Cholesteryl esters in lymph chylomicrons: contribution from high density lipoprotein transferred from plasma into intestinal lymph

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1 Cholesteryl esters in lymph chylomicrons: contribution from high density lipoprotein trnsferred from plsm into intestinl lymph H. C. F. Oliveir,'** K. Nilusen,t H. Meinertz,S nd E. C. R. Quint2o* Lipid Metbolism Lbortory: University of SI Pul Medicl School (LIM-1) nd UNICAMP, SEo Pulo, Brzil; Institute of Medicl Antomy, Section C,t Pnum Institute, University of Copenhgen, Copenhgen, Denmrk; nd Deprtment of Medicine B, Righospitl, 5 University of Copenhgen, Copenhgen, Denmrk Abstrct Most of the cholesterol in intestinl chyle nd chylomicrons is derived from plsm. Our im ws to determine how much plsm low density (LDL) nd high density (HDL) lipoproteins contribute to the cholesterol in chyle nd chylomicrons, nd to exmine how plsm cholesterol becomes ssocited with lymph chylomicrons. Intrvenous injection of rdioiodinted plsm lipoproteins into two chyluric ptients showed tht 82% of the HDL plsm pool trnsferred dily to intestinl chyle, corresponding to 58 % of lymph cholesterol; LDL contributed 18% of its plsm pool, corresponding to 18% of lymph cholesterol. When plsm HDL rdiolbeled in both the protein nd cholesteryl ester moieties ws injected, the isotope rtios of plsm HDL nd lymph lipoproteins were identicl; 85% of the HDL cholesteryl esters trnsferred to triglyceride-rich lipoproteins, while the polipoproteins remined lrgely (7%) in the higher density lipoproteins of the chyle. Incubtions of similrly lbeled plsm HDL showed preferentil trnsfer of cholesteryl esters to rtificil chylomicrons medited by fctor present in lipoprotein-free p1sm.m Thus, sizble portion of plsm HDL enters intestinl lymphtics probbly s intct HDL, nd then trnsfers prt of their cholesteryl esters to chylomicrons, possibly medited by trnsfer proteins. Reverse cholesterol trnsport my therefore include n extrvsculr loop vi lymph chylomicrons nd chylomicron remnnts to the liver.-oliveir, H. C. F., K. Nilusen, H. Meinertz, nd E. C. B. Quintgo. Cholesteryl esters in lymph chylomicrons: contribution from high density lipoprotein trnsferred from plsm into intestinl lymph. J Lipid Res : Supplementry key words cholesteryl ester metbolism low density lipoproteins high density lipoproteins lymph lipoproteins chyluri chylomicron emulsion Lipoproteins in peripherl lymph rise in prt by filtrtion of lipoproteins from plsm (1, 2) nd in prt from locl cellulr synthesis (3-6). The lipoproteins of intestinl lymph similrly consist of both plsm-derived lipoproteins nd of constituents synthesized loclly by the enterocytes (7-15). In ddition, they consist of lipids of dietry nd endogenous origin bsorbed from the intesti- nl lumen. Studies of intestinl lymph lipoproteins in experimentl nimls (8, 16, 17) nd in humn subjects (18, 19) hve yielded closely similr results. Cholesterol trnsported in the intestinl lymph origintes minly in plsm, s shown both in rts (15, 2) nd in chyluric ptients (21, 22). In lymph-cnnulted rts, the intrvenous infusion of rdioiodinted plsm HDL showed tht ll of poe nd prt of poa-i of intestinl lymph lipoproteins were derived from plsm (9, 14, 15). Plsm HDL my therefore be responsible for trnsfer of cholesterol from the circultion to intestinl lymph. In humn subjects, the distribution of cholesterol mong lipoproteins in intestinl chyle is such tht by fr the greter frction is ssocited with the chylomicrons, some is in the very low density lipoproteins (VLDL), nd only limited mounts re in the higher density rnges of LDL nd HDL (18, 19). As no humn studies on the trnsfer of lipoproteins from the blood to intestinl lymph re vilble, the first im of this work ws to exmine the contribution of the mjor cholesterol-crrying lipoproteins in plsm to the lipoproteins of mesenteric lymph in chyluric ptients. The findings indicted tht HDL trnsferred to the intestinl lymph to much greter extent thn did LDL nd thus provided most of the plsmderived cholesterol in intestinl chyle. This rised the question of how cholesterol ws trnsferred from HDL to chylomicrons, nd thus the second im of this study ws to exmine whether this cme bout by fusion of the two kinds of prticles or by selective trnsfer of cholesterol from one type of prticle to the other. Abbrevitions: LDL, low density lipoproteins; HDL, high density lipoproteins; VLDL, very low density lipoproteins. 'To whom reprint requests should be ddressed t: Lipid Metbolism Lb., University of SHo Pul Medicl School, Dr. Amldo, 455 / 3O/ 52, Cep S2o Pulo, Brzil. Journl of Lipid Reserch Volume 34,

2 Mteril MATERIAL AND METHODS Triolein, cholesteryl olete, nd cholesterol were purchsed from Nu-Chek Prep, Inc. (Elysin, MN) nd phosphtidylcholine ws from Lipid Products (South Nutfield, Surrey, UK). Regents for enzymtic determintion of totl nd free cholesterol (Chod Pp) nd of triglycerides were obtined from Boehringer (Mnnheim, Germny) nd Biodignostic Ind. (Prn, Brzil), respectively. Phospholipids were mesured by the Brtlett procedure (23). The following rdioisotopes were obtined from New Englnd Nucler (Boston, MA): NlZ5I, [1,2-3H(N)]cholesterol, [4-14C]cholesteryl olete, [l,2-3h(n)]cholesteryl oleyl ether; N131I ws from IPEN-SBo Pul Atomic Reserch Institute. Iodo-Gen ws from Pierce Chemicl Compny (Rockford, IL) nd Aqusol ws from New Englnd Nucler. Subjects Three mle ptients with chyluri s sequel of filrisis prticipted. Ptient l, 37 yers old nd born in Tnzni, ws studied t Righospitl, University of Copenhgen. Ptient 2, 35 yers old nd born in Brzil, together with ptient 3, 62 yers old nd born in Jpn, were exmined t the University of Slo Pul Medicl School, nd their intestinl lymph-renl fistule were demonstrted by lymphngiogrphy. Ptients 1 nd 2 received liquid formul diets exclusively; ptient 3 te self-chosen food of unknown quntity nd composition during the 4-dy study. Ptients 1 nd 2 hd continuous chyluri with men dily excretion of urinry ft of f 2.8 (SEM) in cse of ptient 1, nd 5.3g *.8 in cse of ptient 2. Plsm volume ws estimted from height nd body weight (24). Lipoprotein trnsudtion studies All subjects received n intrvenous bolus injection of rdiolbeled utologous lipoproteins, which in the cse of ptient 1 consisted of 37 pci 125-lbeled LDL, for ptient 2 ws 9 pci 13lI-lbeled HDL, nd for ptient 3 ws reconstituted HDL lbeled with 1311 (93 pci), [l,2-3h(n)]cholesteryl oleyl ether (18 pci), nd [4-14C]cholesteryl olete (21 pci). To block the incorportion of rdioctive iodine into the thyroid glnd, ll subjects received potssium iodine, 2 mg per dy, orlly from 3 dys prior to nd 4 weeks fter the rdioctive injection. Fsting blood smples were drwn dily nd mixed with EDTA (1.5 mg/ml); the plsm ws dilyzed for 24 h t 4OC ginst physiologicl sline EDTA (1 mm) (1 liter x 5). Plsm lipoproteins were frctionted by sequentil ultrcentrifugtion (ptient 1) (25) or by discontinuous density grdient ultrcentrifugtion (ptients 2 nd 3) (26). For determintion of the specific ctivity of poldl (ptient 1) nd of pohdl (ptient 2), the frctions were respun t their limiting densities. Urine ws collected in EDTA (.1%) nd sodium zide (.1%); 24-h collections were mixed, the volume mesured, nd liquots (1-25 ml) were dilyzed ginst physiologicl sline with EDTA (1 mm) nd sodium zide (.1%) (1 1 x 4) t 4OC bringing the density to 1.6 g/ml. Urinry lipoproteins were then frctionted by sequentil ultrcentrifugtion. Cholesterol nd protein of urinry LDL (ptient 1) nd HDL (ptient 2) were determined. Totl ft, cholesterol, nd protein were mesured by grvimetric (21), gs-liquid chromtogrphic (27), nd colorimetric procedures (28), respectively, in liquots of totl urine nd in lymph chylomicrons of the urine. Trnsudtion of plsm polipoproteins into enteric lymph ws determined for po-ldl (ptient 1) nd po- HDL (ptient 2) s the plsm polipoprotein trnsported in lymph in mg/dy: 24-h urine lipoprotein rdioctivity (dpm) sp ct of plsm polipoprotein (dpm/pg) v ft intke (g/dy) 24-h urinry ft excretion (g) Totl mount of lipoprotein-cholesterol filtered into the lymph ws determined s the polipoprotein trnsudtion multiplied by the cholesterol/polipoprotein (pg/pg) rtio. Lipoprotein lbeling Autologous LDL (d g/ml) nd HDL (d g/ml) were isolted by sequentil ultrcentrifugtion (26), nd the frctions were reisolted t their upper limiting density. The lipoproteins were lbeled with N125I using 1 mci/mg polipoprotein by the modified McFrlne method (29) for LDL nd with Iodo-Gen for HDL. After iodintion, the lipoprotein ws dilyzed ginst physiologicl sline (EDTA 1 mm), 1 1 x 5, for 12 h t 4OC nd sterilized by filtrtion through.22-pm filter (Millipore, Bedford, MA) immeditely prior to intrvenous infusion. Less thn 2% of the rdioctivity ws lipid-extrctble nd 96-98% ws precipitted by trichlorocetic cid. SDS-polycrylmide gel electrophoresis of the 1251-lbeled HDL showed tht.3% of the rdioctivity ws ssocited with contminting lbumin (3). Preprtion of reconstituted, lbeled HDL involved lbeling core lipids with [4-1*C]cholesteryl olete nd [1,2-3H(N)]cholesteryl oleyl ether by modified procedure of Nestler et l. (31), nd of polipoproteins with Briefly, the HDL solution (2 ml) ws delipidted twice with 4 ml cold ethnol-diethyl ether 3:2 (v/v). After removl of solvent from the lipid phse, solution of the rdioctive steroids ws dded, nd the solvent ws removed under N2 t 4OC. The HDL-polipoprotein, diluted with phosphte buffer t ph 7.2, ws dded to the rdiolbeled lipids nd the mixture ws sonicted for 3 min t 7-8 wtts, t 37OC, under N2 (B3 Brnson 173 Journl of Lipid Reserch Volume 34, 1993

3 Sonifier, Brnson Sonic Power Co., Dnbury, CT). This reconstituted 3HP4C-HDL (rechdl), nd prllel, unlbeled HDLrec were purified seprtely by discontinuous density grdient ultrcentrifugtion (Beckmn, SW 41Ti rotor) for 24 h (26). After dilysis, the unlbeled HDLrec ws then lbeled with N13lI (Iodo-Gen procedure), nd both preprtions were dilyzed nd sterilized by Millipore filtrtion (.22 pm). The HDLrec contined 3% cholesterol, 13% cholesteryl ester, 6% triglyceride, 42% phospholipid, nd 36% protein by weight. Rdioctivity mesurement of the iodine lbel ws done directly in the gmm counter, wheres 3H nd 14C required prior chloroform-methnol 2:l (v/v) (plsm) or chloroform (lyophilized urine) extrction. To ensure tht the l3ii did not interfere with counting in toluene-phosphor solution (Beckmn model LS-6 TA), the smples were stored for 8 dys before mesuring 3H nd 14C. Incubtion of plsm HDL with chylomicron-like prticles Bulk HDL (d g/ml) prepred from pooled plsm were lbeled with N'Z5I (Iodo-Gen) or with [1,2-3H(N)]cholesterol by the procedure of Gvish, Oschry, nd Eisenberg (32). Briefly, [3H]cholesterol(5 pci in 1 pl ethnol) ws dded to- the HDL solution together with lipoprotein-free plsm, d > 1.21 g/ml (totl volume 1 ml), nd incubted for 2 h t 37OC. Lbeled HDL ws then seprted from the incubtion mixture by ultrcentrifugtion; nlysis of the HDL-lipids by thin-lyer chromtogrphy showed tht 77-88% of the HDL 3H ctivity ws present s esterified cholesterol. Chylomicron-like, triglyceride-rich prticles were prepred s previously described (33, 34). Briefly, mixture of cholesterol (2%), cholesteryl olete (s), phosphtidylcholine (23%), nd triolein (69%) ws cosonified in NCl solution (2.785 M), ph 7., d 1.11 g/ml, t 7-8 wtts, for 3 min t 37OC under flow of Nz. The chylomicronlike mteril ws collected t d 1.6 g/ml fter discontinuous density grdient ultrcentrifugtion; it contined 4.5% cholesterol, 6.8% cholesteryl olete, 9.5% phosphtidylcholine, nd 8.3% triolein, by weight. HDL preprtions, lbeled with 1251 or with [ 3H]cholesterol, both esterified nd free, were incubted together with chylomicron-like prticles. Incubtion conditions were vrible concentrtions of HDL together with 5 pg triolein in totl volume of 1.1 ml. The incubtion ws terminted by the ddition of hypertonic potssium bromide solution, d g/ml, nd then HDL nd the chylomicron-like mteril were immeditely seprted by discontinuous density grdient ultrcentrifugtion t d g/ml in n SW 41Ti rotor, t 4"C, for 3 min t 4, rpm. The distribution of rdioctivity between the top frction (d < 1.6 g/ml) representing the chylomicron-like mteril nd the infrntnt cor- responding to the HDL ws determined. All ssys were done in duplicte or triplicte, together with blnk incubtions contining no chylomicron-like prticles. Less thn 1.1% of the totl rdioctivity in the blnk incubtions ws found in the top frction (d < 1.6 g/ml). After lipid extrction (chloroform-methnol 2:l) of the two ultrcentrifugl frctions, free nd esterified cholesterol were seprted by thin-lyer chromtogrphy on Silic Gel H in hexne-diethyl ether-cetic cid 7:3:1 (vlvfv). The pproprite frctions, identified by iodine vpor, were scrped into scintilltion vils contining Aqusol for rdioctivity mesurement. The findings were expressed either s percent of totl rdioctivity or s mss tht hd been trnsferred to the other type of prticle. The trnsferred mss ws clculted s rdioctivity (dpm) in the receptor prticle divided by the specific ctivity (dpm/pg) in the donor prticle prior to incubtion. RESULTS Trnsudtion of plsm lipoprotein After n intrvenous injection of lz5i-lbeled utologous LDL (ptient 1) nd HDL (ptient 2), the entry of polipoprotein lbel (1251) into intestinl lymph lipoproteins ws mesured quntittively during the subsequent 4-6 dys. The mount of plsm polipoprotein trnsported dily in the enteric lymph ws clculted from the totl nondilyzble rdioctivity present in the lymph ech dy divided by the specific ctivity of plsm LDL nd HDL polipoprotein on the sme dy. The dt showed tht 4974 * 171 mg (men * SD) of plsm pohdl ws trnsported dily in the lymph of ptient 2, corresponding to 82% of the plsm pool of HDL, s opposed to mg of plsm poldl per dy, representing only 18% of the plsm pool of ptient 1. The mount of polipoprotein-ssocited cholesterol ws clculted from the cholesterol/polipoprotein rtio of plsm LDL nd HDL, respectively. Thus, ssuming tht intct lipoprotein prticles filtered from plsm to intestinl lymph, 489 * 55 mg (ptient 1) nd 892 * 138 mg (ptient 2) of cholesterol in the intestinl lymph originted from plsm LDL nd HDL, respectively. These quntities represented 18% nd 58%, respectively, of the totl cholesterol trnsport in intestinl lymph so tht the combined contribution from the two plsm lipoprotein clsses ws bout 75% of enteric lymph cholesterol. Although these dt were obtined in studies of only two individuls, their generl vlidity ws supported. by our similr findings in previous studies of severl chyluric individuls (21). In plsm, the polipoprotein lbel of HDL nd LDL remined essentilly in the density rnge into which it ws originlly introduced for the durtion of the study (Fig. 1). This ws true for ptients 1 nd 2 who received Oliveiru et ul. Cholesteryl esters in lymph chylomicrons 1731

4 (SI 8 "'1 - APO LDL (PATIENT 1),o le' I - APO nol (PATIENT Is' I - APO HDL REC. (PATIENT PLASMA CHYLE d C d > 1.63 d > 1.6 Fig. 1. Percent distribution of rdioctivity in plsm nd chyle lipoproteins (men i SEM) 4-6 dys fter the intrvenous pulse infusion of W-lbeled poldl (ptient l), 1*5I-lbeled pohdl (ptient 2), nd reconstituted L311-lbeled pohdl (ptient 3). lipoproteins rdioiodinted in their ntive stte, nd lso for ptient 3 who ws injected with reconstituted HDL preprtion. In intestinl lymph, however, more extensive redistribution of the polipoprotein lbel took plce (Fig. 1). In prticulr, the HDL polipoprotein lbel (1251, ptient 2; l3li, ptient 3), of which only trces (1-27) were ssocited with triglyceride-rich lipoproteins (d < 1.6 g/ml) in plsm, shifted in the intestinl lymph so tht bout 3% of the rdioctivity ws in the density rnge of chylomicrons nd VLDL in ptients 2 nd 3. Compred to HDL, the shift in the intestinl lymph of the LDL polipoprotein lbel (lz5i) ws modest in tht the frction ssocited with triglyceride-rich lipoproteins ws 12% (ptient 1). To exmine whether the core lipids of plsm HDL trnsferred in prllel with the polipoprotein to intestinl chyle nd to the individul lymph lipoproteins, utologous reconstituted HDL lbeled in the polipoprotein moiety with 1311 nd in the core lipids with [14C]cholesteryl olete nd [3H]cholesteryl oleyl ether ws injected intrvenously into chyluric subject (ptient 3). The rtios of polipoprotein to core lipid rdioctivity of the lymph lipoproteins were virtully identicl with those of plsm HDL (Tble 1). The simplest interprettion of this finding ws tht the core lipids nd the polipopro- teins pssed from the circulting blood to the intestinl lymph t identicl rtes, most likely s constituents of intct HDL prticles. Once in the lymph, the HDL core lipids ([ 14C]cholesteryl ester nd [ 3H]cholesteryl ether) trnsferred extensively (bout 85%) to the triglyceriderich chylomicrons nd VLDL, nd only modest frction remined in the higher density (d > 1.6 g/ml) lipoproteins (Fig. 2). Thus, the shift of cholesteryl esters from HDL to lymph chylomicrons nd VLDL ws much greter thn tht of the HDL polipoprotein (1311), bout 85% versus 3% (Figs. 1 nd 2), indicting tht cholesteryl esters becme ssocited with triglyceride-rich lipoproteins in intestinl lymph minly by selective lipid trnsfer rther thn by simple fusion of HDL prticles with chylomicrons. In plsm, the HDL core lipids ([ 14C]cholesteryl ester nd [ 3H]cholesteryl ether) likewise trnsferred extensively to lipoproteins of lower densities, but in plsm the mjor frction becme ssocited with LDL rther thn with the triglyceride-rich lipoproteins s in the intestinl lymph (Fig. 2). The enrichment of LDL in plsm with lbeled cholesteryl ester ("C) nd ether (3H) from HDL my explin why the isotope rtio 1311-lbeled pohdl/i4c or 3H of totl plsm lipoproteins ws clerly lower thn tht of plsm HDL nd the lymph lipoproteins (Tble 1). Becuse lipoproteins of lower densities re eliminted fster from circultion thn re those of high density, the rdioctive cholesteryl esters re preferentilly lost from the plsm comprtment compred to the HDL-polipoproteins. The isotope rtio of totl lymph lipoproteins, on the contrry, ws virtully identicl with tht of plsm HDL. This suggests tht the min source of lymph lipoprotein rdioctivity is plsm nd tht plsm HDL reches the intestinl lctels s n intct prticle. Trnsfer of HDL constituents to rtificil chylomicrons In n ttempt to identify fctors in intestinl lymph influencing the trnsfer of HDL constituents to chylomicrons, plsm HDL lbeled in the polipoprotein with 1*51 nd in the lipid moiety with free or esterified [ 3H]cholesterol ws incubted with rtificil chylomicrons. Trnsfer of polipoprotein lbel to triglyceride-rich prticles ws limited to few percent in n incubtion medium of physiologicl sline, nd this process ws even inhibited TABLE 1. Rtios of L311-lbeled HDL to [1r,2r-3H(N)]cholesteryl olevl ether or 14-L*Clcholestervl olete in ptient 3 men f SEA4 Plsm HDL 18.5 c c 1.5 Totl lymph lipoproteins 19.6 c *.9 Totl plsm lipoproteins 12.3 f t Journl of Lipid Reserch Volume 34, 1993

5 c- 2 c e u. z P c 3 E e 1 e z Y Y n 3n-CHOL. OLEYL ETHER PLASMA - I CHYLE I4C-CHQLESTERYL OLEATE DAYS Fig. 2. Percent distribution of rdioctivity in lipoprotein frctions in plsm (pnels A, B: () LDL; () HDL; (x) VLDL) nd urine chyle (pnels C, D: (A) d < 1.6 g/ml; (A) d > 1.6 g/ml) of ptient dys fter the intrvenous pulse infusion of utologous bulk HDL simultneously lbeled with [lor,2or-~h(n)]cholesteryl oleyl ether (pnels A, C) nd [4-1*C]cholesteryl olete (pnels B, D). initilly in the presence of lbumin or lipoprotein-free plsm (d > 1.21 g/ml) (Fig. 3, A, B, C). Free cholesterol trnsferred rpidly nd extensively, unffected by the composition of the incubtion medium (Fig. 3, D, E, F). Trnsfer of HDL cholesteryl ester ws slow initilly nd incresed only grdully with time in sline with or without lbumin, but ws fst nd extensive when the incubtion took plce in lipoprotein-free plsm, presumbly due to the presence of cholesteryl ester trnsfer proteins; bout 8% becme ssocited with chylomicronlike prticles fter 2 h of incubtion (Fig. 3, G, H, I). The mrked difference in trnsfer between HDL polipoprotein nd cholesteryl ester ws independent of the concentrtion of HDL; in sline the frctionl trnsfer of polipoprotein ws less thn one-third tht of cholesteryl ester (5.5% versus 17.7%), nd in lipoprotein-free plsm, polipoprotein trnsfer ws reduced to one-fifth of tht of the sline incubtion, wheres cholesteryl ester trnsfer ws incresed to bout 49% (Fig. 4). DISCUSSION Cholesterol in intestinl chyle is derived from severl sources, nmely intestinl bsorption, de novo synthesis by the enterocytes, nd plsm lipoproteins. Even when intestinl bsorption is gretly incresed by hevy dietry lod, the contribution from plsm lipoproteins remins the mjor source of chylous cholesterol (21, 22). In intestinl chyle the triglyceride-rich lipoproteins, especilly chylomicrons, crry by fr the greter portion of cholesterol, wheres LDL nd HDL ccount for only minor frction (21). This study demonstrtes tht plsm HDL in humn subjects is the mjor source of cholesterol in intestinl lymph, tht HDL cholesteryl esters pss from the circultion to the intestinl lymphtics s prt of HDL prticles, nd tht most of the cholesteryl esters subsequently re trnsferred to lymph chylomicrons nd VLDL without prllel trnsfer of HDL polipoprotein. The contribution of plsm lipoproteins to the cholesterol trnsported in intestinl lymph hs, in the present study of humn subjects, been evluted by the intrvenous injection of rdioctively lbeled plsm HDL nd LDL in chyluric ptients to determine the fte of the lbeled lipoprotein constituents in intestinl lymph. Thus, the trnsfer of cholesterol from plsm to lymph lipoproteins my be viewed s consisting of two discrete steps: the first involves the pssge from circulting blood r i P i 7 D I r n O N E -.s 2 4.s 4 e 24.s 2 4 e - INCUIATION TINE IN HOURS Fig. 3. Trnsfer with time of 1Z51-lbeled pohdl (1 pg) or of [1,2-3H(N)]cholesteryl ester nd [1,2-~H(N)]cholesterol(unesterified) from HDL to rtificil chylomicrons (5 pg "2) in sline (pnels A, D, G), lbumin solution 3.5 g % (pnels B, E, H), or in the plsm d > 1.21 g/ml frctions (pnels C, F, I). In vitro 1251 nd 3H ssys were crried out seprtely. HDL hd been lbeled with (1,2-3H(N)]cholesterol in the presence of the plsm d > 1.21 g/ml frction nd 77-88% ws esterified. In incubted prticles, (3H]cholesterol(unesterified) ws seprted from [3H]cholesteryl ester by thin-lyer chromtogrphy. Mens of duplicte incubtions. Oliueiru et ul. Cholesteryl esters in lymph chylomicrons 1733

6 d: APO HDL I t H-CHOL. ESTER. (yo) Y Y* 1 Fig. 4. Trnsfer to rtificil chylomicrons (5 pg E) of 1251-lbeled pohdl (circles) nd of [1,2-~H(N)]cholesteryl ester (tringles) (SH-CE) from HDL either in sline medium (open symbols) or in plsm d > 1.21 g/ml frction (closed symbols) fter incubtion periods of 3 min (pohdl) or 12 min (SH-CE) t 37OC. Incubtions were done with incresing quntities of rdioctive HDL. Vlues in prentheses indicte percent of [JH]cholesteryl ester nd 125I-lbeled pohdl from the incubtion medium tht ws found in chylomicrons. Results re reported s mens of duplictes. Methods re shown in the legend to Fig. 3. vi interstitil tissue to the lymphtics of the intestinl mucos, nd the second concerns the distribution of cholesterol mong lipoproteins in the lymph. Previous studies in the rt hve shown tht fter intrvenous injection of rdiolbeled plsm HDL, the lbel not only ppers in intestinl lymph (14) but it is recovered in the HDL density rnge of the lymph lipoproteins (9), s n indiction tht HDL is trnsferred s such from the circulting blood to intestinl lymph. Compositionl similrities between HDL in plsm nd lymph in humn subjects (35) nd in pigs (8) hve lso suggested tht plsm HDL is n importnt source of intestinl lymph HDL in these species. In the present study we found tht not only rdioiodinted HDL but lso LDL in plsm pssed into intestinl lymph nd tht the polipoprotein lbels were locted predominntly in the HDL nd LDL density rnges of lymph lipoproteins, indicting trnsfer of plsm HDL nd LDL s such to intestinl lymph. In cse of plsm HDL this conclusion ws further strengthened by our finding tht the frctionl trnsfers of core lipid nd polipoprotein lbels to the intestinl lymph were virtully identicl. In the cse of LDL, previous studies hve shown close similrity between plsm nd lymph lipoproteins in pek flottion rtes in humn subjects (35) nd in composition in the pig (8) in greement with the conclusion tht plsm LDL trnsfers s such to intestinl lymphtics nd is mjor source of lymph LDL. The greter frctionl trnsfer of plsm HDL compred to LDL my well be explined by the smller dimeter of the HDL prticles. The mount of cholesterol crried by HDL nd LDL from plsm to intestinl lymph ws clculted from the mount of trnsferred polipoprotein lbel multiplied by the rtio of cholesterol to polipoprotein mss in plsm HDL nd LDL, ssuming tht this rtio ws vlid for the lipoproteins filtered into the lymphtics. To the extent tht the lipoproteins pssing from plsm to intestinl lymph differ in this rtio from totl plsm HDL nd LDL, the clcultion will be erroneous. However, our finding tht trnsfer of circulting HDL nd LDL together ccounted for bout 75% of totl intestinl lymph cholesterol trnsport grees very well with our previous studies in chyluric ptients, in which the contribution of plsm lipoprotein cholesterol ws determined from the trnsfer of rdioctive sterol from plsm to intestinl lymph (21, 22). Once HDL nd LDL from plsm hve entered the interstitil tissue nd the lymphtics of the intestinl mucos, they encounter lipoproteins of intestinl origin. Our dt show tht both polipoprotein nd core lipid lbels leve their originl density rnges nd become ssocited minly with the predominnt triglyceride-rich lipoproteins of the chyle. In the cse of HDL, the frctionl trnsfer of the core lipid lbels to triglyceride-rich lipoproteins ws nerly complete (85 %) nd lmost three times greter thn tht of the polipoprotein lbel. Thus, most of the cholesteryl ester trnsferred dissocited from polipoprotein, suggesting process medited by cholesteryl ester trnsfer protein. Although trnsfer proteins hve not yet been shown to occur in intestinl lymph, they would be expected to pss esily from circultion to intestinl lymphtics whether they exist in plsm unbound to lipoprotein or in ssocition with HDL becuse their moleculr weight is similr to tht of plsm lbumin (36). If indeed the pssge of cholesteryl ester from HDL to chylomicrons is medited by trnsfer protein, reciprocl trnsfer of triglyceride would tke plce, nd in greement with this, humn HDL from intestinl lymph showed, indeed, higher content of triglycerides nd lower content of cholesteryl ester compred to circulting HDL in the sme ptients. An lterntive explntion for this compositionl difference could, however, be mss ction effect due to the high triglyceride concentrtion in the chylomicrons (35). The presence of trnsfer protein in intestinl lymph might lso medite trnsfer of cholesteryl ester from filtered plsm LDL to triglyceriderich lymph lipoprotein, nlogous to tht from HDL, but we hve no dt to demonstrte such trnsfer. The polipoprotein lbels of plsm HDL nd LDL remined in their originl density clsses while circulting in the blood, but fter entry into intestinl lymph some of the lbels trnsferred to lipoproteins of other densities Journl of Lipid Reserch Volume 34, 1993

7 About 3% of the HDL polipoprotein lbel becme ssocited with the triglyceride-rich lipoproteins, while the corresponding trnsfer of LDL polipoprotein ws only bout 1%. Although we hve no dt on individul polipoproteins, likely explntion for the difference between HDL nd LDL in this respect is tht poc, which is mjor constituent of HDL polipoproteins, hs been shown to trnsfer extensively when lymph chylomicrons from the rt were incubted with serum lipoprotein frctions; some poe trnsferred s well (16). Similrly, humn lymph chylomicrons become enriched in poc nd E when they enter circulting blood (37). How the trnsfer of bout 1% of the LDL polipoprotein lbel to triglyceride-rich lymph lipoproteins nd nother few percent to lymph HDL is to be explined is uncertin in the bsence of detiled polipoprotein nlyses. One possibility is tht poc, E, nd A were responsible for the trnsfer of lbel, s plsm LDL isolted by the presently used procedure generlly contins limited mounts of these polipoproteins (38-4), which re known to trnsfer redily between lipoprotein clsses (37). If, however, the polipoprotein lbel represented pob-1, this would men tht either LDL becme trpped by or fused with lipoproteins of lower nd higher density clsses, or tht lbeled pob-1 in some other wy becme ssocited with other lymph lipoproteins. Although none of these ltter lterntives pper likely, it is noteworthy tht lymph chylomicrons from both humns (41) nd pigs (8) contin pob-1, even though the intestinl mucos my be unble to synthesize this polipoprotein; but even if the pob-1 of lymph chylomicrons is of heptic origin, it is difficult to envision how lbeled pob-1 from plsm LDL could become incorported into lymph chylomicrons. The cholesteryl esters of plsm HDL, originlly derived from cholesterol of peripherl cells, re to considerble extent trnsferred to VLDL nd chylomicrons in plsm, medited by trnsfer protein, in exchnge for triglycerides. They re finlly tken up by the liver vi endocytosis of LDL nd chylomicron remnnts, nd thus they complete the centripetl trnsport of cholesterol from peripherl tissues to the liver. As we hve presently demonstrted, substntil mounts of plsm HDL enter intestinl lymphtics to interct with lrge quntities of triglyceride-rich chylomicrons whereby n extensive trnsfer of HDL cholesteryl esters to chylomicrons occurs. This extrvsculr interction between plsm HDL nd lymph chylomicrons my be of importnce for the efficiency of the reverse cholesterol trnsport becuse reltively more cholesteryl ester in tht mnner ends up in chylomicron remnnts, which re removed more efficiently by the liver thn LDL. A number of unresolved questions remin concerning both the trnsfer of plsm lipoproteins to the intestinl lymphtics nd the interctions in intestinl lymph between lipoproteins of intestinl nd heptovsculr origin. The heterogeneity of prticles in the HDL nd LDL density spectr, in terms of size nd composition, my well result in the selective pssge of some subfrctions from plsm to lymph in preference to others. To wht extent lipoproteins of density below tht of LDL my trnsfer is uncertin; lthough VLDL is generlly considered too lrge to pss the endothelil lining of intestinl blood cpillries, the possibility exists tht some intermedite density lipoprotein might pss. Extensive trnsfer or exchnge of both lipid nd polipoprotein constituents between plsm nd lymph lipoprotein hve been observed in previous nd in the present studies, but informtion on how individul polipoproteins nd phospholipids prticipte is still incomplete. Unresolved lso re questions bout independent or coupled trnsfer of different lipoprotein constituents nd the role of trnsfer proteins for these phenomen. Severl of these questions my be nswered by studies of chyluric ptients by the techniques presently used if extended to include nlysis of rdioctivity nd mss of individul polipoproteins. In summry, we hve provided new evidence tht humn plsm HDL nd LDL re the mjor sources of cholesterol in intestinl chyle nd its mjor lipoproteins, chylomicrons, nd VLDL. The two lipoproteins leve the circultion nd enter the intestinl lymph s LDL nd HDL, respectively. Cholesteryl esters from HDL, nd presumbly from LDL, trnsfer to the triglyceride-rich lymph lipoproteins lrgely dissocited from polipoproteins. As cholesteryl esters of plsm HDL re prt of the trnsport system of cholesterol from the periphery to the liver, the present observtions demonstrte the existence of n extrvsculr loop in this pthwy which my be of importnce for the efficiency of the reverse cholesterol trnsport. l This work ws prtilly supported by grnts from the Dnish Hert Foundtion nd the Stte of SZo Pul Reserch Foundtion (FAPESP). The uthors re thnkful to Miss Jussr C. Roch, SenPri Eguti nd to Miss Ninn Buch nd Ms Hnne Mrete Olsen for expert technicl ssistnce. Mnusmpt nceived 3 Nwember 1992 nd in revised form 7 My REFERENCES Reichl, D., L. A. Simons, N. B. Mynt, J. J. Pflug, nd G. L. Mills The lipids nd lipoproteins of humn peripherl lymph, with observtions on the trnsport of cholesterol from plsm nd tissues into lymph. Clin. Sci. Mol. Med. 45: Reichl, D., A. Postiglione, N. B. Mynt, J. J. Pflug, nd M. Press Observtions on the pssge of poproteins from plsm lipoproteins into peripherl lymph in two men. Clin. Sci. Mol. 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Levy The metbolism of very low density lipoprotein I - pmliminry in vitro nd in vivo observtions. Biochim. Biophyx Act Connelly, P. W., nd A. Kuksis SDS-Glycerol polycrylmide gel electrophoresis of plsm polipoproteins. Biochim. Biophys. Act. 711: Nestler, J. E., M. Bmberger, G. H. Rothblt, nd J. E Struss, Metbolism of high density lipoproteins reconstituted with [3H]cholesteryl ester nd [ 4C]cholesterol in the rt, with specil reference to the ovry. Endocrinology. 117: Gvish, D., Y. Oschry, nd S. Eisenberg In vivo conversion of humn HDL3 to HDL, nd poe-rich HDLl in the rt: effects of lipid trnsfer protein. J. Lipid Res. 28: Oliveir, H. C. E, M. H. Hirt, T. G. Redgrve, nd R. C. Mrnhio Competition between chylomicrons nd their remnnts for plsm removl: study with rtificil emulsion models of chylomicrons. Biochim. Biophys. Act. 958: Zerbintti, C. V., H. C. F. Oliveir, S. Wechsler, nd E. C. R. Quintlo Independent regultion of chylomicron lipolysis nd prticle removl rtes: effects of insulin nd thyroid hormones on the metbolism of rtificil chylomicrons. Metbolism. 4: Andersen, D. W., E. J. Schefer, T. J. Bronzert, E T. Lindgren, T. Forte, T. E. Strzl, G. D. Niblck, L. Zech, nd H. B. Brewer Trnsport of polipoproteins A-I nd A-I1 by humn thorcic duct lymph. J Clin. Invest. 67: Albers, J. J., J. H. Tollefson, C-H. Chen, nd A. Steimetz Isoltion nd chrcteriztion of humn plsm lipid trnsfer proteins. Arteriosclerosis, 4: Hvel, R. J,, J. P. Kne, nd M. L. Kshyp Interchnge of polipoprotein between chylomicrons nd high density lipoprotein during limentry lipemi in mn. J Clzn. Inuest. 52: Lee, D. M., nd P. Alupovic Composition nd concentrtion of polipoproteins in very-low nd low-density lipoproteins of norml humn plsm. Atherosclerosis. 19: Blum, C. B., L. Aron, nd R. Scicc Rdioimmunossy studies of humn polipoprotein E. J. Clin. Invest. 66: Hvel, R. J., L. Kotite, J-L. Vigne, P. Tun, N. Phillips, nd C. G. Chen Rdioimmunossy of humn rginine-rich polipoproteins, poprotein E. J Clin. Invest. 66: Kne, J. P., D. A. Hrdmn, nd H. E. Pulus Heterogeneity of polipoprotein B: isoltion of new species from humn chylomicrons, PTOC. Ntl. Acd. Sci. USA. 77: Journl of Lipid Reserch Volume 34, 1993