Effect of Zinc Deficiency on the Biosynthesis of Phosphatidylcholine in Rat Microsomes

Phosphatidylcholine is the major lipid of all cellular membranes. Phosphatidylcholine biosynthesis in microsomes involves two enzyme pathways, choline phosphotransferase and phosphatidyl-ethanolamine methyltransferase. The present study was designed to examine the effect of zinc deficiency on these two enzymes. Male, weanling Long-Evans rats were fed a biotin-enriched 20% egg white diet deficient in zinc for 15–45 d. The specific activity (pmol phosphatidylcholine formed/min/mg microsomal protein) of choline phosphotransferase, phsophatidylethanolamine methyltransferase, and phos-phatidyldimethylethanolamine methyltransferase was determined. The latter assay measures the third methylation of phosphatidyl-ethanolamine to phosphatidylcholine. Zinc deficiency resulted in a significant increase over controls in the specific activity of phospha-tidylethanolamine methyltransferase and phosphatidyldimethyl-ethanolamine methyltransferase in liver and spleen microsomes. A significant increase in the picomoles of phosphatidylcholine formed by the choline phosphotransferase pathway occurred in liver microsomes of zinc-deficient animals. In the brain microsomes a significant decrease in specific activity of phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase, and choline phosphotransferase occurred among zinc-deficient ani-mals. These data suggest that zinc deficiency alters the biosynthesis of phosphatidylcholine, the major lipid of cellular membranes.     

The effect of hyper and hypothyroidism, hypophysectomy and adrenalectomy on phosphatidylethanolamine methyltransferase, phosphatidyldimethyl-ethanolamine methyltransferase and choline phosphotransferase of rat liver microsomes

The effect of hyper- and hypothyroid, hypophysectomy and adrenalectomy on phosphatidylcholine biosynthetic enzymes, phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of liver microsomes was measured in rats. There was a significant increase in the specific activity of phosphatidylethanolamine methyltransferase in the hyperthyroid rats. There was a significant reduction in the specific activity of phosphatidylethanolamine methyltransferase and phosphatidyldimethylethanolamine methyltransferase in the hypothyroid states. The choline phosphotransferase increased significantly in the hyperthyroid state and decreased in the hypothyroid animals. Hypophysectomy resulted in a significant increase in specific activity of choline phosphotransferase. A reduction in the specific activity of the phosphatidylethanolamine methyltransferase occurred after 28 days of hypophysectomy. Adrenalectomy resulted in a significant stimulation of the specific activity of phosphatidylethanolamine methyltransferase and choline phosphotransferase in liver microsomes.     

Brain and serum levels of choline and lecithin resulting from longterm administration

Choline and phosphatidylcholine tissue concentrations were examined in mice treated with long-term (18–22 month) dietary choline enrichment, choline deficiency, or phosphatidylcholine enrichment. There were no significant differences found in choline levels among the dietary groups in any of the tissues examined: plasma, erythrocytes, cortex, hippocampus, and striatum. In contrast, the concentration of phosphatidylcholine in both the choline enriched and phosphatidylcholine enriched groups were significantly increased in the cortex, compared to the choline deficient group, and in the striatum, compared to control. No differences in phosphatidylcholine concentration were found in the hippocampus or plasma between any of the dietary groups. These results are in contrast to the reported effects of acute or short-term choline and phosphatidylcholine treatment and indicate that there may be differences between the effects of short-term and long-term administration on the blood and brain levels of choline and phosphatidylcholine.     

Phospholipid metabolism in roots and microsomes of sunflower seedlings: Inhibition of choline phosphotransferase activity by boron

The action of boron on phospholipid composition and synthesis in roots and microsomes from sunflower seedlings has been studied. The fatty acid composition and relative amounts of individual molecular species of phospholipids in roots and microsomes were very similar. In both the content of phospholipids was decreased and the relative levels of their component fatty acids changed by treatment with 50 ppm of boron. This concentration of boron in the culture medium was found to inhibit the in vivo [1-^14C] acetate incorporation into root lipids and that of [Me-¹⁴C] choline into phosphatidylcholine of root microsomes. Cytidine-5-diphospho (CDP)-[Me-¹⁴C] choline incorporation into phosphatidylcholine of isolated microsomes was also inhibited by 50 ppm of boron when present in the growth medium of seedlings. These results indicate that the decrease in phosphatidylcholine labelling from [¹⁴C] choline observed when root microsomes were treated with boron would be caused by a decrease in CDP-choline phosphotransferase activity.     

Phosphatidylethanolamine levels and regulation of phosphatidylethanolamine N-methyltransferase

The activity of phosphatidylethanolamine (PE) N-methyltransferase in liver microsomes, measured using endogenous microsomal PE as a substrate, was elevated 2-fold in the choline-deficient state. However, methyltransferase activity assayed in the presence of a saturating concentration of phosphatidyl-N-mono-methylethanolamine or microsomal PE was unchanged by choline deficiency. Accompanying the increase in methyltransferase activity in liver homogenates and microsomes were increased PE concentrations and an increased PE to phosphatidylcholine ratio. The concentration of other phospholipids was unchanged. Immunoblot analysis of choline-deficient and choline-supplemented rat liver microsomes using a rabbit polyclonal anti-PE N-methyltransferase antibody revealed that the amount of enzyme protein was unaltered. The regulation of methyltransferase by PE levels was also investigated in cultured hepatocytes obtained from choline-deficient rat livers. Supplementation of deficient hepatocytes with 200 microM methionine resulted in a 50% reduction in cellular PE levels over a 12-h period. PE N-methyltransferase activity assayed with endogenous PE was also reduced by 50%, but phosphatidyl-N-monomethylethanolamine-dependent activity was unchanged. A 4-h supplementation with choline did not affect PE levels or methyltransferase activity. Either methionine or choline supplementation resulted in net synthesis of cellular phosphatidylcholine. Immunoblotting of membranes from methionine-supplemented hepatocytes revealed no change in enzyme protein, a further indication that enzyme mass was constitutive, and activity was regulated by the concentration of PE.     

Effect of dietary copper and zinc levels on tissue copper,zinc, and iron in male rats

The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 μg/g) and five levels of zinc (1, 4.5, 10, 100, 1000 μg/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 μg Cu, 100 μg Zn/g diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily when dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.     

Marginal copper deficiency and atherosclerosis

Copper is an essential trace element in the maintenance of the cardiovascular system. Copper-deficient diets can elicit, in animals, structural and functional changes that are comparable to those observed in coronary heart disease. In this study, the effect of dietary-induced copper deficiency on aortic lesion development was measured by quantitative image analysis in C57BL/6 mice that are susceptible to diet-induced aortic lesions. The diets administered were severely copper deficient (0.2 mg/kg diet), marginally deficient (0.6 mg/kg diet), or copper adequate (6.0 mg/kg diet). Similarly, increased aortic lesion areas and elevated serum cholesterol were demonstrated in both deficient groups, compared with the copper-adequate group. Evidence for graded differences in copper status among the dietary groups was shown by the dose-response increase in liver copper concentration, copper-zinc superoxide dismutase and cytochrome-c oxidase activities, together with serum caeruloplasmin oxidase with increasing intakes of dietary copper. Despite the difference in copper status between the copper marginal and severely deficient groups, similar lesions found in both groups of mice suggest a threshold effect of copper deficiency on lesion formation.     

Regulation by Lipids of Plant Microsomal Enzymes: III. Phospholipid Dependence of the Cytidine-Diphospho-Choline Phosphotransferase of Potato Microsomes

Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato (Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[¹⁴C]choline into phosphatidylcholine was characterized by the time course of ¹⁴C incorporation and the effect of microsomal protein concentration on choline incorporation.

Potato microsomes were progressively delipidated by treatments (2 min at 0°C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme.

Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory.

These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes.

     

Effects of deoxycholate and phospholipase A2 on choline and ethanolamine phosphotransferases of chicken brain microsomes.

Ethanolamine phosphotransferase (EC 2.7.8.1) and choline phosphotransferase (EC 2.7.8.2) activities were assayed in fresh microsomes from adult chicken brains with either diacylglycerols or alkylacylglycerols. Pretreatment of microsomes with 1.25 mM sodium deoxycholate, a concentration less than the critical micelle concentration, produced a slight inhibition of choline phosphotransferase activity. A deoxycholate concentration (5.0 mM) greater than the critical micelle concentration (3.0 mM) decreased the choline phosphotransferase activity by more than 70% but had no effect on ethanolamine phosphotransferase activity. Inclusion of 1.25 mM deoxycholate in the assay medium decreased choline phosphotransferase activity 35% but increased ethanolamine phosphotransferase activity 50%. The deoxycholate appeared to inactive the choline phosphotransferase. Phospholipase A2 (Vipera russelli) treatments of microsomes removed phosphoglycerides and decreased both phosphotransferase activities to a similar extent. Decreased activities are probably due to disruption of the membrane structure. Choline and ethanolamine phosphotransferase activities are apparently in different enzymes which lack specificity for the type of diglyceride. Thus, the systematic names should include 1,2-diradyl-sn-glycerol instead of 1,2-diacyl-sn-glycerol.     

Copper deficiency depresses rat aortae superoxide dismutase activity and prostacyclin synthesis

Prostaglandin synthesis shows dependence on lipid hydroperoxides and resultant oxygen derived radical formation. In view of the importance of dietary copper in cytosolic copper dependent superoxide dismutase (Cu/Zn SOD) activity and the role of SOD in oxygen radical formation, the influence of dietary copper on prostacylin (PGI2) synthesis and SOD activity in rat aorta was examined. Copper deficient (0.5 micrograms Cu/g diet) rats showed a significant 47% reduction in PGI2 synthesis rates by aortic ring incubations in comparison to copper adequate (6.0 micrograms Cu/g diet) animals. Aortic SOD activity was reduced by 46% in copper deficiency in comparison to copper adequate animals. Marginal dietary copper (1.6 micrograms Cu/g diet) significantly reduced aortic SOD activity by 32% but was without effect on aortic ring incubation PGI2 synthesis. These results indicate that dietary copper deficiency, and the resultant decrease in SOD activity, depresses aortic PGI2 synthesis.     

Choline deficiency induces apoptosis in primary cultures of fetal neurons.

Treatment of rats with choline during brain development results in long-lasting enhancement of spatial memory whereas choline deficiency has the opposite effect. Changes in rates of apoptosis may be responsible. We previously demonstrated that choline deficiency induced apoptosis in PC12 cells and suggested that interruption of cell cycling due to a decrease in membrane phosphatidylcholine concentration was the critical mechanism. We now examine whether choline deprivation induces apoptosis in nondividing primary neuronal cultures of fetal rat cortex and hippocampus. Choline deficiency induced widespread apoptosis in primary neuronal cells, indicating that cells do not have to be dividing to be sensitive to choline deficiency. When switched to a choline-deficient medium, both types of cells became depleted of choline, phosphocholine and phosphatidylcholine, and in primary neurons neurite outgrowth was dramatically attenuated. Primary cells could be rescued from apoptosis by treatment with phosphocholine or lysophosphatidylcholine. As described previously for PC12 cells, an increase in ceramide (Cer) was associated with choline deficiency-induced apoptosis in primary neurons. The primary neuronal culture appears to be an excellent model to explore the mechanism whereby maternal dietary choline intake modulates apoptosis in the fetal brain.     

Diaphragmatic fatigue in normoxia and hyperoxia

The effect of choline deficiency on the lung lipids of actively growing male Sprague-Dawley rats was investigated using a washed soy protein diet deficient in choline and methionine (lipotrophic). The livers from deficient animals had a significantly increased total lipid content and decreased phosphatidylcholine (PC) content and PC-to-phosphatidylethanolamine ratio (P less than 0.01). Although lung free choline levels were decreased 40% compared with controls (P less than 0.05), the PC content of the whole lung homogenate was unchanged. However, disaturated phosphatidylcholine from animals receiving the lipotrophic diet was significantly increased in the lavage and proportionally decreased in the lavaged lung tissue compared with controls (P less than 0.01). This study indicates that, despite decreased lung choline levels as a result of ingesting a lipotrophic diet, and unlike the liver, lung PC content is maintained at normal values. Although the lung total PC levels are maintained, there is a change in the partition of this lipid pool between the tissue and the alveolar space.     

Effect of copper deficiency on the concentrations of catecholamines and related enzyme activities in the rat brain.

Immature rats were made copper deficient by feeding them a low (< 1 p. p. m.) copper diet. During the gestation and lactation periods their dams consumed the same diet. Controls received a dietary supplement of 10 p. p. m. copper. At approx 7 weeks of age, the deficient animals exhibited signs of neurological dysfunction and gross lesions of the brain. Cytochrome oxidase activity and copper content of the liver and brain were used as criteria of copper status and confirmed the existence of severe deficiency. The whole brains minus cerebella of the deficient animals contained approx 30% less dopamine and norepinephrine than those of the controls. The tyrosine hydroxylase activity was depressed more than 25% in the copper deficient brains while the superoxide dismutase activity was lowered more than 35%. There was a high correlation between the chief criterion of copper status, liver cytochrome oxidase activity, and the brain concentrations of dopamine, norepinephrine and tyrosine hydroxylase activity. The decrease in activity of tyrosine hydroxylase was sufficient to account for the lowered concentrations of the catecholamines.     

Female rats are protected against the fructose induced mortality of copper deficiency

Experiments were conducted in copper deficient male and female rats fed diets containing fructose or starch in order to determine whether the same type of interaction between copper status and dietary carbohydrate found in male rats also occurs in the female rat. Mortality occurred only in the male rats fed the fructose diet deficient in copper with 40% of the animals dying during the 8 week study. Only anemia, hypercholesterolemia, increased BUN, heart hypertrophy and reduced body weight were observed in these animals which could be related to their mortality. Despite the increased mortality, plasma ceruloplasmin, erythrocyte SOD and hepatic copper concentrations were reduced to a similar extent in all rats regardless of the sex of the animals or of the type of dietary carbohydrate fed. The results of the present study indicate that although direct measurements of copper status of female rats fed fructose diet deficient in copper are similar to their male counterpart, they are apparently protected from the lethal consequences of the deficiency.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Effects of choline deficiency and methotrexate treatment upon rat liver

Choline deficiency and treatment with methotrexate (MTX) both are associated with fatty infiltration of the liver. Choline, methionine, and folate metabolism are interrelated and converge at the regeneration of methionine from homocysteine. MTX perturbs folate metabolism, and it is possible that it also influences choline metabolism. We fed rats a choline deficient diet for 2 weeks and/or treated them with methotrexate (MTX; 0.1 mg/kg daily). Choline deficiency lowered hepatic concentrations of choline (to 43% control), phosphocholine (PCho; to 18% control), glycerophosphocholine (GroPCho; to 46% control), betaine (to 30% control), phosphatidylcholine (PtdCho; to 62% control), methionine (to 80% control), and S-adenosylmethionine (AdoMet; to 57% control), while S-adenosylhomocysteine (AdoHcy) and triacylglycerol concentrations increased (to 126% and 319% control, respectively). MTX treatment alone lowered hepatic concentrations of PCho (to 48% control), GroPCho (to 69% control), betaine (to 55% control), and AdoMet (to 75% control). The addition of MTX treatment to choline deficiency resulted in a larger decrease in AdoMet concentrations (to 75% control) and larger increases in AdoHcy and triacylglycerol concentrations (to 150% and 500% control, respectively) than was observed in choline deficiency alone. Livers from MTX-treated animals used radiolabeled choline to make the same metabolites as did livers from controls (most of the label was converted to PCho and betaine). In choline deficient animals, most of the labeled choline was converted to PtdCho. Therefore, MTX depleted hepatic PCho, GroPCho, and betaine by a mechanism that was different from that of choline deficiency. MTX increased the extent of fatty infiltration of the liver in choline deficient rats, and choline deficiency and MTX treatment damaged hepatocytes as measured by leakage of alanine aminotransferase activity. Our data are consistent with the hypothesis that the fatty infiltration of the liver associated with MTX treatment occurs because of a disturbance in choline metabolism.     

Copper and ischemic heart disease

Absolute or relative deficiency of copper is hypothesized to be of prime importance in the etiology of ischemic heart disease. According to recent estimates, only 25% of the diets in the United States contain the 2 mg of copper thought to be required daily by adults. Some of these diets have ratios of zinc to copper greater than those that have produced hypercholesteremia in animals. There are many epidemiologic associations between the ratio of zinc to copper and dietary characteristics, organ analyses, clinical status, and environmental features that relate the metabolism of these elements to the anatomy, chemistry, pathology, pharmacology, and physiology of ischemic heart disease. Animals deficient in copper or exposed to a high dietary ratio of zinc to copper, which can produce a relative copper deficiency, are hypercholesteremic and hyperuricemic, and have glucose intolerance and abnormalities of the electrocardiogram. Their hearts and arteries have abnormal connective tissue, lipid deposits, and inflammatory changes; they die suddenly, often with ruptured hearts. Hypercholesteremia and glucose intolerance have been found in men depleted of copper and in children with Menkes’ disease, an inability to absorb copper.     

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.     

Studies of the biochemical basis of steroid sulphatase deficiency--III. Phospholipid composition of microsomes from normal and steroid sulphatase deficient placentas

The phospholipid composition has been determined for placental microsomes from 11 normal and eight pregnancies complicated by steroid sulphatase deficiency. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal placental microsomes, comprising respectively 41.6 +/- 4.6% (mean +/- SD). 30 +/- 5.7% and 22.5 +/- 4.9% of the total phospholipid content. There was no correlation between the steroid sulphatase activity of the microsomes and the content of any of the three phospholipids. Though their contents were significantly decreased. (P less than 0.001) phosphatidylcholine, phosphatidylethanolamine and sphingomyelin similarly constituted the major portion of the total phospholipids in sulphatase deficient microsomes, representing 36 +/- 4.2%, 34 +/- 6.1% and 22.4 +/- 6.7% respectively. Only the percentage of phosphatidylcholine was significantly different (P less than 0.02) from normal microsomes. The results show that the decreased phospholipid content of steroid sulphatase deficient placental microsomes reflects a lower content of all major classes of phospholipids, particularly phosphatidylcholine.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.