W.J. Simmonds

The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat.

A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2 : 1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.

The specificity of bile salts in the intestinal absorption of micellar cholesterol in the rat.

1. Two aspects of cholesterol absorption; (a) the importance of solubilization and (b) the effects of different bile salts on the mucosal metabolism and lymphatic output of cholesterol, have been investigated using two different in vivo techniques.

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

The effect of added monoacylglycerols on the removal from plasma of chylomicron-like emulsions injected intravenously in rats

Lipid emulsions were prepared with compositions similar to the triacylglycerol-rich plasma lipoproteins, but also incorporating added small amounts of monoacylglycerols. Control emulsions without monoacylglycerol were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. The emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the bloodstream, with the removal rates of triacylglycerols faster than those of cholesteryl esters. Much of the removed cholesteryl ester was found in the liver, but only a small fraction of the triacylglycerol, consistent with hepatic uptake of the triacylglycerol-depleted remnants of the injected emulsion. Emulsions incorporating added monooleoylglycerol or stearic acid were metabolized similarly. Added 1- or 2-monostearoylglycerol had no effect on triacylglycerol removal from plasma, but the removal rate of cholesteryl esters was decreased and less cholesteryl ester was found in the liver. These effects are similar to those recently described when emulsions and chylomicrons contained triacylglycerols with a saturated acyl chain at the glycerol 2-position, suggesting that saturated monoacylglycerol produced by the action of lipoprotein lipase may cause triacylglycerol-depleted remnant particles to remain in the plasma instead of being rapidly taken up by the liver.

Uptake and efflux by everted intestinal sacs of micellar cholesterol in bile salts and in non-ionic detergent

Abstract 1. 1. Uptake and efflux of labelled micellar cholesterol by everted sacs of rat jejunum was compared for two detergents, (a) pure sodium taurocholate-sodium taurodeoxycholate (4:1, on molar basis) and (b) a high-molecular-weight non ionic detergent, Pluronic F68. Oleic acid and monoolein were also incorporated in the micellar solutions. 2. 2. Uptake of cholesterol from bile salt micelles was 2–4 times faster than from non-ionic micelles, for incubation periods varying from 10 min to 2 h. 3. 3. Uptake from bile salt micelles increased with temperature (apparent activation energy about 6 kcal/mole cholesterol) but uptake from non ionic micelles did not. Metabolic inhibitors had little effect. 4. 4. When sacs were loaded with isotopic cholesterol from bile salt micellar media, subsequent efflux into buffer, or into unlabelled cholesterol in non-ionic micelles or in bile salt micelles was slower than was efflux into a given medium after loading with isotopic cholesterol from non-ionic micelles. On the other hand, efflux into bile salt micellar medium was faster for a given loading medium than efflux into non-ionic micelles. 5. 5. It is suggested that cholesterol uptake may involve two stages only one of which is temperature sensitive and is promoted by bile salts.

Membrane ATPase and electrolyte levels in marsupial erythrocytes

Abstract 1. 1. Individuals of the Australian marsupial species, the possum ( Trichosurus vulpecula ), differ in erythrocyte cation concentration. ATPase activity was measured in the fragmented membranes of erythrocytes in which potassium predominates (HK cells) and in which sodium predominates (LK cells). A comparison was made with erythrocyte membrane ATPase activity in the marsupial quokka ( Setonyx brachyurus ) in which sodium and potassium are present in approximately equal concentration. 2. 2. All erythrocyte preparations showed basal ATPase activity. Significant (Na + -K + )-activated ATPase was present only in HK possum and quokka cells, absolute values and values relative to basal ATPase being higher in HK possum than in quokka cells. 3. 3. These results suggest that the low erythrocyte potassium concentrations in LK possums may be due to the absence of (Na + -K + )-activated ATPase and pump activity in their cell membranes.

A comparison of absorption of free fatty acid and α-glyceryl ether in the presence and absence of a micellar phase

Abstract Absorption of two lipids from a mixture was compared in unanaesthetized bile diverted rats. The isotopically labelled lipids, oleic acid and glyceryl-1-monoether, were infused intraduodenally in bile salts either as a micellar solution or in an emulsion with no micellar phase present. The two isotopes were absorbed equally into the thoracic duct lymph from micellar solutions. The absorption of both was depressed in the absence of micelles with the monoether label being more affected than the fatty acid label. This suggested a rate-limiting step in the absorptive pathway prior to the appearance of lipid in the lymph. To further localize this step animals were sacrificed at 4 and 6 h and isotope recovered from intestinal lumen and wall as well as lymph. This showed that there was no accumulation of isotope in the intestinal wall but that there was accumulation in the intestinal lumen, suggesting the rate-limiting step to be in the luminal phase of absorption.

The effect of partition of fatty acid between oil and micelles on its uptake by everted intestinal sacs

Abstract 1. 1. Everted sacs of rat jejunum were used to measure the effect on uptake of labelled oleic acid, 1 mM, when an unabsorbable oil (glyceryl-trioleyl ether) was present. The incubation medium also contained monoolein 1 mM, and pure bile salts (sodium taurocholate-sodium taurodeoxycholate, 4:1 on a molar basis). 2. 2. Increasing concentrations of triether had no effect on the low uptake of oleic acid in the absence of a micellar phase, bile salts 1 mM, when virtually all the lipid was in emulsified particles. 3. 3. When most of the oleic acid (and monoolein) was present in a micellar phase, bile salts 4 mM or 5 mM, uptake was greatly accelerated. Addition of increasing amounts of triether now led to partition of an increasing proportion of oleic acid into the emulsified oil particles with a corresponding reduction in concentration of oleic acid in the micellar or isotropic phase. Uptake of oleic acid was reduced in linear proportion to reduction in micellar concentration. 4. 4. The regression of oleic acid uptake on isotropic aqueous concentration was the same in these experiments, in which bile salt concentration remained constant, as in previous experiments in which the isotropic concentration was varied by altering the bile salt concentration. 5. 5. These experiments provide further evidence that uptake in vitro depends on the concentration of fatty acid in the isotropic aqueous phase, and not on the concentration of bile salts per se .

The relationship of the calcium content of smooth muscle to its contractility in response to different modes of stimulation

Abstract The Ca2+ and Mg2+ contents of strips of the circular smooth muscle from the stomach of the toad (Bufo marinus) were measured after varying lengths of exposure to NaCl-Ringer and K2SO4 depolarizing Ringer solutions in which the Ca2+ and Mg2+ concentration was varied. The Ca2+ content of the smooth muscle was correlated with the contractility of the muscle when the tissue was first depleted of Ca2+ to abolish responses and then controlled replacements made. In Ca2+-free depolarizing K2SO4-Ringer the Ca2+ content fell from 0.95 ± 0.02 (mean ± S.E. of mean) to 0.14 ± 0.01 μmole per g wet weight before contractility to acetylcholine was lost. When only 9% of the tissue Ca2+ was replaced by exposure to 0.08 mM Ca2+ in the Ringer solution, full contractility was restored, the muscle then containing 0.23 ± 0.01 μmole per g wet weight. However, contractions produced by alternating current applied in the longitudinal direction of the fibres were practically eliminated when the tissue still contained 0.31 ± 0.01 μmole of Ca2+ per g wet weight. 26% of the maximal contractility was restored when the tissue contained 0.43 ± 0.01 μmole per g wet weight and 42% response when the tissue contained 0.53 ± 0.02 μmole per g wet weight. When the tissue contained supranormal amounts of Ca2+, the contractile response to maximum electrical stimulus equalled that produced by a maximal dose of acetylcholine. The experiments suggest that acetylcholine response depends on a small fraction of Ca2+ located in the cortex of the cell while the effect of alternating current is to displace Ca2+ from myoplasmic sites.

The intestinal uptake and esterification, in vitro, of fatty acid as a diffusion limited process

Abstract The rate of uptake and esterification of fatty acid by everted intestinal sacs is reduced when micellar media of a non-ionic detergent are compared with bile salt micellar media 1 ( Hoffman , Biochim. Biophys. Acta , 196 (1970) 193). In this paper it has been shown that: 1. (1) The reduced rate of uptake correlates with a reduced diffusion coefficient of fatty acid in the non-ionic detergent micellar solution. 2. (2) The sac preparation has a maximal esterifying capacity. 3. (3) This is the same for non-ionic detergent and bile salt micellar media.