Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Lu-YING YANG AND ARNIS KUKSIS' Bunting and Best Department of Medical Research, Universiy of Toronto, Toronto, Ont., Canada

and Department of Biochemistry, University of Toronto, Toronto, Ont., Canada

Received November 3, 1986

YANG, L.-Y., and KUKSIS, A. 1987. Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters. Biochem. Cell Biol. 65: 514-524.

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas-liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phos- phatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B- 100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.

YANG, L.-Y., et KUKSIS, A. 1987. Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters Biochem. Cell Biol. 65: 514-524.

Des rats mdles, dont le canal thoracique est muni d'une canule, ont reGu par intubation de I'huile de mais ou des esters mCthylCs d'acides gras et nous avons recueilli la lymphe entre 2 et 72 h aprks le traitement. Nous avons dttermink par Clectropho~se sur gel de dodkcylsulfate de sodium - polyacrylamine - glycCrol et par focalisation isoClectrique la composition apoprottique (apo) des chylomicrons isolts par ultracentrifugation conventionnelle. Nous avons Ctabli la teneur et la composition des lipides par chromatographie gaz-liquide. Les dimensions des particules sont obtenues par calcul et confirmCes par rnicroscopie Clectro- nique. L'Ctude dkmontre que la biosynthkse des triacylglyctrols par la voie du monoacylglycCrol (ingestion d'huile de mdis) et la voie de I'acide phosphatidique (ingestion des esters mCthylks) donne des chylomicrons dont les profils apoprotkiques sont trbs semblables et comportent les constituants suivants: apo B-48, apo A-IV, apo E, apo A-I et apo C. Une bande prottique correspondant i l'apo B-100 apparait occasionnellement comme composC mineur des chylomicrons des deux groupes d'ani- maux. Les chylomicrons provenant de I'ingestion d'huile de mdis ont des diametres environ deux fois plus grands que ceux provenant des esters mCthylCs. I1 n'existe pas de diffkrences significatives d a q l a composition des apoprottines, mais les particules plus petites ont des rapports apoprotCine/triacylglycCrol deux fois plus ClevCs. Nous avons calculC que la quantitk d'apo B par particule lipidique chez les rats recevant des esters mCthylCs varie de une h huit moltcules et qu'elle est Ctroitement relite aux dimensions des particules. Les rats recevant I'huile de mai's donnent environ trois molCcules d'apo B par particule lipidique sans Cgard aux dimensions des particules. Nous concluons que la voie de biosynthese intestinale des triacylglyc~rols affecte de faqon importante la masse apoprotkique et i un degrC moindre la composition apoprottique et lipidique des chylomicrons. La voie de I'acide phosphatidique produit des particules plus petites et transfkre au torrent sanguin deux fois plus d'apoprottines par gramme de lipide que la voie du monoacylglycCrol qui donne des particules plus grosses. Nous ne pouvons exclure entibrement la contribution de variations possibles dans le site et le taux de biosynthbe des triacylglycCrols.

[Traduit par la revue]

ABBREVIATIONS: apo, apoprotein(s) or apolipoprotein(s); VLDL, very low density lipoprotein(s); CFAME, corn oil fatty acid methyl esters; TFAME, tea-seed oil fatty acid methyl esters; LDL, low density lipoprotein(s); HDL, high density lipoprotein(s); SDS, sodium dodecyl sulfate; TEMED, N,N,N',Nf-tetramethylethylenediamine; D m , dithiothreitol; GLC, gas-liquid chro- matography; TLC, thin-layer chromatography; Da, dalton; IEF, isoelectric focusing; CO, corn oil.

'Author to whom all correspondence should be sent at the following address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ont., Canada M5G lL6.

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YANG AND KUKSIS 515

Introduction It is well known that the intestine is capable of resyn-

thesizing dietary fat via the phosphatidic acid ( I ) and the monoacylglycerol (2) pathways, which under normal conditions account for about 20 and 80%, respectively, of the total intestinal triacylglycerol formation (3,4). It has been observed (5, 6) that the diacylglycerol inter- mediates of the two pathways d o not mix, which is in agreement with histochemical data indicating the asso- ciation of the phosphatidic acid pathway with the rough endoplasmic reticulum and the monoacylglycerol path- way with the smooth endoplasmic reticulum (7, 8). It is therefore possible that the triacylglycerols produced by the two pathways also remain segregated during the secretion and appear in the lymph in the form of dif- ferent lipid particles. In connection it may be noted that inhibition studies have shown that the intestinal chylo- microns and VLDL are formed by two different meta- bolic routes (9), but the relative contributions of the phosphatidic acid and the monoacylglycerol pathways to the biosynthesis of the triacylglycerols of these parti- cles was not determined. In the present study we have compared the size and composition of the lymph lipid particles produced by rats fed either corn oil o r the corresponding fatty acid methyl esters, which would be anticipated to be absorbed essentially via the monoacyl- glycerol and the phosphatidic acid pathways, respec- tively. Although the results demonstrate that the phos- phatidic acid pathway yields lipid particles one half the size of those produced by the monoacylglycerol path- way and significantly higher content of free cholesterol and cholesteryl esters, a clear distinction between chylo- micron and VLDL particles could not be made in the absence of more characteristic metabolic markers.

Materials and methods Animals and diets

Retired male breeder rats of Wistar strain (Charles River Canada, Inc., La Salle, Que.), weighing 380-450 g at surgery were fed formula stock diet ad libitum. Prior to the operation they were fasted overnight (16 h) and under light diethyl ether anaesthesia were given by stomach tube either 3-4 mL of corn oil, or 3-4 mL of CFAME or TFAME plus 2 g of glucose. The corn oil and CFAME contained 16 : 0 (13.5%), 18 : 0 (1.8%), 18 : 1 (25.7%), and 18 : 2 (56.7%), while the TFAME con- tained 16:O (8.8%), 18:O (2.2%), 18: 1 (81.1%), and 18:2 (7.9%). The animals were allowed to recover for 2-3 h and then subjected to surgery as follows.

Surgical procedures The rats were anaesthetized by intraperitoneal injection of

sodium pentobarbital (Abbott Laboratories, Montrkal, Que.) at the dose level of 7.3 mg/ 100 g body weight. The thoracic duct was cannulated as described by Bollman et al. (10). The animals receiving the fatty acid methyl esters had free access to drinking water containing glucose, while all others had access to drinking water alone.

The lymph was collected during the day of operation and over the next 2-3 days. Samples for apo B-100 assay were collected for short periods of time (<1 h). The collection tubes, which were kept on ice, contained a preserving solution prepared as described by Lee et al. (1 1) or 2% EDTA. The lymph flow was variable and averaged < 1 mL/ h.

Isolation of lipoproteins The plasma and lymph lipoproteins were isolated by ultra-

centrifugation (Beckman model L2-65B, rotor40.3) at 10°C as previously described (12). Samples were overlayered with 0.85% NaCl - 0.01% EDTA (pH 7.4) (d = 1.006 g/mL). Chylomicrons and VLDL were recovered following centri- fugation for 30 min and 18 hat 109 000 x g, respectively, both fractions being washed under the same conditions for 18 h. For isolation of LDL (d = 1.006- 1.063 g/rnL) and HDL (d = 1.063-1.21 g/mL), the density was adjusted with solid KBr and the centrifugation was done at 109 000 x g for 18 h and 48 h, respectively. The LDL and HDL fractions were washed once through a KBr solution of appropriate density. All frac- tions were dialyzed against distilled water and 0.01% EDTA (pH 8.2) at 4°C for 18 h. For rapid isolation of chylomicrons, lymph samples were overlayered with 0.01% EDTA (pH 8.2) and the chylomicrons were collected by centrifugation for 20 rnin at 35 000 x g and lyophilized immediately.

Recovery of apoproteins Aliquots of the dialyzed solution were lyophilized and the

lipids were extracted using ethanol - diethyl ether (3 : 1) (13). The apoproteins were dried under a stream of nitrogen and were stored at -4OC for further analyses. Total protein was determined by the method of Lowry (14, 15) using bovine serum albumin as standard.

Gel electrophoresis The SDS-polyacrylamide-glycerol gel electrophoresis

was performed by a modification of the method of Connelly and Kuksis (16). The gel contained 4% acrylamide (acryl- amide-bis-acrylamide, 25: I), 18% glycerol, 0.1% SDS, 0.075% TEMED, and 0.01% ammonium persulfate in 0.1 M phosphoric acid (pH 6.8). The gels were scanned using a Helena densitometer, equipped with an area integrator and a 6 10-nm filter (Helena Company, Beaumont, TX) . The apopro- tein mixtures were run in parallel with the molecular weight standards: bovine thyroglobulin (670 OOO), bovine y-globulin (158 OOO), chicken ovalbumin (44 OW), and horse myoglobin (1 7 000) (Bio-Rad Laboratories, Richmond, CA).

Isoelectric focusing This method was adopted from those described by Uter-

rnann et al. (1 7) and Pagnan et al. (1 8). The apoproteins were focused in 7.5% polyacrylamide gel using an acrylamide-bis- acrylamide ratio of 36 : 1. The gel contained 8 M urea and 2% ampholine. The pH gradient of 4.0-6.5 was obtained by mixing ampholytes pH 4-6 (LKB 1809-1 16, LKB Producter, Brornma, Sweden) and pH 5-7 (Bio-Rad 163-1 152, Bio-Rad Laboratories, Richmond, CA) in a ratio of 4 : 1. The gel was polymerized with riboflavin. For the analysis, the apoproteins of chylomicrons, VLDL, and HDL, containing 100, 150, and 100 pg of total protein, respectively, were dissolved in 200 p L of 0.003 M Tris-HC1 buffer (pH 8.2) containing 8 M urea and 30 mM DTT. The samples were applied and overlayered with

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516 BIOCHEM. CELL BIOL. VOL. 65, 1987

100 p L of 5% sucrose containing 0.8% ampholine. The disc gels were electrofocused for 1 h at 200 V and for 5 h at 450 V. The developed gels were fixed for 18 h in 12% trichloroacetic acid, stained for 3 h in 0.03% Coomassie brilliant blue G-250 in 3.5% perchloric acid (19) and destained in 10% acetic acid. After 1 week the gels were scanned using the Helena densi- tometer as described above.

Lipid analyses Total lipid profiles of the chylomicrons and of the plasma

lipoproteins were determined by high-temperature GLC fol- lowing dephosphorylation with phospholipase C and tri- methylsilylation as previously described (20). The chylo- microns or plasma lipoproteins equivalent to about 0.1 rnL of lymph or plasma were used along with 200-300 kg tri- decanoylglycerol as internal standard.

since the diacylglycerols released by phospholipase C from the phosphatidylcholines overlapped with any free diacyl- glycerols present in the chylomicrons, it was necessary to correct the total diacylglycerol values for the original free diacylglycerol content. This was done by preparing a total lipid extract from another aliquot of the chylomicron fraction and performing a TLC separation prior to the phospholipase C digestion. The plate was developed in a neutral lipid system made up of heptane - isopropyl ether - acetic acid (60 : 40 : 3). The free diacylglycerols and free cholesterol were recovered as a combined fraction from the TLC plate and, after elution with chloroform, were trimethylsilylated as described for the total lipid extract and separated by GLC under the general conditions described for total lipid profiling. The con- tent of the free diacylglycerols was estimated in relation to that of the free cholesterol, which was already known from the determination of the total lipid profile for the sample as described above.

Calculations The average core radii of the chylomicron particles were

determined from the surface to volume ratio of a sphere (21). The calculation assumes that all cholesteryl ester and tri- acylglycerols are located in the lipid core, while all the free cholesterol and the phospholipids are confined to a continuous surface monolayer.

In such a case the surface area = 47rR2 = k(ApL[PL] + AFc[FC]) and the volume of the core = %R3 = k(V,,[TG] + VcE[CE]), where k is a proportionality constant dependent on the number of particles per unit lipid mass; [PL], [FC], [TG], and [CE] are the mole percentages of total phospholipid, free cholesterol, triacylglycerol, and cholesteryl ester, respec- tively. ApL = 69 A2 (1 A = 0.1 nm) and AFC. = 39 A2 are the average cross-sectional areas of the phospholipid and free cholesterol molecules, respectively, while V,, = 1556 A' and VcE = 1068 k are the average molecular volumes of tri- acylglycerol and cholesteryl ester molecules, respectively. No distinction is being made between phosphatidylcholine and sphingomyelin in regards to the molecular dimensions. The total radii of the particles are obtained by adding the thickness of the surface monolayer of 21 A (21). A recalculation assum- ing that 30% of the total free cholesterol was present in the chylomicron interior (22) reduced the radii by 2%, which was not considered significant for the present comparisons.

The average number of apo B molecules per chylomicron

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TABLE 2. Calculated mean radii (A) of lymph chylomicrons of rats fed either corn oil or fatty acid methyl esters

First 24 h Second 24 h

CO CFAME TFAME CO CFAME TFAME

Mean 1265 634* 646* * 710*** 451** 423 * * & SD 299 205 126 63 112 117 n 3 5 5 3 3 3

NOTE: R = M + ~ ( : T R ~ ) / ( ~ T R ~ ) = 3 (vT~[TG1 + Vct'CE1), where Vm and Vc, are 1556 and 1068 A3 and A, and A, APL[PLI + AwtFCl

are 69 and 39 A', and M = 21 A, representing the thickness of the phospholipid protein monolayer of each particle (21). [TG]. [CE], [PL], and [FC] are the mole percentages of triglyceride, cholesteryl ester. total phospholipid, and free cholesterol, respectively. *, P < 0.05 compared with corn oil group. **, P < 0.01 compared with corn oil group. ***, P < 0.05 compared with first 24-h collection of chylomicrons from corn oil group.

particlc were cacalculated from the known a p B mass and the number of chylomicron particles per millilitte of lymph. The number of chylomicron particles per millilitre of lyrnph was calculated from the known average size of the chylomicrons and the content ol aiacylglycerol per millilitre of lymph. For these calculations a density of 0.88 g/mC was assumed for the mixed triacylgtycemls and a molecular mass of 2411 kDa for apo B-48 (1 Da = 1.661 x lo-" g). The radii of the tri- acylglycerol cores of the chylomicrons were calculated as described for the chylomicrons, except that the thickness of the surface monolayer was not included.

Electron microscopy Electron microscopy of the chylomicron particles was car-

ried out after dialysis against 0.01 % EDTA (pH 8.2). One drop from each chylomicron preparation was added to 1 mL of 1% osmium tetroxide to make a final triacylglycerol concentration of <1 mg/rnL (23). The suspensions were fixed for 1 h at room temperature. The freshly prepared samples were exam- ined in Phillips 300 EM at 60 keV accelerating voltage. The particle size was measured by a Durst Enlarger (Durst Labora- tory S-45, Durst SPA, Bolzano-Bozen, Italy) in combination with a computer,

Results Recovery of lymph and of lymph lipids

The volumes of lymph collected and the concentra- tions of the lipids were estimated forthe different groups of rats (five to six animals per group) receiving corn oiI or the fatty acid methyl esters. ' h e lymph was collected (two to three fractions per animal) over 2 days averaging about 7- 12 mL/day. The lipid content of the lymph varied with the water intake of the animals and averaged 4801 + 1836 mgTc of triacylglycerols and 378 * 115 mg% of phospholipids For the corn oil fed group, 3836 +- 1478 mg% of tiacylglycerols and 5 14 r 192 mg% of phuspholipids for the CFAME. and 3540 -1- I770 mg% of triacylglycerols and 388 "- 128 mg% of phos- pholipids for the TFAME fed groups. All groups of rats responded normally to the fatty meals, despite their administration via a stomach tube.

Lipid composition and radii of chylomicrons The quantitative GLC estimates of the triacylgly-

cerol, phospholipid, and free and esterified cholesterol of the chylomicrons are given in Table 1. The GLC elution patterns were essentially identical and character- istic of the lipid composition of normal chylomicrons (24). Table 1 also shows that the content of the cho- lesteryl esters and free cholesterol is significantly higher in the groups of rats receiving the fatty acid methyl esters than in the group of animals receiving corn oil (P < 0.01 or 0.05).

On the basis of the known lipid composition. it was possible to estimate the radii of the chylomicmns (21). Table 2 gives the calculated average radii (A) of the chylomicrons recovered from the three p u p s of rats on consecutive days of lymph coIlection. The average (adii of the lipid particles range from 423 to 1265 A as observed previously (24,25). Corn oil feeding produced particles of about two times larger diameter than those obtained h r n the fatty acid methyl ester feeding. The calculated particle radii were confirmed by electron microscopy (Fig. 1 ). Similar electron micrographs were obtained in three other instances where such compari- sons were made. including the methyl or ethyl esters and triacyI_elycerols. and free fatty acids and the correspond- ing triacyIglycerols.

Identification of apolipoproteins The identification of the apoproteins of the lymph

chylomicrons was based on comparisons with the apo- protein campsition of rat plasma lipoproteins by gel electrophoresis and isoelectric focusing, lo someexperi- ments additional tests were performed as explained below. Figure 2 shows an electropharetogram of the apoproteins of rat plasma lipoproteins and of a chylo- micron sample obtained from corn oil feeding. There is an effective separation of the apo 6-100. apo B-48, albumin, apo A-IV, apo E, apo A-I, and apo C, all of which migrate with rates anticipated on the basis of parallel runs with standard proteins of appropriate molecular weight. On the 4% polyacrylamide gel, the B apoproteins of lymph chylomicrons migrated at rates corresponding to molecular weights of approximately

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FIG. 1 . Electron micrographs of chylomicron particles from rat lymph. (A) Corn oil feeding (first 24-h collection); (B) corn oil feeding (second 24-h collection); (C) CFAME feedin8 (first 24-h collection); (D) CFAME feeding (second 24-h collection). Magnification, 13 184 X . Each bar represents 7584 A. Other conditions are as given in text.

240 000 (apo B-48) and 540 000 (apo B- loo), as did those of rat plasma VLDL and LDL. Apo B of human LDL migrated as a single band corresponding to 540 000 (26-28). On the basis of the mobility of the apoprotein bands in relation to the protein standards, we could also establish the molecular weights of apo A-IV (46 000), apo E (35 000) , apo A-I (26 000) , and apo C (< 12 000) bands. The SDS-polyacrylamide gel pattern of the chylomicron apoproteins resembles that of rat plasma HDL more closely than that of any other rat plasma lipoprotein class, except that the chylomicrons contain also apo B-48.

Effect of dietary fat on apoprotein profiles of chylo- microns

Figure 3 compares the apoprotein profiles of the chy- lomicrons from individual rats fed corn oil and TFAME,

while Fig. 4 shows the apoprotein profiles of the chylo- microns from individual rats fed CFAME. The corre- sponding apoprotein profiles obtained by isoelectric focusing are shown in Fig. 5. The chylomicrons of all three groups of rats have qualitatively identical apopro- tein composition. All of them contain apo B-48, apo A-IV, apo E, apo A-I, and apo C. The amounts of protein applied to the gels ranged from 20 to 100 pg. In this range the bands show up clearly without overload- ing. Only very light bands showed up in the gel positions corresponding to the slower migrating apo B, which indicated that only trace amounts, if any, of apo B-100 were present in the rat lymph chylomicrons at 24 h. The apolipoprotein profiles obtained in these experiments are essentially identical with those previously reported (29, 30). There were differences among individual ani- mals in the contamination of the apoprotein samples

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A - l b - A - I V - W

E - - A-I - c's -

FIG. 2. Electrophoretograms of apolipoproteins of various sources. Sample 1 , lymph chylomicrons from thoracic duct of a rat receiving corn oil; sample 2, VLDL of plasma of a fasting rat; sample 3, LDL of plasma of a fasting rat; sample 4, HDL of plasma of a fasting rat; sample 5, LDL of plasma of a fasting human subject; sample 6, reference standards: 670 000, thy- roglobulin dimer (cow); 330 000, thyroglobulin monomer (COW); 158 000, y-globulin (cow); 44 000, ovalbumin (chicken); 17 000, myoglobulin (horse). Electrophoresis con- ditions and instrumentation are as given in text. Coomassie brilliant blue staining.

with other proteins, which were not related to the pro- portions of the known apoproteins, which did not change.

Table 3 compares the relative percentages of the apr- proteins estimated by density scans of thc .-lsctrophore- tograms. The same percentages of apo B-48 were applied for SDS gels, as well as for IEF gels. There is a good general agreement between the percent composi- tion values derived by the two methods of separation of the apoproteins. According to the SDS gels, the corn oil chylomicrons contain about 8% apo B-48, 19% apo A-IV, 11 % apo E, 28% apo A-I, and 34% apo C. The apoprotein profiles of the chylomicrons from the fatty acid methyl ester feeding contained 1 1 % apo B-48, 14% apo A-IV, 12% apo E, 32% apo A-I, and 30% apo C. There are no significant differences in the ratios of apo B-48 between corn oil and CFAME feeding and only a marginally significant difference between corn oil and TFAME feeding. The lymph chylomicrons from the corn oil and CFAME fed groups had a significantly higher proportion of apo A-IV than those from the TFAME group (P < 0.0 1)

Table 4 gives the apo B concentration of the chylo-

FIG. 3. Electrophoretograms of apolipoproteins of lymph chylomicrons of rats receiving corn oil (A) or TFAME (B). Numbers 1-5 represent the initial 24-h samples from each rat. Coomassie brilliant blue staining of 4% polyacrylamide SDS gels. Other electrophoresis conditions and instrumentation are as given in text.

microns from the corn oil and the fatty acid methyl ester feeding. The apoprotein values were calculated from the total protein measurements and the apoprotein propor- tions were derived from the densitometric scanning of the SDS gels. The areas of the apoprotein bands were linearly related to the mass of the protein applied to the gel (data not shown). The apo B concentrations in the methyl ester fed groups (70.8 pg/mL for CFAME and 69.0 p,g/mL for TFAME) are significantly higher (P < 0.01) than those from the corn oil group (3 1.4 pg/mL). Likewise, the ratios of apo B/triacylglycerol and apo B/phospholipid were significantly higher (P < 0.05-0.01) for the methyl ester fed groups than for the corn oil fed group, as also shown in Table 4. It is seen that, for every gram of chylomicron triacylglycerol, the methyl ester fed group transported into the bloodstream almost twice as much B than did the corn oil fed group.

Figure 6 shows the electrophoretograms of the apo- proteins of lymph chylomicrons as obtained at different times after collection from corn oil (samples 1, 2, 3, 8, and 9) and CFAME (samples 4, 5 , 6, 10, and 11) feeding. Samples 1 and 4 were collected for 1 h and samples 2 and 5 were for 2 h. Samples 3 and 6 were analyzed at 12 h, while samples 8-1 1 were analyzed 4 days after the lymph collection. The proportion of the apo B- 100 species was higher in the initial lymph collec- tions, but decreased to undetectable levels at later times. These results are similar to those of Lee et al. (31), who have claimed that the rat intestine produces apo B- 100 as

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- A-IV- - I

E- - A - I - a

Z: -J . QLr -0 r

FIG. 4. Electrophoretograms of apolipoproteins of lymph chylomicrons from rats receiving CFAME. Numbers 1-5 represent samples from different rats. Samples 3-5 indicate separately the apolipoprotein profiles of the first (left side) and the second (right side) 24-h collection of chylomicrons. Electrophoresis conditions are as given in Fig. 3.

I". t

FIG. 5. Isoelectric focusing of apolipoproteins of lymph chylomicrons of rats receiving corn oil (A) or TFAME (B). Numbers 1-5 represent the initial 24-h samples from each rat. Polyacrylamide (7.5%) gel; 4.0-6.5 pH gradient; Coomassie brilliant blue staining. Other experimental conditions are as given in text.

a minor chylomicron apoprotein. Neither the production nor the disappearance of the apo B-100 appeared to be affected by the nature of the metabolic pathway of triacylglycerol biosynthesis. Since samples 1-6 were

rapidly processed and not washed, there are other pro- tein bands appearing in the area between apo B-48 and apo A-IV, which were not identified.

Figure 7 relates the calculated number of apo B mole- cules per lipid particle to the surface areas of the par- ticles. The correlation coefficients for the CFAME and TFAME derived particles and the surface area are 0.9100 (P < 0.005) and 0.8230 (P < 0.005), respec- tively. It is seen that the particles arising from the CFAME and TFAME feeding (phosphatidic acid path- way). contain one to eight molecules apo B, depending on pahicle size. In contrast, the chylomicrons arising from corn oil feeding (monoacylglycerol pathway) show a very low correlation coefficient (r = 0.4736, P > 0.1) between apo B content and surface area. The corn oil derived chylomicrons of both large and small size con- tain an average of three molecules of apo B per particle. The latter results are not unlike those obtained by Bhattacharya and Redgrave (32), who estimated that lymph chylomicrons from chow fed (monoacylglycerol pathway) rats contained two molecules apo B per par- ticle regardless of the particle size.

Discussion In the present study we have forced two groups of rats

to form triacylglycerols via the phosphatidic acid path- way by feeding them fatty acid methyl esters from either corn oil or tea-seed oil along with glucose. The methyl esters are readily hydrolyzed by pancreatic lipase to free

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TABLE 3. Percent apolipoprotein in the initial sample of lymph chylomicrons of rats fed corn oil or fatty acid methyl esters (mean ? SD)

SDS gel electrophoresis Isoelectric focusing

CO CFAME TFAME CO TFAME (n = 5) (n = 5) (n = 5) (n = 5) (n = 5)

B-48 7.922.9 l l . l k 2 . 8 11.622* 7.9 2 2.9 11.6? 2 A-IV E A-I 28.024.8 26.724.8 31.825.7 30.126.9 33.5k3.1 C-I1 6.3k1.2 5.8+1.0 C-1110 33.822.5 30.5?5.6 30.223.1 9.922.2 10.5k0.8 C-1113 10.3 k2.5 9.92 1.0

NOTE: *, P < 0.05 compared with chylomicrons of corn oil group.

TABLE 4. Apo B concentration of lymph chylomicrons of rats receiving corn oil or fatty acid methyl esters

CO CFAME TFAME

kg/ mL Mean 31.1 70.8* 69.0* * SD 13.2 22.9 18.5 n 8 11 11

Weight ratio, (apo B/triacylglycerols) x 10' Mean 1 .O 2.0** 2.7* 2 SD 0.4 0.8 1.1 n 8 10 10

Weight ratio, (apo B/phospholipids) x lo4 Mean 1.2 1.9** 2.5* + SD 0.4 0.9 0.9 n 8 1 1 11

NOTE: *, P < 0.01 compared with chylomicrons from corn oil group. **, P < 0.05 compared with chylomicrons from corn oil group.

fatty acids (33), which, in the absence of 2-monoacyl- glycerols, become esterified to sn-glycerol3-phosphate as an intermediate step in the triacylglycerol biosyn- thesis via the phosphatidic acid pathway (1, 4-6, 8). Another group of rats received corn oil, which contains a comparable mixture of fatty acids in the form of triacyl- glycerols, and presumably synthesized its chylomicron triacylglycerols largely via the monoacylglycerol path- way (2-4, 7).

On the basis of the apoprotein composition, it would appear that both pathways utilize the same apoprotein complement for triacylglycerol packaging into chylo- microns. There was a statistically significant increase in the relative proportion of apo A-IV in the chylomicrons containing high amounts of linoleate (corn oil and CFAME feeding) when compared with those rich in oleate (TFAME feeding). It is known that the intestinal synthesis and secretion of apo A-IV increases after fat

group. **, P < 0.01 compared with chylomicrons of corn oil

feeding (34, 35), and it has been suggested that it may play a role in the biosynthesis and (or) metabolism of intestinal lipoproteins. Apo A-IV is abundantly repre- sented in rat and human lymph chylornicrons, rat plasma HDL, and in the d = 1.21 g/mL fraction of human plasma (36), but its relationship to the fatty acid com- position of the lipoprotein lipids has not been assessed.

From the chemical analyses and measurements of the particle size, it is clear, however, that the chylomicrons containing triacylglycerols generated via the phospha- tidic acid pathway are smaller and more like VLDL. Like VLDL, they contain more cholesteryl ester and free cholesterol than chylornicrons (37), but in the absence of specific markers it is not possible to distinguish between small chylomicrons and large VLDL.

The particles produced via the phosphatidic acid path- way showed a regular increase in the number of apo B molecules with increasing particle size. It was calcu- lated that particles with average diameters of 75 nm contained one to two and those with average diameters of 150 nm about four to six apo B molecules. Glickman and Kirsch (38) have also claimed that apo B content increases with increasing particle size for chylomicrons of unknown biosynthetic origin isolated from human chylous fluid. In our study the chylomicrons generated via the monoacylglycerol pathway of triacylglycerol biosynthesis apparently contained a more or less fixed number (three) of apo B molecules per particle, regard- less of particle size. This observation agrees with that of Bhattacharya and Redgrave (32), who estimated a fixed number (two) of apo B molecules per chylomicron gen- erated via the monoacylglycerol pathway (rat chow feeding). Similarly, Mjos et al. (39) reported a fixed mass of apo B corresponding to five molecules per chylomicron from the monoacylglycerol pathway (corn oil feeding). The latter results were derived from elec- tron microscopic measurements of particle diameter and volume calculations, which tendedto underestimate the number of the larger particles present (see Ref. 32) and

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FIG. 6. Electrophoretograms of apolipoproteins of lymph chylomicrons from rats receiving corn oil (samples 1-3, 8, 9) or CFAME (samples 4-6, 10, 1 1) as obtained at different times of storage and processing. Samples 1 and 4, 1-h lymph collection; samples 2 and 5, 2-h lymph collection; samples 3 and 6, 12-h lymph collection. Samples 1-6 were centrifuged for 20 min to separate chylomicrons, which were lyophilized without prior washing and dialyzing. Samples 8-1 1 were washed for 18 h and dialyzed for 18 h. Sample 7 represents rat lymph LDL reference standard of apo B-100 and B-48. Electrophoresis conditions are as given in Fig. 3.

CFAME

,ArnME

Surface Area of Chylom~cron P a r t ~ c l e ( x l o 6 i 2 )

FIG. 7. Correlation between average number of molecules of apo B and average surface areas of chylomicron particles from rats receiving corn oil, CFAME, or TFAME. (0) Corn oil (CO) feeding: r = 0.4736; n = 8; P > 0. I; Y = 7.197 x IO-'X + 2.8378. (A) CFAME feeding: r = 0.9100; n = 11; P < 0.005; Y = 7.238 x IO-'X - 0.135. (A) TFAME feeding: r = 0.8230; n = l I ; P < 0.005; Y = 6.259 X 1 O - l ~ + 0.783. The calculations of the number of apo B molecules and the chylomicron surface areas are described in Materials and methods.

therefore exaggerated the amount of apo B associated with individual particles. In the present work the particle size was obtained from the neutral and polar lipid ratio, assuming that all polar lipids (free cholesterol and phos- pholipids) were located in the surface monolayer and all the neutral lipids (triacylglycerols and cholesteryl esters) were in the interior of the particle.

The discrepancy in the apoprotein content of the two types of particles is due to the difference in their total surface area, which is related to the particle size. The apoprotein/total surface area ratios remain constant. The reason for the difference in the size of the lipid particles arising via the two different pathways of intes- tinal triacylglycerol biosynthesis is not clear. It is possi- ble that this is related to the much more rapid absorption and resynthesis of the triacylglycerols via the mono- acylglycerol pathway, and to their more rapid transport, which may be limited only by the rate of synthesis of apo B. Under the more moderate rates of triacylglycerol formation and secretion via the phosphatidic acid path- way, the supply of triacylglycerol rather than apo B might be limiting and the available apo B would be carried by many small particles. The biosynthesis of the phosphatidic acid itself is known to take place at a site different from that responsible for the acylation of the monoacylglycerols (7, 8). Alternatively, the chylo-

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YANG AND KUKSIS 523

microns from the methyl ester feeding could have been smaller because of a slower rate of absorption of the fatty acids owing to their slower release from the methyl when compared with the glyceryl esters. Also, chylo- microns formed at the peak of fat absorption are known to be larger than those formed at the beginning and towards the end of the absorption period (40). In the present study, however, differences in particle size were seen between the two pathways at all times of sampling. Furthermore, the differences in the particle size were also seen between free fatty acid and triacylglycerol feeding (L.-Y. Yang and A. Kuksis, unpublished results), which should not have been influenced by any difficulty in hydrolyzing the methyl esters.

In conclusion, this study demonstrates that the meta- bolic pathway of triacylglycerol biosynthesis affects the composition of the apoproteins of the chylomicron and VLDL-like particles of the lymph. Stored chylomicrons from both pathways contained B-48 as the sole apo B species, while freshly collected chylomicrons in both instances contained also a small proportion of the apo B-100 species. While it is possible that apo B-100 is produced by the rat intestine as suggested by Lee et al. (31), its origin by filtration from blood has not been completely excluded. Further evidence for an intestinal synthesis of this apoprotein must be sought in the jejunal villus cells, which already have given evidence of the presence of mRNA for apo B- 100 (4 1). Furthermore, apo B-100 has recently been demonstrated to be formed in human intestine in vitro (42). The physiological sig- nificance of the transfer of higher amounts of intestinal apoproteins per gram of fat to the bloodstream by the smaller lipid particles resulting from fatty acid methyl ester feeding remains to be established.

Acknowledgments These studies were supported by funds from the Heart

and Stroke Foundation of Ontario, Toronto, Ont., and the Medical Research Council of Canada, Ottawa, Ont. L.-Y. Y. was the recipient of an Ontario Graduate Scholarship.

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