Competition between chylomicrons and their remnants for plasma removal: a study with artificial emulsion models of chylomicrons

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Competition between chylomicrons and their remnants for plasma removal: a study with artificial emulsion models of chylomicrons
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  Bmhrmica et Biophysics Acta 958 (1988) 211-217 Elsevier 211 BBA 52724 Competition between chylomicrons and their remnants for plasma removal a study with artificial emulsion models of chylomicrons Helena C.F. Oliveira ‘, MArio H. Hirata a, Trevor G. Redgrave ’ and Raul C. MaranhZo a u The Heart Institute (INCOR) of the SZo Paul0 University Medical School Hospital and Faculty of Pharmaceutical Sciences, ScTo Paula University, Scio Paula (Brazil) and ’ Universrty of Western Australia, Nedlands (Auwulia) (Received 22 September 1987) Key words: Chylomicron remnant: Model chylomicron; Cholesteryl ester metabolism: (Rat lymph) zyxwvutsrqponmlkjihg In previous studies, protein-free emulsions of defined lipid composition were shown capable of simulating either the metabolism of chylomicrons chylomicron-like emulsion) or their remnants remnant-like emul- sion), depending on the content of free, unesterified cholesterol. To validate further the assumption that remnant-like and chylomicron-like emulsion have metabolic pathways in common with their natural counterparts, studies of competition for plasma removal were undertaken: the remnant-like emulsion labeled with [3H]triolein was injected sequentially twice in the carotid arteries of rats to compare the clearance of remnant-like emulsion of the second injection with the first control). Prior to the second injection, a large bolus of the chylomicron-like emulsion or rat lymph chylomicron was injected, to check the hypothesis that remnant generated from chylomicron-like emulsion or natural chylomicrons could compete with and displace remnant-like emulsion particles from their tissue receptor sites. Experiments were also performed in rats treated with Triton WR-1339, to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB lock the generation of remnants. Results showed that remnants derived from either natural chylomicrons or chylomicron-like emulsion both strongly competed with the remnant-like emulsion. In contrast, when transformation of remnants was prevented by Triton, the undegraded particles of chylomicron-like emulsion or natural chylomicron were unable to compete with or displace remnant-like emulsion from its sites of removal from the plasma. In agreement with plasma clearance data, the hepatic uptake of the remnant-like emulsion the spleen uptake was unaffected by was inhibited by the surplus dose of natural chylomicrons. In contrast, it. Introduction As recently stated [l], chylomicrons from lymph undergo continuous change while circulating in Abbreviations: PPO, 2,5-diphenyloxazole: dimethyl POPOP, 1.4-bis-(4-methyl-5-phenyl-2-oxazolyl)benzene. Correspondence: R.C. MaranhBo, Faculdade de Ci&ncias Farmac&uticas da Universidade de o Paulo. Departamento de Analises Clinicas e Toxicolhgicas (B 17). Rua Lineu Prestes 580. Caixa Postal 30786, SZo Paulo, 05508 Brazil. the bloodstream. Hydrolysis of their tri- acylglycerol moiety by lipoprotein lipase is the key event, accompanied by net transfer of phospholi- pid to HDL and changes in their apolipoprotein profile. The resulting chylomicron remnants, with depleted triacylglycerol but with preserved cholesteryl ester content are finally taken up by the tissues, mainly the liver. Recently, we described two types of emulsions composed of triolein, cholesteryl oleate, cholesterol and phosphatidylcholine that were shown to re- produce the metabolism of chylomicrons 0005-2760/88/ 03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)  212 (chylomicron-like emulsion) and their remnants (remnant-like emulsion). Remnant-like emulsions had a much higher cholesterol/phospholipid molar ratio than chylomicron-like emulsions (1.70 and 0.40, respectively) [3]. When injected into carotid arteries of rats, the chylomicron-like emulsion had the two-step pat- tern characteristics of chylomicron metabolism, i.e., (a) triacylglycerol was removed faster than cholesteryl ester from the plasma compartment and (b) the particles trapped by the liver had most of the injected cholesteryl ester but only residual triacylglycerol [2]. On the other hand, as expected for remnants, remnant-like emulsion had the same plasma removal rates for triacylglycerol and cholesteryl ester, indicating that no lipolysis oc- curred, Also, as expected for remnants, remnant- like emulsion particles were trapped by the liver faster than the chylomicron-like emulsion. In other work [4] we have demonstrated that Triton WR-1339 blocked triacylglycerol hydrolysis of the chylomicron-like emulsion, leading to im- paired removal of these emulsion particles from the plasma. However, Triton had no effect on the remnant-like emulsion removal. These results were consistent with the behavior of natural chylomicrons and remnants, and also illustrate the usefulness of Triton as a device to inhibit lipolysis, without effect on the tissue uptake mechanism. Remnants were shown to have higher affinity for liver receptors than native chylomicrons in hepatocyte monolayers [5-71 and liver plasma membrane assays [8]. In isolated perfused liver system, remnants were removed faster than their chylomicron precursor [9-141. Nonetheless, some other studies show at least some intact chylomicrons were removed by the liver [15-181. In the present work, we measured the rates of removal from plasma, to test for competition be- tween the remnant-like emulsion and remnants from the chylomicron-like emulsion or natural chylomicrons. In another set of experiments Tri- ton was injected to inhibit lipolysis; remnants generation from both chylomicron-like emulsion and natural chylomicrons was therefore prevented. Then, competition with remnant-like emulsion for removal was again tested with intact chylomicron particles, natural as well as artificial. The competi- tion between chylomicrons and the remnant-like emulsion was also documented in tissue uptake experiments. The aims of these studies were to show whether the artificial remnant-like emulsion leave the plasma compartment through the same tissue up- take mechanisms as the remnants generated from chylomicron-like emulsion or natural chylomi- crons, and also to estimate the capacity of intact, non-metabolized particles to displace remnants from their receptor sites. Materials and Methods zyxwvutsrqponmlkjihgfedcbaZ Preparation of emulsions Triolein, cholesteryl oleate and cholesterol were purchased from Nu-Chek Prep. (Elysian, MN) and egg phosphatidylcholine from Lipid Products (Surrey, U.K.), each more than 99 pure by thin-layer chromatography. Two different lipid mixtures (total mass, 100 mg) were prepared to obtain the chylomicron-like and remnant-like emulsions. Chylornicron-like emulsion mixture had 2 cholesterol, 23 phos- phatidylcholine, 6 cholesteryl oleate and 69 triolein ( weight), Remnant-like emulsion mix- ture had 24.5 cholesterol, 24.5 phosphati- dylcholine, 10.3 cholesteryl oleate and 40.7 triolein. The lipids were dispensed from stock solutions into vials. In the making of the remnant-like emulsion [ 3 Hltriolein (Amersham) or [‘4C]cholesteryl oleate was added. The specific activity of the triolein was 2.21 . lo6 cpm/mg and of the cholesteryl oleate was 4.85 . lo6 cpm/mg. After evaporation of solvents under nitrogen, the vials were placed overnight in a vaccum desiccator at 4’ C to eliminate residual solvent. Lipids were emulsified by sonication in 8 ml of 2.785 M NaCl solution (density, 1.101 g/ml) with a Branson Cell Disruptor (Danbury, CT) at ap- prox. 55°C using a 1 cm probe with continuous output of 70-80 W. Purification of crude emul- sions was by ultracentrifugation in discontinuous gradients of NaCl solutions with densities of 1.065, 1.020 and 1.006 g/ml. The ultracentrifugation procedure was performed differently for the two types of emulsion. The chylornicron-like emulsion was initially centrifuged at 12000 rpm for 15 min in an SW 41 Beckman rotor at 22°C. The rem- nant-like emulsion was centrifuged at 10000 rpm  213 for 20 min. After this first run, the coarse lipid that floated to the top of the 1.006 g/ml solution was removed by aspiration and replaced with its corresponding volume of fresh 1.006 g/ml solu- tion. The chylomicron-like emulsion was again centrifuged, now at 36000 rpm for 25 min, and the remnant-like emulsion at 38000 rpm for 30 min at 22” C. The emulsion particles floating to the top of the gradient were aspirated and used for analysis and for competition studies. The lipid composition of the emulsion was determined by standard laboratory procedures [19-211. Both em- ulsions had been previously characterized by nega- tive staining electron microscopy [2,3]. Preparation of chylomicrons Intestinal lymph was collected over ice with added EDTA (final concentration, 1 mM) for 24 h from male Wistar rats weighing 300-400 g, through a cannula implanted in the mesenteric lymph duct. After surgery the rates were maintained in re- striction cages and a constant infusion of cotton seed oil (0.035 ml/h) was delivered through a gastrostomy tube. Water drinking was ad libitum. Saline (5 ml) (pH 7.0) d= 1.006 g/ml contain- ing EDTA (1 mM) was layered on lymph (5 ml) into the Beckman SW 41 rotor tube and centri- fuged at 24 500 rpm for 20 min at 20°C. Chylomicrons were recovered from 1.5 ml of the creamy top layer aspirated from each tube and assayed as described above. They were used in the experiments within the ensuing 24 h. Competition studies The rats utilized in this study were non-fasted, non-anesthetized male Wistar rats weighing 250-300 g. Emulsions, lymph chylomicrons, saline solution and Triton were injected in a bolus through a polyethylene cannula (Intramedic PE 50) inserted under diethylether anesthesia into the right carotid artery, according to the protocols described below. Clotting was prevented by previ- ous treatment of the cannula with silicon (Clay Adams, Parsippany, NJ). The animals were al- lowed to recover from anesthesia for 2-3 h in individual cages. The remnant-like emulsion (0.3-0.5 mg of its triolein moiety) labeled with radioactive triolein was injected sequentially twice into the bloodstream of the rats to compare the plasma clearance rates of the second injection with the first one (control injection). The second injection was made 20 min after the first and 10 min after the intraarterial administration of isotonic saline solution (0.5 ml) (Experiment 1), chylomicron-like emulsion (30 mg of total lipids) (Experiment 2) or rat lymph chylomicrons (30 mg of total lipids (Experiment 4). The above experiments were re- peated after the injection of Triton WR-1339 (600 mg/kg of body weight, [22]) 10 min before the first injection, to inhibit lipoprotein lipase activity (Experiments 3 and 5). In these experiments, only the triolein moiety was labeled, but from our previous work [4] it is known that for remnant-like emulsion the triolein and cholesteryl oleate plasma clearances are identical. After the two injections of labeled remnant-like emulsion, 0.2-ml blood samples were drawn at 2 min intervals for 10 min. After the experiments, animals were killed by air embolization. 100 ~1 of blood plasma were extracted with chloroform/ methanol (2 : 1, v/v) [23] and neutral lipid classes separated by TLC in the solvent system hexane/diethyl ether/acetic acid (70 : 30 : 1, by vol.) Bands corresponding to triacylglycerols were then scraped into counting vials for radioactivity measurement in 10 ml of scintillation solution (0.59 PPO/59 dimethyl POPOP/333 ml Triton X-100/667 ml toluene) [24] in a Beckman LS 100 C Spectrometer. Plasma clearance kinetics (frac- tional clearance rate) of [3H]triolein were com- puted from monoexponential curves fitted by the least-square procedures. In another set of competition experiments, the remnant-like emulsion labeled with [ I4 Clcholes- teryl oleate was injected, as described above, in control and rats that had been given 30 mg of total lipids of lymph chylomicrons administered as a bolus 10 min before hand. 3 min after the labeled remnant-like mulsion injection, the animals were killed by air emboliza- tion and their livers, spleens, lungs, hearts, kidneys and adrenals as well as samples of their muscle and adipose tissue were quickly excised to de- termine the radioactive label uptake by these organs. Lipids from approx. 1 g of the tissues were extracted by chloroform/methanol (2 : 1, v.v) [23] and processed as described for plasma samples.  214 TABLE I COMPOSITION OF THE EMULSIONS AND LYMPH CHYLOMICRONS Results are given as meanf SE. The composition ( ) of the initial sonicated lipid mixture is given in parentheses. Constituents ( by weight) Chylomicron emulsion Remnant emulsion (low-cholesterol) (high-cholesterol) (n=8) (n=5) Lymph chylomicrons (n =l) Cholesterol Phospholipid Cholesteryl ester Triacylglycerols Cholesterol/phosphohpid (molar ratio) Cholesteryl ester/triacylglycerol (molar ratio) 1.9 +0.3 (2) 21.3 * 2.7 (24.5) 0.7 10.4 k 1.3 (23) 18.7 k 2.4 (24.5) 4.0 11.2 k3.0 (6) 18.7 k 5.4 (10.3) 4.5 76.5 k4.1 (69) 41.4 + 3.4 (40.7) 90.8 0.39 + 0.06 2.4 kO.5 0.13 * 0.01 0.67 + 0.2 zyxwvutsrqponmlkjihgfedcbaZYXWV Results Composition of emulsions prepared as de- scribed from their respective lipid mixtures and of the lymph chylomicron are shown on Table I. Experiment 1 (Table II) was a control for the general design of the current experiment. Compar- ing the fractional clearance rates of the two injec- tions of remnant emulsion, removal from the sec- ond injection was slowed by 1.4-fold. Neverthe- less, this baseline difference can be considered as rather minor when compared to the results of the ensuing experiments, in which saline was replaced by the chylomicron-like emulsion or natural chylomicrons. When injected after a load of chylomicron-like emulsion (30 mg of total lipids) in Experiment 2 (Table II), labeled remnant-like emulsion had its fractional clearance rate diminished 4.8-fold com- pared with the control first injection. This finding indicates that the remnant particles generated from chylomicron-like emulsion were capable of com- peting with the labeled remnant-like emulsion par- ticles from their tissue receptor sites, thus prevent- ing the plasma removal of remnant-like emulsion. In the ensuing Experiment 3, generation of re- TABLE II COMPETITION FOR PLASMA REMOVAL BETWEEN REMNANT-LIKE EMULSION AND CHYLOMICRON-LIKE EM- ULSION OR NATURAL CHYLOMICRONS Fractional clearance rates of two sequential injections of [3H]triolein labeled remnant-like emulsion are compared when the second injection was preceded by the administration of either saline solution, chylomicron-like emulsion (30 mg of total lipid) or natural chylomicrons (30 mg). The experiments were also performed with Triton WR-1339 (600 mg/kg of body weight, intraarterially 10 min. prior to first injection) to prevent remnant generation from chylomicron-like emulsion or natural chylomicrons. Results are meanf S.E. Fractional clearance rate = ln2/f,,,. n = 7 in every case, except for saline (n = 4). n.s., not significant. Experiment Fractional clearance rate of remnant-like emulsion (mm’) zyxwvutsrqponmlkjihgfedcbaZ  a first injection second injection 1. Saline 0.24 * 0.01 0.17 * 0.01 i 0.02 2. Chylomicron-like emulsion 0.29 k 0.04 0.06 + 0.01 < 0.001 3. Chylomicron-like emulsion, Triton treatment 0.28 + 0.05 0.30+0.03 n.s. 4. Natural chylomicrons 0.27 k 0.02 0.07 & 0.01 < 0.001 5. Natural chylomicrons, Triton treatment 0.25 + 0.04 0.22 f 0.03 n.s. a Comparison between first and second injection (Student’s l-test).  215 mnants from the chylomicron-like emulsion load was blocked by inhibition of the lipoprotein lipase action with Triton WR-1339. In this case, intact, non-metabolized chylomicron-like emulsion par- ticles were not effective competitors with remnant-like emulsion for the receptor sites, as shown by the unaltered clearance of the chylomicron-like emulsion. In experiments 4 and 5 (Table II) the name 30 mg lipid mass of natural chylomicrons obtained from lymph of donor rats was injected. The results were very similar to those following the injection of chylomicron-like emulsion: the fractional clearance rate of the remnant-like emulsion de- creased approx. 4-fold when natural chylomicrons were administered alone. Also, like the chylomicron-like emulsion, when the natural chylomicrons were injected in association with Triton, the plasma removal of remnant-like emul- sion was not altered. Table III shows the uptake of the labeled re- mnant-like emulsion by several tissues (liver, spleen, lung, heart, kidney, adrenal, muscle and TABLE III UPTAKE OF THE [‘4C]CHOLESTERYL OLEATE MOIETY OF THE REMNANT-LIKE EMULSION BY DIF- FERENT TISSUES IN CONTROL AND RATS SUPPLIED WITH A PREVIOUS SURPLUS DOSE OF NATURAL CHYLOMICRONS 30 mg of total lipids of the natural chylomicrons were adminis- tered intraarterially 10 min before the injection of the labeled emulsions. Organs were excised for lipid extraction and radio- activity determination 3 mitt after the injection of the emul- sion. Results are mean +S.E. The number of experiments is given in parentheses. The total organ mass of muscle and adipose tissue was calculated from Refs. 32 and 33. Tissues Organ uptake of [“‘Clcholesteryl oleate (W of injected dose) control supplied with chylomicrons Liver 42.54k3.50 (11) 30.92 * 1.5 (7) a Spleen 5.97 f 0.70 (10) 5.30 * 0.70 (7) Lung 1.07 f 0.26 (5) 1.00 * 0.20 (8) Muscle 0.77 k 0.13 (6) 0.84*0.19(7) Heart 0.10+0.03 (6) 0.14 + 0.03 (8) Kidney 0.13kO.02 (6) 0.13 * 0.03 (8) Adipose tissue 0.07 + 0.02 (6) 0.06 k 0.01 (8) Adrenal 0.01 k 0.00 (6) 0.01 * 0.00 (7) a P i 0.025 by f-test. c 4 & ‘v zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM   ,’ 1 I 0” 0 2 1 zyxwvutsrqponmlkjihgfedcbaZYXWV  3 4 mg of total lbplds of chylomlcrons Fig. 1. Effects of increasing the injected dose of chylomicrons on the plasma clearance of the radioactive remnant-like emul- sion. The chylomicron dose was supplied intraarterially 10 min before the injection of the remnant emulsion. Each point corresponds to 5-9 experiments of plasma clearance de- termination. Bars represent standard errors of the means. FCR, fractional clearance rate. adipose tissue) measured 3 n-tin after its injection. Whereas 42 and 6 of the injected cholesteryl ester moiety was taken up by the liver and the spleen, respectively, the uptake of the other tissues were 1 or less. When the remnant emulsion was injected after previous administration of the surp- lus mass of lymph chylomicrons, in the liver only the emulsion uptake was diminished by the com- petition with the remnants generated from the natural chylomicrons. The effect of the previous injection of increas- ing chylomicron doses on the plasma disap- pearance kinetics of the remnant emulsion (dose- response curve) is shown in Fig. 1. zyxwvutsrqponmlkjihgfe Discussion A protein-free emulsion of defined lipid com- position can model chylomicron metabolism. After injection, the apolipoprotein necessary for their metabolism is gained rapidly from apolipoprotein that is free in plasma or by exchange with other lipoproteins. Their surface lipid composition will determine the profile of adsorbed apolipoproteins, which in turn modulate the metabolic pathway of the emulsions. Accordingly, we showed in previ- ous work [3] that cholesterol-enriched remnant-like emulsion bound less apolipoproteins A-I, A-IV and C and relatively more apolipoprotein E than
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