Extraction of phospholipids and analysis of phospholipid molecular species.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S J Robins
Publications and source records attributed to S J Robins.
Analysis of lipids by high performance liquid chromatography. Part II: phospholipids.
Explore the source record for details and available documents.
Journal Article↗
Analysis of lipids by high performance liquid chromatography: part I.
Explore the source record for details and available documents.
Journal Article↗
Effect of bile salt on phosphatidylcholine composition and secretion of hepatic high-density lipoproteins.
Bile salts are necessary for the secretion of phosphatidylcholines (PCs) in bile and result in the selective secretion of highly hydrophilic molecular species of PC that contain a 16:0 acyl group. To determine the effect of bile salt on the secretion of PCs in lipoproteins, isolated rat livers were perfused with and without taurocholate. The PC composition of very-low-density lipoproteins (VLDL), newly synthesized by the liver, precisely mirrored the composition of PCs in the whole liver and was not changed with the administration of taurocholate. In contrast, both the composition of PCs in high-density lipoproteins (HDL) and the absolute amount of newly synthesized HDL were markedly affected by the administration of taurocholate. With taurocholate the PC content of HDL was increased, HDL was enriched, like bile, in 16:0 molecular species of PC, and the amount of HDL that was recovered in the perfusate was 2.5-fold greater than without taurocholate (P less than 0.001). These findings suggest that VLDL and HDL are differently derived from within the liver, that the PCs of HDL and bile originate from the same hepatic pool or by the same mechanism, and that both the secretion of bile and HDL from the liver are susceptible to regulation by bile salt.
Animals↗
The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal.
Studies were undertaken to assess the contribution of lipoprotein cholesterol to bile and to determine whether already-existent hepatic free cholesterol and the free cholesterol which is newly generated from the hydrolysis of hepatic cholesteryl esters are equally available for secretion into bile or constitute metabolically separate pools. Rats with a bile fistula were injected with an intravenous bolus of high-density lipoprotein recombinants containing free [14C]cholesterol and [3H]cholesteryl esters. Results showed (1) that bile free [14C]cholesterol secretion was a constant and linear proportion of the whole liver free [14C]cholesterol pool, (2) that secretion into bile of free [3H]cholesterol was in direct proportion to the rate of hydrolysis of hepatic [3H]cholesteryl esters, and (3) that rates of biliary cholesterol secretion were very similar when secretion was calculated using the specific activity of free [14C]cholesterol and free [3H]cholesterol in the entire liver to 'label' the precursor free cholesterol pool. Furthermore, rates of secretion that were calculated using either isotope closely approximated the mass of free cholesterol that was directly measured in bile. Results thus indicate that because of equilibration and extensive dilution by the large pool of already-existent free cholesterol, the transport of isotopic cholesterol from lipoproteins cannot be used to directly assess the contribution of lipoprotein cholesterol to the cholesterol that is secreted in bile. These studies further suggest that the totality of preformed free cholesterol in the liver is in metabolic equilibrium in one single kinetic pool and that all hepatic free cholesterol is potentially available for secretion into bile.
Animals↗
Response of serum triglycerides of endogenous origin to the administration of triglyceride-rich lipid particles.
Studies were performed in rats to quantitate the changes in the concentration of serum triglycerides (TGs) of endogenous and exogenous origin after the acute intravenous injection of TG-rich emulsion particles. Emulsions were prepared to approximate chylomicrons and to contain a TG with a single fatty acid that could be traced during its clearance from the serum. After injection of emulsions, there was a rapid increase of not only the emulsion TG but TGs that contained a variety of other fatty acids of endogenous origin. Endogenous TGs were cleared from the serum at a slower rate than the emulsion TG and accounted for the major increase in serum TGs, especially during the latter phase of the clearance period. The increase in endogenous TGs was completely abolished by hepatectomy, which had no effect on the clearance of the emulsion TG. Results thus show that TGs of hepatic origin accumulate in the serum in response to the introduction of new TG-rich lipoproteins. Feeding rats a specific TG produced a similar result, with a pronounced rise in endogenous TGs that, like the changes after emulsion administration, was particularly evident once the TG that was fed was largely cleared from the serum. These findings are consistent with a process in which the preferential clearance of chylomicron TGs interrupts the clearance of very low density lipoprotein TGs that are produced by the liver. Consequently, the composition of serum TGs that accumulate after a meal may not reflect the composition of the meal itself.
Animals↗
Effect of different molecular species of phosphatidylcholine on the clearance of emulsion particle lipids.
Studies were performed to assess the effect of changes in the molecular species composition of phosphatidylcholine (PC) on the clearance of emulsion particles that were made to approximate chylomicrons in size and lipid composition. Emulsions were prepared with free [14C]cholesterol, [3H]cholesteryl oleate, triolein, and one of four single PCs that differed in hydrophilic strength (as assessed by the relative rate of elution of these PCs from a reverse phase column). Emulsions were injected as an intravenous bolus into unanesthetized rats and the clearance of lipids was determined at 2-min intervals for 10 min. All emulsion lipids were cleared from the serum in parallel and in an order that closely corresponded to the relative hydrophilic strength of the particular PC that was administered. Fractional rates of clearance, calculated from log-linear plots, were 2- to 10-fold greater for all lipids for the emulsion that was made with the most hydrophilic PC compared to the least hydrophilic PC. Although hepatectomy, performed in acutely anesthetized animals, generally slowed the clearance of lipids, hepatectomy did not abolish differences in the clearance of triolein or specific PCs from emulsions prepared with the most and least hydrophilic PCs. These results indicate that a change in the composition of emulsion particle PCs, independent of any other change in the lipid composition of these particles, can significantly change the metabolism of the whole emulsion particle in the live animal. More specifically, these studies show that the rate of clearance of all emulsion lipids closely corresponds to the hydrophilic strength of the PCs that occupy the emulsion particle surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Influence of different molecular species of phosphatidylcholine on cholesterol transport from lipoprotein recombinants in the rat.
Studies were performed to determine to what extent phosphatidylcholines (PCs) of different composition influence the turnover of lipoprotein cholesterol. Lipoprotein recombinants with the composition and structure of spherical high density lipoproteins (HDL-R) were prepared with apoproteins, 14C-labeled unesterified cholesterol (UC), a [3H]cholesteryl ester (CE), and one of four single molecular species of PC. PCs were selected to include relatively hydrophilic species (16:1-16:1 and 16:0-18:2 PCs) and relatively hydrophobic species (18:0-18:2 and 20:1-20:1 PCs). PCs were also selected to include molecules with novel acyl group pairs (16:1-16:1 and 20:1-20:1 PCs) that would permit the whole molecule to be traced during its clearance from the serum. Rats were injected with HDL-R as an intravenous bolus, and serum, liver, and bile samples were obtained for up to 2 h. The clearance from the serum of each PC was monoexponential with the two most hydrophilic species much more rapidly cleared than either of the two less hydrophilic species. Clearance of specific PCs was not accompanied by PC remodeling (i.e. transacylations), and in the main could not be attributed to the action of lecithin-cholesterol acyltransferase (LCAT). In incubations designed to simulate in vivo conditions, no more than 15% of the disappearance of 16:1-16:1 PC, one of the most rapidly cleared PCs, was due to the action of LCAT. With 20:1-20:1 PC, one of the least rapidly cleared PCs, no LCAT activity could be detected. The clearance of radiolabeled UC was multiexponential and closely corresponded to the rate of disappearance of each PC. The clearance of radiolabeled CE was linear and, in contrast to UC, was the same with the administration of different PCs. Uptake of radiolabeled UC by the liver and excretion of radiolabeled UC into bile took place in parallel and corresponded to the rapidity of turnover of UC (and PCs) in the serum. With administration of 16:1-16:1 PC, complete equilibration of serum, liver, and bile UC was achieved by about 90 min, whereas with 20:1-20:1 PC, serum UC had not equilibrated by the end of the study. These findings demonstrate that, in the live animal, the kinetic pattern of transport of different lipids from an HDL recombinant is highly disparate, the rate of PC clearance is more rapid with molecular species of greater hydrophilic strength, and the rates of PC and UC clearance are closely coordinated and largely independent of the clearance of CE.
Animals↗
Separation of phospholipid molecular species by high performance liquid chromatography: potentials for use in metabolic studies.
Different molecular species of phospholipids exhibit distinctly different patterns of biologic behavior. In this minireview, the utility of HPLC for analysis of molecular species of phospholipids is illustrated in studies in which it has been demonstrated that molecular species are selectively synthesized, selectively transported, and selectively participate in enzymatic reactions. HPLC appears to be more adaptable for routine use than older procedures used to separate phospholipid molecular species. Since the metabolism of intact molecules can be characterized with HPLC, this procedure promises to provide particularly novel information with respect to changes in composition brought about by remodelling reactions during the biologic life of specific phospholipids.
Animals↗
Evidence for separate pathways of transport of newly synthesized and preformed cholesterol into bile.
Marked kinetic differences were observed when hepatic newly synthesized cholesterol and preformed cholesterol were separately radiolabeled and separately traced into bile. Whereas newly synthesized cholesterol was not evenly distributed throughout the liver but was preferentially secreted into bile, preformed cholesterol was in near-complete equilibrium in the whole liver and bile. Furthermore, whereas newly synthesized cholesterol in bile originated from the interior of the hepatocyte, results suggest that biliary preformed cholesterol may be transported directly from the blood through the plasma membrane of the hepatocyte and secreted from the canaliculus without first entering the interior of the cell and mixing with newly synthesized cholesterol.
Animals↗
Origin of plasma lysophosphatidylcholine: evidence for direct hepatic secretion in the rat.
In the plasma, lysophosphatidylcholine (LPC) is formed by the action of lecithin-cholesterol acyltransferase (LCAT) when a fatty acid is removed from plasma phosphatidylcholine (PC) and transferred to cholesterol. To determine whether plasma LPC might also be generated by the hydrolysis of hepatic PC, we assessed phospholipid production by the isolated perfused rat liver. Bile duct-cannulated livers were perfused with bile salt and a recirculating, lipid-free medium containing albumin. We found that LPC accumulated in the perfusate to a greater extent than any other phospholipid, exceeding the accumulation of PC (the second most prevalent phospholipid) twofold. We further found that perfusate LPC was not formed by hydrolysis of PC in the perfusate and was not dependent on the presence of infused bile salt. LPC that accumulated in the perfusate was highly unsaturated and markedly dissimilar to the more saturated LPC that results from the activity of LCAT. Results thus indicate that the isolated liver directly secretes LPC, which is presumably generated from hydrolysis of hepatic PC. Because plasma LPC is to a great extent unsaturated in the live rat, these findings suggest that direct hepatic secretion is a quantitatively important source of plasma LPC.
Animals↗
Cholesterol exchange and synthesis in the live rat.
The turnover of plasma cholesterol and de novo cholesterol synthesis were measured simultaneously in the live rat, immediately after administration of [3H]water together with a large volume exchange transfusion of whole blood prelabeled with [14C]cholesterol. It was possible to separate the exchange of unesterified cholesterol from the uptake and secretion of lipoprotein cholesteryl ester, and also to assess the impact of plasma cholesterol exchange on the measurement of in vivo rates of cholesterolgenesis by individual tissues. Cholesterol was measured by an HPLC procedure that effectively separated cholesterol from other structurally similar sterols, and synthesis was determined by the incorporation of [3H]water into cholesterol. Plasma unesterified cholesterol turnover was multiphasic and exceedingly rapid (initial T1/2, 4.1 min) in contrast to the near linear and much slower turnover of plasma cholesteryl ester (initial T1/2, 59.4 min). Plasma unesterified cholesterol equilibrated with different tissues at different rates, with the liver and adrenal equilibrating most rapidly. Full equilibration of plasma unesterified cholesterol was not achieved with any tissue during the course of this study. For rapidly exchanging tissues like the liver, which was responsible for about 60% of plasma unesterified cholesterol exchange, unesterified cholesterol appeared to be kinetically compartmentalized into rapidly, and much less rapidly, exchangeable pools. After [3H]water administration, the content of newly synthesized cholesterol was greatest in the liver, adrenal, and intestine, and appreciably lower in all other tissues studied. Hepatectomy and intestinal resection resulted in a profound reduction of newly synthesized cholesterol in the plasma and adrenal, but no certain change in the already low amounts at other sites. Thus, while it is clear that appreciable amounts of newly synthesized cholesterol in the adrenal were derived from the plasma by exchange, it was not possible to make this assessment for other selected individual tissues. When, however, newly synthesized cholesterol was determined in the total mass of all extrahepatic and extraintestinal tissues together, exchange could be calculated to account for close to 50% of the new cholesterol recovered in the carcass (in studies of 60 min duration). After correcting for exchange, the liver accounted for 82% of all newly synthesized cholesterol, the intestine for about 10%, and the remaining tissues of the body for just 9%. These results are in marked contrast to recent findings of others and demonstrate that in the live rat cholesterol synthesis is principally confined to the liver.
Animals↗
Influence of lecithin acyl chain composition on the kinetics of exchange between chylomicrons and high density lipoproteins.
The kinetics of lecithin exchange between native lipoproteins was characterized for individual molecular species of lecithins of rat mesenteric lymph chylomicrons and rat plasma HDL. Studies were performed in the absence of lipid transfer proteins. Donor (chylomicrons) and acceptor (HDL) particles were present in ratios of 1:1 and 1:10 with respect to total phospholipid. Biphasic exchange kinetics were observed for all major lecithins common to chylomicrons and HDL at both proportions of donor to acceptor particles. During the early rapid phase of exchange, complete in about 30 min, 40-60% of the total lecithin pool was exchanged. Initial exchange rates were most rapid for the more hydrophilic species of the major lecithins normally present in both lipoproteins. Calculated activation energies correspondingly were least for a diunsaturated lecithin (18:1-20:4), intermediate for lecithins were 16:0 in position-1 (16:0-18:2 and 16:0-20:4), and highest for analogous lecithins with 18:0 in position-1. A 10-fold increase in the ratio of acceptor to donor particles affected neither the biphasic nature of the exchange nor the rates of exchange of individual molecular species (consistent with exchange by diffusion rather than by particle collisions). Total equilibration of individual molecular lecithin species was achieved by 24 hr (37 degrees C, donor to acceptor ratio of 1:1) with only a small change in the relative mass of lecithins in chylomicrons and HDL. Novel lecithins containing 18:3, incorporated into chylomicrons, were found to exchange exceedingly rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Utilization of individual lecithins in intestinal lipoprotein formation in the rat.
To determine the molecular species composition of lecithins of different nascent lipoproteins, high density lipoproteins (HDL), very low density lipoproteins (VLDL), and chylomicrons (CM) were isolated from the mesenteric lymph of rats. Lymph was collected at 0 degrees C with 5,5'-dithiobis-2-dinitrobenzoic acid added to inhibit lecithin-cholesterol acyl transferase. CM were separated by ultracentrifugation and HDL from VLDL by dextran SO4-MG+2 precipitation. Molecular species of lecithin were directly isolated by reverse phase high performance liquid chromatography. In fasted animals, the lecithin compositions of lymph HDL and VLDL were virtually the same and closely resembled the lecithin composition of intestinal mucosa. When bile lecithin was eliminated (by bile diversion), there was a marked change in lecithin composition of all lipoprotein and mucosal samples, which was most notable for a reduction in 16:0-species (which are predominant in bile) and a relative increase in the corresponding 18:0-species. Feeding unsaturated triglycerides (triolein, trilinolein, or a combination of triolein and trilinolein) also resulted in a change in HDL and VLDL lecithin composition. The effect was similar whether bile lecithin was present or eliminated and was notable for a reduction in 16:0-species, an increase in 18:0-species, and the emergence of large amounts of diunsaturated lecithins that corresponded to the fatty acid composition of the triglycerides fed (i.e., 18:1-18:1, 18:2-18:2, and 18:1-18:2 lecithins). When bile-diverted rats were infused via the duodenum with a mix of [14C]choline-labeled lecithins (isolated from the bile of other rats), the incorporation of infused lecithins into different lymph lipoproteins was distinctly different. Individual lecithins were incorporated to a variable extent into each lipoprotein. In fasted rats the specific activities of all major molecular species of lecithin were relatively greater in VLDL than HDL, indicating that HDL derived proportionately more of its lecithins from an endogenous pool than did VLDL. Feeding triolein changed the specific activities of more of the lecithin species of VLDL than of HDL. The specific activities of lecithins in CM were more similar to VLDL than to HDL after triolein feeding. Results thus indicate that, although the lecithins of different mesenteric lymph lipoproteins are similar and may be derived from membrane sites with the same lecithin composition, lecithins incorporated into different lipoproteins originate from different metabolic pools and/or by different mechanisms.
Animals↗
Lipids of pigment gallstones.
The lipids of pigment gallstones were analyzed. In contrast to previous reports, pigment stones were found to contain a wide variety of free fatty acids. In addition, pigment stones contained unhydrolyzed phospholipids. Both free fatty acids and phospholipids were present in much higher concentrations in a brown stone obtained from a patient with a biliary tract infection than in a black stone obtained from a patient with sterile bile and a long-standing hemolytic anemia. However, the phospholipids in both kinds of stone consisted primarily of phosphatidylcholine. Separation of stone and bile phosphatidylcholines into their individual molecular species by high performance liquid chromatography indicated that the phosphatidylcholines in stones closely resemble those in bile. The data suggest that both the free fatty acids and the phosphatidylcholine of pigment stones derive from bile phosphatidylcholine, but that the extent of bile phosphatidylcholine participation in pigment stone formation may be variable.
Adult↗
Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography.
Isocratic high-performance liquid chromatography methods are described for separating the major classes of phospholipids and for isolating the individual molecular species of phospholipids. Fractionation of a total lipid extract of rat liver on a silica column resulted in quantitative recoveries of all major phospholipids with preservation of their fatty acid composition. Rat liver phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were then each chromatographed on a C18 reverse phase column to isolate individual molecular species. Component peaks were identified by their fatty acid composition and quantitated by phosphorus determination. Using this method we found that for each of these phospholipids from 30 to 35 different molecular species can be routinely identified and reproducibly quantitated. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on a reverse phase column.
Animals↗
Origin of biliary cholesterol and lecithin in the rat: contribution of new synthesis and preformed hepatic stores.
The contribution of de novo synthesis to the secretion of cholesterol and lecithin in bile was assessed in isolated rat livers, perfused with a lipid-free medium. Cholesterol and lecithin synthesis were measured by the incorporation of tritiated water and [14C]-choline, respectively. Taurocholate stimulated the secretion of biliary lipids to the same extent in perfused livers and in live rats. During the first hour of perfusion, and when hepatic synthesis was active, newly synthesized cholesterol accounted for about 10% of biliary cholesterol and newly synthesized lecithin for 3% of biliary lecithin. Fasting reduced the contribution of newly synthesized cholesterol in bile to less than 1% but did not change the rate of biliary cholesterol secretion. After 2 hours of perfusion, newly synthesized biliary cholesterol accounted for only 4% of total hepatic sterol synthesis. Biliary lecithin, synthesized hepatic lecithin. We conclude that new synthesis makes only a small contribution to biliary cholesterol and lecithin secretion, and that, in the absence of perfusate lipids, both biliary cholesterol and lecithin must be predominantly mobilized from a preformed hepatic pool.
Animals↗
Effect of organic anions on biliary lipids in the rat.
Bile salts enhance the biliary secretion of phospholipid and cholesterol. Other amphipilic molecules, organic anions, are secreted into bile as well. We studied the effects of bilirubin and iodipamide, two chemically dissimilar organic anions, on biliary lipid secretion in the rat. We infused bile salt pool-depleted rats with a stepwise infusion of taurocholate and a constant infusion of organic anion. Both organic anions markedly inhibited the biliary secretion of phospholipid and cholesterol without affecting bile salt secretion. This inhibition, at least with iodipamide, was dose-dependent and fully reversible. Using tritiated water as a precursor, we measured hepatic and biliary cholesterol synthesis in the presence or absence of an iodipamide infusion to see if decreased lipid synthesis could explain decreased secretion. Despite the marked reduction in biliary cholesterol secretion, the specific activity of biliary cholesterol was unchanged during an iodipamide infusion. We suggest that organic anions interfere with the assembly of the biliary mixed micelle resulting in micelles that are deficient in phospholipid and cholesterol.
Animals↗