The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

Is there an entero-hepatic circulation of the bile phospholipids?

Bile previously labeled with tritiated oleic acid (the main radioactivity was on bile phospholipids) was mixed with pure isolated phospholipids previously labeled with¹⁴C oleic acid; this mixture was perfused during 6 or 23 hr into the duodenum of test rats bearing a bile fistula. At the time of decapitation, in the small intestine a large hydrolysis of the¹⁴C phospholipids was found. In contrast no bile phospholipid hydrolysis was observed. In the collected bile samples of the test rats, no¹⁴C could be detected (this means a very large decrease of the¹⁴C fatty acids specific activities by the body fatty acids), and the tritiated fatty acids specific activities were only 2.5–12 times lower than in the perfused bile. These results can be explained, assuming that the bile phospholipids enter in an entero-hepatic circulation and are preserved from the dilution in a large pool of lipids.     

In vitro and in vivo effects of exogenous lipids on the enzymatic hydrolysis of rat bile phospholipids

The addition of total phospholipids, phosphatidylcholines, triglycerides, cholesterol or glycerol to incubation media containing rat pancreatic juice and bile labeled with [9,10³H₂] oleic acid (90% of the radioactivity present as phospholipids) had no effect on the hydrolysis of bile endogenous phospholipids. The introduction of 2 or 10 mg of phosphatidylcholines and 0.5 ml of bile (≈ 1.5 mg of phospholipids)into the rat upper duodenum decreased the rate of absorption of rative bile phospholipids. It was not followed by an increase of free fatty acids released from biliary phospholipids in the intestinal lumen. The introduction of bile (0.5 ml) and small amounts of triolein (1.4–3.5 mg) into the duodenum had little effect on the rate of hydrolysis and absorption of native bile phospholipids, but caused a reduced absorption of the free fatty acids released or those coming from initial nonphosphorus biliary lipids. The introduction of bile (0.5 ml) and large amounts of triolein (30 mg) into the duodenum increased the rates of hydrolysis and absorption of endogenous bile phospholipids. These observations suggest that luminal lipid components can modify the organization of luminal micelles and, consequently, the action of the pancreatic phospholipase A₂ and the absorption of bile lipids.     

Hydrolysis of biliary phospholipids in the upper small intestine of the chick

Chick endogenous phospholipids were doubly labeled by an intravenous injection of [³²P] phosphate and [1-¹⁴C] oleic acid, and the free fatty acid and phospholipid fraction of gall bladder bile and in contents of upper small intestine were analyzed 4 days later. There was evidence of hydrolysis of biliary phosphatidylcholine to lysophosphatidylcholine in the duodenum and jejunum, but this did not account for the pronounced increase in the¹⁴C radioactivity of the free fatty acids relative to the³²P phospholipid radioactivity between bile and upper intestinal segments. It is suggested that phosphatidylcholine is largely absorbed in the duodenum of the chick while the remainder is progressively hydrolyzed and absorbed.     

Lipid products formed during desaturation of [1-14C] stearyl CoA by hen liver microsomes

A number of lipid products are formed during the desaturation of stearyl CoA by hen liver microsomes. This article presents an analysis of the products formed when [1-¹⁴C] stearyl CoA is incubated with hen liver microsomes for various time periods. [1-¹⁴C] Oleyl CoA was the first radioactive unsaturated product formed. Synthesis of phospholipids containing [1-¹⁴C] oleic acid occurs only after the desaturase activity has begun to decline. The specific radioactivity of [1-¹⁴C] oleyl CoA was similar to the specific radioactivity of [1-¹⁴C] stearyl CoA at all time periods tested. The specific radioactivities of [1-¹⁴C] oleic acid and phospholipids containing [1-¹⁴C] oleic acid were much lower than that of the [1-¹⁴C] stearyl CoA.     

Discharge of newly-synthesized dolichol and ubiquinone with lipoproteins to rat liver perfusate and to the bile

An effective system for perfusing rat liver using complete tissue culture medium and washed calf erythrocytes as oxygen carriers was devised. Infusion of taurocholate and glucose proved necessary to maintain stable metabolic activity an bile secretion during a 6-hr period. Perfusate pO₂, pCO₂ and pH values were monitored continuously and found to be stable. Electron microscopic examination revealed the maintenance of normal hepatic structure, even after 6 hr. Normal rates of protein and urea synthesis, no leakage of cytoplasmic enzymes, and continuous bile acid production demonstrated the functional integrity of this system. Using [³H]mevalonic acid as precursor, dolichol, dolichyl phosphate, ubiquinone and cholesterol were found to be continuously synthesized in this perfusate, indicating discharge through the ER-Golgi system. The lipoproteins of the perfusate were isolated by ultracentrifugation and characterized with respect to size distribution and lipid composition. Dolichol was found in VLDL, LDL and HDL fractions, with the highest concentration present in the latter. In rat and human blood plasma this lipid was mainly associated with HDL. The ubiquinone in the perfusate was primarily associated with the VLDL fraction, while in rat plasma it was found more evenly distributed among all the three lipoprotein fractions studied. Dolichol, ubiquinone and cholesterol were also discharged to the bile, whereas dolichyl phosphate was not. Thus, newly-synthesized dolichol and ubiquinone are transported out of the hepatocyte to the blood and to the bile.     

Analytik plasmalogenhaltiger Phosphatide aus Herzgewebe mittels extracellulärer Phospholipase A2

The Analysis of Phospholipids from Cardiac Membranes by Phospholipase A₂. Phospholipase A₂ from snake venom has been employed to analyse the positional distribution of fatty acids in glycerolipids from guinea pig and pig cardiac membranes. It is known that phospholipase A₂ hydrolyses the fatty acids in sn-2 position of 1,2 diacylglycerophospholipids. In cardiac membranes phosphatidylcholine and phosphatidylethanolamine contain along with diacyl esters the corresponding alkenylether analogues of phospholipids. In the present experiments the alkenylether moieties were slowly hydrolysed by phospholipase A₂ from snake venom. We therefore separated by TLC lysophosphatides from fatty acids and unattacked phospholipids. The latter were the plasmalogens. The separated lipids were characterized by GLC. In some experiments the phospholipids were labelled with ¹⁴C-fatty acids before they were hydrolysed by phospholipase A₂. The alkenyl ether chain of plasmalogens seems to negativly influence the hydrolysis of fatty acids in sn-2 position of those phospholipids.     

Incorporation of [1-14C] acetate into lipids of soybean cell suspensions

Suspension cultures of soybean cells incorporated [1-¹⁴C] acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 min; more than 75% of this label was found to be in the oleic acid of the phosphatidylcholine. After longer periods of incubation, the proportion of¹⁴C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively,bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglycerols, and the glycolipid fraction. Contribution no. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977.     

Bile acid metabolism in mammals: VII. Studies on sex differences in deoxycholic acid metabolism in isolated perfused rat liver

The hepatic metabolism of deoxycholic acid was studied using the isolated perfused rat liver technique. In 20 perfusions, 10 involving the livers of male rats and 10 involving the livers of female rats, 30 μmoles deoxycholic acid was added to the perfusion medium. In 10 perfusions, 5 male and 5 female, 1 μmole deoxycholic acid was added to the perfusion medium. In 10 of the high dose studies and in the 10 low dose studies, 1 μCi deoxycholic acid-C-24-C¹⁴ also was added. The deoxycholic acid was added to 100 ml perfusion medium after 2 hr of baseline perfusion, and the studies were continued another 3 hr. Biliary bile acids were analyzed by combined thin layer and gas chromatography, and the radioactivity content of the perfusion medium and liver was documented. Although there was no sex difference in total bile acid secretion in the high dose studies, there were sex differences in the bile acid secretion rate and in the quantitative secretion of individual bile acids. The biliary secretion of deoxycholic acid and cholic acid was immediate in the female studies and delayed in the male, and the amounts of cholic acid and sulfated deoxycholyl-taurine secreted were considerably greater in the male studies. In the low dose studies the isolated perfused liver of the female rat converted more deoxycholic acid to cholyl-taurine than did that of the male rat. There are sex differences in the hepatic metabolism of deoxycholic acid. In contrast to those found in the case of chenodeoxycholic acid, these sex differences are not impressive when physiological amounts of deoxycholic acid are presented to the liver.     

Specificity of polyunsaturated fatty acid release from rat brain synaptosomes

Release of specific polyunsaturated fatty acids from cell membranes may have a significant implication in biological function, considering the involvement of various fatty acids in cell signal transduction. In the present study, release of polyunsaturated fatty acids from rat brain synaptosomes by endogeneous synaptosomal lipase activity was examined in comparison to that by cobra venom phospholipase A₂ (Naja naja naja). Cobra venom phospholipase A₂ (Naja naja naja) preferentially hydrolyzed docosahexaenoic acid (22∶6n−3) from both synaptosomes and lipid muxtures containing similar classes of lipids commonly found in the brain. Arachidonic acid (20∶4n−6) and oleic acid (18∶1n−9) were also hydrolyzed; however, monoene species was hydrolyzed slower than were polyenoic species in synaptosomes. Phosphatidylethanolamine was the most preferred phospholipid class for release of 22∶6n−3 fatty acid from both lipid mixtures and synaptosomes. In contrast to hydrolysis by cobra venom phospholipase A₂, endogenous synaptosomal lipase activity preferentially hydrolyzed 20∶4−6 from rat brain synaptosomes, despite the high abundance of 22∶6n−3 in synaptosomal membranes. Preferential release of 20∶4n−6 was observed over a wide range of pH values and calcium concentrations. Synaptosomal 22∶6 species appeared to be resistant to hydrolysis even after stimulation with various agents such as phorbolmyristate, suggesting that physiological importance of 22∶6−3 in neuronal membranes may not be as the release fatty acid.     

Composition and positional distribution of fatty acids in phospholipids isolated from pork muscle tissues

The composition and positional distribution of fatty acids in phospholipids isolated from four locations of a hog carcass is presented. Variations in fatty acid composition of phospholipids were found depending upon the location in the carcass. The total unsaturated fatty acid content averaged 34.3 mole % for lecithin, 52.5 mole % for phosphatidylethanolamine, 40.3 mole % for phosphatidylserine and 41.3 mole % in sphingomyelin. The cephalins had a much higher percentage of polyunsaturated fatty acids than lecithin. The chief saturated fatty acid in lecithin and sphingomyelin was palmitic and in cephalins it was stearic. A snake venom enzyme preparation(Crotalus adamanteus) hydrolyzed primarily unsaturated fatty acids in phosphoglycerides and the higher the percentage of unsaturation within the fatty acid the higher percentage of hydrolysis occurred. The unsaturated fatty acids were found chiefly at the theβ-position and the saturated fatty acids at thea-position in the phosphoglycerides. Michigan State Agricultural Experiment Station Publication No. 3389. Supported by the U.S. Public Health Service Research Grant No. GM 08801-03.     

Source of lung surfactant phospholipids: Comparison of palmitate and acetate as precursors

The phospholipids and the fatty acid compositions of major phospholipids in rat lung parenchyma, microsomes, lamellar bodies and alveolar wash were quantified. Adult rats were injected simultaneously with [³H] palmitate and [¹⁴C] acetate into the femoral vein. The appearance of labeled phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) in each lung fraction was measured during short periods of time (5 min to 2 hr) after isotope administration. Relatively more PC, DSPC and PG labeled with acetate radioactivity in lung microsomes entered lamellar body and alveolar wash fractions than those labeled with palmitate radioactivity. However, there was no difference between palmitate and acetate labeled phospholipids in the transport from microsomes to lamellar bodies by phospholipid exchange proteins. On the other hand, prior injection of colchicine resulted in decrease in the transport of PC from microsomes to alveolar space to a relatively greater extent in the acetate radioactivity than in the palmitate radioactivity.     

Nutrition and metabolic studies of methyl esters of dimeric fatty acids in the rat

Methyl esters of dimeric fatty acids were prepared by fractionating a mixture of conjugated linoleic and oleic acids that was heated for 24 hr at 300 C in the absence of air. Rats fed diets containing less than 1% dimers showed no significant difference (P<0.05) in the growth rate, feed efficiency, liver: body weight ratio, and lipid: liver weight ratio from those fed normal diets. A lymph cannulation study using¹⁴C labeled dimers showed that ca. 0.4% of the dimers fed were absorbed within 12 hr and were transported as free acids in the lymph. Within a 28 hr period, 2% of the labeled dimers fed by gastric intubation were oxidized to¹⁴CO₂, and 1% radioactivity was recovered from the urine. The metabolism of methyl oleate appeared normal for rats prefed diets containing dimers.     

Effect of phospholipase a upon brain cholesterol ester formation

On addition of snake venom phospholipase A to homogenates of rat brain we have been able to show increased ester formation. Within certain limits, the amount of ester formed was dependent upon the amount of phospholipase added. The fatty acid patterns of free fatty acid released and of cholesteryl ester fatty acid formed during 18 hr incubation were compared. Although fatty acid patterns were different, the data were still consistent with the hypothesis that, in demyelination, a neural phospholipase A may play a role in cholesteryl ester deposition.     

Thermal oxidation of model molecules to reveal vegetable oil polymerization studied by NMR spectroscopy and self-diffusion

Oxidative polymerization of plant oils and lipids is poorly understood yet widely encountered. Oils and fats are renewable resources providing biofuels and polymers. Oil oxidation is accelerated at high temperatures, typically above 110°C, where triacylglycerides are converted into toxic compounds and viscous deleterious polymers. Polymerization of mono-unsaturated oil (210°C, 3 h, open to air) was investigated by comparing four similar sized molecules with different functional groups: oleic acid, methyl oleate, trans-7-tetradecene, and stearic acid. Non-volatile products identified by NMR spectroscopy are minor ketones for saturated fatty acid (stearic acid), epoxides for acyl chains without acid groups (methyl oleate, tetradecane) and copious oligomerization, through ester cross-links, for acyl chains with acid, and olefinic groups (oleic acid). Long range C H coupling clearly shows ester (not ether) cross-links, contradicting long-held beliefs. Chain fragmentation also occurs for heated oleic acid as revealed by formation of a species with a methylene group bonded to oxygen of an ester,  CH₂ O C(O) . Large size (slow diffusion) of the first oligomer (trimer) formed from oleic acid, used to represent hydrolyzed vegetable oil, was evidenced by DOSY (diffusion-ordered spectroscopy). Combined NMR results show oligomers found in heated oleic acid are fatty acid estolides. Model oil reactions demonstrate why olefin and carboxylic acid groups are required for polymerization.     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

The effect of lipids on taurocholate absorption from intestinal loops in the rat

The rates of uptake and serosal trans-fer of [¹⁴C]-labelled taurocholate (7.77 mM in bicarbonate buffer, pH 6.5) were determined in situ in ligated segments of rat intestine in the presence of lipids. Oleic acid, monoolein, lecithin, and lysolecithin enhanced taurocholate uptake and transfer in the jejunum, each lipid exhibiting and optimal concentration at which the bile acid fluxes were maximal. The maximal rates of bile acid uptake observed with the various lipids were close to four times the uptake rates found with the lipid-free taurocholate medium, whereas serosal transfer rates under optimal conditions were enhanced about six-fold. The optimal concentrations differed widely among the various lipids, being inversely related to the lipids’ polarity. Simultaneous measurement of taurocholate and [³H]-labelled oleic acid showed that under optimal conditions, when the molar concentration of oleic acid was about equal to that of the bile acid, the fatty aicd and bile acid also exhibited closely similar rates of absorption. At other fatty acid concentrations, the fractional rate of absorption of the bile acid was much lower than that of the fatty acid. The rates of uptake and serosal transfer of pure taurocholate by the ileum exceeded those of the jejunum by factors of about 7 and 15, respectively, but in the presence of lipids this difference in absorptive capacity for bile acid between the distal and proximal segment largely disappeared.     

Tamoxifen-induced modification of serum lipoprotein phospholipids in the cockerel

The administration of tamoxifen (Tam), a nonsteroidal antiestrogen, or of a diphenyl-methane derivative of Tam that does not bind to the estrogen receptor (DPPE) of cockerels results in a marked decrease in the concentration of serum lipoprotein constitutents with an apparent alteration in phospholipid composition. To establish the nature of changes in phospholipids, the molecular species of phosphatidylcholine (PC) and sphingomyelin (Sph) were isolated and characterized. Between 9 and 18 hr following the administration of Tam or DPPE, there was a marked decrease in the proportion of molecular species of serum PC containing C16 and C18 fatty acids, but there was an increase in the proportion of molecular species containing C20 and C22 polyunsaturated fatty acids. Fatty acid analyses revealed that this change was due to an increase in arachidonic and docosaxaenoic acids at the expense of oleic and linoleic acids. These proportional changes were due to an absolute decrease in serum of PC molecular species containing palmitic and stearic acids in association with oleic and linoleic acids with very little change in the absolute concentration of molecular species containing arachidonic and docosahexaenoic acids. By contrast, the composition of Sph, which contained palmitic acid as the major fatty acid, was not altered during treatment. It is concluded that the short-term effect of Tam and DPPE on plasma phospholipids of the cockerel is due to a selective conservation of PC containing long chain polyunsaturated fatty acids.     

Uptake and metabolism of α-monopalmitin by rat lung in vitro

CO2 production from and uptake of alpha-glyceryl mono (palmitate-1-14C) were studied in an in vitro system using minced rat lung. Monoglyceride radioactivity was readily incorporated into lung tissue lipids. In a time course of 5-120 min, ca. 2.9-21.9% of the initial medium 14C-radioactivity was recovered in tissue lipids, including free fatty acid and monoglyceride, per one g of tissue. From 93 to 72% of the initial radioactivity remained in the medium during the same incubation periods. The ratio of tissue neutral lipid to phospholipid radioactivity decreased from 2:1 at 5 min to ca. 1:2.1 at 120 min. Most of the phospholipid-14C was in phosphatidyl choline, and this accounted for 80% of phospholipid-14C. Analysis of the tissue lipid radioactivity pattern revealed that during early periods of incubation (5-15 min) there was a rapid accumulation of 14C in monoglycerides and free fatty acids, which decreased with increasing incubation time concomitant with increase in radioactivity of tissue phospholipids and triglycerides. During the same time course, 6.5-85.3% of medium-14C was in free fatty acid, indicating the presence of an active alpha-monopalmitin-hydrolyzing system. After 2 hr of incubation, only 1.8% of the initial medium-14C had been oxidized to CO2. Under the same experimental conditions, 14C-alpha-monopalmitin and palmitate-1-14C were almost equally utilized and the patterns of lipid incorporated from both substrates were similar. It is suggested that rat lungs can utilize alpha-monopalmitin in a similar manner as palmitate after the former is hydrolyzed.     

Absorption and biliary secretion of intraperitoneally injected 3β-methoxycholest-5-ene-4-14C in the rat

Following ip injection of 1 mg amounts of methoxycholestene-4-¹⁴C into normal intact rats, the amount of label in the whole liver was measured at different time intervals in different animals. These studies indicated that most of the injected compound was absorbed by or before 12 hr. In bile duct-cannulated rats the secretion of label from ip injected methoxycholestene-4-¹⁴C or cholesterol-4-¹⁴C into bile was usually less during the first 24 hr than during the second 24 hr after injection. Studies on the nature of the labeled compounds in the bile following ip injection of methoxycholestene-4-¹⁴C indicate that most of the label is present in metabolites, 47% of the label in cholic acid and 31% in chenodeoxycholic acid.