactant during essential fatty-acid deficiency.

Previous studies (Kyriakides, E.C., Beeler, D.A. and Balint, J.A. (1974) Clin. Res. 22, 717a, and Burnell, J.M. and Balint, J.A. (1975) Fed. Proc. 34, 426) have indicated that essential fatty-acid deficiency in rats resulted in significant reduction of palmitate content of lung tissue and lavage phosphatidylcholines. Experiments were, therefore, undertaken to confirm and further characterize these changes and to examine the reversal of these alterations when essential fatty acid deficient rats were fed fat-free diets supplemented with linoleate for 1-14 days. Analysis of the fatty acid composition of liver lipids was used to confirm the presence of essential fatty-acid deficiency in rats that were fed a fat-free diet supplemented with 4% by weight of tripalmitoylglycerol for 14 weeks. Phosphatidylcholines from lung tissue and lavage fluid of essential fatty-acid deficient rats contained significantly less palmitate and significantly more palmitoleate and oleate than those rats receiving linoleate. These changes in fatty acid composition were reflected in a significant reduction of disaturated phosphatidylcholines (predominantly dipalmitoyl) in lung tissue and lavage fluid from essential fatty-acid deficient rats, while the total phosphatidylcholine content remained unchanged. On feeding the diet containing linoleate to the deficient rats, a reversal of these changes began after one day and was nearly complete by 7-14 days.

The pathways of lecithin biosynthesis in essential fatty acid deficiency in hamsters.

Abstract 1. 1. Male hamsters were fed fat-free and chow (control) diets for 12 weeks. At this time they were injected with an isotopic mixture containing 32 P i , [ Me - 14 C]methionine and [ Me - 3 H]choline and killed 1, 2 and 4 h afterwards. The incorporation of these labels into bile and the various fractions of liver phospholipids were studied. 2. 2. In the hamsters receiving the fat-free diet, the incorporation of all three isotopes into hepatic lecithins was increased. There was no change in the pool size of lecithin or of its water soluble phosphorylated precursors. 3. 3. The monoene and triene fractions of hepatic phospholipids from experimental animals were metabolically similar to the diene and tetraene fractions, respectively, of control animals. 4. 4. Cholesterol gallstones were observed in eleven of fifteen hamsters receiving the fat-free diet. None were observed in a similar number of controls. 5. 5. The normal diene lecithin fraction of bile was replaced by a monoene lecithin in the fat-free animals. Biliary phospholipids attained higher specific activities than any hepatic lecithin in control animals while the reverse was the case in the experimental group.

Water-soluble phospholipid precursor pool-sizes in quick-frozen and unfrozen rat livers

A more complete understanding of the various pathways of hepatic phospholipid biosynthesis and their possible importance in whole body metabolism could be gained from a quantitative rather than a qualitative description of the pathways. An initial step in the construction of a quantitative model of phospholipid biosynthesis should involve accurate determination of in vivo pool-sizes of phospholipid precursors. Published values of water-soluble phospholipid precursor pool-sizes in rat liver were found to vary over a wide range. This study was therefore undertaken with a twofold purpose: to determine accurate values for pool-sizes of water-soluble phospholipid precursors in rat liver and to examine postmortem changes in pool-size that could account for differences in published values.

Morphological alterations in type II alveolar cells in essential fatty acid deficiency

Abstract Type II epithelial cells from rats on a diet deficient in essential fatty acids demonstrated ultrastructural alterations not heretofore described in this tissue. Mitochondria became enlarged, rounded in outline, and cristae packed the intramitochondrial space. Mitochondrial vesiculation also occurred. The changes did not occur in other cell types, including bronchiolar (Clara) cells, in lung tissue. These events when correlated with biochemical and biophysical data concerning the alveolar surface materials from these animals suggests that a state of essential fatty acid deficiency provides an excellent experimental tool for further study of structural-functional relationships related to synthesis, storage, and secretion of the surface active material from Type II cells onto the alveolar surface.

Desaturation of endogenous and exogenous palmitate in lung tissue in vitro

Lung slices from rats fed a fat-free diet supplemented with safflower oil (control) or tripalmitoyl-glycerol (essential fatty acid [EFA]-deficient) were incubated with [14C] acetate, [14C] palmitate, or [14C] stearate. Of the14C recovered in phospholipids after incubation with [14C] acetate, more than 87% was in 16-carbon fatty acids. Desaturation, as assayed by the percentage of radioactivity in monoenoates in phospholipid fatty acids, was generally double in EFA-deficient slices compared to control slices, regardless of substrate. Desaturation was significantly greater in slices incubated with acetate or octanoate compared to palmitate, indicating that endogenously synthesized palmitate was desaturated more actively than that derived from an exogenous source.