Nucleation of cholesterol crystals from native bile and the effect of protein hydrolysis

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1 Nucletion of cholesterol crystls from ntive ile nd the effect of protein hydrolysis N. R. Pttinson nd K. E. Willis Gstroenterology Reserch Unit, hristchurch School of Medicine, hristchurch Hospitl, hristchurch, New Zelnd Astrct Nucletion time represents the terminl step in in vitro studies exmining ile lithogenicity. Becuse of the concern tht residul microcrystls, left fter ultrcentrifugtion, my e responsile for the rpid nucletion time of gllldder ile from ptients with cholesterol gllstones, we hve included finl filtrtion step. However, we found this procedure to considerly lengthen the nucletion time of norml iles. In view of the centrl importnce of the nucletion ssy we compred the effect of three commonly used gllldder ile pretretment regimes (designed to remove endogenous crystls) on nucletion time. They were: U) immedite filtrtion of ile (0.22 pm filter); 6) ultrcentrifugtion; nd c) ultrcentrifugtion followed y filtrtion. The respective nucletion times were: U) 9.3 * 3. dys, n = 6; 6) 2.9 f 0. dys, n = 10; G) dys, n = 11. To determine whether the drmtic chnge in nucletion time ws due to the removl of components other thn seed crystls, we exmined the mucus content, the totl lipid composition of ile, nd tht of its cholesterol trnsport components following the different pretretments. No significnt difference in totl lipid, percentge cholesterol crried y the trnsport components, or their cholesterol/phospholipid rtio were found. Ultrcentrifugtion lone ws sufficient to removl ll detectle lrge moleculr weight mucus glycoprotein. Although nucletion time of the norml gllldder smples ws extended in the ultrcentrifuged/filtered iles, it ws still significntly different (P < 0.01) from tht of norml gllldder iles, confirming n intrinsic difference etween norml nd norml iles, in cholesterol metstility. We lso exmined the effect of protein digestion on the nucletion time of ntive iles. Proteolysis of iliry proteins hd little effect on the nucletion time of either norml gllldder or norml gllldder nd common duct iles. The puttive fctors, if they re proteins, pper resistnt to proteolytic clevge or re of minor importnce. Pttinson, N. R., nd K. E. Willis. Nucletion of cholesterol crystls from ntive ile nd the effect of protein hydrolysis. J. Lipid Res : 52. Supplementry key words glycoprotein cholesterol gllstone disese mucus Nucletion time (i.e., the ppernce of cholesterol monohydrte crystls) represents the terminl step in in vitro studies exmining ile lithogenicity (1, 2). Bsed on such mesurements it hs een determined tht the for mtion of cholesterol crystls is not only dependent on the cholesterol sturtion of ile nd totl lipid concentrtion (13) ut lso on the presence or sence of certin proteins with either pronucleting or ntinucleting ctivity (12). However, recent communiction (13) hs rised some douts over when nucletion time of ntive iles cn e considered s unequivocl. Moreover, recent work in our lortory hs pointed to yet nother potentil prolem with this in vitro ssy, in tht the type of pretretment of the ntive ile (designed to remove endogenous cholesterol crystls) prior to setting up the nucletion ssy cn lso gretly influence the ssys outcome. Becuse of the concern tht residul microcrystls, left fter ultrcentrifugtion, my e responsile for the rpid nucletion time of gllldder ile from ptients with cholesterol gllstones, we felt it prudent to include finl filtrtion step fter ultrcentrifugtion (1). This hd the dded dvntge of not only ensuring removl of ny residul crystls, ut lso of mintining sterility of the ile. In ddition, previous studies (, ) hve indicted tht filtrtion fter ultrcentrifugtion did not ffect nucletion time. However, we hve found this pretretment regime to considerly prolong the nucletion time of norml gllldder iles compred with the vlues reported y other groups. In this study, therefore, we hve compred the effect of three gllldder ile pretretment regimes (commonly used y different lortories) on nucletion time, nd hve exmined whether the differences found could e explined y either the removl of mucus or chnges in the lipid composition of the iliry trnsport components responsile for cholesterol soluiliztion. In ddition, the effect of pronse digestion of iliry proteins on nucletion time ws exmined in gllldder nd heptic iles from gllstone ptients nd in gllldder iles from ptients without cholesterol gllstones. Arevitions: SI, cholesterol sturtion index. Downloded from y guest, on August 26, 2018 Journl of Lipid Reserch Volume 32,

2 Bile collection METHODS Gllldder ile ws otined from ptients with gllstones t the time of elective cholecystectomy. ommon duct ile ws lso otined from numer of these ptients y introduction of ctheter vi the cystic duct prior to performing n opertive cholngiogrm. Norml gllldder ile ws otined from ptients y needle puncture spirtion nd removl of the entire content during dominl surgery for conditions other thn those ffecting the livedpncres or iliry tree [ gstrectomy (), sigmoid colectomy (3), pyloroplsty, nd vgotomy (l)]. Approvl of the protocol used in this investigtion ws otined from the Ethicl ommittee of our Institution. All ptients gve written consent prior to entering the study. The sterility of ll iles (prior to processing) ws ssured y eroic culture on lood pltes nd Roinson's cooked met medium, nd neroic culture on supplemented gr pltes (vitmin K 10 pg/ml; hemin 5 pg/ml) using stndrd methods. Nucletion time: effect of pretretment To determine the rte of de novo cholesterol monohydrte crystl formtion, crystlfree frctions of norml gllldder iles re required. To this end series of 11 gllldder ile smples were sujected to the following pretretment regimes. ) Filtrtion through sterile 0.22pm Flowfore D microdisc filter (Srtorius, West Germny) (11, 15). Prior to filtrtion the ile ws leled for 30 min t 3O with stock micellr solution (20 pl/ml ile) contining [ 1]cholesterol nd [ 3H]phosphtidylcholine. The 10ml stock solution contined 00 pmol turocholic cid, 25 pmol egg lecithin, 5 pi diplmitoyl phosphtidylcholine (cholinemethyl 3H) (New Englnd Nucler, Boston, MA), nd 20 pi [1]cholesterol (Amershm L., Englnd). Rdiolel rpidly exchnged with endogenous lipid giving identicl specific ctivities in ech lipid trnsport form within 30 min. However, leling of sumicroscopic nd microscopic crystls would e unlikely within this time period. Becuse of the high mucus content of ll the norml iles, immedite filtrtion of ile ws very difficult. Thus, we were only le to otin sufficient ile for nucletion studies nd lipid nlysis in of the 11 iles studied. ) Ultrcentrifugtion t 100,000 g for 2 h (Beckmn L Ultrcentrifuge, rotor TI 0.1; 38K) (1, 2,, 5,, 16). The vrition in ultrcentrifugtion time ws necessitted y the high mucus content of numer of ile smples. The superntnt ws then isolted nd mixed thoroughly prior to chemicl nlysis nd ssy of nucletion time. c) The sme procedure s in () ut the isolted superntnt ws filtered immeditely fter spirtion, through sterile 0.22pm Flowfore D microdisc filter (,, 1). After microscopic exmintion of the processed ile for the sence of cholesterol monohydrte crystls, liquots were removed for lipid nlysis nd gel filtrtion. Smpling of iles for ssy prior to pretretment ws impossile ecuse of the high mucus content in ll norml gllldder ile smples. Bile smples (0.5ml volume) were then incuted t 3O in sterile 1.5ml screwtopped tues nd exmined dily under polrizing microscope for the first ppernce of cholesterol crystls. Oservtion of one crystl in single dy ws not considered s sufficient evidence for nucletion unless the result ws repeted on t lest 2 consecutive dys. The nucletion time ws then tken s the dy on which the first crystl(s) ppered. Nucletion time: effect of pronse digestion Biles from ptients with nd without cholesterol gllstones were treted s per regime c). Aliquots of 0.5 ml were incuted t 3O in sterile tues in the presence nd sence of dded pronse (Streptomyces griseus, 2.5 mg/ml doule distilled H20; 20 pv0.5 ml ile; 3.5 U). Hydrolysis of pronsesensitive iliry protein is essentilly complete within 2 h s determined y silver stining, fter SDSpolycrylmide gel electrophoresis under reducing conditions (1) (Fig. 1). Tht the pronse ws ctive in ll ile smples ws ssured y plcing n liquot of smple on Xry film covered with 1% grose. After 2h incution t room temperture, the film ws thoroughly wshed. In ll smples contining pronse the film cking hd een digested wy. Biochemicl methods Gelfiltrtion chromtogrphy. Bile (0.2 ml) ws chromtogrphed t room temperture on Sephrose LB200 (Sigm hemicl o., St. Louis, MO) column, 0.8 cm x 12 cm, preequilirted with nd eluted with phosphteuffered sline, ph., 0.0% sodium zide, contining 6 mm turocholte. Eluted frctions were monitored for [ ']cholesterol nd [3H]phosphtidylcholine using Pckrd Tricr Scintilltion counter. The scintillnt contined 5.5 g PPO, 0.2 g POPOP, 330 ml Triton X100, nd 660 ml toluene. olor quenching nd crossover of I counts into the 3H chnnel were corrected for y the externl stndrd method. The composition of the ile trnsport components ws determined from the clculted specific ctivity of cholesterol nd phospholipid (18). hemicl nlysis of the iliry components ws s previously descried (1). Mucus glycoprotein ssy. Humn gllldder mucus glycoprotein (in three ile smples) ws determined essentilly y the method of Person et l. (19). The following smples were exmined: n liquot of whole ile diluted 1:5 with phosphteuffered sline contining 30 mm turocholte; n liquot tken fter ultrcentrifugtion; nd n liquot of ultrcentrifuged ile tht hd een Downloded from y guest, on August 26, Journl of Lipid Reserch Volume 32, 1991

3 / were compred etween norml nd the nongllstone ptients, nd etween thenorml gllldder nd heptic iles using ttests. After these tests, stepwise discriminnt nlysis ws used to see whether some liner comintion of the vriles would etter seprte the norml/norml gllldder iles. All mens re presented with stndrd errors (SE). RESULTS Effect of ile pretretment of nucletion time Pretretment of the supersturted gllldder iles gretly ffected their nucletion time. Men nucletion times for the vrious pretretment regimes were s follows: ) immedite filtrtion, dys, n = 6; ) ultrcentrifugtion, dys, n = 10; c) ultrcentrifugtion/filtrtion, dys, n = 11. Nucletion time of ile given pretretment ) ws very rpid s previously documented (1, 2). Pretretment c) significntly incresed (P < 0.01) the time for cholesterol monohydrte crystl formtion compred with ). No significnt difference on nucletion time ws found etween pretretments ) nd c). The difference etween ) nd ) just filed to rech significnce. * pssed through 0.22pm microdisc filter. Smples were sujected to Sephrose LB200 gel chromtogrphy (20 x 1. cm; flow rte 8.25 ml/h; frction volume, 1.1 ml) in column preequilirted nd eluted with phosphteuffered sline contining 30 mm turocholte. Void volume mucus glycoprotein, seprted from free protein, ile cids, nd pigment, ws quntitted y the periodic cidschiff ssy. Sttistics Sttisticl comprisons of nucletion times were mde using Survivl Anlysis with themontelox sttistic. omprisons ssessing the influence of pretretments on the lipid compositions were mde using pired ttests or ANOVA when the composition estimtes for ech pretretment smple involved repliction. SI ndg/dl levels * Effect of ile pretretment on iliry lipid composition nd mucus content To determine whether the filtrtion step ws removing lile lipid component of ile, we compred the totl lipid composition of the iles fter ech pretretment. No significnt difference etween cholesterol, phospholipid, ile cid, totl lipid concentrtion, or cholesterol sturtion index etween pretretments ws found (Tle 1). However, since the lile entity need not necessrily e lrge percentge of the whole, we tried toincrese the sensitivity of our mesurements y seprting the vrious iliry cholesterol trnsport components y gel filtrtion. Gel filtrtion ofwhole ile on Sephrose LB200 seprtedoutthree cholesterolcontining frctions: high moleculr weight component in thevoid volume corresponding tothe nonmicelle/vesiculr component (found predominntly in diluteheptic iles), nd smller vesiculr component (18) seen s shoulder leding up to the mjor trnsport component in gllldder ile, the mixed micelle. The percentge distriution of cholesterol etween these three componentsfor five of the gllldder ile smples isgiven in Tle 2. No difference in the percentge distriution of cholesterol resulted from the vrious pretretments, irrespective of the nonmicelldvesiculr content. The cholesterol/phospholipid rtio of the trnsport forms is lso given in Tle 2. Agin, no significnt differences were seen etween pretretment regimes. Direct mesurements of cholesterol, retined on the filters (fter hexne extrction), reveled no Pttinson nd Willis Nucletion ndproteinhydrolysis Downloded from y guest, on August 26, 2018 Fig. 1. Pronse di.vstion of ile: time dependence. Gllldder ile ws incuted with pronse (Strcptomycesgrkek) s descried in Mterils nd Methods nd sujected to SDSpolycrylmide gel electrophoresis; lne 1, moleculr weightmrkers (myosin, 205,000; fl glctosidse, 116,000; phosphorylse B, 9,00; ovine serum lumin, 66,000; ovlumin, 5,000; cronic nhydrse, 29,000); lne 2, ile inthe sence of pronse; lne 3, pronse (2.5 mglml); lne, digestion for 30 min; lne 5. digestion for 1 h; lne 6, digestion for 2 h; lne, digestion for h; lne 8, digestion for 6 h; lne 9, digestion for 12 h; lne 10, digestion for 2 h; lne 11, digestion for 8 h. Ech smple ws diluted 1:3 in reducing uffer nd 30 pl ws pplied per lne. *

4 TABLE 1. Bile lipid composition Ptient Pretretment RA PL H onc SI Nucletion Time AL TO RO H FN BL 1 RS HO O ED mm mht mnf s/dl dys The effect of ile pretretments (, filtered immeditely;, ultrcentrifugedsuperntnt; c, ultrcentrifugedfiltered superntnt) on iliry lipid composition nd nucletion time; BA, ile cid; PL, phospholipid; H, cholesterol; SI, cholesterol sturtion index. mesurle levels (phospholipid nd mucin were not mesured). In ddition, we exmined the effects of ultrcentrifugtion nd filtrtion on mucus glycoprotein concentrtion. Mucus glycoprotein ws seprted on column of Sephrose LB200 nd mesured y the periodic cid Schiff ssy with sornce t 555 nm. High levels of sornce were found in the void volume frction of the column with whole ile smples (diluted 1:5) prior to pretretment (sornce 0.20.). No mesurle void volume glycoprotein ws found in ultrcentrifuged smples (undiluted) or those tht hd een ultrcentrifuged nd filtered. Nucletion time Anorml ile duct nd gllldder iles nd gllldder iles from nongllstone ptients were otined nd sujected to identicl pretretments, i.e., ultrcentrifugtiodfiltrtion, prior to setting up the nucletion ssy. Nucletion time, SI, nd concentrtion (g/dl) for the norml gllldder nd common duct nd norml gllldder iles re given in Tles 3,, nd 5. Gllldder ile from cholesterol ptients nucleted significntly (P < 0.01) more rpidly thn from nongllstone ptients ( dys, n = 25 vs dys, n = lo), lthough there ws considerle overlp. We ttempted to seprte the groups using liner comintion of the three vriles: nucletion time, SI, nd concentrtion using stepwise discriminnt nlysis. Only one vrile (nucletion time) met the entry criteri (F >.0). A cutoff point etween 1316 dys on nucletion time gve 68.8% successful clssifiction overll; 80% nongllstone formers; 6% gllstone formers. Anorml gllldder ile nucleted Downloded from y guest, on August 26, Journl of Lipid Reserch Volume 32, 1991

5 ~ ~ ~ TABLE 2. holesterol soluiliztion % H HIPL Ptient Ves SVes Mic Ves SVes Mic FN c RS HO c O c ED c The effect of ile pretretment (, filtered immeditely;, ultrcentrifugedsuperntnt ; c, ultrcentrifugedfiltered superntnt) on cholesterol soluiliztion y its respective trnsport forms seprted y gel filtrtion chromtogrphy (Sephrose B); Ves, vesicle; Mic, micelle; H, cholesterol; PL, phospholipid; SVes, smll vesicle. significntly more rpidly thn heptic ile (P < O.Ol), despite the ltter hving significntly higher men SI (Tles 3 nd 5). Effect of pronse digestion on nucletion time Becuse iliry proteins hve een implicted s hving importnt roles in oth preventing nd ccelerting nucletion, we exmined the effect of proteolysis on cholesterol monohydrte crystl formtion. Although the gret mjority of iliry protein ws hydrolyzed within 2 h, not ll proteins were susceptile to pronse digestion (Fig. 1). Proteolysis of iliry protein from nongllstone ptients reduced nucletion time in only one of nine iles. Similrly, protein hydrolysis hd no significnt effect on the nucletion time of gllldder or heptic iles from gllstone ptients (Tles 3,, nd 5). DISUSSION The results of this study demonstrte tht the wy gllldder ile smples re hndled prior to setting up nucletion studies drmticlly lters the time for cholesterol monohydrte crystls to form. Although we found s did others (1, 2), tht gllldder iles of similr cholesterol sturtion collected from ptients with cholesterol gllstones nucleted more rpidly thn those without, the mgnitude of the difference ws considerly reduced under our conditions such tht it ws impossile to completely discriminte etween the two popultions on this sis. As nerly ll gllldder ile smples from ptients with cholesterol gllstones lredy contin cholesterol crystls, it is criticl to ensure their complete removl prior to incution for new crystl growth. It is possile, however, tht the filtrtion step rther thn removing residul crystls could hve removed some other criticl structure, such s liquid crystls, vesicles (or their ggregtes), or mucus. Vesicle ggregtion hs een shown to precede nucletion (16); however, we were unle to show ny quntittive chnge in the gel filtrtion profile of the lipid components s result of the vrious pretretment regimes. Tht the difference in in vitro nucletion time could e due to the incomplete removl of mucus glycoprotein lso seems unlikely, s we found tht ultrcentrifugtion lone ws sufficient to remove ll lrge moleculr weight (void volume) periodic cidschiffpositive mteril. Direct ttempts to mesure precipitted cholesterol crystls on the microdisc filters were mde ut we filed to find mesurle levels. This my e due to our inility to mesure with ny ccurcy smll yet potentilly criticl quntities of lipid mteril. For the sme reson, role for quntittively smll vesiculr frction cnnot e totlly excluded. TABLE 3. Gllldder ile from cholesterol gllstone ptients Nucletion time Ptient SI onc. No Pronse Added Pronse g/dl d.u. WI HU R GR os SP JO DO VH K NI AV PR BE HD FI HP MI FN BL L RS HO O ED Men i SE Nucletion time of gllldder ile from gllstone ptients in the presence or sence of the proteolytic enzyme, pronse SI, cholesterol sturtion index. Downloded from y guest, on August 26, 2018 Pttinson nd WiIlis Nucletion nd protein hydrolysis 9

6 TABLE. Gllldder ile from nongllstone ptients Nucletion timc Ptient SI onc. No Pronse Added Pronse PA DE ME WO SM LO MO ES MU M Men * SE 1.oo g/dl dys N? Nucletion time of gllldder ile from ptients without cholesterol stones in the presence or sence of the proteolytic enzyme, pronse (Streptomyces griseus); SI, cholesterol sturtion index. 'Pigment stone. Previous work y Holzch nd collegues (6) suggested tht the delyed onset of nucletion oserved in norml gllldder ile ws produced y fctors present in the iliry protein frction. When ile proteins were digested with pronse nd susequently recomined with model ile, the oserved extension of nucletion times seen with intct iliry protein ws lost. We therefore nticipted, prticulrly in iles from ptients without cholesterol gllstones, tht pronse digestion would cce TABLE 5. ommon ile duct ile from cholesterol gllstone ptients Nucletion time Ptient SI onc. No Pronse Added Pronse BL R SP JO VH NI MO AV PR HE BR Men * SE g/dl dys Nucletion time of common ile duct ile from cholesterol gdlstone ptients in the presence or sence of the proteolytic enzyme, pronse (Streptomyces gsm); SI, cholesterol sturtion index. Pigment stone. lerte the nucletion time in these iles. In only one out of nine norml gllldder iles did pronse digestion cuse more rpid nucletion. onversely, in the norml gllldder smples we expected pronse digestion to extend nucletion time. A slight ut nonsignificnt decrese ws oserved. These results suggest either tht protein is not n importnt determinnt of nucletion time or tht the puttive fctors re resistnt to pronse digestion. Although the gret mjority of iliry protein ws hydrolyzed y pronse, silver stining did revel some intct protein nds (Fig. 1). Recent work would indicted tht the pronucleting fctor identified y Groen et l. (10) is indeed resistnt to proteolytic digestion. On the other hnd, the ntinucleting fctor(s) descried y Holzch et l. (6) were pronsesensitive. A role for iliry protein s ntinucleting gents would therefore pper negligile. The difference etween norml nd norml gllldder iles with respect to their nucleting times is, therefore, likely to e due to the inctivity or sence of pronucleting fctor in the former, rther thn the presence of n ntinucleting fctor. Although pronse digestion of iliry protein did not significntly chnge the nucletion time, we did oserve n pprent increse in oth the numer nd size of cholesterol crystls. Thus role for ntinucleting proteins in slowing down susequent crystl growth cnnot e excluded. In conclusion, our results would indicte tht, depending on the pretretment regime chosen to remove endogenous cholesterol crystls, the outcome of the nucletion ssy will vry mrkedly. No significnt chnge in lipid composition or mucus content could e shown to ccount for this phenomenon. We suggest tht only fter ultrcentrifugtion nd filtrtion cn one solutely exclude the possiility of residul cholesterol crystls eing responsile for the very rpid nucletion seen in these norml iles. The results indicte need to stndrdize the ssy procedure. Although nucletion time of the norml gllldder smples ws extended in the ultrcentrifuged/filtered iles, it ws still significntly different from tht of norml gllldder iles, confirming n intrinsic difference etween norml nd norml iles in cholesterol metstility. Proteolysis of iliry proteins hd little effect on nucletion time of either norml or norml gllldder iles. The puttive pro/ntinucleting fctors, if they re proteins, pper unffected y proteolysis or re of minor importnce. I The uthors wish to thnk the New Zelnd Medicl Reserch ouncil for finncil support, Mr. P. Bgshw nd Associte Professor J. Bxter for the provision of ile smples, nd Dr.. Frmpton for performing the sttisticl nlyses. Mnuscript received 1 Novemer 1989, in reuisedform June 1990, nd in rerevised form 10 Octoer Downloded from y guest, on August 26, 2018 ' 220 Journl of Lipid Reserch Volume 32, 1991

7 REFERENES 1. Holn, R. T., R. T. Holzch, R. E. Hermnn, A. M. oopermn, nd W. J. lffey Nucletion time: key fctor in the pthogenesis of cholesterol gllstone disese. Gstroenterology. : Gollish, S. H., M. J. Burnstein, R. G. Ilson,. N. Petrunk, nd S. M. Strserg Nucletion of cholesterol monohydrte crystls from heptic nd gllldder ile of ptients with cholesterol gllstones. Gut. 2: Kie, A., M. A. Dudley, Z. Hlpern, M. P. Lynn, A.. Breuer, nd R. T. Holzch Fctors ffecting cholesterol monohydrte crystl nucletion time in model systems of supersturted ile. J. Lipid Res. 26: Burnstein, M. J., R. G. Ilson,. N. Petrunk, R. D. Tylor, nd S. M. Strserg Evidence for potent nucleting fctor in the gllldder ile of ptients with cholesterol gllstones. Gstroenterology. 85: Gllinger, S., P. R.. Hrvey, nd S. M. Strserg Evidence for nucletion defect in ile from gllstone ptients. Hepto1o.v. : 1s19s. 6. Holzch, R. T., A. Kie, E. Thiel, J. H. Howell, M. Mrsh, nd R. E. Hermn Biliry proteins: unique inhiitors of cholesterol crystl nucletion in humn gllldder ile. J. lin. Invest. 3: 355. Gllinger, S., R. Tylor, P. R.. Hrvey,. N. Petrunk, nd S. M. Strserg, Effect of mucous glycoprotein on nucletion time of humn ile. Gstroenterology. 89: Groen, A. K., J. P. J. Stout, J. A. G. Drpers, F. J. Hoek, R. Grijm, nd G: N. J. Tytgt holesterol nucletioninfluencing ctivity in Ttue ile. Heptofogy. 8: Groen, A. K., *J. P. J. Stout,. Noordm, E J. Hoek, P. L. M. Jnsen, nd G. N. J. Tytgt Isoltion of potent cholesterol nucletionpromoting fctor from humn gllldder ile. Gstroenterology. 92: 13 (strct). 10. Groen, A. K., J. P. J. Stout,. Noordm, A. Korsen, F. J. Hoek, P. L. M. Jnsen, nd G. N. J. Tytgt Isoltion nd prtil chrcteriztion of cholesterol nucletionpromoting fctor from humn ile. Heptofogy. : 1023 (strct). 11. Drpers, J. A. G., A. K. Groen, J. P. J. Stout,. Noordm, E J. Hoek, P. L. M. Jnsen, nd G. N. J. Tytgt Quntifiction of cholesterol nucletionpromoting ctivity in humn gllldder ile. lin. him. Act. 165: Levy, P. F., B. E Smith, nd J. T. Lmont Humn gllldder mucin ccelertes nucletion of cholesterol in rtificil ile. Gstroenterology. 8: Peled, Y When does nucletion time relly occur? Gstroenterology. 95: Pttinson, N. R., nd B. A. hpmn Distriution of iliry cholesterol etween mixed micelles nd nonmicelles in reltion to fsting nd feeding in humns. Gstroenterology. 91: Whiting, M. J., nd J. McK Wtts Supersturted ile from oese ptients without gllstones supports cholesterol crystl growth ut not nucletion. Gstroenteml OD. 86: Hlpern, Z., M. A. Dudley, A. Kie, M. P. Lynn, A.. Brewer, nd R. T. Holzch Rpid vesicle formtion nd ggregtion in norml humn iles. A timelpse video enhnced contrst microscopy study. Gstroenterology. 90: Lemmli, U. K levge of structurl proteins during the ssemly of the hed of cteriophge T. Nture. 22: Pttinson, N. R., K. E. Willis, nd. M. Frmpton omprtive nlysis of cholesterol trnsport in ile from ptients with nd without cholesterol gllstones. J. Lipid &S. 32: Person, J. P., R. A. J. Kur, W. Tylor, nd A. Allen The composition nd polymeric structure of mucous glycoprotein from humn gllldder ile. Biochim. Biophys. Act. 06: Downloded from y guest, on August 26, 2018 Pttinson nd Willis Nucletion nd protein hydrolysis 2