I \ \ \ I held in Washington, D, C. Mav 1983,, 2To whom correspondence ~hould be addressed: Mo

Transcription

1 JOUR:-AL OF SUR,,,',,,,L ;',.c.:,',.h 39, ) The Effect of Portacaval Shunt on Hepatc Lpoproten Metabolsm n Famlal Hypercholesterolema 1 JEFFREY M. HOEG, M.D.,2 STEPHEN J. DEMOSKY, JR., B.A., ERNST J. SCHAEFER, M.D., THOMAS E. STARZL, M.D.,* KENDRCK A, PORTER, M.D.,t AND H. BRYAN BREWER, JR., M.D. Molecular Dsease Branch, Natonal Hearl, Lung, and Blood nslrule, NUlonalnsltutes o{health, Bethesda, Maryland; and "The Department o/surgery, Unversty of Pttsbwgh School of Medcne, Pllsburgh, Pennsylvana: and tsr Mary's Hospzal School of Medcne, London, Unted Kngdom { f l r t Submtted for publcaton Aprl 16, 1984 The hyperlpdema observed n famlal hypercholesterolema can be reduced by portacaval anastomoss, We report the effects of a portacaval shunt on hepatc morphology and bosynthetc pathways crucal to hepatc cholesterol homeostass n homozygous receptor-negatve famlal hypercholesterolema, Portacaval anastomoss was assocated wth a dramatc change n hepatocyte morphology, 28% reducton n plasma' low-densty lpoproten concentraton, and a decrease n hepatc total and free cholesterol content by 27 and 75%, respectvely. Furthermore, the rate-lmtng enzyme n cholesterol bosynthess, 3-hydroxy-3- methylgutaryl coenzyme A reductase was decreased by 56%, Fnally, the reduced bndng oflow-densty lpoprotens to hepatc membranes preoperatvely was ncreased followng the portacaval shunt. These com bned results ndcate that the changes n crculatng lpoproten concentratons observed after portacaval shunt are due to alteratons n the metabolc consequences of the defectve recognton of lowdensty lpoprotens by the lver of famlal hypercholesterolemc subjects, Academc Press, nc, NTRODUCTON The mammalan lver plays a central role n lpd and lpoproten metabolsm. t s the prmary ste for endogenous cholesterol and lpoproten bosynthess as well as the prncpal organ for cholesterol excreton through ble and ble acd formaton [11, 18,48]. Studes n rats [9,19,54], swne [33], and rabbts [27, 34] ndcate that a sgnfcant porton of an njected dose of radolabeled low-densty lpoprotens 3 s removed by the mammalan A prelmnary account of ths work was presented at he Amercan Federaton for Clncal Research Meetngs held n Washngton, D, C. Mav 1983,, 2To whom correspondence ~hould be addressed: Mo llecular Dsease Branch, Natonal Heart, Lung, and Blood lnsttute, Natonal nsttutes of Health, Buldng 10, Room 7N117, Bethesda, Md, 20205, J Abbrevatons used: FH, famlal hypercholesterolema; LDL, low-densty lpoprotens; SE, standard error of the 1 mean; PBS, phosphate-buffered salne; BSA, bovne serum ~butnn; TrsO, trs hydroxymethyl)amnomethane;!2s_, DL, 12s_labeled low-densty lpoprotens; VLDL, very-, OW-densty lpoprotens; LPDS, lpoproten-defcent serum; HMO-eoA reductase, 3-hydroxy-3-methylglutaryl toenlyffe A reductase; apob. apolpoproten B. lver. The ntal step n the cellular uptake of crculatng LDL s the bndng of the lpoproten to a receptor n the plasma membrane [8, 15], solated hepatc membranes from a number of anmal speces have been shown to contan a receptor whch [1, 23, 26, 28, 55] specfcally bnds LDL. Recently, hepatc membranes from normal adult humans have also been demonstrated to bnd LDL [16, 22]. Thus, coordnate control of lpoproten uptake, catabolsm, and synthess may occur n normolpdemc man. Famlal hypercholesterolema s an autosomal domnant dsorder characterzed clncally by hypercholesterolema, xanthomas, and premature atheroscleross [24]. By analyss of skn fbroblasts from patents homozygous for FH, Brown and Goldsten determned that FH was due to one of several mutatons n the gene codng for the cellular receptor for LDL [7, 51]. The most frequent allelc mutaton resultng n FH s the loss of the functonal hghaffnty receptor for LDL and s referred to as receptor-negatve FH [51]. We have recently demonstrated that the loss of the fbroblast /85 $1.50 Copyrght 1985 by Academc Press. nc, All rghts of reproducton n any form reserved.

2 _.. _..._ JOURNAL OF SURGCAL RESEARCH: VOL. 39, NO.5, NOVEMBER , ~!: LDL receptor n FH homozygotes s paralleled by a defect n the hepatc membrane recognton of LDL [22]. Therefore, the profound hypercholesterolema and accelerated atheroscleross observed n FH may occur as a result of the loss of the hepatc LDL receptor and the resultng changes n hepatc lpd and lpoproten metabolsm. Patents wth FH frequently are refractory to all lpd lowerng drug regmens. However, the use of a portacaval anastomoss to shunt blood flow from the lver has been shown to be effectve n lowerng the plasma lpds n these patents [42]. The present studes were performed on hepatc tssue n a receptor-negatve FH subject to evaluate the effect of the portacaval shunt on both the bndng ofldl to hepatc membranes as well as to assess the mpact of the portacaval shunt on hepatc cholesterol metabolsm. METHODS Patent hstory. The patent studed was frst noted to be hypercholesterolemc at the age of 13. Despte therapeutc trals of cholestyramne, nacn, hydroxymethylglutamc acd, and neomycn, the plasma cholesterol ranged from 915 to 1210 mg/dl and she developed symptomatc coronary artery dsease. The patent's mother age 40) had a plasma cholesterol of 462 mg/dl and an LDL cholesterol of 359 mg/dl. The patent's father was not avalable for study. Analyss ofldl bndng n skn fbroblasts ofth demonstrated no detectable receptor for LDL [22]. Therefore, the patent's genetc, clncal, and bochemcal profles were consstent wth the dagnoss of receptor-negatve FH. At age 21, after sufferng two myocardal nfarctons and recevng a double coronary artery bypass graft, the patent receved a therapeutc portacaval anastomoss at the U nversty of Pttsburgh n an effort to lower the plasma lpd concentratons. A lver bopsy taken at the tme of the anastomoss provded the bass for the bomedcal and hstopathologc studes referred to as "preshunt" values. Three months after the ntal surgery, hepatc tssue was obtaned; nformed consent had been gven. Bochemcal and morphologc studes performed on ths tssue are referred to as "postshunt" values. The hepatc tssues from normolpdemc control subjects were obtaned at the tme of laparotomy for kdney donaton for renal transplantaton. All hepatc bopses were performed after nformed consent had been gven and the protocol used for hepatc bopsy and portacaval anastomoss was approved by the human expermentatons commttee at the U n versty of Pttsburgh School of Medcne. Lpoproten preparaton. Preparaton of human LDL Cd ) was from 500 ml plasma collected n 0.01% EDT A by plasmapheress from fastng, healthy volunteers. Lpoprotens were separated by preparatve ultracentrfugaton at 4 DC for ll [17] usng KBr for densty gradent adjustment [36]. These subfractons were then dalyzed 34 hr at 4 DC aganst 150 vol of phosphatebuffered salne ph 7.0) GBCO, Grand sland, N. Y.). Each solated lpoproten fracton was sterlzed by 0.45-j.Lm Mllpore fltraton Mllpore Corp., Bedford, Mass.) and used wthn 1 month of preparaton. 125_LDL was prepared by the odne monochlorde method [31] as modfed for lpoprotens [4]. A 25-30% effcency of odnaton was obtaned and less than 6% of the radoactvty was soluble n the organc phase followng a chlorofonnmethanol extracton. After dalyss over 24 hr at 4 DC aganst vol PBS, specfc actvtes ranged from 2.7 to 4.6 X 10 9 Bq/ml LDL proten. The LDL proten concentraton was determned by the method of Lowry et at [30] usng bovne serum albumn standard. After Mllpore fltraton, 1251_LDL was stored at 4 C and used wthn 2-3 weeks of preparaton. Lver membrane preparaton. Bopsy spec mens were mmedately placed n a beaker and all processng occurred at 4 C smlar to that descrbed by others [1. 2]. A.fter mncng the tssue wth a razor blade. t was washed wth an ce-cold buffer contanng 0.9% w/v) NaC!. 1 rru'-1 EDT A. and 10 mm TrsCl ph 8.0). Homogenzaton was performed by sx

3 .. HOEC ET!'L.: PORTACAVAL SHUNT 371 c d le )f al :r- ~n ld he he e. of,00 as- ~rs.. lye 17}.ent zed atelston ton lsed was thod 25- and.uble lon ~4 hr e acq/mj aton et at. dard. tored repa- spec,eaker lar to.ncng ashed w/v) :1 p1 by sx strokes of a motor-drven Teflon pestle n a buffer contanng 0.25 M sucrose, 1 mm EDTA, and 10 mm TlsCl ph 8.0). The ho-, rnogenzed preparatons 10 mg/ml) were c:ntrfuged for 10 mn at 1000g. The supernatant soluton was recentrfuged for 25 mn at O,OOOg, followed by ultracentrfugaton at 100,OOOg for 60 mn. The pellet from ths ultracentrfugaton was resuspended n a buffer contanng 150 mm NaCl, 10 mm TrsCl ph 8.0), and flushed through a 22-gauge needle, O tmes. These membranes were recentr fuged for 15 mn at 100,OOOg and the mem- brane pellets were then frozen n dry ce and. stored n lqud ntrogen untl used for bndng. assays and quanttaton ofhmg-coa reduc- lase actvty. The 10,000g supernatant was used for cholesteryl esterase assays. Lpoproten quanttaton. Blood was ob taned n 0.01% EDT A from patent TH after a 12- to 14-hr overnght fast, and the plasma was separated at 4 DC n a refrgerated centr { fuge. Plasma cholesterol and trglycerdes were ~ quanttated on a Glford 3500 usng prevously descrbed enzymatc methods [32, 52]. HDL [ cholesterol was determned followng dextran sulfate precptaton of plasma. Plasma was ultracentrfuged g/ml) for 18 hr at 39,000 rpm 4 C) n Beckman 40.3 rotors Beckman, Fullerton, Calf.) and the VLDL was separated from the other plasma lpopro tens by tube slcng [17]. The cholesterol con. centraton n the gjml nfranate was l measured, and the VLDL and LDL cholesterol were calculated. Hepatc cholesterol and cholesteryl ester de. termnaton. Lver bopsy samples from nor mal and famlal hypercholesterolemc sub jeets were weghed, extracted three tmes wth chloroform-methanol 2/1 v/v) at 41 0e. The samples were then extracted overnght wth Chloroform-methanol 2/1 v/v) at 30 e. The. chloroform-methanol extracts were blown to, dryness and the lqud was resuspended n 2- Propanol J. T. Baker Chemcal Co., Phllpsburg, N. J.). Free and total cholesterol were then measured by the enzymatc, fluormetrc method of Heder and Boyette [20J. Esterfed, cholesterol was determned as the dfference between tu!j.] and,ree cholesterol. The nanomoles of cholesterol extracted from the bopsy samples were normalzed to ntal sample weght. Hepatc en::yme actvtes. The actvty of HMG-CoA Reductase EC ) was quanttated n the 100,000g pellet, as prevously descrbed [3]. The cholesteryl ester hydrolase actvty EC ) was determned n the cytosolc fracton at both ph 4.0 and ph 7.0 [21]. Bndng of 125J_LDL to lver membranes. Hepatc membrane bndng of 125_LDL was assessed usng prevously descrbed methods [1, 22, 28]. Brefly, frozen lver membrane preparaton was thawed and resuspended n 50 rnm NaCl, 30 rnm Trs-HCl ph 7.5) buffer mg/ml) and passed through a 22-gauge needle. Membranes were then soncated by fve 4-sec pulses at the 55-W settng usng an ultrasoncs mcrotp Heat Systems Ultrasoncs, nc., Planvew, N. Y.). From 100 to 200 p.g membrane proten was then added to a 50 mm NaCl, 20 mm Trs-Cl ph 7.5) buffer contanng 1 mm CaCl _LDL was added at the ndcated concentratons wth or wthout unlabeled LDL wth a total assay mxture volume of 0.1 ml. ncubatons were carred out at 37 C for 30 mn at whch tme prelmnary studes had demonstrated that equlbrum had been reached. Bound 1251_ LDL was separated from free lgand by a 3 mn, 100,000g centrfugaton of 50 p.l of the assay mxture through 125 p.l PBS n a 30De angle rotor n an ar-drven ultracentrfuge Beckman, Palo Alto, Calf.). The supernatant was removed from the pellet by vacuum aspraton, and the pellet was washed once wth 125 ll of PBS. The cellulose ntrate tube tps contanng the membrane pellet were slced and the radoactvty n the pellet quanttated n a Bogamma scntllaton counter Beckman). Specfcally bound 125_LDL was defned as the dfference n 125_LDL quanttated n samples whch were ncubated wth and wthout 390 p.g of unlabeled LDL. Hstopathologc studes. One sample of each lver specmen was fxed n 10% neutral formaln and was then processed for examnaton

4 372 JOURNAL OF SURGCAL RESEARCH: VOL. 39, NO.5, NOVEMBER 1985 by lght mcroscopy. A second pece of the bopsy tssue was fxed n buffered glutaraldehyde, postfxed n osmum tetroxde, and then embedded n epoxy resn Epon 812). Some of the lver postshunt tssue was also postfxed n osmum and processed for electron mcroscopy. Ultrathn sectons were staned wth lead ctrate and examned n a Phllps 300 electron mcroscope. The szes of the mdzonal hepatocytes before and after portal dverson were determned on hematoxyln and eosn-staned sectons by a method prevously descrbed [43]. Md-zonal hepatocytes dentfed n 1.0-,um-thck epoxy resn sectons were also used for measurng the length of rough endoplasmc retculum per area of cytoplasm by a morphometrc method [29]. Statstcal methods. Statstcal comparsons of pared and unpared lpoproten determnatons and bochemcal assays were made usng two-taled t tests assumng the samples were ndependent [39]. RESULTS The effects of portacaval shunt on the lpd and lpoproten concentratons n the plasma n FH are summarzed n Table 1. All of the lpd and lpoproten concentratons decreased followng the portacaval shunt. The declne of 291 mg/dl n total cholesterol and 266 mg/dl n the LDL cholesterol represented a 28% change P < 0.01). Although the concentratons of VLDL, HDL, and total trglycerdes also declned, these changes were not statstcally sgnfcant. These changes n lpoproten concentraton were paralleled by strkng alteratons n hepatocyte morphology Fg. 1). Before portacaval dverson the hepatocytes were enlarged and ther cytoplasm was vacuolated. Lpd deposts were demonstrated by stanng frozen sectons wth Sudan V. Ultrastructurally, the cytoplasmc droplets possessed a double membrane. The amounts of rough and smooth endoplasmc retculum were normal. Free polyrbosomes were also present n the. cytoplasm. Glycogen was abundant. After portacaval shunt, the sze of the hepatocytes was nearly halved and the amount of fat n the cytoplasm of the lver cells decreased. Ultrastructurally, the lpd droplets remaned enclosed n a double membrane. Morphologc analyss showed that the area of rough endoplasmc retculum was reduced to 47% of the quantty found n the preoperatve bopsy. The amount of smooth endoplasmc retculum was reduced to 55% compared to the frst bopsy. Free rbosomes were abundant. Glycogen partcles were rare. Therefore, marked alteratons n hepatocyte morphology, ndependent of any autolytc artfact, were observed both before and after surgery. TABLE THE EFFECT OF PORTACAVAL SHUNT ON PLASMA LpD AND LPOPROTEN CONCENTRATONS N FAMLAL HYPERCHOLESTEROLEMA Cholesterol mg/dl) Trglycerdes mgjdl) Total VLDL LDL HDL mgfdl) Normal a range Preshunt Postshunt % Change ± ± 37* 28* ± ± ± ± ± ± 43* 23 ± ± 43 28* Note. Plasma lpd and lpoprotens were measured fve tmes preshunt and four tmes postshunt. The values represent the mean ± SE. a "onnal values are those reported for the 0-90 percentle for females ages from The Lpd Research Clncs Prevalence Study [49]. These values represent a Sgnfcant dfference from preshunt values P < 0.05).

5 HOEG ET AL.: PORTcCA" AL SHUNT ~,.f f D - -, {, :e ues tfch PRESHUNT POSTSHUNT FG.. Electron mcroscopy of hepatc bopsy specmens taken before portacaval shunt left panel) and. after portacaval shunt rght panel). The ncreased cytoplasmc hepatocyte lpd accumulaton preshunt decreased after portacaval shunt. Magnfcaton n both specmens s X,400. The POrtacaval shunt was al", assocated wth major changes n hepatc cholesterol content Table 2). Before the portacaval shunt, ', the total cholesterol content n the FH lver was 1.7 tmes that of normal lver. Although the portacaval shunt reduced total hepatc cholesterol by 27%, the postshunt cholesterol content was stll 21 % hgher than normal. The most strkng change ncluded the 75% declne n free cholesterol that was observed after the operaton. As the free cholesterol declned, the amount of cholesterol n the esterfed form mcreased by 79%. The normal lvers had 24% of the cholesterol esterfed whle the FH lver preshunt had 32% of the cholesterol esterfed. By performng the portacaval anastomoss, the fracton of hepatc esterfed cholesterol n FH mcreased to 77%. Thus, the portacaval shunt bad a profound effect not only on the absolute cholesterol content n the lver ofth, but also on the cholesterol dstrbuton between free and esterfed forms. TABLE 2 EFFECT OF PORTACAVAL SHUNT ON HEPATC CHOLESTEROL CONTENT N FAMLAL HYPERCHOLESTEROLEMA Hepatc cholesterol nmole/mg wet tssue) Total Free Esterfed Normal 5.80 ± ± ± 0.52 Famlal hypercholesterolema Preshunt 9.61 ± ± ± 0.32 Postshunt 7.04 ± ± ± 0.29 Note. Three replcate samples of hepatc bopses were measured and the values represent the mean ± SE.

6 374 JOURNAL OF SURGCAL RESEARCH: VOL. 39, NO.5, NOVEMBER 1985 ", " t tj l~,/,. '" :~ - Enzymes central to hepatc cholesterol metabolsm were quanttated n lver bopsy specmens taken before and after surgery Table 3). The actvtes of the enzymes HMG CoA reductase, acd cholesteryl ester hydrolase, and neutral cholesteryl ester hydrolase n the preshunt lver bopsy were comparable to the actvtes observed n normal lver. Although the portacaval shunt had no apparent effect on neutral esterase actvty, changes n both HMG-CoA reductase and acd esterase actvtes were observed Table 3). A 56% declne n HMG-CoA reductase actvty was paralleled by a 220% ncrease n acd esterase actvty. Thus, portacaval shunt of blood flow from the lver apprecably altered the enzyme actvtes relevant to hepatc cholesterol homeostass. The ablty of hepatc membranes to specfcally bnd LDL was drectly determned n membranes solated from the FH lver before and followng portacaval anastomoss Fg. 2). There was a sgnfcant reducton n LDL bndng to the FH hepatc membranes compared to bndng to hepatc membranes from normolpdemc subjects. After the portacaval shunt, however, the specfc bndng of 125 1_ LDL to the hepatc membranes was sgnfcantly ncreased compared to preshunt values. Thus, portacaval shunt enhanced the hepatc membrane bndng of 125_LDL. TABLE 3 THE EFFECT OF PORTACAVAL SHUNT ON HEPATC ENZYMA TC ACTVTES N F AMLlAL Preshunt Postshunt HYPERCHOLESTEROLEMA HMG CoA 129 :!: ± 4 Enzymatc actvty % control) Acd esterase 110 ± 3 249:!: 19 Neutral esterase 92 ± ± 5 Note. The enzymatc actvty n the FH hepatc tssue was compared to that from lver taken from three norrnolpdemc subjects. The control specfc actvnes expressed as pmole/mn/ mg proten were HMG-CoA reductase 3.24 ± acd esterase 71.5:!: and neutral esterase 11.4 ± 2.1. Values represent the mean ± SE of three or four replcate samples....j Cl...J "0'. LDL l'g/ml) FG. 2. Specfcally bound 12s_LDL to hepatc memo branes from normal subjects and a famlal hypercholesterolema before and after portacaval shunt. Membranes were prepared from hepatc bopsy specmens from three normal subjects 0, N = 3), and famlal hypercholester_ olemc hepatc tssue preshunt b.), and postshunt.). From 100 to 200 Lg of membrane proten was added to a 75 mm NaO, 150 mm TrsO, mm CaCl 2 ph 7.5) buffer wth the ndcated concentratons of 12s_LDL n the presence and absence of excess unlabeled LDL. Specfcally bound 2s_LDL, defned as the dfference n 1251_ LDL bound n the presence and absence of excess unlabeled LDL, was normalzed to the amount of membrane proten n the sample. Values represent the mean ± SE of trplcate determnatons. DSCUSSON The extreme hypercholesterolema and rapdly progressve premature cardovascular dsease observed n famlal hypercholesterolema have prompted the search for an effectve, defntve treatment. After observng a marked declne n serum lpd concentratons wth a portacaval shunt for Type glycogen storage dsease [47], Starzl and co-workers successfully employed ths procedure to lower the plasma cholesterol levels n a patent wth famlal hypercholesterolema [42]. Subsequent observatons oflowered LDL cholesterol levels, xanthoma regresson, and safety of the procedure have been reported by Starz as well as several other nvestgators [6, 10, 12, 13, 25,40,41,44,54]. Thus, the use of portacaval anastomoss appeared to be one of the few successful hypocholesterolemc maneuvers 10 patents homozygous for FH. The 28% reducton n total cholesterol and LDL observed n TH after portal dverson was typcal of the 20-55% declne n these values l.. ' p { 4' fa pc oj { st t th m m sy fo re ro Ll '" de tho Sl he L to an tal th the we pa str rot un 43 an, dn we tac alt, Ch tae qu tet COl ch to en: ree lov SYr the

7 HOEG ET AL.: PORTACAVAL SHUNT -,--.., J J ~ J ~ 500 patc mem- ypercholes- Membranes 5 from three lcrcholester- stshunt.). vas added to Ch ph 7.5) 1251_LDL n rl LDL. Spe- :rence n m_. excess unla- Jfmembrane nean ±SEof lema and ovascular ~holesterolor an effec Jbservng a lcentratons : glycogen co-workers ureto lower patent wth 42]. Subse L cholesterol safety of the Starzl as well 10 12, 13, ~fp~rtacaval e of the few naneuvers n olesterol and dverson was 1 these values prevously reported [6, 10, 12, 13.25,40-42, 44,54]. Ths declne could occur because ofa fall n the synthess of cholesterol contanng lpoprotens, an enhanced clearance of the lpoprotens from the plasma, or a combnaton of these two possbltes. Metabolc turnover studes of radolabeled lpoprotens ndcated that the fractonal catabolc rate of FH homozygotes was sgnfcantly less than n normal ndvduals [5, 37, 38, 50] and that the synthess rate of apob nto LDL was two- to fourfold normal [5, 37, 38]. Blhemer et al. reported that portacaval shunt n a patent homozygous for FH enhanced the clearance of j LDL from the crculaton by 17% and a 48% declne was measured n the rate of LDL synthess n a patent homozygous for FH [5]. A smlar response has been reported n a patent heterozygous for FH [14]. Thus, the fall n LDL observed after portacaval shunt appeared to result from changes n both LDL synthess and removal. Snce the mammalan lver may be mportant for both cholesterol and lpoproten syn thess as well as degradaton, a dsrupton n the blood supply rch n hepatotrophc factors l r,,, would be antcpated to alter these bochemcal 1 pathways. As n the present case, the cellular structures nvolved wth lpd and lpoproten metabolsm have consstently been shown to undergo profound morphologc changes [35, 43, 45, 46]. The reducton n hepatocyte sze and the development of cytoplasmc lpd. droplets n the FH lver after portacaval shunt! were smlar to the changes nduced by por- tacaval shunt n the dog [45]. These anatomc alteratons are paralleled by bochemcal changes observed n the lver of TH after por tacaval shunt. Frst, quanttatve as well as. qualtatve changes n hepatc cholesterol content were observed. Total hepatc cholesterol COntent decreased; however, the fracton of cholesterol n the free form decreased from 68 l to 23%. Second, the actvty of the rate-lmtng enzyme for cholesterol synthess, HMG-CoA reductase, was reduced by more than half fol. OWng the portacaval shunt. Thus, the bosynthetc. pathway of cholesterol synthess n the FH lver was shown for the frst tme to declne n parallel wth the fall n hepatc content of total and free cholesterol followng portacaval shunt. The dstrbuton and hydrolyss of cholesteryl ester wthn the FH hepatocyte may reflect aberrant metabolsm of cholesteryl estercontanng lpoprotens. The LDL receptormedated uptake mechansm leads to lysosomal localzaton and degradaton of LDL [8, 15 J. Snce TH had no hgh-affnty LDL receptor, delvery of LDL to cells could only occur through a pnocytotc or an alternate pathway for LDL uptake. Such an alternate pathway may lead to neffectve delvery of cholesteryl ester-rch lpoprotens to approprate subcellular compartments. The exstence of such an alternate pathway has recently been shown to be the major source of LDL delvery to the lver of the Watanabe hertable hyperlpdemc WHHL) rabbt, the only exstng anmal model for FH [34]. n TH, the portacaval shunt was assocated wth an ncrease n the "receptor-ndependent" bndng ofldl te the hepatc membranes Fg. 2). An enhancement ofldl transport, though a less effcent alternate pathway, could account for the strkng modfcatons observed n hepatc cholesterol metabolsm. n summary, portacaval shunt n FH has been shown to ncrease hepatc LDL recognton, markedly alter the ntracellular enzymes central to cholesterol metabolsm, and modfy hepatc cholesterol concentraton and dstrbuton. These changes parallel the ncreased clearance of plasma LDL and reduced LDL synthess observed n FH patents after portacaval shunt. By manpulatng the metabolc consequences of the hepatc LDL receptor loss n FH, more effectve therapeutc approaches to the hypercholesterolema and accelerated atheroscleross present n FH can be developed. ACKNOWLEDGMENTS We thank Dr. Lee, Dr. Warty, and Dr. Sanghvj at the Unversty of Pttsburgh for the use of ther laboratory facltes for a porton of these studes. We are also grateful to Dr. Dana Fowlkes for hs effort n facltatng acquston of postshunt tssue.

8 ------,-~-----' JOURNAL OF SURGCAL RESEARCH: VOL. 39, NO.5, NOVEMBER 1985 REFERENCES heterozygous famlal hypercholesterolema: Effect of portacaval shuntng. Bochrn. Bophys. Acta 712: Bachork, P. S., Kwterovch, P.O., and Cook, J. C ' -' solaton of a porcne lver plasma membrane fracton 15. Goldsten,). L., and Brown, M. S. Bndng and deg_ that bnds low densty lpoprotens. Bochemstry 17: radaton of low densty lpoprotens by cultured hu- 5287, man fbroblasts. J. Bo. Chern. 210: 5153, Basu, S. K., Goldsten, J. L., and Brown, M. S. Char- 16. Harders-Spengel, K., Wood, C. B., Thompson, G. R., acterzaton of low densty lpoproten receptor n Myant, N. B., and Soutar, A. K. Dfference n saturable membranes prepared from human fbroblasts. J. Bo. bndng oflow densty lpoproten to lver membranes Chern. 253: 3852, from normocholesterolemc subjects and patents wth 3. Beg, Z. H., Stonk, 1. A., and Brewer, H. B.. Jr. heterozygous famlal hypercholesterolema. Proc. 3-Hydroxy-3-methylglutaryl coenzyme A reductase: Natl. Acad. Sc. USA 78: 6355, Regulaton of enzymatc actvty by phosphorylaton 17. Havel. R. 1.. Eder. H. A.. and Bragdon, J. H. The and dephosphorylaton. Proc. Nat!.. lead. SC. USA dstrbulon and chemcal composton of ultracen- 75: 3678, trfugally separated lpoprotens n human serum Blhemer, D. W., Esenberg, S., and Levy, R. 1. The Cln. nvest. 34: 1345, metabolsm of very low densty lpoprotens. 1. Pre- 18. Havel, R. 1., Goldsten, J. L., and Brown, M. S. n 3- lmnary n 'lro and n vvo observatons. Bochrn. P. K. Bondy and L. E. Rosenberg Eds.), Metabolc Bophys. Acta 260: 212, Control and Dsease. Phladelpha: Saunders, ~ " 3lhemer, D. W., Goldsten, 1. L, Grundy, S. M., pp and Brown, M. D. Reducton n cholesterol and low 19. Hay, R. Y., Pottenger, L. A., Rengold, A. L., and j.. densty lpoproten synthess after portacaval shunt Getz, G. S. Degradaton of 12S-labeled serum low, surgery n a patent wth homozygous famlal hyper- densty lpoproten n nonnal and estrogen-treated 35 ~ cholesterolema. J. Cln. nvest. 56: 1420, male rats. Bochern. Bophys. Res. Cornrnun. 44: 1471, ~: 6. Blanchard, H., Bensoussan, A. L, Colln, P. P., Des ",. jardns,1. G., Oumet, A., and Letarte, 1. Tennno- 20. Heder, J. G., and Boyette, R. L. The pcomole deb lateral portacaval anastomoss n Type homozygous tennnaton of free and total cholesterol n cells n ~ famlal hypercholesterolema. Chld. Pedatr. 20: 221, culture. J. Lpd Res. 19: 514, Hoeg, J. H., Demosky, S. J., and Brewer, H. B., Jr. e.., 7. Brown, M. S., and Goldsten, J. L. Famlal hyper- Characterzaton of neutral and acd ester hydrolase cholesterolema: Defectve bndng of lpoprotens to n Wolman's Dsease. Bochrn. Bophys. Acta 711: E 1J cultured fbroblasts assocated wth mpared regula- 59, ~) ton 3-hydroxy-3-methylglutaryl coenzyme A reduc-,' 22. Hoeg, J. M., Demosky, S. J., Jr., Schaefer, E. J., Starzl,... tase actvty. Proc. Natl. Acad. Sc. USA 71,788, T. E., and Brewer, H. B., Jr. Characterzaton of he 38. ~ 8. Brown, M. D., Kovanen, P. T., and Goldsten, 1. L. patc low densty lpoproten bndng and cholesterol.. Regulaton of plasma cholesterol by lpoproten re- metabolsm n normal and famlal hypercholesterceptors. Scence Washngton, D. C.) 212: 628,1981. olemc subjects, J. Cln. nvest. 73: 429, Carew, T. E., Pttman, R. C, and Stenberg, D. Tssue 23. Hu. D. Y., nnerarty, T. A., and Mahley, R. W. Lstes of degradaton of natve and reductvely meth- poproten bndng to canne hepatc membranes; 39. ylated ['4C]sucrose-labeled low densty lpoproten n metabolcally dstnct ApoE and ApoB, E receptors. rats. J. Bo. Chern. 257: 800, J. Bo. Chem. 256, 5646, Cywes, S., Daves, M. R. Q., Louw, J. H., Berger, 24. Khachaduran, A. K. The nhertance of essental fa 40. G. M. B., Bonnc, F., and Joffe, H. S. Portacaval shunt mlal hypercholesterolema. Amer J. Med. 37: 402, n two patents wth homozygous Type hyperlpo protenema. S. Afrcan Med. J. 50: 239, Korgh, L., and Wckens, J. T. Portacaval shunt for. Detschy, J. D. M., and Wlson, 1. R. Regulaton of hypercholesterolema. S. Afrcan Med. J. 48: 2302, 4. cholesterol metabolsm. N. Eng!. J. Med. 282: 1128, , Kovanen, P. T., Blhemer, D. W.. Goldsten, J. L., 12. Farraux, J. P., Rbet, M., Bertrand, M., Hggns, M., Jaramllo, J., and Brown, M. S. Regulatory role for 42. Mazzuca, M., and Luychy, A. Treatment of Type hepatc low densty lpoproten receptors n vvo n famlal hypercholesterolema wth portacaval anas- the dog. Proc. Natf. Acad. Sc. USA 78: 194, tomoss. Arch. Fr. Pedatr. 33: Kovanen, P. T., Brown, M. S., Basu, S. K., Blhemer Fonnan, M. B., Baker, S. G., Meny, D. J., et al. D. W., and Goldsten, J. L. Saturaton and suppresson Treatment of homozygous famlal hypercholesterol- of hepatc lpoproten receptors: A mechansm for the emla wth portacaval shunt. Atheroscleross 41: 349, hypercholesterolema of cholesterol-fed rabbts. pro' Natf.,~cad. Sc. US,../, 78: 1396, L 14. Gnsberg, H., Davdson, N.. Le. N, A., Gbson Kovanen, P. T.. Brown, M. S.. and GoldsteT, J.. ' Ahrens, E. H., and Brown. W. V. Marked overpro- ncreased bndng of low densty 17 a-ethnylestradto. ducton of low densty lpoproten B n a subject wth 1. Bo. Chern. 254: 11367, 1979.

9 HOEG ET AL.: PORTAe A VAL SHUNT 377 ~9, Loud. A, V, A quanttatve sterologcal descrpton of the ultrastructure of nonnal rat lver parenchymal cells, J Cell Bo. 37: 27, 1968, 30, Lowry, 0, H" Rosebrough, N, j" Farr, A. L.. and Randall. R, 1. Proten measurement wth the Foln phenol reagent, J Bo. Chelll 193: 265, MacFarlane, A. S, Effcent trace labelng of protens wth odne. NalUre London) 182: 53, 1958, 32, Pesce, M, S" and Bodouran, S. H. Enzymatc rate method for measurng cholesterol n serum. Cln. Chern 22: 2042, Pttman, R. C. Atte, A, D., Carew, T. E., and Stenberg, D. Tssue stes of degradaton of low densty lpoproten: Applcaton of a method for determnng the fate of plasma protens. Proc. Natl. Acad. Sc, USA 76: 5345, Pttman, R. C, Carew, T. E" Atte, A. D.. Wtztum, j, L., Watanabe, Y., and Stenberg, D. Receptor-dependent and receptor-ndependent degradaton oflow densty lpoprotens n normal rabbts and n receptordefcent mutant rabbts. 1. Bo. Chern, 257: 7997, Putnam, C. W., Porter, K. A., and Starzl, T. E. Hepatc encephalopathy and lght and electron mcrographc changes ofthe baboon lver after portal dverson. Ann. Surg. 184: 155, Reddng, C. M., and Stenberg, D. Studes on the synthess and secreton of serum lpoprotens by rat lver slces. j, Cln. nvest. 39: 1560, Rechl, D, L., Smons, L. A" and Myant, N. B. The metabolsm of low densty lpoproten n a patent wth famlal hyperbetalpoprotenema. Cln. Sc. Mol. Med. 47: 635, Smons, L. A., Rechl, D. L., Myant, N. B., and Mancn, M. The metabolsm of the apoproten of plasma ' low densty lpoproten n famlal hyperbetajpoprotenema n the homozygous form. Atheroscleross 21:, 283, Snedecor, G. W" and Cochran, W. G. Comparson of Two Samples. Statstcal Methods. Ames, owa: owa State Unv. Press, Pp Soutar, A. K., Myant, N. B., and Thompson, G. R. Measurement of apopoproten B turnover n very low and low densty lpoproten n famlal hypercho- r lesterolema. Atheroscleross 28: 247, Starzl, T. E" Chase, H. P., Ahrens, E. H., et a, Por tacaval shunt n patents wth famlal hypercholesterolema. Ann Sllrg, 198: Starzl, T. E., Chase, H. P., Putnam, C W., and Porter, K. A. Portacaval shunt n hyperpoprotenerna. Lancet 2: 940, Starzl, T. E., Francavlla, A., and Halgrmson, C G. The orgn. hormonal nature and acton hepatotrophc substances n portal venous blood. Surg. Gynecol. Obslet. 137: 179, 1973, 44, Starzl, T. E.. Koef. p, J., and Wel, R.,. Portacaval shunt for Type hyperlpdema. Atheroscleross 1. Proceedngs of the Ffth nternalonal Symposum of Alherosc!eross, Berln: Sprnger-Verlag, 1980, P. 450, 45. Starzl, T. E,. Lee,. Y., Porter. K, A., and Putnam, C. W, The nfluence of portal blood upon lpd metabolsm n normal and dabetc dogs and baboons. Surg, Gyneco. Obstet, 140: 381, 1975, 46. Starzl, T. E. Porter, K. A., Watanabe, K., and Putnam, C. W, The effects of nsuln. glucagon, and nsuln/ glucagon nfusons upon lver morphology and cell dvson after complete portacaval shunt n dogs. Lancetl: 821, Starzl, T. E.. Putnam, C W., Porter, K. A., Halgrmson, C G., Corman, J.. Brown, V, L., Gotln, R, W" Rodgerson, D.O., and Greene, H. L. Portal dverson for the treatment of glycogen storage dsease n humans. Ann. Surg. 178: Sten, Y., and Sten, O. n G. Scheltler and A. Wezel Eds,), Atheroscleross Berln: Sprnger-Verlag, Pp The Lpd Research Clnc Populaton Studes Data Book: The Prevalence Study, NH Publcaton No ,1980. Pp , 50. Thompson, G. R., Soutar, A. K., Spengel, F. A., Jadhav, A., Gavgan, S. J. P., and Myant, N. B. Defects of receptor-medated low densty lpoproten catabolsm n homozygous famlal hypercholesterolema and hypothyrodsm n vvo. Proc, Natl. A cad. Sc. USA 78: 2591, Tolleshaug, H., Hobgood, K. K., Brown, M. S., and Goldsten, J. L. The LDL receptor locus n famlal hypercholesterolema: Multple mutatons dsrupt transport and processng of a membrane receptor. Cell 32: 941, Wahlenfeld, A. N, Methods of Enzymatc Analyss. New York: Academc Press, P Warner, T. G., Dambach, L. M., Shn, J. H., and O'Bren, J. S. Separaton and characterzaton of the acd lpase and neutral esterases n human lver. Amer, 1. Hum. Genet. 32: 869, Weglck, W. B., Ganda, O. P., Soeldner, J. S., Murawsk, B. J., Cohn, L. H., and Couch, N. P. Portacaval dverson of severe hypercholesterolema. Arch. Surg, 112: 634, Wndler, E. E, T" Kovanen, P. O. T., Chao, Y. S., Brown, M. D., Havel, R. J., and Goldsten, J. L. The estradol stmulated lpoproten receptor of rat lver. j, Bo. Chern, 255: 10464, 1980.