Increased plasma bile alcohol glucuronides in patients with cerebrotendinous xanthomatosis: effect of chenodeoxycholic acid

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1 Inresed plsm bile lohol gluuronides in ptients ith erebrotendinous xnthomtosis: effet of henodeoxyholi id Ashok K. Btt,' Gerld Slen, Srh Shefer, G. Stephen Tint, nd Mnju Btt Deprtment of Mediine nd the Smmy Dvis J. Ntionl Liver Institute, University of Mediine nd Dentistry of Ne Jersey-Ne Jersey Medil Shool, Nerk, NJ 73; Veterns Administrtion Medil Center, Est Ornge, NJ 79; nd the Cbrini Medil Center, Ne York, NY 3 Ahtrt Lrge quntities of Cz7 bile lohols hydroxylted t C- 5 re exreted in the bile nd urine of ptients ith erebrotendinous xnthomtosis, lipid storge disese tht results from defetive bile id synthesis. The presene of both biliry nd urinry bile lohols reflets impired bile id synthesis. After tretment of smples ith &gluuronidse, plsm bile lohols ere quntitted by gs-liquid hromtogrphy-mss spetrometry. 58- Cholestne-3,7,l,5-tetrol (334 Fg/dl) s found to be the mjor bile lohol, folloed by 5/3-holestne-3,7,,3R,5- pentol(65 pg/dl), nd 5@-holestne-3,7,l,4(R nd S),5- pentols (6.5 Fg/d nd 64.5 Fgldl, respetively) in the plsm of these ptients. hen ompred to biliry nd urinry bile lohol exretions, the plsm pttern resembled bile here 5&holestne-3,7,,5-tetrol gluuronide predominted. In ontrst, urinry bile lohols ere omposed hiefly of 58-holestnepentol gluuronides ith only smll mounts of 58-holestne-3,7,,5-tetrol gluuronide. Tretment ith henodeoxyholi id, hih suppresses bnorml bile id synthesis in these ptients, redued plsm bile lohol onentrtions drmtilly. m These results sho tht lrge quntities of bile lohol gluuronides, prtiulrly 5/3-holestne-3,7,l,5- tetrolgluuronide, irulte in plsm of ptients ith erebrotendinous xnthomtosis. The plsm bile lohols losely reemble biliry bile lohols hih indites their hepti origin. The lrge quntities of polyhydroxylted bile lohols in the urine my suggest their formtion, t lest in prt, from 5&holestne-3,7Y,,5-tetrol by renl hydroxylting mehnisms. Tretment ith henodeoxyholi id inhibits hepti bile lohol synthesis nd redues plsm onentrtion of bile lohol gluuronides.- Btt, A. K., G. Slen, S. Shefer, G. S. Tint, ndm. Btt. Inresed plsm bile lohol gluuronides in ptients ith erebrotendinous xnthomtosis: effet of henodeoxyholi id.j. Lipid Rs : 6-. Supplementry key ords urinry bile lohols plsm bile lohols in fees, free bile lohols re exreted presumbly due to bteril hydrolysis (). The mjor biliry nd fel bile lohols exreted in CTX re 5~-holestne-3,7,,5- tetrol nd 5~-holestne-3,7,l,3~,5-pentols long ith substntil mounts of 5~-holestne-3,7,,4R,5- pentol (, 3). In ontrst, olthers et l. (4) nd Btt et l. (5) hve found tht the urine of these ptients ontins 5-holestnepentols s the mjor bile lohols ith only smll mounts of 5~-holestne-3,7,r,5-tetrol. The isoltion of lrge mounts of bile lohol gluuronides from the urine suggests plsm pool of these ompounds in CTX. The purpose of this investigtion s to mesure plsm bile lohol omposition nd ompre the plsm pttern ith tht in urine nd bile. Also, the effet of henodeoxyholi id (CDCA), hih suppresses bnorml hepti bile id synthesis in CTX, s determined. Mterils EXPERIMENTAL PROCEDURES Sep-Pk C rtridges ere purhsed from ters Assoites (Milford, MA). The etone poder of holylglyine hydrolse (from C. perfringens (elhii)) (from Hek pomtzh) ere from Sigm Chemil Co. (St. Louis, MO). Chenodeoxyholi id s obtined from G. Phrmix, Miln, Itly. The bile lohols, 5~-holestne-3r,7,l,5-tetrol nd 5~-holestne-3,7,l,4(R nd S)5-pentols ere syn- Donloded from.jlr.org by guest, on June 4, 8 Cerebrotendinous xnthomtosis (CTX), fmilil sterol storge disese, results from blok in bile id synthesis () nd is hrterized by the umultion of holesterol nd holestnol in most tissues (). Lrge mounts of bile lohols hydroxylted t C-5 hve been deteted s gluuronides in bile nd urine of CTX ptients (3,4) hile, Abbrevitions: CTX, erebrotendinous xnthomtosis; CDCA, henodeoxyholi id; TLC, thin-lyer hromtogrphy; GLC, gs-liquid hromtogrphy; TMSi, trimethylsilyl. The folloing trivil nmes ere used: henodeoxyholi id, 3,7-dihydroxy-5fl-holnoi id; holesterol, 5-holesten-38-; holestnol, 5u-holestn-3&ol. 'To hom reprint requests should be ddressed t: Deprtment of Mediine, UMDNJ-Ne Jersey Medil Shool, Nerk, NJ Journl of Lipid Reserh Volume 8, 987

2 ~ thesized from holi id ording to Dyl et l. (6). 5fl- Cholestne-3,7,l,3R,5 pentol s isolted from the bile of CTX ptient s desribed by Btt et l. (5). The gluuronide of Sfl-holestne-Jr,7,,5-tetrol s prepred ording to Hoshit et l. (7). Ptients Nine untreted CTX ptients (five mle nd four femle, ged beteen 8 nd 6 yers) ere exmined. All exhibited tendon xnthoms, dementi, nd trts. Elevted plsm holestnol levels ere present in ll ses. Smples Bile nd plsm ere olleted from fsting CTX subjets before nd fter feeding g/dy of henodeoxyholi id for month nd stored t - OoC until used. Tenty four-hour urine olletions from these ptients ere obtined before nd fter bile id therpy nd stored t -OOC until nlyzed. Thin-lyer hromtogrphy The TLC of the urinry, biliry, nd plsm bile lohol gluuronides s performed on sili gel G pltes in solvent system of hloroform-methnol-eti id-ter 6::4: (v/v/v/v) (7). The spots ere visulized by sprying the plte ith % HS, folloed by solution of 3.5% phosphomolybdi id in isopropnol nd then heting the plte t llo C for min. Alterntively, the plte s spryed ith.% solution of nphthoresorinol in ethnol-phosphori id 9:l (dv) folloed by heting t llo C for 5 min. The gluuronides gve blue spots. Syntheti 5~-holestne-3,7,,5-tetrol-3-gluuronide s used for TLC omprison. The identifition of the vrious bile lohol gluuronides s mde fter their extrtion from the respetive bnds on the TLC plte ith methnol, hydrolysis ith 8-gluuronidse, nd gs-liquid hromtogrphy of the liberted unonjugted bile lohols s their trimethylsilyl ether derivtives. These methodologies re desribed belo. Gs-liquid hromtogrphy The vrious bile lohols in the plsm, bile, nd urine ere silylted ith pl of Sil-Prep (Applied Siene Lbs), for 3 min t 55OC. After evportion of solvents under NP, the silyl ethers ere dissolved in pl of hexne nd -5 pl s injeted into Helett Pkrd 588A gs hromtogrph equipped ith split/splitless devie for pillry olumns. A fused sili CP-Sil-5 pillry olumn (5 m), i.d..-. mm s employed nd helium s used s the rrier gs ith flo rte of.9 ml/min. The GLC operting onditions ere s follos: injetor nd detetor tempertures ere set t 6OOC nd 9OoC, respetively. Folloing injetion, the oven temperture s kept t C for min, then progrmmed t rte of 5OC/min to temperture of 65OC, nd subse- quently t rte of.5oc/min to finl temperture of 8 C (4). The retention times of some of the stndrd bile lohols reltive to tht of 5-holestne (internl stndrd) re given in Tble. The retention times of the bile lohols ere reproduible nd, for mounts of bile lohols vrying from ng to 4 ng injeted onto the olumn, the detetor response s shon by the integrtor s liner (the retention time of the bile lohol slightly inresed hen reltively lrge mount of the TMSi ether of the bile lohol s injeted). The response ftors of the vrious bile lohols (5@-holestnetetrol nd 58-holestnepentols) ere found to be similr nd the orretion ftor orresponding to the pek re of 5-holestne s found to be of the order of.5 for 5/3-holestne-3,7,l,5- tetrol s ell s the 58-holestnepentols. This orretion ftor s used for the quntittion of the bile lohols in the plsm, bile, nd urine of the ptients. Mss spetr The mss spetr ere obtined from Vrin MAT-, gs hromtogrph-mss spetrometer (Vrin Assoites, Plo Alto, CA) s desribed previously (8). Isoltion of plsm bile lohols Plsm ( ml) s sloly pssed through preshed Sep-Pk C 8 rtridge nd the bile ids nd bile lohols ere eluted from the Sep-Pk ith methnol s desribed before (5). Methnol s evported, the residue s dissolved in ml of. M sodium ette buffer, ph 5., nd.5 mg of 8-gluuronidse s dded nd the produts ere inubted t 37OC for 8 hr. After nother filtrtion through Sep-Pk, the bile ids nd bile lohols ere eluted ith methnol. Methnol s evported nd the residue s dissolved in ethyl ette (5 ml). The ethyl ette solution s shed ith % sodium hydroxide solution ( ml x 3) folloed by ter shing ( ml x 3). The TABLE. 5&Cholestne- GLC retention times of the trimethylsilyl ethers of bile lohols" GLC (RR?) 3~,7~~,5-triol.54 3,7, J5-tetrol.56 3,7,lZ,6-tetrol (5R + 5s).6 3~,7,,3R,5-pentol.78 3,7,,4S,5-pentol.83 3, 7,,4R,5-pentol.84 "Operting onditions for GLC ere s follos: olumn, fused sili CP-S-5 pillry olumn (5 m), i.d.,.-. mm; rrier gs, He, t flo rte of.9 mllmin; injetor temperture, 6 C; detetor temperture, 9 C; oven temperture, lc for min, then progrmmed t rte of 5"C/min to temperture of 65'C, nd subsequently t rte of.5"c/min to hl temperture of 8 C. bretention time reltive to tht of 5-holestne (retention time,. min). RRT of holesterol under these onditions is.3. Donloded from.jlr.org by guest, on June 4, 8 Bth d l. Inresed plsm bile lohols in CTX 7

3 residue obtined fter evportion of solvent s silylted, tken up in pl of hexne, nd -5 pl s injeted onto GLC olumn. by ter shing ( x ml). The orgni lyer s evported nd the produts ere silylted nd used for GLC. Isoltion of biliry bile lohols Bile (. ml) s diluted ith ml of. M sodium ette buffer, ph 5., nd inubted ith.5 mg of - gluuronidse t 37OC for 8 hr. The bile lohols ere then isolted s desribed bove for the isoltion of plsm bile lohols. The finl residue s similrly silylted nd used for GLC nlysis. Isoltion of urinry bile lohols To milliliters of entrifuged urine s sloly filtered through Sep-Pk nd the methnol solution of the bile ids nd bile lohols obtined s subjeted to hydrolysis s desribed bove. The bile lohols ere isolted nd used for GLC nlysis s desribed for the plsm bile lohols. In order to determine the reovery of the bile lohols nd their gluuronides during the Sep-Pk nd P-gluu- ronidse tretment, SP-holestne-3,7,,5-tetrol-3- gluuronide (.5 mg) s dded to ml of plsm from helthy ontrol before pssing through Sep-Pk. -Gluuronidse tretment of the produts folloed by nother Sep-Pk filtrtion resulted in the reovery of 86% of the gluuronide s free 5P-holestne-3,7,l,5-tetrol. Isoltion of bile lohol gluuronides In order to isolte the bile lohol gluuronides, the plsm ( ml), bile (.4 ml), or urine ( ml) s filtered through Sep-Pk nd the produts ere eluted ith methnol. The residue fter evportion of methnol s either used s suh for TLC (bile lohol gluuronides plus bile id onjugtes) or s first subjeted to holylglyine hydrolse s follos. The residue s dissolved in ml of. M sodium ette buffer, ph 5.6, nd inubted ith.5 mg of holylglyine hydrolse for 8 hr t 37%. The produts ere gin pssed through Sep-Pk nd the bile id plus bile lohol frtion s eluted ith methnol. The residue fter evportion of methnol ontined bile lohol gluuronides plus unonjugted bile ids nd s used for TLC exmintion. In order to determine hether unonjugted bile lohols ere present in the plsm, ml of the plsm s pssed through Sep-Pk nd the produts ere pplied s bnd on TLC plte nd the plte s developed in hloroform -etone-methnol 7:5:7 (9). Stndrds of 5-holestne-3,7,lZ,5-tetrol nd 5P-holestne-3,7,l,4(R nd S) 5-pentols ere pplied on the sides of the plte. Sili gel s srped from the plte orresponding to the res here the unonjugted bile lohols ppered nd the produts ere eluted ith methnol. The residue, fter evportion of methnol, s dissolved in ethyl ette (5 ml) nd shed ith % NOH ( ml) folloed RESULTS The plsm bile lohols in the CTX ptients ere isolted by the sme method used for the isoltion of the urinry bile lohols in these ptients (5). Thus, the plsm from the CTX ptients s pssed through Sep-Pk. After shing the Sep-Pk ith ter, bile ids nd bile lohols ere eluted ith methnol. In this y, proteins nd over 9% of the holesterol nd holestnol ere removed. The bile lohol gluuronides in the methnol extrt ere hydrolyzed ith P-gluuronidse, nd fter seond Sep- Pk tretment, the free bile lohols ere seprted from the bile id onjugtes by extrtion of the ltter ith sodium hydroxide. The bde lohol frtion s then silylted nd subjeted to gs hromtogrphy. Fig. la, shos the hromtogrm of the TMSi ethers of the bile lohols in the plsm of n untreted CTX ptient (A) nd in Tble re shon the mounts (pgldl) of the bile lohols present in the plsm of nine untreted ptients. As seen from the figure, severl peks ere observed in the region of tetr- nd penthydroxy bile lohols. Hoever, the mjor pek orresponded ith the retention time due to the TMSi ether of 5P-holestne- 3,7,,5-tetrol (pek ). In ddition, smll mounts of 5~-holestne-3,7,l,3R,5-pentol (pek b), 5- holestne-3,7,,4s,5-pentol (pek ), nd 5p-holestne-3,7,l,4R,5-pentol (pek d) ere lso present. The mjor frgments in the mss spetr of the TMSi ether derivtives of these ompounds re given in Tble 3. The frgmenttion pttern s ompletely onsistent ith the strutures of these ompounds. The detiled interprettion of the mss spetr of these ompounds hs been previously desribed by us (8). As seen from Tble, the plsm onentrtion of 5- holestne-3,7,l,5-tetrol s pproximtely tie tht of the totl of the three 5P-holestnepentols (334 * 74 pgldl S@-holestnetetrol vs. 9.8 i 48 pg/dl 5P- holestnepentols). 5P-Cholestne-3,7,l,5-tetrol s lys the predominnt plsm bile lohol nd lthough the rtio of 5B-holestnetetrol to the 5P-holestnepentols vried from ptient to ptient, it s lys found to be greter thn. Bile lohols ere not deteted in the plsm from three ontrol subjets hen ml of the plsm s nlyzed in n identil y. Figs. B nd C sho the GLC pttern of the TMSi ethers of bile lohols present in the bile nd urine, respe- tively, of ptient A. 5/3-Cholestne-3,7,l,5-tetrol s gin the mjor biliry bile lohol ompnied ith smller mounts of 5@-holestne-3,7,l,3R,5-pentol nd 5@-holestne-3u,7,,4R,5-pentol. The proportions of the vrious bile lohols ere similr in the hro- Donloded from.jlr.org by guest, on June 4, 8 8 Journl of Lipid Reserh Volume 8, 987

4 L P v) e V P I -#I Ib I5 5 TIME( MINUTES) L V n I I L TIME ( MINUTES) b b B A C mtogrms of the biliry nd the plsm bile lohols. In ontrst, 58-holestnepentols ere the predominnt urinry bile lohols nd 5~-holestne-3,7r,,5-tetrol s only minor onstituent. The urine lso ontined severl more polr bile lohols (probbly 58-holestnehexols nd 58-holestneheptols) (4). Thus, the plsm mirrored the biliry bile lohol pttern rther thn the urinry bile lohol pttern in tht 5/3-holestne-3,7,l,5- tetrol s the mjor bile lohol in both plsm nd bile hile this bile lohol s present only in smll quntities in the urine. Also, the rtios of 5~-holestnepentols:5flholestne-3,7,r,5-tetrol in the plsm nd bile ere similr hile the urine ontins muh lrger proportion of the penthydroxy bile lohols. The bile lohols isolted from the plsm ere present s gluuronides ith no ppreible mounts of unonjugted bile lohols, s s demonstrted by diret TLC exmintion of plsm smple fter onentrtion of the bile lohols nd bile ids by Sep-Pk nd hydrolysis of bile id onjugtes ith holylglyine hydrolse. A omprison ith the biliry bile lohol gluuronide frtion similrly obtined shoed n identil pttern beteen the plsm nd the bile in tht the 58-holestnetetrol gluuronide s the mjor ompound nd the 58-holestnepentol gluuronides ere omprtively less. On the other hnd, the TLC of the urinry bile lohol gluuronides shoed preponderne of 58-holestnepentol gluuronides ith only smll proportion of the gluuronide of 5~-holestne-3,7,l,5-tetrol. The effet of henodeoxyholi id feeding on the plsm bile lohol levels in six CTX subjets is lso shon in Tble. A omprtive gs hromtogrm of the bile lohols in the plsm, bile nd urine of CTX ptient A fter month of gldy of henodeoxyholi id feeding is shon in Figs. ZA, ZB, nd ZC. After month, plsm bile lohol gluuronide onentrtions delined mrkedly (Tble ). Similrly, urinry nd biliry exretion of bile lohols lso diminished (Figs. A-C). Donloded from.jlr.org by guest, on June 4, 8 n I I O I b 5 5 DISCUSSION The results of the present investigtion sho tht substntil onentrtions of bile lohol gluuronides irulte in the plsm of ptients ith CTX. The lrge quntities of bile lohol gluuronides tht result from defet in the bile id synthesis re not only exreted in the bile but lso overflo into the plsm nd re then exreted into the urine. Of importne s the demonstrtion tht the urinry bile lohol pttern differed mrkedly from the plsm nd biliry bile lohol pttern. Although the preise bile id syntheti defet in CTX remins ontroversil, it is greed tht holi id biosynthesis in ptients ith this disese ours vi 5-hydroxylted intermedites (). As result, lrge mounts of 5~-holestne-3,7r,,5-tetrol Bdt et. Inresed plm bile lohols in CTX 9

5 ~~~~ ~ ~~ ~~ TABLE. Bile lohols in the plsm of CTX ptients before nd during CDCA feeding" Bile Alohol: 5@-Cholestne-3,7, -* Ptient 5-Tetml 3,5-Pentol 4R,5-Pentol 4S,5-Pentol A 35 (6)' 7 () 7 (9) 6 (6) B 8 (8) (-)d 8 (-) 5 (-) C 6 (89) 65 () 6 (- 56 (-) D 35 () 4 () 47 (-) 47 (- E () 5 (5) 8 (- 44 (- F 3 () 5 (5) (-) (-) G H I Men 334 (36) i 76 ( i 3) 65 (9) f 8 ( i 9) 63 (5) i 66 (i ) 65 (3) i 48 ( f 7) P <.' P <. P <.5 P <. "Fsting plsm smples ere obtined before nd fter month of g/dy CDCA tretment. Plsm s nlyzed by pdhry GLC fter hydrolysis ith &gluuronidse. Vlues re lulted s bg/d plsm. For GLC operting onditions see Tble, footnote (). *Smll mounts of 5/3-holestne-3,7,l,3-tetrol (5 + pg/dl) ere lso found in the plsm of ll ptients. 'Vlues shon in prentheses re those obtined fter feeding CDCA. dnot deteted. 'Signifine levels lulted for first six subjets only (pired Student's I-test). re produed fmm 5~-holestne-3,7r,-triol. Aording overfloing into the plsm here it rehes the kidney. to Slen et l. (ll), defet in hepti mirosoml 4S- In previous studies, it s demonstrted tht beteen 3 hydroxyltion bloks the trnsformtion of 5-holestne- nd mgldy of bile lohol gluuronides is exreted 3,7,,5-tetrol into 5/3-holestne-3,7,l,4S,5- in the urine nd only bout mgldy of the bile lohols pentol nd holi id. As result, the 5P-holestnetetrol is exreted in the fees (, 4, 5). Thus, the urine represents umultes in the liver here it undergoes gluuronid- the mjor pthy for the elimintion of the bile lohol tion. A portion of the nely formed 5/3-holestnetetrol gluuronides. Hoever, the mjor bile lohol gluuronides gluuronide is sereted into the bile ith the reminder exreted in the urine re 56-holestnepentols nd probbly m - e TABLE 3. Perent reltive intensity for mjor frgments in the mss spetr of the trimethylsilyl ethers of bile lohols Frgmenttion 74 M'.5 68 M' M' M* M'- (9 + 3) 577 M' (9 + 45) 544 M' - ( x 9) 5 M'- ( x 9 + 3) 487 M+- ( x ) 454 M*- (3 x 9) 4 4 M' - (3 x 9 + 3) 397 M'- (3 x ) 5~-Cholestne-3,7r, - 5-Tetrol" 3E,5-Pentolb 4S,5-PentolC 4R,5-Pentold % reltive intensiq M' - (4 x 9) M' - ( x 9 + side hin) 3 M'- (4 x 9 + 3) M'- (4 x ) M' - (3 x 9 + side 'hin) 48 3 (CH3)3 Si--C' (CH,)? Donloded from.jlr.org by guest, on June 4, 8 "GLC pek ''. *GLC pek 'b'. 'GLC pek ''. dglc pek 'd'. Journl of Lipid Reserh Volume 8, 987

6 UJ z UJ z Q UJ Q v) z. UI I- O ID ZU TIME ( MINUTES) TIME( MINUTES) TIME( MINUTES) Pig.. GLC of the bile lohols in the plsm, bile nd urine of CTX ptient A fter feeding g/dy of henodeoxyholi id for month. A, Plnm, B, bile; nd C, urine. For GLC operting onditions, see Tble. For identifition of the GLC peks, see legend to Fig.. 5-holestnehexols nd -heptols (4, 5). Thus, the urinry bile lohol pttern differs qulittively from tht of the plsm nd bile here 5~-holestne-3,7,l,5-tetrol gluuronide is the mjor omponent. To possible explntions exist. Sine smll mounts of 5-holestnepentols re present in the bile nd plsm, the urine my seletively serete these penthydroxy (nd more polr) bile lohol gluuronides. Alterntively, 58-holestnetetrol gluuronide, hih is produed in the liver nd irultes in the plsm, my be hydroxylted by renl hydroxylses nd exreted in prt in the urine, hile portion my be rebsorbed from the kidney nd irulte in the plsm to be exreted by the liver. The importnt point is tht substntil mount of 5~-holestne-3,7,l,5-tetrol gluuronide tht is produed in the liver is trnsported in the plsm but is not found in the urine. Existene of bile lohol gluuronides in the plsm hs importnt dignosti nd pthogeni implitions. In some ptients ith CTX, plsm holestnol levels re only modestly elevted. The demonstrtion of bile lohol gluuronides in the plsm ill led to more definite dignosis of CTX. Furthermore, in ertin ses of liver disese, plsm holestnol onentrtions tend to be reltively high (). The simultneous determintion of plsm bile lohols helps estblish the orret dignosis. The sme plsm smple n be used for the determintion of the bile lohol gluuronides tht s used for sterol determintion, fter extrtion of the sterol frtion ith hexne. Tretment ith henodeoxyholi id redues plsm holestnol levels to ner norml in the CTX ptients (3) nd virtully elimintes both biliry nd urinry bile lohols (4, 5). A similr effet on plsm bile lohol gluuronide levels s seen fter feeding henodeoxyholi id. As shon in Tble, plsm bile lohol gluuronide onentrtions ere mrkedly suppressed in just month of henodeoxyholi id therpy (56 pg/d plsm to 5 pg/dl plsm, determined s the free bile lohols). Figs. A-C sho gs hromtogrms of the plsm, biliry, nd urinry bile lohols in CTX ptient A fter month of henodeoxyholi id therpy. A omprison of Figs. nd lerly shos the mrked redution in the plsm nd biliry s ell s urinry bile lohol gluuronides in the ptient fter CDCA tretment. Thus, determintion of plsm bile lohol gluuronides gives n dditionl prmeter for the evlution of the effet of henodeoxyholi id therpy. The presene of bile lohols in the plsm my lso hve pthologil signifine. Normlly the blood-brin brrier is highly impermeble to plsm lipoproteins (4, 5). It is possible tht the lrge plsm bile lohol gluuronides pool plys role in the bnorml blood-brin brrier permebility in these ptients, sine e hve reently found inresed mounts of lbumin nd polipoprotein B in the erebrospinl fluid of eight untreted CTX ptients (Btt, A. K., et l., unpublished observtions). This my led to the inresed trnsport of holestnol nd holesterol in the brin, sitution tht my be orreted by feeding henodeoxyholi id sine the plsm levels of bile lohol gluuronides re gretly redued ith this tretment. In summry, e hve demonstrted the presene of bile lohol gluuronides in the plsm of CTX ptients. The mjor plsm bile lohol gluuronide is 58-holestne- Donloded from.jlr.org by guest, on June 4, 8 Bth et l. Inresed plsm bile lohols in CTX

7 3,7,,5-tetrol nd the pttern is very different from tht in the urine here 5-holestnepentols predominte, but losely resembles the biliry pttern. e hypothesize 6. tht renl hydroxyltion of 5/3-holestne-3~~,7~~,,5- tetrol to the 5P-holestnepentols, -hexols nd -heptols is responsible for this differene. The presene of plsm bile lohol gluuronides my be useful not only in estblishing the dignosis of CTX but my lso ply role in ffeting blood-brin brrier permebility. Tretment ith henodeoxyholi id, hih suppresses bnorml bile id formtion in CTX, redues plsm bile lohol gluuronide onentrtions. I This ork s supported by U.S. Publi Helth Servie Grnts No. AM-877, HL-788, AM-6756, nd the Hermn Goldmn Foundtion. Mnusnipt reeived Deember 986 nd in revisedform Februry 987. REFERENCES. Setoguhi, T., G. Slen, G. S. Tint, nd E. H. Mosbh A biohemil bnormlity in erebrotendinous xnthomtosis ssoited ith inomplete degrdtion of the holesterol side hin. J Clin. Invest. 53: Slen, G. 97. Cholestnol deposition in erebrotendinous xnthomtosis: possible mehnism. Ann. Intern. Med. 75: Shefer, S., B. Dyl, G. S. Tint, G. Slen, nd E. H. Mosbh Identifition of penthydroxy bile lohols in erebrotendinous xnthomtosis: hrteriztion of 5~-holestne-3,7,l,4~,5-pentol nd 58-holestne- 3,7,,3,5-pentol. J Lipid Res. 6: olthers, B. G., M. Volmer, J. vn der Molen, B. J. Koopmn, A. E. J. de Jger, nd R. J. ttereus Dignosis of erebrotendinous xnthomtosis (CTX) nd effet of henodeoxyholi id therpy by nlysis of urine using pillry gs hromtogrphy. Clin. Chim. At. 3: Btt, A. K., S. Shefer, M. Btt, nd G. Slen Effet of henodeoxyholi id on biliry nd urinry bile ids nd bile lohols in erebrotendinous xnthomtosis; monitoring by high performne liquid hromtogrphy. J. Lipid Res. 6: Dyl, B., S. Shefer, G. S. Tint, G. Slen, nd E. H. Mosbh Synthesis of 5/3-holestne-3,7,l,5- tetrol nd 5/3-holestne-3,7,l,4~,5-pentol. J Lipid Res. 7: Hoshit, T., M. Ysuhr, M. Une, A. Kibe, E. Itog, S. Kito, nd T. Kurmoto. 98. Ourrene of bile lohol gluuronides in bile of ptients ith erebrotendinous xnthomtosis. J Lipid Res. : 5-. Tint, G. S., B. Dyl, A. K. Btt, S. Shefer, F.. Cheng, G. Slen, nd E. H. Mosbh Gs-liquid hromtogrphy-mss spetrometry of trimethylsilyl ethers of bile lohols. J. Lipid Res. 9: Btt, A. K., G. Slen, nd S. Shefer. 98. Thin-lyer hromtogrphy of the bile lohols, bile ids nd onjugted bile ids. J. Liq. Chromtogr 3: Slen, G., S. Shefer, nd V. M. Berginer Fmilil diseses ith storge of sterols other thn holesterol: erebrotendinous xnthomtosis nd sitosterolemi ith xnthomtosis. In The Metboli Bsis of Inherited Disese. J. B. Stnbury, J. B. yngrden, D. S. Fredrikson, J. L. Goldstein, nd M. S. Bron, editors. MGr-Hill Book Co., Ne York Slen, G., S. Shefer, F.. Cheng, B. Dyl, A. K. Btt, nd G. S. Tint Choli id biosynthesis. The enzymti defet in erebrotendinous xnthomtosis. J Clin. Invest. 63: Koopmn, B. J., J. C. Vn der Molen, B. G. olthers, A. E. J. de Jger, R. J. terreus, nd C. H. Gip Cpillry gs-hromtogrphi determintion of holestnol/ holesterol rtio in biologil fluids. Its potentil usefulness for the follo up of some liver diseses nd its lk of speifiity in dignosing CTX (erebrotendinous xnthomtosis). Clin. Chim. At. 37: Slen, G., T.. Meriether, nd G. Niolu Chenodeoxyholi id inhibits inresed holesterol nd holestnol synthesis in ptients ith erebrotendinous xnthomtosis. Biohem. Med. 4: Brdbury, M.. B The struture nd funtion of the blood-brin brrier. Federtion Pro. 43: Roheim, P. S., M. Crey, T. Forte, nd G. L. Veg Apolipoproteins in humn erebrospinl fluid. Pro. Ntl. Ad. Si. USA. 76: Donloded from.jlr.org by guest, on June 4, 8 Journl of Lipid Reserh Volume 8, 987