Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption

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1 Inheritnce of cholesterol metolism of pronds with high or low cholesterol sorption Helen Gylling* nd Ttu A. Miettinen 1, Deprtment of Clinicl Nutrition,* University of Kuopio, nd Kuopio University Hospitl, Kuopio, Finlnd; nd Division of Internl Medicine, Deprtment of Medicine, University of Helsinki, Helsinki, Finlnd Astrct Heredity of cholesterol sorption nd synthesis ws studied in silings of hypercholesterolemic pronds with low nd high serum cholestnol to cholesterol rtio (ssumed to indicte low nd high sorption of cholesterol, respectively). Cholesterol synthesis ws ssyed with sterol lnce technique nd mesuring serum cholesterol precursor to cholesterol rtios (synthesis mrkers of cholesterol), nd cholesterol sorption with mesuring dietry cholesterol sorption percentge nd serum plnt sterol nd cholestnol to cholesterol rtios (sorption mrkers of cholesterol). In the silings of the low sorption fmilies, cholesterol sorption percentge nd rtios of sorption mrkers were significntly lower, nd cholesterol nd ile cid synthesis, cholesterol turnover, fecl steroids nd rtios of synthesis mrkers significntly higher thn in the silings of the high sorption fmilies. The rtios of sorption nd synthesis mrkers were inversely interrelted, nd they were correlted with cholesterol sorption nd synthesis in the silings. In ddition, low sorption ws ssocited with high ody mss index, low HDL cholesterol, nd serum sex hormone inding gloulin levels, suggesting tht low sorption ws ssocited with metolic syndrome. Intrfmily correltions were significnt for serum synthesis mrkers, cholestnol, triglycerides, nd lood glucose level. In conclusion, cholesterol sorption efficiency nd synthesis re prtly inherited phenomen, nd they cn e predicted y the rtios of non-cholesterol sterols to cholesterol in serum. Gylling, H., nd T. A. Miettinen. Inheritnce of cholesterol metolism of pronds with high or low cholesterol sorption. J. Lipid Res : Supplementry key words cholesterol synthesis non-cholesterol sterols lthosterol cmpesterol sitosterol cholestnol coronry rtery disese Owing to smll sorption of dietry plnt sterols, serum contins low concentrtions of these sterols, minly cmpesterol nd sitosterol. In ddition, serum lso contins low concentrtion of cholestnol, 5 derivtive of cholesterol, which is prtly synthesized in the ody, ut is lso present in diet in very smll mounts. These three sterols re trnsported y serum lipoproteins, minly y LDL like cholesterol. The rtio of the three sterols to cholesterol correltes positively to sorption efficiency nd negtively to synthesis of cholesterol (1, 2). Accordingly, their incresed serum rtio points to high sorption percentge of cholesterol nd low cholesterol synthesis. Furthermore, low cholesterol synthesis decreses the relese of cholesterol precursor sterols, most likely minly from the liver to circultion, such tht the rtios of squlene, methyl sterols, cholestenol, desmosterol, nd lthosterol to cholesterol re lso reduced (1). In generl, the rtios of the sorption mrkers, nd synthesis mrkers of cholesterol re negtively relted to ech other (3). Accordingly, selection of sujects from popultion with high rtios of the sorption mrkers predicts high sorption nd low synthesis of cholesterol in these cses (1). On the other hnd, the higher the rtios of the synthesis mrkers, e.g., lthosterol, the higher is cholesterol synthesis. In ddition, seline synthesis rte of cholesterol predicts to some extent lowering of cholesterol to sttins, inhiitors of cholesterol synthesis. For instnce, in the highest cholestnol qurtile of the Finnish sugroup to the Scndinvin Simvsttin Survivl Study (4S), serum cholesterol lowering ws slightly ut significntly lower thn in the lowest qurtile with high cholesterol synthesis (3). From mong the non-cholesterol sterols, methyl sterol rtios to cholesterol exhiited significnt intrpir correltions in serum of monozygotic ut not dizygotic twins (4). However, concentrtions of demethylted precursor sterols in plsm of lrge numer of fmilies nd twins showed ssocitions, e.g., with poe polymorphism (5), ut no fmilil studies hve een performed. Our routine non-cholesterol sterol nlyses hve reveled severl fmilies with high serum plnt sterol rtios ssocited with low rtios of synthesis mrkers, suggesting heredity of vriles of cholesterol metolism. To this end, this study ws plnned to show the possile ssocition of cho- Mnuscript received 9 April 2002 nd in revised form 21 My DOI /jlr.M JLR200 1 To whom correspondence should e ddressed. e-mil: ttu..miettinen@helsinki.fi Copyright 2002 y Lipid Reserch, Inc Journl of Lipid Reserch Volume 43, 2002 This rticle is ville online t

2 lesterol sorption nd synthesis in silings of pronds with high versus low rtios of serum sorption mrker sterols for cholesterol, suggesting high versus low cholesterol sorption. For this pupose, the first-degree reltives of those pronds were studied for serum precursor sterols, cholesterol sorption percentge, nd cholesterol synthesis with the sterol lnce technique. MATERIALS AND METHODS Prond popultion From lrge numer (n 330) of coronry sujects with seline mesurement of serum non-cholesterol sterols, six nd seven sujects with respective lowest nd highest rtios of serum cholestnol to cholesterol were selected s pronds. Definition for low nd high sorption fmilies ws performed y the serum cholestnol to cholesterol rtios, such tht the respective rtios were 98 5 (men SE) nd mmol/mol of cholesterol with no overlpping etween the groups. Siling popultion Pronds sisters nd rothers were questioned y mil out their willingness to prticipte in the study. Of the 75 silings 37, 14 from the low nd 23 from the high sorption fmilies, responded positively nd were sent n invittion letter to prticipte in the study. None of them took lipid-lowering drugs or other products known to ffect specificlly cholesterol metolism. A lood smple ws drwn fter n overnight fst, nd creful clinicl exmintion ws performed, nd personl nd fmily histories were recorded. The prticipnts were dvised to keep their norml hitul diet nd possile drugs unchnged. They were sked to keep food diry for the next week, nd consume cpsule (contined chromic oxide, C 14 -leled cholesterol nd tritited sitostnol) three times dy with ech mjor mel. During the lst three dys of the week, the prticipnts were sked to provide stool smples to test tues nd ring them to lortory, nd give nother lood smple. Owing to vrying ge nd willingness, cholesterol sorption could e performed only in ten prticipnts.the prond group hd een studied erlier, nd most of them hd een given sttin tretment y doctors t the Outptient Deprtment fter the seline lood smples, such tht sorption nd fecl studies could not e performed. sujects volunteered to the study, the protocol of which ws ccepted y the Ethics Committee of the hospitl. Lortory methods Tle 1 shows the demogrphic dt of the pronds with low nd high cholesterol sorption nd of their respective silings. Dietry diries were clculted for different vriles using recent ntionl method for their computerized nlysis (6). Serum lipid nlysis for totl, HDL cholesterol (HDL-C) nd LDL-C, totl triglycerides, nd phospholipids were mde with the routine commercil kits (Boehringer Mnnheim, Germny; Roche, Switzerlnd; Wko Chemicls, Germny). Ultrcentrifugtion ws performed in the siling s smples ccording to elier methods (7) seprting VLDL, IDL, LDL, nd HDL followed y mesurement of totl, free, nd esterified cholesterol, phospholipids, nd triglycerides with commercil kits. In ddition, lood glucose (hexokinse method; Roche, Switzerlnd), fsting serum insulin (rdioimmunossy; Phrmci & Upjohn, Sweden), nd serum sex hormone inding gloulin (SHBG), n indictor of insulin resistnce (8) (fluoroimmunossy, Wllc, Finlnd), were quntified with routine commercil kits. Plsm totl homocysteine level ws ssyed with high-pressure liquid chromtogrphy. Serum cholesterol, squlene, nd non-cholesterol sterols mere mesured y gs-liquid chromtogrphy (GLC) on 50 m long cpillry column (9) from non-sponifile mteril in serum. Ech GLC run seprtes cholesterol, squlene, cholestenol, desmosterol, lthosterol (three sterols reflecting cholesterol synthesis), cholestnol, cmpesterol, sitosterol, nd vensterols (four sterols reflecting cholesterol sorption). The squlene nd non-cholesterol sterol levels were stndrdized y cholesterol of the sme GLC run, ecuse this procedure elimintes differences cused y vrile lipoprotein contents trnsporting cholesterol nd non-cholesterol sterols. Thus, the vlues re expressed in terms of 10 2 mmol/ mol of cholesterol nd expressed in the text s rtio. Cholesterol sorption percentge nd fecl sterols of oth cholesterol nd plnt sterol origin, nd ile cids were mesured from pooled smple of the three dily stool smples y erlier methods (10 13). Men dily intestinl cholesterol pool ws clculted dividing fecl neutrl sterols y (1-frctionl sorption of cholesterol). Biliry cholesterol secretion ws the difference etween the intestinl pool nd dily dietry cholesterol. Frctionl sorption multiplied y the intestinl pool, iliry secretion, or dietry cholesterol reveled the sorption of totl, iliry, nd dietry cholesterol, respectively. Cholesterol synthesis ws clculted y the sterol lnce technique sutrcting dietry cholesterol intke from the fecl sum of neutrl sterols of cholesterol origin plus ile cids. Cholesterol turnover ws cholesterol synthesis plus sored dietry cholesterol. vlues re expressed in terms of mg/dy. TABLE 1. Demogrphic dt, vriles of glucose metolism, nd plsm totl homocysteine level of study popultion Pronds Silings Vriles Numer of ptients Age, yers Weight, kg BMI, kg/m Blood glucose, mmol/l Serum insulin, pmol/l Serum SHBG, nmol/l Plsm totl homocysteine, mol/l Men SE. BMI, ody mss index; SHBG, sex hormone inding gloulin. Significntly different from pronds. Significntly different from low sorers. Gylling nd Miettinen Inheritnce of cholesterol metolism 1473

3 TABLE 2. Serum nd lipoprotein lipids of study popultion Pronds Silings Vriles (n 6) (n 7) (n 13) (n 14) (n 23) (n 37) Serum cholesterol (C), mmol/l HDL-C, mmol/l ,c Serum triglycerides, mmol/l LDL-C, mmol/l VLDL-C, mmol/l IDL-C, mmol/l c LDL-C, mmol/l HDL-C, mmol/l c Serum phospholipids, mg/dl c VLDL-PL, mg/dl IDL-PL, mg/dl LDL-PL, mg/dl HDL-PL, mg/dl c Men SE. Enzymtic nlyses from serum. The other lipoprotein lipid vlues re from ultrcentrifuge frctiontion without recovery corrections. Significntly different from pronds. c Significntly different from low sorers. Sttistics Mens nd SEs were clculted nd the differences etween the four groups were nlyzed y ANOVA nd Student s twosided t-test. Correltion coefficients were clculted y Person s product-moment correltion. P 0.05 ws considered sttisticlly significnt. RESULTS Tle 1 shows the demogrphic vlues for pronds nd silings in the low nd high sorption groups. The silings were 8 yers younger thn the pronds, ut the difference ws significnt only in the high soring fmilies. Body mss index ws higher in the low soring pronds, nd higher overll in pronds thn in silings. Both distolic nd systolic lood pressure vlues were higher in the pronds thn silings, ut the low nd high sorers did not differ from ech other. Blood glucose nd serum insulin vlues were similr in the sugroups, even though SHBG vlues were high in the silings, especilly in the high soring ones. Plsm concentrtion of totl homocysteine did not differ etween the groups. Lipids nd lipoproteins Tle 2 shows the mjor lipids of pronds nd silings nd ultrcentrifugtion nlysis of the silings. It cn e seen tht serum totl nd LDL-C levels did not differ etween the low nd high sorers, even though they were higher in the pronds, ut HDL-C level ws significntly lower in the low thn high sorers. However, no consistent difference in triglycerides ws seen etween low nd high sorers, even fter ultrcentrifugtion (dt not shown), such tht they only tended to e higher in the low thn high sorers, especilly in VLDL nd IDL. In contrst to cholesterol, serum phopholipids were lower in the low thn high sorers due to difference in HDL. Esterifiction percentge of cholesterol ws significntly lower only in HDL frction of the low thn high sorers. Squlene nd non-cholesterol sterol rtios Squlene. The rtio of serum squlene only tended to increse in silings s compred with pronds, ut the differences were not significnt. The levels were not ffected y the sorption stte (Tle 3). Cholesterol precursors. The rtios of serum cholestenol, desmosterol, nd lthosterol were significntly higher in the low thn high sorers of oth the pronds nd silings, respectively. The low soring silings tended to hve lower rtios thn their pronds (significnt for des- TABLE 3. Serum squlene nd non-cholesterol sterol to cholesterol rtios (10 2 mmol/mol of cholesterol) in study popultion Compounds Group Squlene Prond Siling Cholestenol Prond Siling Desmosterol Prond Siling Lthosterol Prond Siling , Cholestnol Prond Siling Cmpesterol Prond Siling , Sitosterol Prond Siling Avensterol Prond Siling Men SE. Significntly different from pronds. Significntly different from low sorers Journl of Lipid Reserch Volume 43, 2002

4 mosterol), while in the high soring silings the lthosterol rtio ws higher thn in the respective pronds. Asorption sterols. In ddition to the rtio of serum cholestnol, those of cmpesterol nd sitosterol, ut not of vensterol, were higher in the high thn low sorers in oth the pronds (y definition for cholestnol) nd silings, respectively. In the low sorers, the rtios of serum cholestnol nd cmpesterol were lower in the pronds thn silings, this difference eing significnt only for serum cmpesterol in the high sorers. In generl, s in the pronds, the rtios of the sorption mrkers were negtively relted to those of the synthesis mrkers, ut in the silings their respective rtios to serum cholesterol, for instnce, were positive (e.g., r for cmpesterol; P 0.01) nd negtive (e.g., r for cholestenol; P 0.05). Intrfmily correltions were significnt for the rtios of cholestenol (P 0.047), desmosterol (P 0.014), lthosterol (P 0.009), triglycerides (P 0.043), nd glucose (P 0.037), ut not for squlene, totl or HDL-C, or ody mss index, nd only tended to e significnt for plnt sterol rtios (P for cmpesterol), ut ws tht for cholestnol (P 0.015). Vriles TABLE 4. Cholesterol sorption nd metolism in silings of low nd high sorers (n 23) (n 14) Cholesterol sorption, % Dietry cholesterol, mg/d Fecl steroids, mg/d Neutrl sterols Bile cids Totl Cmpesterol Sitosterol Cholesterol synthesis, mg/d Cholesterol turnover, mg/d Intestinl cholesterol, mg/d Totl pool Biliry pool Cholesterol sorption, mg/d Totl mss Dietry Biliry Men SE. Significntly different from high sorers. TABLE 5. Correltion coefficients of cholesterol synthesis (mg/d) nd sorption (%) with rtios of serum mrkers of cholesterol sorption nd synthesis (10 2 mmol/mol of cholesterol) in silings of high nd low sorption pronds Vriles Cholesterol Synthesis Fecl nd diet dt Cholesterol sorption efficiency ( % vs %) ws significntly higher in the high thn low sorers, finding supporting the rtios of the cholesterol sorption sterols in serum (Tle 4). Correspondingly, fecl output of cholesterol s neutrl sterols of cholesterol origin nd s ile cids ws significntly incresed in the low sorers, resulting in incresed cholesterol synthesis (increses lso ile cid synthesis from newly formed cholesterol), s compred with the high sorers. Despite the low cholesterol sorption efficiency in the low sorers, their dietry intke of cholesterol ws lower thn in the high sorers, ut the respective dietry intke of plnt sterols ws lso higher. Owing to mrkedly high iliry secretion of cholesterol in the low sorers, their solute cholesterol sorption ws within the limits of the high sorers despite low solute sorption of dietry cholesterol. Cholesterol sorption efficiency ws positively relted to the rtios of the serum sorption mrkers (P 0.05 to P 0.001), indicting tht the higher the serum plnt sterol nd cholestnol rtios, the higher is cholesterol sorption efficiency (Tle 5). Cholesterol synthesis (mg/d) ws correspondingly positively relted to the rtios of the synthesis mrkers (P 0.01 to P 0.001), nd negtively to the sorption mrkers (P 0.01) nd cholesterol sorption efficiency (P 0.001). Thus, the higher the rtios of the synthesis mrkers the higher is solute synthesis of cholesterol, nd the lower is cholesterol sorption in oth siling groups. It is owing to smll numers tht the correltions were seprtely less frequently significnt in the high or low soring silings. DISCUSSION Cholesterol Asorption Cholesterol sorption Squlene Cholestenol Desmosterol Lthosterol 0.828, Cmpesterol Sitosterol , Cholestnol 0.709, correltion coefficients (n 37), except etween squlene nd sorption, P Significntly different for high sorers. Significntly different for low sorers. The present results show convincingly tht cholesterol sortion efficiency nd cholesterol synthesis re inherited phenomen. Second, our erlier interprettion tht the high serum cholestnol, cmpesterol, nd sitosterol rtios to serum cholesterol (or low serum precursor sterol rtios) indicte high cholesterol sorption efficiency nd low cholesterol synthesis ws proven to e true. The findings on vriles of cholesterol metolism in these coronry fmilies re proly regulted not only y heredity, ut environmentl fctors my lso ply some role. It remins to e shown whether, in rndom norml popultion, selection of sujects into sorption ctegories using high versus low rtios of serum sorption mrkers (or low vs. high synthesis mrkers) of cholesterol lso indicte identifiction of fmilies with respective high versus low sorption (or low vs. high synthesis) of cholesterol, s in the present hypercholesterolemic coronry Gylling nd Miettinen Inheritnce of cholesterol metolism 1475

5 popultion. However, despite differences in synthesis nd sorption of cholesterol, serum totl nd LDL-C levels were quite similr e.g., in the two siling groups, indicting tht removl nd/or synthesis of LDL pob pprently regulted LDL-C level. The lck of intrfmily correltion of totl nd LDL-C might e explined y the smll numer of sujects, since usully lipid ssocition is found in fmily studies (14). On the other hnd, lower HDL-C level nd trend of higher serum triglyceride vlue nd ody weight in low compred with high sorers confirm our erlier findings in lrge numer of coronry ptients (15), nd resemle the findings oserved in metolic syndrome (16). In ddition, low serum SHBG levels support the presence of metolic syndrome in the low sorers. The intke of ft, cholesterol, nd plnt sterols, the three dietry vriles interfering with cholesterol metolism, were different in the two siling groups. Dietry ft intke ws, however, due to different ody weight, nd the ft intke ws comprle in terms of g/kg of ody weight. er dietry chlesterol intke in the low thn high sorers lowered its sorption nd could hve enhnced cholesterol synthesis. It is difficult to imgine, however, tht the increse of synthesis y lmost 900 mg/dy in Tle 4 could e cosequence of out 70 mg/dy reduction of dietry cholesterol sorption. Additionlly, low sorers were eting lrger mounts of plnt sterols, which could hve decresed cholesterol sorption nd, ccordingly, incresed cholesterol synthesis. Adjusted for ody weight, plnt sterol intke ws, however, similr in the two groups, suggesting tht its contriution to different cholesterol sorption ws proly miniml. It hs een shown erlier tht weight reduction in oese sujects with dietes increses the t seline low cholesterol sorption efficiency (17), suggesting in concordnce with the present results tht low cholesterol sorption efficiency seems to e ssocited with insulin resistnce. Wht will then e the finl explntion: primrily ltered sorption or ltered synthesis of cholesterol, for the inheritnce of cholesterol metolism, remins unknown. Current oservtions suggest tht cholesterol sorption is regulted y ABG G/5 G/8 genes (18) through the expression of sterolins (19), which then regulte t lest plnt sterol, nd pprently lso cholesterol sorption. NOTE ADDED IN PROOF It hs recently een shown (Berge et l J. Lipid Res. 43: ) tht vrition in the plsm concentrtions of noncholesterol sterols is highly heritle, nd polymorphism in ABCG8 contriutes to genetic vrition in the plsm plnt sterol concentrtions. The study ws supported y grnt from the Finnish Hert Reserch Assocition. The expert technicl ssistnce of Ms. Leen Kipiinen, Pi Hoffström, Anne Honkonen, nd Ritv Nissilä is gretly cknowledged. REFERENCES 1. Miettinen, T. A., R. S. Tilvis, nd Y. A. Kesäniemi Serum plnt sterols nd cholesterol precursors reflect sorption nd synthesis in volunteers of rndomly selected mle popultion. 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Lipid Reserch Clinics Progrm, DHEW puliction no. NIH/ Ntionl Institutes of Helth, Wshington DC Hffner, S. M Sex hormone-inding protein, hyperinsulinemi, insulin resistnce nd noninsulin-dependent dietes. Horm. Res. 45: Miettinen, T. A., nd P. Koivisto Non-cholesterol sterols nd ile cid production in hypercholesterolemic ptients with ilel ypss. In Bile Acids nd Cholesterol in Helth nd Disese. G. Pumgrtner, A. Stiehl, nd W. Gerok, editors. MTP Press, Lncster, PA Crouse, J. R., nd S. M. Grundy Evlution of continuous isotope feeding method for mesurement of cholesterol sorption in mn. J. Lipid Res. 19: Miettinen, T. A Gs-liquid chromtogrphic determintion of fecl neutrl sterols using cpillry column. Clin. Chim. Act. 124: Grundy, S. M., E. H. Ahrens, Jr., nd T. A. Miettinen Quntittive isoltion nd gs-liquid chromtogrphic nlysis of totl fecl ile cids. J. Lipid Res. 6: Miettinen, T. A., E. H. Ahrens, Jr., nd S. M. Grundy Quntittive isoltion nd gs-liquid chromtogrphic nlysis of totl dietry nd fecl neutrl steroids. J. Lipid Res. 6: Hmsten, A., L. Iselius, G. Dhlen, nd U. De Fire Genetic nd culturl inheritnce of serum lipids, low nd high density lipoprotein cholesterol nd serum polipoproteins A-I, A-II nd B. Atherosclerosis. 60: Miettinen, T.A., H. Gylling, T. Strnderg, nd S. Srn, for the Finnish 4S Investigtors Bseline serum cholestnol s predictor of recurrent coronry events in sugroup of Scndinvin simvsttin survivl study. BMJ. 316: Moller, D. E., nd J. S. Flier Insulin resistnce-mechnisms, syndromes nd implictions. N. Engl. J. Med. 325: Simonen, P., H. Gylling, A. N. Howrd, nd T. A. Miettinen Introducing new component of the metolic syndrome: low cholesterol sorption. Am. J. Clin. Nutr. 72: Berge, K. E., H. Tin, G. A. Grf, L. Yu, N. V. Grishin, J. Schultz, P. Kwiterovich, B. Shn, R. Brnes, nd H. H. Hos Accumultion of dietry cholesterol in sitosterolemi cused y muttions in djcent ABC trnsporters. Science. 290: Lee, M. H., K. Lu, nd H. Yu Identifiction of gene, ABCG5, importnt in the regultion of dietry cholesterol sorption. Nture Genetics. 27: Journl of Lipid Reserch Volume 43, 2002