T. G. Redgrave' and L. A. Carlson

Transcription

1 Changes in plasma very low density and low density lipoprotein ontent, omposition, and size after a fatty meal in normo- and hypertriglyeridemi man T. G. Redgrave' and L.. Carlson King Gustaf V Researh Institute, Karolinska Hospital, Stokholm, Sweden bstrat Four subfrations of plasma haraterized by dereasing Sf value and LL were isolated by density gradient preparative ultraentrifugation from normotriglyeridemi (NTG) and hypertriglyeridemi (HTG) (type IV) subjets in the fasting state and after a fatty meal. Chemial analysis and omputation of numbers of partiles in eah fration showed that the hyperlipidemia of type IV subjets was aounted for by an inrease in total numbers of and a shift in the distribution of towards partiles of larger diameter. Postprandial hyperlipidemia was due to the presene of hylomiron remnants rather than intat hylomirons, and was aounted for by an inrease in partile diameter of the largest subfration rather than by an inrease in partile numbers. Postprandial hyperlipidemia was aompanied by a shift in the distribution of towards partiles of larger diameter in both NTG and HTG subjets, probably beause of ompetition for the triglyeride-depletion proess between hylomirons and hepati. Most hylomiron remnants were removed from the irulation without degradation to smaller or to LL, but some remnants were suffiiently small to ontribute to smaller subfrations. The LI. of type IV subjets ontained more apoprotein than those from NTG subjets, and this differene was assoiated with inreases in diameter, moleular weight, density, and the ratio of pr0tein:phospholipid in LL from type IV subjets. efetive degradation of large to small, and of to LL may be related to this alteration in apoprotein ontent of the lipoproteins in type IV subjets-redgrave, T. G., and L.. Carlson. Changes in plasma very low density and low density lipoprotein ontent, omposition, and size after a fatty meal in normoand hypertriglyeridemi man. J. Lipid Res : Supplementary keywords apoprotein. hylomirons hylomiron remnants. plasma triglyerides bsorption of a fatty meal adds triglyeride-rih intestinal partiles to the plasma lipoprotein pool, but postprandial hyperlipidemia is usually of modest proportions (1-3) despite onsiderable fluxes of absorbed dietary lipids through the plasma ompartment. Removal rates of hylomiron triglyeride and holesterol from the plasma are very rapid in man and in experimental animals, but are redued in hypertriglyeridemia (3-6), raising the possibility that hylomirons or their ataboli remnants may persist in the plasma and ontribute to hyperlipidemia. Evidene that suh a mehanism ontributes to hyperlipidemia has been obtained in the holesterol-fed rabbit (7), in diabeti and hypothyroid rats (S), and in obese and alohol-fed rats (5, 6). The present study had two objetives. The first was to define the nature of postprandial hyperlipidemia in terms of lipoprotein number, size, and moleular omposition by analyzing the hemial omposition of subfrations. The postprandial hanges in normo (NTG)- and hypertriglyeridemi (HTG) subjets were ompared to establish if hylomiron remnants ontribute to the plasma pool. Seond, we were interested in omparing the omposition of subfrations and of LL from subjets with type IV HTG with those from NTG individuals. MTERILS N METHOS Subjets and proedures Seven NTG and seven HTG adult men or women were studied. The NTG subjets had normal blood lipids exept for one healthy man lassified as having type I1 (9) with plasma holesterol of 7.8 mmol/l (302 mg/dl). The HTG subjets were all lassified as having type IV hyperlipoproteinrmia (9). Healthy volunteers or patients who had previous myoardial infartion were hosen for the study, exluding bbreviations: LL, low density lipoprotein;, very low density lipoprotein: NTG, normotriglyeridemi; HTG, hypertriglyeridemi; ET, ethylene diamine tetraaeti aid; IL, intermediate density lipoprotein; HL, high density lipoprotein; TG, triglyeride. Visiting sientist on leave from the epartment of Physiology, University of Melbourne, Parkville, Vitoria 3052, ustralia. ddress orrespondene to r. Redgrave at this address. ownloaded from by guest, on pril 17, 2018 Journal of Lipid Researh Volume 20,

2 r lml0i en 1.06 z 1.04 u) S F: O R volume (ml) Fig. 1. Shape of the density gradient. The densities at 20 C of 1-ml frations from the entrifuge tube were measured. Points are means of dupliate entrifugations. 0-Not entrifuged, stood on benh for 18 hr; -entrifuged 28,300 rpm for 43 min (spin ); U-spin plus 37,000 rpm for 18 hr (spin ). subjets with either diabetes or other known metaboli disease, or under treatment likely to affet lipid metabolism. blood sample was taken on the morning of the test after an overnight fast. Subjets then onsumed a test meal of 1OOg of soybean oil seasoned with lemon juie. The oil ontained 10.8% palmiti, 3.6% steari, 21.4% olei, 54.8% linolei, and 8.9% linoleni aids, giving an average moleular weight for triglyerides of 872. The test meal was tolerated well and no subjet was nauseated. dditional blood samples were taken after 3 and 6 hr. lood was taken into tubes ontaining 1.4 mg/ml ET. Plasma was separated as soon as possible by entrifugation for 20 min at loo0g at room temperature (20-23"C), plaed into tubes ontaining 0.8 mg/ml ofp-hloromeruriphenylsulfoni aid (lo), and stored at 4 C for not longer than 48 hr before lipoprotein separation. Methods density gradient ultraentrifugation proedure was used to subfrationate the plasma lipoproteins. Plasma was adjusted to density 1.10 g/ml by adding solid Kr, 0.14 g/ml. Gradients onsisting of 4 ml of 1.10 glml plasma, 3 ml eah of g/ml and g/ml, and 3.4 ml of g/ml salt solutions were formed as desribed previously (1 1) and entrifuged in the SW40 rotor of the ekman L5-75 ultraentrifuge at 20 C. was subfrationed by umulative rate entrifugation, as desribed by Lindgren, Jensen, and O Hath (12), to float partiles of diameter > 75 nm (S, > 400, fration ), nm (Sf , fration ), nm (S, , fration C), and nm (S, , fration ) to the top of the tube. Calulated requirements for these flotations at 20 C were 4.5 x lo6, 17.5 x lo6, 31.2 x lo6, and 152 x lo6 g-min. In the SW 40 rotor, after orretion for aeleration and deeleration fores and previous runs, entrifugation was for 43 min at 28,300 rpm (fration ), then for 67 min at 40,000 rpm (fration ), then for 71 min at 40,000 rpm (fration C), and lastly for 18 hr at 37,000 rpm (fration 1). Times given are from swith-on to swith-off of drive power. Maximum aeleration was used, and the brake was not used. Eah fration was arefully aspirated from the top of the tube, and density g/ml salt solution was used to refill the tube before the next run. The shape of the gradient is shown in Fig. 1. fter the final entrifugation, the major LL fration was easily identified as a yellow band about 5 mm wide, loated between 4 and 5 m from the bottom of the tube. This band, whih ontained 67% (SEM 4.0, t/ = 7) of total plasma LL, was also aspirated from the tube at the end of the proedure. nalysis Triglyeride (13) and holesterol (14) onentrations in plasma and separated frations were measured on a Tehnion uto-nalyzer I after lipid extration (15). Lipid phosphorus was measured (16) after digestion of samples and the values were multiplied by 25 to give phospholipid ontent. Protein ontent was determined (1 7) after extrating with hloroform the turbidity due to lipids. poprotein ontent of lipoprotein frations was estimated by differene after extration of soluble apoproteins with equal volumes of 2-propanol (18) and then l-pentanol. Extration with 2-propanol quantitatively preipitates apoprotein (18) and the subsequent extration with 1-pentanol removes lipids and produes an aqueous phase onentrated by a fator of 1.48, whih greatly failitates determination of the nonapo-, soluble apoproteins in the lipoprotein subfrations. High salt onentrations were initially redued by dialysis to avoid interferene with the protein assay. Esterified and free holesterol were separated on olumns of Sephadex LH-20, modifying the desribed proedure (19) to employ olumns 1 m in diameter and 20 m high. Eletrophoresis in agarose gel was aording to Noble (20). is gel eletrophoresis (21) was performed in 8 M urea at ph 9.1 on the peptides soluble after extration with 2-propanol and 1-pentanol. ownloaded from by guest, on pril 17, Journal of Lipid Researh Volume 20, 1979

3 Plasma albumin onentrations were measured by roket immunoeletrophoresis (22) to enable orretions for shifts of body water. nalytial ultraentrifugation was performed in the Spino model E, with a 1.2-ml double setor ell in the N- rotor. Runs in g/ml salt solution at 26 C were at speeds of 10,000 rpm for fration, at 20,000 rpm for frations and C, at 30,000 rpm for fration, and at 40,000 rpm for LL. F values were orreted to zero onentrations (12) to give the Sf values. ensities were measured at 20 C with a alulating digital density meter (Model M 45 nton Paar KG Graz, ustria). For analysis of the fatty aid omposition the frations were extrated by hloroform-methanol ( 15). fter separation by thin-layer hromatography (23) the triglyeride fatty aids were methylated and separated on a 5720 gas hromatograph (Hewlett- Pakard) as desribed (24). Calulations Lipoprotein TG moleular weight was taken to be 860 and that of holesteryl ester 1.68 X 387. Partile densities were alulated from the partial speifi volumes of the onstituents; for TG; for holesteryl ester; for free holesterol; and for phospholipids (25). The partial speifi volume for apoprotein is not known, but it was estimated for apoprotein from the bakground density of LL isolated from gradients as desribed. akground density was measured after ultrafiltration of LL by membrane filters. ssuming that the LL band was at its equilibrium density, the partial speifi volume of its apoprotein was alulated from measured lipoprotein omposition and by employing the known partial speifi volumes of the other onstituents (rhimedes priniple). value of t (SEM, n = 6) was obtained and was used for all apoprotein moieties. This value should be ompared with those that an be alulated (26) from published (27) amino aid and arbohydrate ontents of the apoproteins, viz for apoprotein ; and 0.743, 0.730, and for apoproteins C-I, C-11, and C-111, respetively. The radius, rl, of the partile was alulated assuming spherial partiles with all apolar lipids in the ore with radius r2. s the total volume, V1, of all onstituents of a fration and the volume, Vz, of the apolar onstituents an be alulated, e.g., per ml in the isolated frations, the ratio r2/r, = C = (V$ V1)1 3 an be obtained. Sine the data of Sata, Havel, and Jones (25) and MjSs et al. (28) indiate that all partiles have a onstant thikness of the polar surfae shell of 2.15 nm it follows that rl - r2 = 2.15 and hene rl was alulated from the relation rl - rl*c = 2.15 or rl = 2.15/(1 - C). Partile Sr and moleular weight were alulated assuming Stokes spheres as desribed by Lindgren et al. (12) viz. Sf = z ( d)/1.847 and MW = X 3 x d where is the diameter in nm and d is the partile density. Partile moleular onstituents in daltons were then alulated as the produt of weight fration and moleular weight. Partile numbers were then alulated by dividing the volume of a partile of diameter into the total lipoprotein volume. ll results were orreted for dilution due to Kr solution (4%) and for shifts in body water (0-6%). RESULTS Charateristis of isolated frations The desribed ultraentrifugal proedure divided plasma into four subfrations of progressively dereasing diameter, inreasing density, and dereasing moleular weight (Table 1). lthough the alulated moleular parameters of the separated subfrations were within the predited range of partile dimensions, analytial ultraentrifugation was used to gain an independent assessment of the average size of the frations. y this method fration had diameter 74 nm, fration 42.8 nm, fration C 37.0 nm, fration 33.6 nm, and LL 17.9 nm (means of determinations on two separate samples from fasting HTG subjets), thus onfirming the general validity of the alulations, but also showing that alulated diameter is overestimated by 15% on average, perhaps beause the alulations assume a uniform population whereas, in fat, eah fration is a ontinuum. The orrelation oeffiient between the omputed values for diameters obtained by analytial ultraentrifugation and the ratio (volume of apolar onstituents)/(total partile volume) was iret measurements by eletron mirosopy also gave diameters lose to the alulated ones, with means of 98 nm for fration, 61 nm for fration, 46 nm for fration C, and 35 nm for fration (Fig. 2). The material of fration did not enter the gel on agarose gel eletrophoresis (Fig. 3). Frations, C, and all had pre-p mobility that was slightly slower postprandial in fration C and slower in fration at all times. is-gel eletrophoresis in polyarylamide gels (Fig. 4) showed that the proteins soluble after extration with 2-propanol and I-pentanol were approximately similar in all frations, but fration on- ownloaded from by guest, on pril 17, 2018 Redgruve and Curlson Changes in plasma lipoproteins after a fatty meal 219

4 TLE 1. Moleular parameters of lipoprotein frations t t t t t t t ? rnszty q/m/ t t f f t f t C 0.003" 0.933? t t t t t t t t t LL LL LL 54.4 t t t t t t t t t t t t t t t t t t 0.20 inmrl<u iim 159 t f t f If- 0.68' 44.2 rt ? f f ? 0.29".LlolPrular iwrghl x 1 OP 1678? f t f 1.05' t t f " 130 t 5.gd t t t 1.00" 639 t 76.5d 50.5 t t t t 0.50b 176 t 15.5d 54.5 t t 2.12' 31.5 t ? 0.50" 1698 t 471.8d 51.0t t 2.59' r t 0.15* Results are means t SEM from five observations in eah ase exept due to sample loss. The fasting sample of fration was insuffiient for analysis. For eah fration sample 0 hr is fasting sample, 3 hr and 6 hr are times after the fatty meal. Statistial analysis by Student's t test between NTG and HTG group. " P < *P < Statistial analysis by paired t test to ompare pre- and postprandial results within eah group. P < P < tained relatively more C-I and arginine-rih peptides, and less C-II peptide. Lipoproteins in the fasting state, NTG vs. HTG The triglyeride onentration was higher in plasma and all frations in HTG subjets in the fasting state (Table 2). ll other omponents of as well as the number of partiles were higher in eah fration in HTG than NTG subjets (Tables 2 and 3). There was a shift towards relatively more larger partiles for the HTG group (Fig. 5). There was no differene, however, between NTG and HTG subjets with regard to density, diameters, and moleular weights for the four frations. The perentage of apoprotein ontent (measured as protein insoluble in 50% 2-propanol plus l-pentanol) inreased progressively from fration to fration in both NTG and HTG subjets (Table 2). In LL, 97% (SEM 0.26, n = 15) and 96% (SEM 0.53, n = 7) of protein was aounted for by apoprotein in NTG and HTG subjets respetively. The major LL band was signifiantly less (P < 0.001) in HTG subjets. verage reovery of total plasma holesterol in the subfrations plus the LL band was 57% in NTG and 67% in HTG subjets. Unaounted holesterol in IL and HL was not measured in this study. The moleular mass of all lipoprotein omponents dereased drastially with dereasing partile diameter exept the mass of apo-peptide (Table 4). The far greatest fall was for daltons of triglyeride, from around lo8 in -fration to 3 X lo5 in LL. Table 4 shows that LL of HTG subjets ontained as muh phospholipid but more protein than NTG subjets, hene the mean ratio of LL protein:phospholipid was >1 in the HTG group, and <1 in the NTG group (Fig. 6, right-hand side). The major LL fration from HTG subjets was more dense and slightly larger than in NTG subjets. The mean moleular weight of LL of HTG subjets was 14% greater than in NTG subjets. This differene was statistially signifiant (P < 0.01) when observations at all times were pooled. The individual values for apoprotein omplement ownloaded from by guest, on pril 17, Journal of Lipid Researh Volume 20, 1979

5 : :C Z. 100 nm : LL Fig. 2. Eletron mirosopi photographs of the lipoprotein subfrations and of LL from a HTG subjet at 0 hr. Mirosope magnifiation ~48,000. Measurements of partile diameter of the visualized frations gave the following values (mean and range, nm), 98 (66-159); R, 61 (41-97); C. 46 (35-62);, 35 (24-45). in frations, C, and are plotted in Fig. 6. While there is no obvious differene in apoprotein ontent of from NTG or HTG subjets, it should be noted that the overall mean of the data in Table 4 for NTG subjets is 0.66 x lo6 daltons of apoprotein, ompared with 0.74 X lo6 daltons in HTG subjets. For LL, however, there is learly more apoprotein in HTG subjets viz (SEM 0.03, n = 8) X lo6 daltons ompared with 0.65 (SEM 0.01, n = 15) for NTG subjets. This differene is highly signifiant (P < 0.001) by Student's t test). Effets of the fatty meal Table 2 shows the hanges produed in plasma lipids and in the subfrations after the fatty meal. The postprandial inrease in plasma TG was aounted for mainly by the inrease in TG of fration, but frations and C showed small butsignifiant inreases in NTG subjets. The rise in triglyeride onentration of fration was due to an inrease in the size of the partiles, not of the numbers. The volume inreased about 10 times for HTG subjets (Table 1). It is note- ownloaded from by guest, on pril 17, 2018 C LL """"" CII Clll I Fig. 3. garose gel eletrophoresis of subfrations and LL from a NTG subjet. Fration does not enter the gel. Frations, C, and show pre-p mobility, slightly slower in fration C and slower in fration at all times. 0,3 hr, and 6 hr indiate elapsed time after fatty meal. - C C C I" 0 3h 6h Fig. 4. Polyarylamide dis-gel eletrophoresis of subfrations from a HTG subjet. The soluble apoproteins of fration ontain more C-I and arginine-rih peptides, and less C-I1 peptide postprandially. 0.3 hr, and 6 hr indiate elapsed time after fatty meal. Redgrave and Carlson Changes in plasma lipoproteins after a fatty meal 221

6 TLE 2. Lipid and protein ontents of plasma and lipoprotein frations NTG HTG 0 hr 3 hr 6 hr 0 hr 3 hr 6 hi- Triglyeride onentration (pmovml) Plasma 1.70? ? ? 0.04 C 0.26 t ? 0.05 Cholesterol onentration (pmovml) Plasma C LL Phospholipid onentration (pmovml) 0.05? 0.01 C 0.09? ? 0.04 Protein (pg/ml) C 2.0? ? O.2Ib 5.4? ? ? 1.08b 0.24? ? O.lOb 0.30? t t O.3Cib 0.23? t 0.07" 1.70? ? ? t ? 0.04" 1.33 t t t ? ? ? ? ? ? ? ? ? ? ? t ? ? 0.02* 0.08? ? b 0.06? ? ? 0.02" 0.51? ? ? ? t ? 0.02" 0.59 t t k ? ? t t t t ? ? ? ? t t ? t ? apoprotein (% total protein) 3.4? C 35.4? ? ? ? ? t t ? t ? ? 1.4b 17.5 t ? 4.5" 33.1? ? 4.2" 92.7? ? ? t t ? ? t t ? ? ? t t t ? ? % ? ? O.ll'l 7.1? ? t 5.3* 127.9? ? ? ? ? t t t t ? t ? t ? ? ? t ? ? t 2.87 etails and symbols as in Table 1. Plasma results are from seven subjets eah. eause TG and PL were assayed as glyerol and phosphorus, molar units are given. pproximate onversion fators assume moleular weights of 860 for TG, and 775 for PL. The average perentages of holesterol esterified were 73% in fration, 53% in fration, 54% in fration C, 58% in fration, and 76% in LL. Statistial analysis by paired t test to ompare pre- and postprandial results within eah group. " P < ownloaded from by guest, on pril 17, 2018 worthy that HTG subjets showed in plasma and fration an inrease of muh greater magnitude than in NTC subjets. Total plasma holesterol was not signifiantly inreased in this study, but holesterol in fration showed a marked postprandial inrease in NTG and HTG subjets. Cholesterol was also inreased by a small amount in frations and C in NTC subjets. Phospholipids of the subfrations showed hanges similar to those for TG and holesterol, but the inreases were not statistially signifiant. How- ever, in fration phospholipid was signifiantly dereased 6 hr postprandial. s shown in Table 2, the protein ontent in the subfration showed hanges very similar to those for lipoprotein lipid, with hanges most pronouned in fration. The relative amount of apoprotein C-I1 was onsistently redued whereas arginine-rih peptide was inreased in postprandial samples in fration (Fig. 4). rginine-rih apoprotein was variably present, and was more onsistently found in NTG than in HTG subjets. There was no ap- 222 Journal of Lipid Researh Volume 20, 1979

7 TLE 3. Numbers of partiles in the lipoprotein subfrations ( x 10-'z/ml of plasma) ~ NTG HTG 0 hr 3 hr 6 hr 0 hr 3 hr 6 hr fration 0.3 f ? rf: f ? f ? ? f ? 20.8 C 9.4? ? ? f f ? ? rf: " LL 595? f ? ? f rf: 33.5 Statistial analysis by paired t test to ompare pre- and postprandial results within eah group. 'P < parent qualitative differene between the two groups studied. espite the hanges observed in the hemial ontent of the subfrations after the fatty meal, as shown in Table 3, the alulated numbers of lipoprotein partiles in frations and C did not hange signifiantly, although a tendeny for inreased numbers was apparent. In fration, numbers were signifiantly redued 6 hr after the fatty meal in HTG subjets. Table 4 shows that partiles in fration have a muh greater moleular mass postprandially for all lipid onstituents, in parallel with the observed inrease in partile dimensions, but other frations were not hanged by the fatty meal. Unlike all other onstituents the mean moleular mass of apoprotein was remarkably onstant in the different frations. Fration appeared to ontain more apoprotein in HTG subjets, but beause of the diffiulty of measuring the very small amounts of protein in fration this figure may be unreliable, although a similar high value was observed in rat large lipoproteins (28). The relative amounts in triglyerides of the two most harateristi fatty aids of the fatty meal, linolei (18:2) and linoleni (18:3) aid, present in 54.8 and 8.9%, respetively, in the fat, are given in Table 5. In fration the perentage of 18:2 and 18:3 inreased 2- to 3-fold postprandially in both NTG and HTG subjets. t 6 hr the relative ontent of 18:2 and 18:3 in fration had approahed the orresponding values of the fatty meal. The relative amounts of these fatty aids rose also in the other frations but to a lesser degree and the perentage figures for 18:2 and 18:3 did not reah one-fourth of that for the ingested fat. Furthermore, the rise was smaller the smaller the partiles were. While the inrease in relative amount of 18:2 and 18:3 was similar in fration for NTG and HTG subjets, it was smaller for HTG than NTG individuals in the frations to. This differene beame more pronouned the smaller the. For instane, in fration the perentage of 18:3 rose by about 1 % for NTG but only by 0.1 % for HTG subjets. ISCUSSION Our findings onfirm and extend many previous studies of postprandial hyperlipidemia after a fatty meal (1-3). more omplete quantitative desription of postprandial hyperlipidemia than before has been obtained by our subfrationation and hemial haraterization of the lipoproteins. In partiular our experimental design permitted observations of the possible ontribution of hylomirons and their remnants to smaller diameter frations of the plasma lipo- a 801 n C C C 0 3h 6h Fig. 5. Perentage distribution of partile numbers (a) and TG mass (6) in subfrations. 0 NTG, HTG (data from Tables 2 and 3). Compared with NTG subjets, HTG subjets show a relative shift towards partiles of larger size. fter a fatty meal, both groups show a shift towards larger partiles, without an inrease in total partile number. The mass of TG is distributed more evenly than partile numbers, but similar differenes between NTG and HTG subjets are apparent, as are hanges indued by the fatty meal. ownloaded from by guest, on pril 17, 2018 Redgrave and Carlson Changes in plasma lipoproteins after a fatty meal 223

8 TLE 4. Moleular mass of lipoprotein onstituents (daltons X 10F) I.(; H1.G 0 hr :1 Ill. 6 hr tll 6 hr Triglyeride : ? t ? * ? t , ? i t ? LL> 0.2? f t Cholesteryl ester t i t ? t t ? t t LL i t 0.20 Free holesterol LL Phospholipid LL Soluble apoprotein LI, poprotein 1.8 t t ? ? t t t ? t i ? I i ? t & ? ? ? & f ? f * f * f * t t t ? t t t t t & t ? t t ? ? ownloaded from by guest, on pril 17, 2018 LI t ? t f t P < 0.01, omparison by Student s t test proteins, and of differenes in hylomiron learanes between NTG and HTG subjets. The data of Tables 2,3, and 4 show that almost all of postprandial hyperlipidemia is aounted for by inreases in mass in fration. espite the hylomironemia the inrease in hemial onstituents of fration is aounted for by an inrease in partile size (Tables 1 and 4), not partile numbers. The small numbers of partiles in fration during fat absorption requires explanation. However it an readily be alulated that 100 g of fat produes only 2.6 x 10l6 hylomirons of diameter 200 nm. This represents an influx of 3.6 x 1Olo min- ml-i of plasma if the fat load is assumed to be absorbed uniformly over 4 hr. If hylomiron t% = 5 min in NTG subjets (3, 4, 29) then turnover is 0.14 min-, and the alulated influx rate orresponds to a steady-state onentration of 0.26 x 1 OI2 partiledml, 224 Journal of Lipid Researh Volume 20, 1979

9 whih agrees with our observations (Table 3). Similarly in HTG subjets if hylomiron t% = 15 min the steady-state plasma ontent an be alulated to be TLE 5. Content of linolei (18:2) and linoleni (18:3) aid in the triglyerides of the four frations and of LL from two subjets in eah group 0.78 x 10l2 partiles/ml, again in aord with our ob- NTti HTG servations (Table 3). Chemial omposition of fration Ohr 6hr Ohr 6hr shows that in NTG subjets the 'hylomiron' fration ontains about 10% holesteryl ester, whih is more onsistent with a TG-depleted hylomiron rem nant than a hylomiron. Remnants still are suf fiiently large to float in this fration (30). In HTG subjets, fasting fration also has a high holesteryl ester ontent, but postprandial samples show less TG (; depletion than in NTG subjets. Nevertheless, fration analysis shows onsiderably more holesteryl ester than the 1.4% of human lymph hylomirons (31) The high proportion of holesterol esterified (73%) LL in fration is onsistent with its origin from intestinal lipoproteins, as is its ontent of linolei and linoleni aids (Table 5) whih in postprandial samples approahed that of the ingested fat both in NTG and H'TG subjets. poproteins are present in hylomirons from lymph (31) but were not observed in fration of postprandial samples so are presumably lost when hylomirons are degraded to remnants. The inreases in mass of lipoprotein in frations and C in NTG subjets are aompanied by small inreases in partile numbers of these frations, but the hanges are not statistially signifiant. In fat fration shows a derease in partile numbers, whih anels possible inreases in numbers of the smaller frations so that total numbers of lipoprotein partiles do not inrease in the total (Table 3). However, Linolei aid, %, fration Linoleni aid, % fration : LL as shown in Fig. 5a, the distribution of partiles is different between HTG and NTG subjets, and the differene is aentuated after a fatty meal. oth groups show a shift in the population towards larger partiles after the fatty meal but this is not refleted in individual subfrations (Table 1). There is onvining evidene that the smaller partiles (fration ) are derived by triglyeride depletion from larger (32). The different shapes of the populations in the fasting state suggest that depletion is less effiient in HTG subjets ompared with ontrols (Fig. 5u). When the ataboli load on the lipolyti pathway is inreased after the fatty meal, both C a@ protein:p-l NTG and HTG subjets show a shift in the popula-._.._._.._.._ LL.._.... tion towards larger sizes, most likely as a onsequene -~ ~..- Fig. 6. poprotein moleular omplement and protein: phospholipid ratio. Individual values for apoprotein omplement of frations, C, and and LL are shown, with the means (bar). The LL from HTG subjets ontain about one-third more than LL from NTG subjets but, beause variability is greater, subfrations show no differene in ontent of apoprotein between NTG and HTG subjets. The LL protein:phospholipid ratio in HTG is greater than in most NTG subjets. Note that the right-hand axis refers only to the protein:phosphoepid ratio on the extreme right-hand side of the figure. Of 'Ompetition for lipolysis between hy1omirons and large (33). y omparing the distribution of lipoprotein numbers and triglyeride mass ( ~ i % ~. and b)* it is Seen that hypertriglyeridemia in HTG subjets is due to a shift in the population toward larger partiles, together with an inrease in partile numbers (Tab1e 3). Two disontinuities are apparent in the otherwise ownloaded from by guest, on pril 17, 2018 Redgave and Carlson Changes in plasma lipoproteins after a fatty meal 225

10 smooth deline of the moleular masses of onstituents (exept apoprotein ) as one proeeds from larger to smaller partiles (Table 4). The first disontinuity is in postprandial samples between fration and fration, indiating that fration might not be progressively depleted of onstituents until it attains the moleular proportions of fration, but instead might be removed diretly from the plasma while still retaining the moleular harateristis of fration. This interpretation is onsistent with removal of hylomiron remnants by the liver as in the rat (30) when only SO-SO% of partile triglyeride has been removed. On the other hand the data of Table 5 show lear inreases in the proportions of linolei (18:2) and linoleni (18:3) aid in the smaller frations, suh that newly ingested triglyeride onstitutes about one-third of frations and C in NTG subjets, but less than one-tenth of these frations in HTG subjets. Smaller proportions appear in fration. Three different mehanisms ould aount for this observation. First, hylomiron remnants might be degraded suffiiently to appear in these frations (vide infra). Seond, hylomiron remnant triglyeride fatty aids might be resereted by the liver after their initial learane. Third, onstituent fatty aids released by the ation of lipoprotein lipase in peripheral tissues might be reyled as hepati within the time ourse of the experiment. The relative signifiane of these mehanisms is unknown. The seond disontinuity ours between fration, the smallest fration of, and LL and is most apparent for TG, phospholipid, and soluble apoprotein (Table 4). It is a onsequene of the ataboli onversion of to LL, through IL. lthough IL was not studied in these experiments it has a hemial omposition between that of fration and LL (34-36). The moleular mass of apoprotein within eah fration of (exluding fration ) plus LL is remarkably onstant (Table 4). This finding extends those of Hammond and Fisher (37), MjQs et al. (28), and Eisenberg and Rahmilewitz (36). In all ases, and LL partiles ontain about 0.7 x 10' daltons of apoprotein, and this value is similar in rat and man. Our study onfirms this finding for and LL in man. Values of x lo6 reported for human by Eisenberg et al. (38) appear too low, in part, apparently, beause of inorret assumptions about partile hydrated density used to alulate moleular weight. For example, a partile of Sf 147 was assumed to have a hydrated density of 0.94 g/ml, but our measurements indiate a hydrated density of 0.96 g/ml. Our data allow a omparison of apoprotein ontent between NTG and HTG groups. There is a small but signifiant differene between the groups for LL but not for. Nevertheless, on average from HTG subjets ontain 0.74 x 10' daltons of apoprotein ompared with 0.66 X lo6 daltons in NTG subjets. For LL however, HTG subjets ontain 0.89 x 10' daltons of apoprotein ompared with a mean of 0.65 x lo6 daltons for the NTG group. Our data should be ompared with values of x 10' daltons for LL from type IV subjets and x lofi daltons for LL from normal or type 11 subjets reported by Hammond and Fisher (37) and Fisher, Hammond, and Warmke (39). Our data onfirm the differene in LL omposition, for whih one possible explanation ould be the addition of eight monomeri units of apoprotein, whih has a reported moleular weight of 27,500 (40). Hene LL from N'TG subjets would ontain 24 monomeri units of apoprotein (0.66 x 10' daltons) whereas LL from HTG subjets may ontain 32 monomeri units (0.88 x 10' daltons). lso plausible is the possibility that LL from NTG subjets ontains three subunits of apoprotein with a polypeptide hain of 0.22 x 10' daltons, whereas LL from HTG subjets ontains four suh subunits. This possibility agrees well with the size of the polypeptide hain reported by Simons and Helenius (41) and Smith, awson, and Tanford (42). Our data suggest, but do not prove, that from HTG subjets also ontains more apoprotein than from NTG subjets, whih would be onsistent with the generally aepted onept that LL partiles are derived from partiles without loss of this peptide. Table 1 shows that the prinipal LL band from HTG subjets also differs in other ways. It has a slightly higher hydrated density, larger moleular diameter, and greater moleular weight than LL from NTG subjets. eause of the inrease in protein ontent the ratio of protein to phospholipid is reversed (Fig. 6) independently of any assumptions that ould overestimate LL moleular weight. The relationship established between surfae and ore onstituents for larger partiles might not be ompletely appliable to LL although reently a spherial model for LL has been proposed, with a thikness of the polar surfae shell of 2.13 nm (43). Furthermore our alulations ignore partile hydration (44) and assume uniform spherial partiles. Nevertheless alulated LL moleular weight is in fair agreement with reent hydrodynami measurements by Fisher et al. (39,44) ( x lo6) and Nelson et al. (45) (2.9 x lo6) using sedimentation equilibrium. Hydrated density found by these workers agrees well with our measure- ownloaded from by guest, on pril 17, Journal of Lipid Researh Volume 20, 1979

11 ments. If LL moleular weight were somewhat overestimated by our alulations, omparisons between NTG and HTG subjets would not be invalidated. The ontent of apoprotein appears to be greater in HTG partiles ompared with NTG ontrols. This differene may underlie the defetive atabolism in HTG subjets of to smaller partiles that is apparent in our results. It may also explain the smaller numbers of LL partiles found in HTG subjets (Table 3). Furthermore, suh a hange in the atabolism of may be related to our previous finding that there are differenes in the surfae material of sublasses between NTG and HTG so that HTG have a redued ratio apo C-II/apo C-I11 (46). It will be of interest to know if the differenes in LL omposition and in apoprotein ontent persist after treatments of HTG subjets with lipid-lowering regimes (47). Our onlusions onerning the atabolism of hylomirons should be ompared with those of Hazzard and ierman (48) who studied the appearane of radioative vitamin in hylomirons and (Sf ) 6 hr and 24 hr after a fatty meal. t 6 hr most vitamin was in the hylomiron fration, whih is onsistent with our onlusion that hylomiron remnants ontribute largely to fration, and are removed from this fration without extensive degradation to smaller partiles. However on the basis of their reovery of most vitamin radioativity in Sf after 24 hr, Hazzard and ierman (48) onluded that hylomiron remnants persisted as small partiles in the plasma. Two onsiderations make this explanation unlikely. First, a hylomiron of diameter 200 nm that ontains 1.4% holesteryl ester (31) ontains about 58 x lo3 nm3 of holesteryl ester, whereas a partile from fration (Table 1) with diameter 54 nm has a total volume of only 82 X lo3 nm3. eause the holesteryl ester ontent of fration is not more than 11% (Tables 2 and 3), partiles ontaining about 70% holesteryl ester learly do not ontribute signifiantly to fration nor to smaller subfrations. Table 4 shows that the moleular mass of holesteryl ester inreases only in fration after the fatty meal. The numbers of holesteryl moleules in the partiles an be readily alulated from the data of Table 4. This alulation serves to emphasize the different harater of the partiles in fration, whih ontain 15- to 80-fold more holesteryl ester moleules than partiles of fration or LL. Partiles of frations and C ontain up to three times more than fration or LL, but still very muh less than fration. Frations and C hene show evidene for only a minor ontribution of hylomiron remnants by this riterion, also. Seond, beause absorption of triglyeride from the intestine will be ompleted within 24 hr, hylomirons or their remnants will not persist in the plasma by virtue of their well-doumented brief lifetimes in the irulation. eause of these onsiderations, the appearane of dietary linolei and linoleni aid (Table 5) in the smaller subfrations annot be taken as unequivoal evidene that hylomiron remnants are atabolized so ompletely within the vasular ompartment. s disussed earlier, reseretion of by the liver of fatty aids released peripherally or after hepati uptake of remnant triglyeride ould ontribute to this observation. The simplest interpretation of our results is that in man, as in the rat (30), most hylomiron remnants are removed rapidly by the liver without appreiably ontributing to smaller subfrations or LL.M This work was supported by grants from the Swedish Medial Researh Counil (19x-204) King Gustaf V 80th irthday Foundation, Margarinindustrins naringsfysiologiska forening and the Nordi Insulin Foundation. Manusript reeived 4 pril 1978; aepted 2 ugust REFERENCES 1. Havel, R. J Early effets of fat ingestion on lipids and lipoproteins of serum in man.]. Clin. Invest. 36: ngervall, G On the fat tolerane test. ta Med. Sand. 176: suppl Grundy, S. M., and H. Y. I. Mok Chylomiron learane in normal and hyperlipidemi man. Metabolism. 25: Nestel, J. J., M.. enborough, and J. O'ea isposal of human hylomirons administered intravenously in ishemi heart disease and essential hyperlipidemia. Cir. Res. IO: Redgrave, T. G Catabolism of hylomiron triaylglyerol and holesteryl ester in genetially obese rats. J. 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