The effect of bleomycin on the uptake and incorporation of [14C] choline into phospholipids in hamster lung tissue slices

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [¹⁴C] choline into phospholipids of lung slicesin vitro. The stimulatory effect is opposite to the results obtained previously using [¹⁴C] acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.     

Effect of chlorphentermine on incorporation of [14C]choline in the rat lung phospholipids

The effect of chlorphentermine (CP) treatment (50 mg/kg/ day, per os [po]) on the incorporation of [¹⁴C]choline into rat lung phospholipid was studied.Total phospholipid content was increased 2.0-fold and 1.7-fold after seven and 14 days, respectively, compared with the pair-fed rats. The incorporation of [¹⁴C]choline into phosphatidylcholine (PC) was significantly inhibited by either seven or 14 days of CP treatment. Nevertheless, the PC content was significantly increased by day 7 and stayed elevated at day 14 of CP treatment. Choline and phosphorylcholine contents were significantly decreased by the CP treatment. These results suggest that the higher accumulation of PC is due to inhibition of enzymes involved in the hydrolysis of phospholipids rather than to a stimulation of the phospholipid synthesis. Presented in part at the SOT Meeting, Atlanta, GA, March 1984 (abstracted inThe Toxicologist 4[1], 64).     

The incorporation of14C-glycerol into different species of diglycerides and triglycerides in rat liver slices

The relative rates of de novo synthesis of species of diglycerides and triglycerides from¹⁴C-glycerol were examined in rat liver slices. Diglycerides containing one or two double bonds per molecule and triglycerides containing four or more double bonds per molecule represented 70% and 60% respectively of the newly synthesized diglycerides and triglycerides. The newly synthesized triglycerides were more unsaturated than the endogenous triglycerides. Our results suggest that a nonrandom synthesis of species of diglycerides occurred followed by an almost random utilization of the various diglyceride species for the biosynthesis of triglycerides.     

The incorporation of phosphorylethanolamine into the phospholipids of brain microsomes in vitro

Synthesis of ethanolamine phosphoglycerides (EPG) from labeled¹⁴C-phosphorylethanolamine (PE) and cytidine triphosphate (CTP) has been studied in vitro in particles and in soluble fractions of chicken brain. The microsomal membranes can carry out this conversion, but supplementing the microsomes with an enzymic fraction derived from the particle-free supernatant results in a noticeable increase of the rate of EPG synthesis. Mitochondria are almost inactive, in this connection. The conversion of PE to lipid is very low, in no case exceeding 0.5–0.6%, even in the presence of diacyl glycerols or 1-alkenyl 2-acyl glycerol. No stimulation occurs by supplementing the incubation system with natural lipid acceptors, intermediates of lipid synthesis, energy-producing cofactors or monoacylsn-glycero-3-phosphorylethanolamine (GPE), monoacylsn-glycero-3-phosphorylcholine (GPC) and other lipid material. From these and other results, the conclusion is made that the PE:CTP cytidylyltransferase (E.C. 2.7.7.14) must display very low activity in vitro, thus limiting the overall rate of synthesis from PE. Diacyl GPE is the only lipid which has been found labeled after incubation with PE of the brain preparations. A small synthesis of alkenyl acyl GPE takes place, only when PE is incubated with suitable concentrations of a plasmalogenic diglyceride.     

In vitro incorporation of acetate-1-14C into the phospholipids of rabbit and human endometria

Endometria from nonpregnant and 6-day pregnant rabbits and from humans in the proliferative and secretory phases were incubated with 1-¹⁴C-acetate.¹⁴CO₂ was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of¹⁴C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of¹⁴C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria. Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolamine of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease. This investigation was supported by a grant from the Ford Foundation.     

The relative incorporation of arachidonic and eicosapentaenoic acids into human platelet phospholipids

The incorporation of arachidonic acid (AA) as compared to eicosapentaenoic acid (EPA) into human platelet phospholipids was tested by incubating washed platelets with a known mixture of [³H]AA and [¹⁴C]EPA. Following incubation, the platelet lipids were extracted, the individual phospholipids—phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE)—were separated by thin layer chromatography, and their corresponding [³H]/[¹⁴C] ratios were determined. Based on a [³H]/[¹⁴C] ratio of unity for the substrate mixture, the PC, PS, PI and PE exhibited ratios of 0.55, 0.93, 1.12 and 0.74, respectively, which were significantly different from 1.00 in all instances except in the case of PS. These results indicate that PC and PE selectively incorporated EPA, while PI showed preference toward AA. These selectivities may account partly for the differing AA/EPA mass ratios that have been observed among the individual phospholipids of human subjects consuming fish oils.     

Uptake of [14C] choline and incorporation into lung phospholipid by the isolated perfused rat lung

We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 mM. The rate of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extracellular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Km.     

3-甲基吲哚的合成工艺研究

3-甲基吲哚是一种重要的精细化工原料,可用于日用香精及食用香精的调配,作为定香剂使用.作者以丙醛和苯肼为原料,经缩合反应脱水生成丙醛苯腙,丙醛苯腙在高温下发生环化反应,最终得到产品3-甲基吲哚,分别考察了缩合过程中反应物投料比、环化反应过程中投料比及催化剂用量等条件对反应的影响.实验结果表明,当缩合反应n(丙醛):n(...     

Effects of omega-3 fatty acids on vascular smooth muscle cells: Reduction in arachidonic acid incorporation into inositol phospholipids

A rapid increase in arachidonic acid incorporation into phosphatidylinositol (PI) occurred following exposure of cultured porcine pulmonary artery smooth muscle cells to calcium ionophore A23187. This response was specific for PI and phosphatidic acid; none of the other phosphoglycerides showed any increase in arachidonic acid incorporation. The incorporation of [³H]inositol also was increased, indicating that complete synthesis of PI rather than only fatty acylation occurred in response to the ionophore. The presence of omega-3 fatty acids, especially eicosapentaenoic acid (EPA), reduced arachidonic acid but not inositol incorporation into PI. Stimulated incorporation of EPA also occurred under these conditions, suggesting that EPA replaces arachidonic acid in the newly synthesized pool of PI. Although much less arachidonic acid was incorporated into the polyphosphoinositides following exposure to the ionophore, arachidonic acid incorporation into these phosphorylated derivatives also decreased when EPA was present. These findings suggest that when omega-3 fatty acids are available, less arachidonic acid is channeled into the inositol phospholipids of activated smooth muscle cells because of replacement by EPA. This may represent a mechanism whereby omega-3 fatty acids, especially EPA, can accumulate in the metabolically active pools of inositol phospholipids and thereby possibly influence the properties or responsiveness of vascular smooth muscle.     

Dietary fat effect on incorporation and release of lipids and cholesterol by rat intestinal slices

The effect of saturated and unsaturated fats on in vitro formation and release of lipids and cholesterol from¹⁴C acetate by rat intestinal tissue was investigated. The rats were fed a basal diet enriched with either 25% corn oil or lard and then sacrificed after a 10- or 25-day feeding period. It was observed that a similar¹⁴C lipid content but a greater¹⁴C cholesterol content was found in the intestinal tissue of rats fed corn oil than in rats fed lard for 10 days. After a longer period of feeding of 25 days, the intestinal tissue¹⁴C cholesterol level was decreased in the corn oil fed rats without any significant effect on other lipids. These data suggest that corn oil in some way influences cholesterol biosynthesis depending upon its degree of unsaturation and the period of time for which it is fed. The decrease at the later time might involve some mechanism which aids in getting rid of accumulated tissue cholesterol. Less¹⁴C lipid and¹⁴C cholesterol were released by the intestinal tissue of rats fed the unsaturated fat as compared with those fed the saturated fat, suggesting a possible role in vivo in reducing blood lipids and blood cholesterol levels. Robert A. Welch Foundation.     

Fatty acid distribution in lipids and32P incorporation into phospholipids during early amphibian development

Several aspects of lipid composition and³²P incorporation were studied during early embryogenesis of the toad,Bufo arenarum, Hensel. The surveyed stages ranged from unfertilized oocyte to neural tube formation. The fatty acid distribution in polar and neutral lipids, as well as in acetone eluate from Unisil columns was similar in unfertilized oocyte and late blastula stage. There was no significant effect of cell cleavage on the fatty acid composition of these lipid fractions. Neutral lipids represent ca. 67% of the total lipids. The main components of the phospholipids were phosphatides of choline and ethanolamine. The total lipid and phospholipid content does not change through the studied stage of neurula. However a large increment in the phospholipid's specific radioactivity occurs when³²P is injected along with the hormone to induce ovulation. It is suggested that this may reflect changes in turnover rates rather than net biosynthesis. Since a large amount of cell membranes is being formed during the early development and because the level of phospholipids remains constant, an explanation is offered regarding membranogenesis. Active phospholipid biosynthesis may take place during oogenesis. These lipids may be stored in the yolk platelet, and fertilization may regulate the functioning of a transport mechanism to corresponding membrane sites. The increased incorporation of³²P may reflect changes in the activity of new membranes.     

Studies on the lipids of sheep red blood cells. II. The incorporation of phosphorus into phospholipids of HK and LK cells

The incorporation of inorganic phosphate (as NaH₂PO₄) into the phospholipids of sheep red blood cells was studied in vitro in blood samples from five highpotassium (HK) and five low-potassium (LK) sheep. The erythrocytes from HK sheep incorporated more activity in 4 hr than those from the LK sheep. However no activity was incorporated into the major phospholipids of the cells (phosphatidyl ethanolamine, phosphatidyl serine, and sphingomyelin) of either group. The phosphatidic acid fraction was labeled in both groups and to a significantly greater extent in the HK samples. However the highest activity in the phospholipid of sheep red-cells was located in three unknown compounds not previously detected. Their specific activities were the same in the HK and the LK samples although they were present in slightly larger amounts in the HK samples. In general, incorporation was at a rather low level, and from stoichiometric considerations it was concluded that the metabolism in the red-cell phospholipids could not be directly involved in the active transport of ions across the cell membrane. This work also confirmed a previous report that no quantitative differences exist among the major phospholipid classes in the two types of cells.     

Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents

Chronic feeding of 1-O-octadecyl-sn-glycerol (batyl alcohol) to patients suffering from congenital deficiency in tissue ether glycerolipids showed an increase in the plasmalogens content of their erythrocytes. However, nothing is known about the ether lipid content of other tissues in these patients. Feeding 1-O-heptadecyl-sn-glycerol to young rats showed that this uncommon ether lipid was incorporated to a high extent into the plasmalogens of all tissues except brain. Comparative studies with other precursors, such as 3-O-heptadecyl-sn-glycerol, heptadecanol and heptadecanoic acid, indicated a stereospecific incorporation of the dietary 1-O-alkyl-sn-glycerols into tissue plasmalogens without cleavage of the ether bond. Dietary ether lipids were also shown to be transferred from mothers to suckling rats, but not from pregnant rats to fetuses. The implication of these results to possible dietary ether lipid therapy for patients suffering from peroxisomal disorders is discussed.     

Glycerolipid biosynthesis in rat adipose tissue: VIII. Effect of obesity and cell size on [14C]acetate incorporation into lipids

[¹⁴C]Acetate incorporation into different lipid fractions was measured as a function of adipocyte size by using the larger and smaller adipocytes derived from Sprague-Dawley rats. In both the larger and smaller adipocytes, [¹⁴C]acetate was incorporated into phospholipid, diacylglycerol, free fatty acid and triacylglycerol fractions. Although the rates of lipid formation were significantly higher in the larger adipocytes compared to the smaller ones, the proportions of the various lipids formed from [¹⁴C]acetate did not change significantly as a function of cell size. In some experiments, isolated adipocytes derived from obese Zucker rats were fractionated further to isolate an adipocyte preparation which was similar in size to those obtained from lean animals. The matching adipocytes derived from lean and obese animals did not differ significantly with respect to lipid formation from [¹⁴C]-acetate. These studies suggest that the larger adipocytes are more active in lipogenesis from [¹⁴C]-acetate than the smaller ones and that the increased capacity of lipogenesis in obese adipose tissue noted proviously (Biochem. J., 170, 153–160, 1978) is not an intrinsic property of all the obese adipocytes, but is limited mainly to the larger adipocytes.     

Measurement of the incorporation of orally administered arachidonic acid into tissue lipids

The applicability of a stable isotope method to monitor the mixing of dietary arachidonic acid with endogenous arachidonic acid in tissue lipids was evaluated. Rats were fed octadeuterated arachidonic acid during a 20-day period, and the entry of the dietary acid into lipid esters of various tissues was examined by gas chromatographymass spectrometric (GC-MS) analysis of their fatty acids. The rats were maintained on a fat-free diet from weaning until 63 days old to enhance the ratio of the dietary acid to endogenous arachidonate. Three separate forms of eicosatetraenoic acid in the tissue lipids could be distinguished by GC-MS: octadeuterated arachidonic acid (recent dietary origin), unlabeled arachidonic acid (maternal origin) and unlabeled, 4,7,10,13-eicosatetraenoic acid (originating from palmitoleic acid). The total eicosatetraenoic acid in the tissue lipids contained about 90% arachidonate from recent dietary origin in lung, kidney, heart and fat, 70% in muscle and liver and 27% in brain. The n−7 isomer of eicosatetraenoic acid was estimated to make up 6% or less of the total eicosatetraenoic acid in lung, kidney, brain, muscle and heart tissue lipids, but it comprised around 15% of the total eicosatetraenoic acid in liver. The unlabeled arachidonic acid of maternal origin thus comprised only about 10% of the eicosatetraenoic acid in all tissues examined except muscle and brain, where it was 24% and 70% of the eicosatetraenoic acid, respectively. The relative amounts of the three forms of eicosatetraenoic acid are consistent with a limited access of dietary arachidonate to the brain tissue and with a competition between the dietary n−6 isomer and the endogenous n−7 isomer for esterification in the liver. Because most muscle mass would have formed after weaning, the high proportion of maternal arachidonate in the muscle lipids suggested that maternal arachidonate may have been displaced from other tissues to muscle, from which it equilibrated slowly with dietary arachidonate acid. The combination of deuterated arachidonic acid and GC-MS analysis thus furnished more detailed information about the composition and origin of eicosatetraenoic, acid in tissue lipid esters than that previously available from radiotracer studies or GC-MS analyses alone.     

Brain cholesterol. XII. The incorporation of 1-14C-acetate into baboon sterol

The incorporation of 1-¹⁴C-Acetate into tissue cholesterol of the baboon was measured. Using this indicator gray matter of the cerebrum indicated greater metabolic activity than did white matter. Other tissues besides neural tissue were examined. The peak of radioactivity occurred between 3 and 4 hr. The highest incorporation of radioactivity was measured in the adrenal gland. Liver, spleen and kidney values were of intermediate order.     

Incorporation ofcis-octadecenoic acids into the rat liver mitochondrial membrane phospholipids and adipose tissue triglycerides

The incorporation of the dietarycis 18∶1 (n−12) andcis 18∶1 (n−10) into liver mitochondrial membrane phospholipids and adipose tissue trigly cerides was studied in 4 groups of rats fed diets containing 10 weight percent (wt%) of fat with the following contents of octadecenoic acids: 50%cis 18∶1(n−12) +9%cis 18∶1 (n−9); 25%cis 18∶1 (n−12)+32%cis 18∶1 (n−9); 50%cis 18∶1 (n−10)+10%cis 18∶1 (n−9); or 54%cis 18∶1 (n−9). Dietary linoleic acid was 3 wt% in all 4 groups. In the mitochondrial membranes, the isomeric octadecenoic acids were primarily incorporated into the 1-position of phosphatidylcholines and phosphatidylethanolamines at the expense of saturated fatty acids. The maximal incorporations observed in the 1-position of phosphatidylethanolamines were 4.8% 18∶1 (n−12) and 8.9% 18∶1 (n−10). No effects on the contents of polyunsaturated fatty acids in the phospholipids were seen. In the adipose tissue, the isomeric octadecenoic acids were incorporated at a level of 13%cis 18∶1 (n−12) or 23%cis 18∶1 (n−10), paralleled by a reduction in the content of oleic acid. Presented in part at the 9th Scandinavian Symposium on Lipids, Visby, Sweden, June 1977.     

Enhanced incorporation of exogenous arachidonic acid into phosphatidylinositol and other phospholipids during the early stages of thrombin-induced aggregation in gerbil platelets

The degradation of platelet phospholipids via phospholipase activity is known to occur during thrombin-induced platelet aggregation. Both phosphatidylinositol and phosphatidylcholine are considered to be sources of the released arachidonic acid which becomes a substrate for prostaglandin and thromboxane A₂ formation. In this work, the effect of thrombin on the incorporation of exogenous arachidonic acid into platelet membrane phospholipids was studied. Suspensions of gerbil platelets were incubated in aggregometer cuvettes with [¹⁴C] arachidonic acid in the absence or presence of thrombin, and product formation was monitored by thin layer chromatography and scintillation counting. Within 30 sec, the entry of arachidonic acid into phosphatidylinositol was increased by 165% in thrombin-stimulated platelets over controls. Under identical conditions, the incorporation into phosphatidylcholine was increased by only 57%. These results suggest that the incorporation of exogenous arachidonic acid via lysophosphatidylinositol and lysophosphatidylcholine acyltransferase activities may be intimately associated with thrombin-induced platelet aggregation in the gerbil. Presented in part at the 73rd annual AOCS meeting, Toronto, 1982.     

Effect of dietary α-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants

The fractional conversion rates of plasma phospholipid α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) to docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6), respectively, and the fractional rates of incorporation of 22:6n-3 and 20:4n-6 into plasma phospholipids were determined in 27 healthy 3-wk-old term infants who had received formulas with ≈16% of fat as 18:2n-6 and 0.4% (n=6), 1.0% (n=11), or 3.2% (n=10) as 18:3n-3 from birth. The infants were given a single dose of both [U-¹³C] 18:2n-6 and [U-¹³C]18:3n-3 with a feeding, and blood samples were collected 8, 12, and 24 h afterward for determination of the isotopic enrichments of the [M+18] isotopomers of plasma phospholipid fatty acids by negative chemical ionization gas chromatography/mass spectrometry. A simple precursor/product compartmental model was used to estimate fractional rates of conversion and incorporation. All infants converted 18:3n-3 to 22:6n-3 and 18:2n-6 to 20:4n-6. Although the fractional rate of conversion of 18:3n-3 to 22:6n-3 did not differ among groups, the fractional rate of incorporation of 22:6n-3 into the plasma phospholipid fraction was greater in infants who received 3.2% vs. 0.4% or 1.0% 18:3n-3 (4.1±2.2 vs 1.6±1.5 or 2.0±1.0% of the plasma phospholipid 22:6n-3 pool daily). The fractional rate of conversion of 18:2n-6 to 20:4n-6 was less in infants who received the 3.2% 18:3n-3 intake (0.4±0.3% of the plasma phospholipid 18:2n-6 pool daily vs. 1.1±0.7% and 0.8±0.5% in those who received 0.4 and 1.0% 18:3n-3, respectively). The fractional rate of incorporation of 20:4n-6 into plasma phospholipid also was less in the 3.2% vs. the 0.4 and 1.0% 18:3n-3 groups (2.7±1.4% vs. 5.9±2.6 and 4.4±1.7%, respectively, of the plasma phospholipid 20:4n-6 pool daily).     

Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on fatty acid absorption, incorporation into serum phospholipids and postprandial triglyceridemia

Fourteen healthy volunteers were randomly allocated to receive 4 g highly purified ethyl esters of eicosapentaenoic acid (EPA) (95% pure, n=7) or docosahexaenoic acid (DHA) (90% pure, n=7) daily for 5 wk in supplement to their ordinary diet. The n−3 fatty acids were given with a standard high-fat meal at the beginning and the end of the supplementation period. EPA and DHA induced a similar incorporation into chylomicrons which peaked 6 h after the meal. The relative uptake of EPA and DHA from the meal was >90% compared with the uptake of oleic acid. During absorption, there was no significant elongation or retroconversion of EPA or DHA in total chylomicron fatty acids. The concentration of EPA decreased by 13% and DHA by 62% (P<0.001) between 6 and 8 h after the meal. During the 5-wk supplementation period, EPA showed a more rapid and comprehensive increase in serum phospholipids than did DHA. DHA was retroconverted to EPA, whereas EPA was elongated to docosapentaenoic acid (DPA). The postprandial triglyceridemia was suppressed by 19 and 49% after prolonged intake of EPA and DHA, respectively, indicating that prolonged intake of DHA is equivalent to or even more efficient than that of EPA in lowering postprandial triglyceridemia. This study indicates that there are metabolic differences between EPA and DHA which may have implications for the use of n−3 fatty acids in preventive and clinical medicine.