Sterol Composition of Normal Human Bile

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1 GASTROENTEROLOGY 98;8:- Sterl Cmpsitin f Nrmal Human Bile Effects f Feeding (Marine) Sterls DON S. LIN, WILLIAM E. CONNOR, and BEVERLEY E. PHILLIPSON Sectin f Clinical Nutritin and Lipid Metablism, Department f Medicine, L, The Oregn Health Sciences University, Prtland, Oregn T examine the sterl cmpsitin f nrmal human bile and the effects f dietary cmpnents frm certain shellfish upn bile cmpsitin, we fed subjects diets rich in shellfish fr wk fllwing a typical American diet. The ttal chlesterl, bile acid, and phsphlipid, and the individual sterls and bile acids f the bile samples during each dietary perid were measured. In the bile f subjects cnsuming the typical American diet, nine different neutral sterls, in additin t chlesterl, were identified. Similar patterns f these sterls have been fund in human gallstnes and in plasma after shellfish feeding. The five shellfish sterls (- dehydrchlesterl, -methylene chlesterl, brassicasterl, isfucsterl, and a C- sterl) increased frm 0.% t a ttal f.% (p < 0.00) f ttal sterls. A cmparisn f the ratis f varius shellfish sterls t chlesterl in plasma and in bile suggested selectively greater excretin f shellfish sterls relative t chlesterl. Our data demnstrated that human bile cntains a mixture f sterls and that its sterl cmpsitin can be readily altered by dietary changes. The lithgenicity f the bile based n the rati f chlesterl, bile acids, and phsphlipids, and the bile acid cmpsitin was nt affected by the presence f shellfish sterls. Hwever, since sme f these sterls ccur in human gallstnes, their lithgenic capacity cannt be ruled ut. Received April 8, 98. Accepted Octber 0, 98. Address requests fr reprints t: William E. Cnnr, M.D., Sectin f Clinical Nutritin and Lipid Metablism, Department f Medicine, L, The Oregn Health Sciences University, Prtland, Oregn 90. This wrk was supprted by research grants AM 8 frm the Natinal Institute f Arthritis and Metablic Disease; HL,8 frm the Natinal Heart, Lung and Bld Institute; and RR frm General Clinical Research Center f the Natinal Institutes f Health. Beverley E. Phillipsn was a Research Fellw, Natinal Heart, Lung and Bld Institute Training Prgram HL by the American Gastrenterlgical Assciatin 00-08/8/$.00 Human gallstnes cntain a number f unusual sterls besides the predminant chlesterl (). Amng these varius sterls are sme that culd nly be derived frm dietary surces and sme that riginated frm the metablism f chlesterl. Amng the dietary sterls fund in gallstnes are - dehydrchlesterl and -methylene chlesterl, which are sterls cmmn in certain shellfish (clams, ysters, and scallps) (). Questins lgically develped abut the absrptin and transprt f these shellfish sterls and the significance f their presence in gallstnes. There was n infrmatin abut their intestinal absrptin and their excretin in human bile. As regards the significance f their presence in gallstnes, we suggested three pssibilities. Perhaps shellfish sterls had been precipitated frm bile alng with chlesterl. sterls in bile might have increased the lithgenicity f bile by altering the rati f chlesterl, bile acid, and phsphlipids r the bile acid pattern in the bile, r bth. sterls culd affect the slubility f chlesterl and ther sterls in the biliary micellar cmplex. In a recent study, we fed a diet rich in clams, scallps, and ysters t human subjects and fund that shellfish sterls were indeed absrbed and were incrprated int different plasma lipprteins much as is chlesterl (). They had, hwever, little effect upn the plasma lipid levels per se (). As a lgical cntinuatin f these previus studies, we have psed the fllwing inquiries in a new study f bile after shellfish feeding. (a) What is sterl cmpsitin f the bile f nrmal humans cnsuming a typical American diet, especially with reference t shellfish sterls? (b) Will the feeding f shellfish affect the sterl cmpsitin f bile? (c) If shellfish sterls in bile are increased by dietary Abbreviatins used in this paper: GLC, gas-liquid chrmatgraphy; TLC, thin-layer chrmatgraphy.

2 LIN ET AL. GASTROENTEROLOGY Vl. 8. N. Table. Sterl Cmpsitin f the Diet a sterls Plant sterls Other sterls Isfuc- C- b -Dehydr- -Methylene Brassica- Sub- Campe- Stigma- Sit- Sub- Chle- Chle- Lath- Ttal Diet sterl sterl chlesterl chlesterl sterl ttal sterl sterl sterl ttal sterl stan I sterl sterls Cntrl a In milligrams per day. b -Trans--nr-chlesta-.-dien-,B-l. shellfish feeding, will the lithgenicity f bile be changed? (d) Will the structural differences between shellfish sterls and chlesterl affect their transprt frm bld t bile? With these questins in mind, we have analyzed the ttal chlesterl, bile acid, and phsphlipids and the individual sterls and bile acids f bile samples frm subjects cnsuming a typical American diet and then a diet rich in shellfish. Methds Seven subjects ( nrmal subjects and type V hyperlipidemic patient, - yr ld, cnsumed a baseline typical American diet fllwed by a diet cntaining clams, ysters, and scallps fed fr wk. The cntrl diet cntained 8 mg sterls ( mg plant sterls and 8 mg chlesterl). The experimental r shellfish diet cntained 0 g f shellfish. It was similar t the cntrl diet in ttal sterl cntent (8 mg) and in plant sterl cntent ( mg), but it had less chlesterl ( mg) and additinal shellfish sterls ( mg), bth f which ttaled mg. The detailed sterl cmpsitin f the cntrl and shellfish diets is shwn in Table. The mlecular structure f these sterls with different side chains is depicted in Figure. Befre and at the end f the wk f shellfish feeding, dudenal bile samples were cllected by the fllwing prcedure. After vernight fasting, an intestinal tube was passed int the secnd psitin f the dudenum f the HO~ R = I""J Chlesterl = ~ -Dehydrchlesterl = ~ C- Sterl ~H = II - Methylene Chlesterl II- CH = ~ Isfucsterl CH, -H = ~ Campesterl C HS... H --~ _.. I Stigmasterl C~... H = ~ Sitsterl I:!, /CH, = ~ Brassicasterl Figure. The mlecular structure f sterls with different side chains fund in human bile. experimental subject with the assistance f flurscpy. The bile sample was cllected by aspiratin after intravenus injectin f Kinevac (E.R. Squibb & Sns, Inc., Princetn, N.J.). In rder t investigate the rate f change f biliary sterl cmpsitin at different times after the shellfish diet, additinal bile samples were cllected after wk f shellfish feeding frm fur f these subjects. Aliquts f freshly cllected bile samples were taken immediately fr lipid analysis. The neutral sterl cntent f the bile and diet was analyzed by the methds described previusly (,,,). They invlved sapnificatin extractin, digitnin precipitatin, thin-layer chrmatgraphy (TLC), gas-liquid chrmatgraphy (GLC), and mass spectrmetry. Authentic standards f the sterls were used fr calibratin. Fr separating the cmplex shellfish sterls, an additinal GLC clumn with SP-000 packing was used, thus enabling us t separate brassicasterl frm lathsterl, campesterl frm -methylene chlesterl, and sitsterl frm isfucsterl (). Chlestanl was separated frm ther sterls by argentatin TLC and quantified by GLC with a QF- clumn (). The bile acids were identified and quantified as trimethyl silyl ether and trifluracetate derivatives by GLC with SE-0 and QF-l clumn, respectively (,8). The phsphlipids were measured clrimetric ally by the methd f Bartlett (9). The preceding prtcl had been apprved by the Human Research Cmmittee. Infrmed written cnsent frm all subjects had been btained. Results Table dcuments the neutral sterl cmpsitin f bile frm the subjects wh cnsumed a typical American diet during a cntrl perid and then a diet rich in shellfish fr wk. In the cntrl diet, chlesterl accunted fr >9% f the ttal neutral sterls, and chlestanl cmprised.%. Lathsterl amunted t 0.% and plant sterls.0% f ttal bile neutral sterls. Sitsterl was the majr plant sterl present. Small amunts f shellfish sterls were fund in the cntrl bile. After the incrpratin f shellfish int the diet, the neutral sterl cntent f the bile changed significantly (Table ). sterls increased frm 0.% t.% f ttal sterls and chlesterl decreased t 90.%. Of the shellfish sterls, -methylene chlesterl increased the mst, frm 0.% t.0%; hwever, -dehydrchlesterl, brassicasterl, the C- sterl, and isfucsterl all increased

3 Table. Effects f Dietary Upn the Neutral Sterls in Human Bile: A Cmparisn With a Cntrl Dietary Perid a Neutral sterl cmpsitin (%) sterls Plant sterls Other sterls Subjects ± SO Diet Cntrl Cntrl Cntrl Cntrl Cntrl Cntrl Cntrl ±0.0 C- sterl -Dehydr- -Methylene chlesterl chlesterl ± ±0.0 Isfucsterl Brassicasterl Subttal ±0. Campe- Stigma- Sit- Subttal sterl sterl sterl ±0. ±0. ± 0.8 ± ± Chlesterl Chlestanl Lathsterl ±0. ± ±SD 0. ± ± ±0..00 ±.0 0. ±0.0. ± ±0.9 ± 0.0 ± 0. ± ± ±0. ± 0. p-value (cntrl vs. shellfish) p < 0.0 P < 0.00 P < 0.00 p < 0.00 P < 0.00 P < 0.00 NS NS NS NS P < 0.00 NS NS a Cntrl diet was a typical American diet and precede d the shellfish diet. NS = nt significant. Neutral sterl cntent (JLg/ml bile) ± ±0 NS > '0 ::!.... ~ CXl.;...., til > ~ C/l ::r: tl r r -.: en ::c > z t::tj r t'r. n : "d C/l ::j z cr>... w

4 LIN ET AL. GASTROENTEROLOGY Vl. 8, N. Table. Isfucsterl Bile/Plasma Ratis G f Varius Sterls in Subjects Fed sterls - Dehydr- -Methy- Plant sterls C- chles- lene ch- Brassicasterl Campesterl terl lesterl Ttal sterl Sitsterl Ttal Other sterls. ±..8 ±.. ± ±.9 9. ± 8..9 ±.. ±..0 ± ±..8 ±..0 ±.0 (SD) a These ratis were calculated by taking the rati f a given sterl t chlesterl in bile versus bld. This apprach must be fllwed because f the invariable and incnstant dilutin f the bile samples by gastric and intestinal secretins. Chlestanl Lathsterl manyfld. There were n changes in chlestanl, lathsterl, and the three plant sterls (sitsterl, campesterl, and stigmasterl). Weekly bile samples were cllected frm subjects after beginning the shellfish feeding. The cmpsitin f biliary neutral sterls after and wk f shellfish feeding was similar. This indicated a rather rapid change f neutral sterl cmpsitin in respnse t dietary sterl intake. The rati f shellfish sterls t chlesterl was 0.0 in the bile and 0.0 in the plasma. These data suggested that the shellfish sterls were selectively cncentrated, abut fivefld, in bile (Table ). When we cmpared the ratis f individual sterls in bile and bld, we nted that the sterls with a methyl r ethyl grup at the carbn psitin were especially cncentrated in the bile. Fr example, the rati fr sitsterl (a-ethyl) was times higher in the bile than in the bld, while it was nly times higher fr -dehydrchlesterl, which des nt have an ethyl r methyl grup at the th carbn (Table ). The bile-plasma rati f camp ester I (amethyl), brassicasterl (~, /-methyl), and - methylene chlesterl were als high, being, 9, and 0, respectively. The rati was fr chlestanl,. fr isfucsterl (-ethylidine), and.0 fr lathsterl. The divergent ratis btained frm sterls with different structure are interesting and nt yet fully understd. The rati f stigmasterl (~, a-ethyl) culd nt be btained because f its extremely lw cncentratin in plasma. In the cntrl diet perid, % f the ttal bile Table. Cmpsitin f Bile Acids in the Bile f Subjects Wh Cnsumed a Cntrl Diet G and a Diet Rich in Bile acid cmpsitin (percent f ttal) Bile acid f,a- Chen- Urs- a-hydrxy- cntent Lith- Dihydrxy- Dexy- dexy- dexy- -ket Uniden- (f-tg/ml Subjects Diet chlic chlanic chlic chlic chlic Chlic chlanic tified bile) Cntrl , Cntrl Cntrl ,80 Cntrl ,8 Cntrl ,89 Cntrl ,9 Cntrl , , ± SD ±. ± 0. ±. ±. ± 0. ±. ± 8, , , , , , , , , ± SD ±. ± 0. ±. ±. ± 0. ±.8 ± 9,8 p-value (cntrl vs. shellfish) p < 0.0 NS NS NS NS a Cntrl diet was a typical American diet and preceded the shellfish diet. NS = nt significant.

5 April 98 DIETARY SHELLFISH AND BILE COMPOSITION Table. Mlar Percentage and Rati f Lipids f the Bile f Subjects Wh Cnsumed a Cntrl Diet and a Diet Rich in Subjects Diet Bile acids Cmpsitin (% ttal ml) Mlar rati (chlesterl/bil e Phsph- acids + phslipids Chlesterl phlipids) Cntrl Cntrl Cntrl Cntrl Cntrl Cntrl Cntrl ± SD. ± ±.8 ±.0 ± ± SD. ± ±. ± p-value (cntrl) vs. shellfish) NS NS NS NS NS = nt significant. acids were primary bile acids (chlic acid.%, chendexychlic acid.%) (Table ). The secndary bile acids (dexychlic acid.9%, lithchlic acid.%) cnstituted %. Minr bile acids included {,a-dihydrxy chlanic acid (0.%) and ursdexychlic acid (0.%). After wk f the shellfish diet, there was n significant change in either bile acid cntent r bile acid cmpsitin in the bile. The mlar percentages f these three lipid cmpnents (Table ) (bile acids, phsphlipids, and chlesterl) were.%,.%, and.9% fr the cntrl diet and.%,.%, and.% after the shellfish diet. The mlar ratis f chlesterl t bile acids plus phsphlipids were nt significantly different between the cntrl and shellfish diets (0.0 ± 0.0 vs. 0.0 ± 0.0). The ttal lipid cntent f the bile averaged. ±. and. ±. g/dl fr the cntrl and the shellfish diets, values which were similar statistically. Discussin The results f the present study clearly demnstrated fr the first time the influence f dietary factrs frm certain shellfish upn the lipid cmp- sitin f human bile. When subjects cnsumed a diet rich in certain shellfish fr wk, the neutral sterl cmpsitin f their bile was significantly altered by the influx f shellfish sterls frm the diet int the plasma. sterls, which include five different majr sterls, increased frm 0.% f the ttal biliary neutral sterls in the cntrl diet t.% f the ttal neutral sterls in the shellfish diet perid, a -fld increase (p<o.ool). In ur previus study, we fund that these shellfish sterls are absrbed by the human intestine, are transprted in the plasma by the lipprteins, and readily assciate themselves with certain membranes f the bdy (). The data f this study further demnstrated the transprt f these shellfish sterls thrugh the liver int bile. While shellfish sterls cntributed.% f the neutral sterls in the bile f the subjects fed shellfish, they nly amunted t - % f the ttal sterls in their plasmas. We had reprted previusly similar values in the plasmas f 8 nrmal subjects fed shellfish (). The shellfish sterls, therefre, appeared t be selectively cncentrated in the bile. In a study f the metablism f sitsterl in humans, Salen et al. (0) nted that there was als a preferential rapid secretin f this sterl int the bile. When we cmpared the rati f individual sterl t chles-

6 LIN ET AL. GASTROENTEROLOGY Vl. 8, N. terl in bth bld and bile, we fund that the cmpunds with methyl r ethyl grups at the carbn psitin tended t be mre cncentrated in the bile. The mechanism by which the liver cell distinguishes between shellfish sterls and sitsterl, which has an ethyl grup frm chlesterl, is unclear. Althugh there have been numerus reprts abut the lipid cmpsitin f human bile, infrmatin derived frm mdern analytical techniques is scanty abut its sterl cmpsitin. In the present study, we have reprted fr the first time the presence f nine different sterls in additin t chlesterl in the bile f nrmal subjects cnsuming a typical American diet. These sterls may be derived frm dietary surces r may arise endgenusly. Fr example, chlestanl in bile culd be derived frm sme fds f animal rigin, such as eggs and liver cntaining chlestanl. Chlestanl is absrbed by the human intestine (unpublished data). It is als cnverted frm chlesterl in the bdy (). Salen () reprted that 0.% f the biliary sterl was chlestanl. Lathsterl is fund in fdstuffs, i.e., butter, egg ylk, and liver (unpublished data) and is als a precursr in chlesterl bisynthesis (). Its presence in human bile has nt been reprted befre. Campesterl, stigmasterl, and sitsterl are the characteristic sterls f plants and are particularly abundant in vegetable ils. They are absrbed by the human intestine in small quantities. Salen et al. (0) indicated the presence f sitsterl in human bile, but did nt reprt its quantity nr that f the ther plant sterls. Plant sterls have als been fund in the bile f patients with the genetic diseases, f-sitsterlemia and xanthmatsis (unpublished data). In this study, shellfish sterls were demnstrated in the human bile fr the first time. Althugh the quantity was small, fur f the five majr shellfish sterls were detected. The varius sterls identified in the bile f subjects cnsuming a typical American diet have als been fund in human gallstnes (). Althugh these sterls were fund in bth the bile and gallstnes, the relative amunts f these sterls in these tw specimens were different. Fr example, while the rati f chlestanl t chlesterl in gallstne and bile was the same, that fr lathsterl was three times higher in gallstnes than in the bile. The relative cncentratin f the sitsterl was six times higher in the bile than in gallstnes (.% vs. 0. %). Such divergent values suggest the pssibility that the slubility f the nnchlesterl sterls in the biliary micellar cmplex may differ frm sterl t sterl. In cntrast t the greatly different neutral sterls after shellfish feeding, the bile acid cmpsitin did nt change frm the cntrl diet. Seven individual bile acids were identified. Minr bile acids were present because f the actin f intestinal micrrganisms upn the primary bile acids (,). The rati f the three majr bile acids (chliclchendexychlicldexychlic) was similar t that previusly reprted in yung subjects n ad libitum diets (.:.0:0.) (). The liver did nt synthesize unusual bile acids frm the diverse grups f nnchlesterl sterls in clams, ysters, and scallps. The absence f unusual bile acids synthesized frm shellfish sterls, hwever, des crrelate well with the earlier wrk f Salen and clleagues (0) n the metablism f sitsterl, which is structurally similar t sme f the shellfish sterls. There was n enzymatic discriminatin between chlesterl and sitsterl in their cnversins t bile acids. Sitsterl was cnverted t the same primary bile acids as chlesterl. At what stage the ethyl g~up at the th carbn is remved, hwever, remains t be determined. Furthermre, unlike sitsterl, brassicasterl has an alkyl grup at the psitin in the f cnfiguratin. The C- sterl (-trans--nrchlesta-,-dien-f-0l) has ne less carbn n the side chain. The effect f these structural differences n bile acid frmatin is nt knwn. T evaluate the relative lithgenicity f bile under the tw dietary regimens, the results f the present study were expressed as mlar percent fr each cmpnent and as the mlar rati f chlesterl t bile acids and phsphlipids (,8). Since previus studies have shwn that bile frm gallstne patients frequently cntains a greater amunt f chlesterl than can be slubilized by bile acids and phsphlipids (9,0)' the term "lithgenic bile" has been used fr the bile that is supersaturated with chlesterl. There is disagreement, hwever, as t the precise limits f chlesterl slubility in bile (9- ). In the present paper, the term "lithgenic" was used in a cmparative sense. Our results shwed that bth f the mlar percentages and ratis were similar between the cntrl and shellfish-rich diets and that ttal lipid cncentratins, anther imprtant factr affecting the slubility f biliary chlesterl (), were als similar. Accrding t the critical tables develped by Carey (), we calculated the chlesterl saturatin f the biles f these subjects fed cntrl and shellfish diets. The percent f chlesterl saturatin was als nt different (0 ± % vs. 0 ± 8%). We thereby cncluded that, based upn these parameters, shellfish-rich diets did nt change the lithgenicity f the bile. Nnetheless, as demnstrated in ur study, the neutral sterls in the bile included up t 0% f nnchlesterl sterls after shellfish feeding. Their different mlecular structures culd well change the slubility f chlesterl and f ther sterls in bile. The fact that certain f

7 April 98 DIETARY SHELLFISH AND BILE COMPOSITION the shellfish sterls are fund in human gallstnes des nt necessarily mean that they are lithgenic; they may simply have precipitated ut f bile with chlesterl because they were cnstituents f bile. References. Ilias AM, Cnnr WE, Cry HT, Lin OS, Daves GO Ir, Krippaehne WW. Sterls f human gallstnes: the recent identificatin f eight different digitnin precipitable sterls. Gastrenterlgy 980;9:9-.. Austin J. The sterls f marine invertebrates and plants. In: Briggs MO, ed. Advances in sterid bichemistry and pharmaclgy. New Yrk: Academic, 90:-9.. Cnnr WE, Lin OS. Absrptin and transprt f shellfish sterls in human subjects. Gastrenterlgy 98;8:-8.. Cnnr WE, Lin OS. The effect f shellfish in the diet upn plasma lipid levels in humans. Metablism 98;:0-.. Lin OS, Cnnr WE. The lng term effects f dietary chlesterl upn the plasma lipids, lipprteins, chlesterl absrptin, and sterl balance in man: the demnstratin f feedback inhibitin f chlesterl bisynthesis and increased bile acid excretin. I Lipid Res 980;:0-.. Lin OS,!lias AM, Cnnr WE, Caldwell RS, Cry HT, Daves GO Jr. Cmpsitin and bisynthesis f sterls in selected marine phytplanktns. Lipids 98;:88-.. Nrdby HE, Nagy S. An evaluatin f recent gas-liquid chrmatgraphic liquid phases fr reslutin f acetylated plant sterls. I Chrmatgr 9;: Lin OS, Cnnr WE, Naptn LK, Heizer RF. The sterids f 000 year-ld human cprlites. J Lipid Res 98;9:-. 9. Bartlett GR. Phsphrus assay in clumn chrmatgraphy. J BiI Chern 99;: Salen G, Ahrens EH Ir, Grundy SM. Metablism f f-sitsterl in man. I Clin Invest 90;9:9-.. Werbin H, Chaikff IL, Imada MR. a-chlestan-f-0: its distributin in tissues and its synthesis frm chlesterl in the guinea pig. I Bii Chern 9;:0-.. Salen G. Chlestanl depsitin in cerebrtendinus xanth- matsis. A pssible mechanism. Ann Intern Med 9 ;: 8-.. Gdman OS, Avigan J, Steinberg O. Studies f chlesterl bisynthesis. V. The time-curse and pathway f the later stages f chlesterl bisynthesis in livers f intact rats. / BiI Chern 9;8:8-9.. Haslewd GAD. Bile salts. Lndn: Methuen & C., Ltd., 9.. Bergstrm S, Oanielssn H. Frmatin and metablism f bile acids. In: Cde CF, Heidel W, eds. Bile, ruminal physilgy. Handbk f physilgy. Sectin : The alimentary canal. Vl.. Baltimre, Md.: Williams & Wilkins, 98:9-0.. Sjvall J. Bile acids in man under nrmal and pathlgical cnditins. Clin Chim Acta 90;:-.. Carey MC, Small OM. The physical chemistry f chlesterl slubility in bile: relatinship t gallstne frmatin and disslutin in man. / Clin Invest 98;: Grundy, SM. Effects f plyunsaturated fats n lipid metablism in patients with hypertriglyceridemia. I Clin Invest 9; : Admirand WH, Small OM. The physic-chemical basis f chlesterl gallstne frmatin in- man. J Clin Invest 98;: Isakssn B. On the lipid cnstituents f bile frm human gallbladder cntaining chlesterl gallstnes. Cmparisn with nrmal human bladder bile. Acta Sc Med Lip Sal 9;9:-9.. Hlzback RT, Marsh M, Olszewski M. Hlan K. Chlesterl slubility in the bile. Evidence that supersaturated bile is frequent in healthy man. I Clin Invest 9;:-9.. Mufsn O. Meksuwan K. Zaremb JE. Ravin L/. Chlesterl slubility in lecithin-bile salt systems. Science 9;: 0-.. Hegardt FG, Dam H. The slubility f chlesterl in aqueus slutins f bile salts and lecithin. Z Ernahrungswiss 9;0:-.. Neiderhiser OH, Rth HP. Chlesterl slubilizatin by slutins f bile salts and bile salts plus lecithin. Prc Sc Exp Bii Med 98;8:-.. Carey Me. Critical tables fr calculating the chlesterl saturatin f native bile. / Lipid Res 98;9:9-.

DATA RELEASE: UPDATED PRELIMINARY ANALYSIS ON 2016 HEALTH & LIFESTYLE SURVEY ELECTRONIC CIGARETTE QUESTIONS

DATA RELEASE: UPDATED PRELIMINARY ANALYSIS ON 216 HEALTH & LIFESTYLE SURVEY ELECTRONIC CIGARETTE QUESTIONS This briefing has been specifically prepared fr the Ministry f Health t prvide infrmatin frm this