Rapid transient absorption and biliary secretion of enantiomeric cholesterol in hamsters

To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of cholesterol (ent-cholesterol) and cholesterol were labeled with deuterium, gavaged into hamsters, and measured by negative ion mass spectrometry. Initial uptake of both tracers into the intestinal mucosa at 30 min was similar but cholesterol was temporarily retained there, whereas mucosal ent-cholesterol declined rapidly with concomitantly increased enrichment in both the systemic circulation and the gut lumen. In a 3 day fecal recovery study, ent-cholesterol was quantitatively recovered in the stool, whereas cholesterol absorption was 53.2%. ent-Cholesterol given by intracardiac injection was selectively secreted into bile, and the ratio of ent-cholesterol to cholesterol tracers in the gut lumen increased down the length of the small bowel, with the largest value being found in stool. ent-Cholesterol is efficiently taken up by the intestinal mucosa and undergoes transient enterohepatic recirculation, but it is quantitatively eliminated over 3 days as a result of selective secretion into bile and selective enrichment within the lumen of the intestine. These findings suggest that cholesterol absorption is structurally specific and likely to be mediated by enantiospecific cellular proteins. To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of cholesterol (ent-cholesterol) and cholesterol were labeled with deuterium, gavaged into hamsters, and measured by negative ion mass spectrometry. Initial uptake of both tracers into the intestinal mucosa at 30 min was similar but cholesterol was temporarily retained there, whereas mucosal ent-cholesterol declined rapidly with concomitantly increased enrichment in both the systemic circulation and the gut lumen. In a 3 day fecal recovery study, ent-cholesterol was quantitatively recovered in the stool, whereas cholesterol absorption was 53.2%. ent-Cholesterol given by intracardiac injection was selectively secreted into bile, and the ratio of ent-cholesterol to cholesterol tracers in the gut lumen increased down the length of the small bowel, with the largest value being found in stool. ent-Cholesterol is efficiently taken up by the intestinal mucosa and undergoes transient enterohepatic recirculation, but it is quantitatively eliminated over 3 days as a result of selective secretion into bile and selective enrichment within the lumen of the intestine. These findings suggest that cholesterol absorption is structurally specific and likely to be mediated by enantiospecific cellular proteins. Abbreviations NPC1L1Niemann-Pick C1-Like 1 Niemann-Pick C1-Like 1 The intestinal absorption of dietary and endogenous biliary cholesterol is a key regulatory control point for cholesterol homeostasis. In contrast to other nutrients, only approximately half of intestinal cholesterol is normally absorbed, and excretion of the unabsorbed fraction constitutes the principal pathway for cholesterol catabolism (1Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol..J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar). Reducing cholesterol absorption is a potentially effective way to reduce LDL-cholesterol levels and cardiovascular risk. However, the molecular mechanisms underlying cholesterol absorption are complex and not yet fully understood. Although cholesterol absorption can be completely blocked by biliary diversion or bile salt deficiency, attempts to disrupt cholesterol absorption selectively while maintaining the absorption of other nutrients have been only moderately successful. Phytosterols reduce cholesterol absorption by ∼35% when added to the diet (2Ostlund Jr., R.E. Phytosterols in human nutrition..Annu. Rev. Nutr. 2002; 22: 533-549Crossref PubMed Scopus (548) Google Scholar), whereas the new drug ezetimibe displays maximum inhibition of 54% (3Sudhop T. Lutjohann D. Kodal A. Igel M. Tribble D.L. Shah S. Perevozskaya I. von Bergmann K. Inhibition of intestinal cholesterol absorption by ezetimibe in humans..Circulation. 2002; 106: 1943-1948Crossref PubMed Scopus (0) Google Scholar). Many of the proteins that may play a role in cholesterol absorption have been identified, including ABCA1, ABCG5, and ABCG8 (sterol efflux pumps), Niemann-Pick C1-Like 1 (NPC1L1) (a target of ezetimibe), and ACAT-2 (the intestinal form). However, single-gene knockout of each of these candidates has produced incomplete reduction in cholesterol absorption efficiency (4Wang H.H. Afdhal N.H. Gendler S.J. Wang D.Q. Lack of the intestinal Muc1 mucin impairs cholesterol uptake and absorption but not fatty acid uptake in Muc1−/− mice..Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G547-G554Crossref PubMed Scopus (25) Google Scholar, 5Davis Jr., H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis..J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 6Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Buchler C. et al.ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism..Gastroenterology. 2001; 120: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr., R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice..Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (300) Google Scholar, 8McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. et al.High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1..Proc. Natl. Acad. Sci. USA. 2000; 97: 4245-4250Crossref PubMed Scopus (486) Google Scholar). Likewise, overexpression of NPC1L1 in nonenterocyte cells failed to generate active cholesterol transport, suggesting that additional subunits or cofactors may be required (9Altmann S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption..Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1471) Google Scholar). Therefore, although the hypothesis that cholesterol is absorbed by a specific protein or proteins is attractive, the evidence to support it is still incomplete. Cholesterol absorption is inherently a low-affinity process acting on millimolar amounts of cholesterol in the intestine. Thus, the specificity of cholesterol absorption cannot be studied effectively by traditional kinetic methods using a tracer and its competing unlabeled ligand. An alternative approach is to use the unnatural enantiomer of cholesterol (ent-cholesterol) in metabolic studies. As shown in Fig. 1, ent-cholesterol is the mirror image of cholesterol and is chemically synthesized with the opposite configuration at each of the eight chiral centers. ent-Cholesterol has physical properties identical to those of cholesterol with a different three-dimensional configuration, making it likely to be recognized differently by cell proteins. By contrast, the interactions of cholesterol and ent-cholesterol with other lipids are identical (10Mannock D.A. McIntosh T.J. Jiang X. Covey D.F. McElhaney R.N. Effects of natural and enantiomeric cholesterol on the thermotropic phase behavior and structure of egg sphingomyelin bilayer membranes..Biophys. J. 2003; 84: 1038-1046Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Westover E.J. Covey D.F. Brockman H.L. Brown R.E. Pike L.J. Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers..J. Biol. Chem. 2003; 278: 51125-51133Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Because enantiomers are not separable by conventional, nonchiral chromatographic methods and have identical mass spectra, the use of differential deuterium labeling and mass spectrometry for their detection is required. In this work, we gavaged hamsters with a mixture of cholesterol labeled with seven deuterium atoms and ent-cholesterol labeled with two deuterium atoms and measured tracer enrichment with negative ion mass spectrometry. ent-Cholesterol was recognized differently from cholesterol and, despite rapid transient absorption into the systemic circulation, was quantitatively excreted in the stool over 3 days. Moreover, ent-Cholesterol tracer given by intracardiac injection was selectively secreted into bile, enriched in the intestinal lumen, and eliminated in the stool. [25,26,26,26,27,27,27-2H7]cholesterol was synthesized by CDN Isotopes (Montreal, Quebec, Canada). Deuterated ent-cholesterol was synthesized by reduction of ent-desmosterol (ent-Δ24-cholesterol) using a platinum/carbon catalyst and deuterium gas as described (12Westover E.J. Covey D.F. First synthesis of ent-desmosterol and its conversion to ent-deuterocholesterol..Steroids. 2003; 68: 159-166Crossref PubMed Scopus (28) Google Scholar). The actual distribution of deuterium incorporation was 38.2% none, 35.7% one, 16.2% two, 6.4% three, 2.3% four, 0.9% five, and 0.3% six, similar to previous reports of sterol side chain labeling reactions (13Kirk D.N. Varley M.J. Makin H.L.J Trafford D.J.H Synthesis of [26,27-2H6]cholesterol and derivatives substituted in the side chain..J. Chem. Soc. Perkin Trans. 1983; I: 2563-2567Crossref Google Scholar). [5,6,22,23-2H4]sitostanol was purchased from Medical Isotopes (Pelham, NH). F1B male hamsters weighing ∼120 g were obtained from Charles River Breeders (Boston, MA) and housed individually in a room maintained at 24°C with a 12 h light/12 h dark cycle. Animals were fed Purina Rodent Chow 5053 containing 4.5% fat, 20.0% protein, and 54.8% carbohydrate. Procedures were approved by the Washington University Animal Studies Committee. ent-Cholesterol and cholesterol tracers were mixed in ethanol, dried under vacuum, and redissolved in soybean oil. For time-course experiments, food was removed at the beginning of the light cycle at 6 AM; the animals were fasted for 2 h and then gavaged with 0.6 ml of soybean oil only (for time 0 controls) or containing 0.34 mg of cholesterol-d7 and 1.06 mg of ent-cholesterol-d2. Hamsters (three for each time point) were euthanized by carbon dioxide asphyxia at 0, 0.5, 1, 2, and 4 h afterward. Blood was collected by heart puncture, and the liver and small intestine were removed. The small intestine was divided into four equal segments that were analyzed separately. Lumenal contents were collected by washing with ice-cold PBS, and then the mucosa was separated from the bowel wall and extruded by exerting pressure with a microscope slide. Lipids were by the of and E.G. rapid of total and J. Physiol. PubMed Scopus Google and The were into and to Jr., R.E. D.L. of cholesterol tracers by gas ion mass PubMed Scopus Google Scholar). In experiments, mucosal and cholesterol were analyzed separately. To of the total were added 3 of and 3 of as The mixture was taken up in ml of and to 3 ml with were with 6 ml of in and were then collected in 6 ml of in The were then and for For 3 day cholesterol absorption hamsters were gavaged with ml of soybean oil containing mg of mg of and mg of control hamsters ml of soybean oil were collected from for a 3 day was obtained by and stool were as described For the intracardiac injection ent-cholesterol and cholesterol tracers were in and then mixed with Jr., R.E. cholesterol absorption in and with tracers and negative ion mass Lipid Res. Full Text Full Text PDF PubMed Google Scholar). only ml for time 0 controls) or containing mg of cholesterol-d7 and mg of was to hamsters by intracardiac injection 2 h of and animals 3 for each time point) were euthanized at time 0 and 4 or were collected as for the was only to animals for the h time molecular Jr., R.E. D.L. of cholesterol tracers by gas ion mass PubMed Scopus Google Scholar), were separated on a with The was at for 2 increased at a of to a of and for The was analyzed in mass using negative ion ion was at for and for cholesterol and ent-cholesterol and at and for The enrichment of tracers was from ion measured at and The mass was it in ent-cholesterol tracer and the of unlabeled natural a of the tracer at and were for the of unlabeled natural cholesterol and for from each that ent-cholesterol cholesterol tracer was given to its detection but that are as the ratio of tracer with to natural cholesterol of tracer tracer The of and cholesterol-d7 in the liver and stool were from each tracer recovered to its total are The of intestinal and of tracer were analyzed using a was to tracer Cholesterol absorption is a process that uptake into the mucosa from the intestinal lumen, the and into the systemic To the phase of absorption, hamsters were gavaged with a mixture of cholesterol-d7 and ent-cholesterol-d2. levels in the intestinal lumen and and liver were then by at 0.5, 1, 2, and 4 of the small bowel was analyzed separately. are as tracer enrichment in and to specific or cholesterol in using that mucosal uptake of both tracers was similar at time The cholesterol tracer level increased in over time the intestinal segments In contrast, the level of ent-cholesterol tracer at h and then this was similar for four of the small intestine These suggest that ent-cholesterol is rapidly taken up and then eliminated from the by efflux into the intestinal lumen or by the side of the or The of sterol was studied by the of each tracer that was at 1, 2, and 4 h tracer in the of the intestinal mucosa By were of both time and tracer However, the of both tracers was time-course of cholesterol-d7 and in the mucosal hamsters were gavaged with a mixture of cholesterol-d7 and as described in and was collected and analyzed for and sterol by mass spectrometry. of each of the cholesterol enantiomers was as a of the total and By were of time and tracer are in a new hamsters were gavaged with a mixture of cholesterol-d7 and as described in and was collected and analyzed for and sterol by mass spectrometry. of each of the cholesterol enantiomers was as a of the total and By were of time and tracer are the small the contents of the lumen were collected using a were from the contents and analyzed by that the level of cholesterol tracer was over 4 h in each of the intestinal lumen. However, for given was cholesterol tracer in of the ent-cholesterol tracer levels increased with down the bowel the ratio of ent-cholesterol to cholesterol tracer increased with down the intestine for the ratio was in 4 at 4 differential of the sterol tracers by the To the side of the the tracer of the was small amounts of the cholesterol tracer were found in the over the 4 h time The accumulation of the cholesterol tracer in the mucosa and the small enrichment in a in the systemic absorption of cholesterol In contrast, amounts of ent-cholesterol tracer were found in the as as 30 By 4 was a enrichment of ent-cholesterol tracer with cholesterol tracer in the small amounts of the cholesterol tracer were found in the liver for of the cholesterol tracer was in the liver at 4 By contrast, a of the ent-cholesterol tracer of the was in the liver at 4 h The enrichment of the ent-cholesterol tracer was that of the cholesterol tracer in the liver at 4 bile was and analyzed for sterol but cholesterol ent-cholesterol tracer was found in amounts within the time of these not traditional of sterol absorption was by the tracers and from the animals for the of the were analyzed for tracer on of the h stool cholesterol absorption was as similar to the of found in Jr., R.E. cholesterol absorption in and with tracers and negative ion mass Lipid Res. Full Text Full Text PDF PubMed Google Scholar). In contrast, of the ent-cholesterol was recovered in the stool. cholesterol-d7 was in but ent-cholesterol tracer was not not day absorption of cholesterol and ent-cholesterol by fecal in hamsters were gavaged with a mixture of cholesterol-d7 and as described in and were collected for h and analyzed for sterol by mass spectrometry. of each of the cholesterol enantiomers was by to unabsorbed not in a new hamsters were gavaged with a mixture of cholesterol-d7 and as described in and were collected for h and analyzed for sterol by mass spectrometry. of each of the cholesterol enantiomers was by to unabsorbed not To the by was from cholesterol and a mixture of cholesterol and ent-cholesterol tracers was to the systemic circulation by intracardiac levels in were then by at 4 or The levels of cholesterol and ent-cholesterol were similar at 4 h intracardiac injection However, enrichment of both tracers at h time with the cholesterol-d7 being to suggesting rapid of ent-cholesterol-d2. with was found in the liver and liver enrichment of both tracers over time of the was found in the liver at 4 h of at the were enrichment to cholesterol-d7 in the bile for the tracer a selective excretion of the The enrichment of in the stool was that of cholesterol-d7 at 4 h injection and at h As a of the the amounts of increased from at 4 h to at whereas the amounts of cholesterol-d7 were and These results that was selectively excreted over the h of this intracardiac were of time and in the enrichment of cholesterol-d7 but in in the mucosa The ratio of ent-cholesterol to cholesterol tracers in the mucosa and over time In the intestinal lumen, were of time for cholesterol-d7 enrichment and for and for cholesterol-d7 and for the ratio of ent-cholesterol to cholesterol tracers in the lumen increased but over of the cholesterol tracer as a was absorbed over 3 days. result is with previous reports in Jr., R.E. cholesterol absorption in and with tracers and negative ion mass Lipid Res. Full Text Full Text PDF PubMed Google Scholar). In contrast, 3 the ent-cholesterol tracer was not in the it was quantitatively recovered in the stool. ent-Cholesterol was not in this cholesterol absorption of tracers have shown that of are absorbed over days Jr., R.E. Zeng Covey D.F. J. absorption and of and in J. Physiol. 2002; PubMed Scopus Google Scholar). Because were not in this for ent-cholesterol was to by the process it is that the process of cholesterol absorption not only cholesterol from from cholesterol and but cholesterol from its only in Because cholesterol and ent-cholesterol in three-dimensional configuration, chiral as proteins have the to the two The that hamsters completely cholesterol and ent-cholesterol evidence that cholesterol absorption is and likely to be with on or the of systemic the time-course that ent-cholesterol was taken up by the and The mucosal levels of cholesterol and ent-cholesterol tracers were similar the 30 min the 4 h cholesterol to in the However, this tracer was found only in small amounts in the or liver by 4 h systemic absorption of cannot the that cholesterol be efficiently to results are with other reports that cholesterol tracers given may days to a in Jr., R.E. cholesterol absorption in and with tracers and negative ion mass Lipid Res. Full Text Full Text PDF PubMed Google Scholar, G. C. M. H. D. cholesterol is secreted in in J. Nutr. 2001; PubMed Scopus Google Scholar, J. K. of cholesterol the intestinal Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). By contrast, mucosal levels of ent-cholesterol four at 1 h and then declined rapidly a rapid uptake of ent-cholesterol of the small intestine at 1 h by have shown that rapid but transient accumulation in M. Giesa U. Lutjohann D. von Bergmann K. of the intestinal uptake of and in Lipid Res. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Although its role is still NPC1L1 to play role in the uptake of both cholesterol and from the intestinal lumen into the Jr., H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis..J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, S.W. Davis Jr., H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption..Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1471) Google Scholar), and results suggest that NPC1L1 may not In the and a enrichment of ent-cholesterol over cholesterol was as as 30 min The ratio of ent-cholesterol to cholesterol enrichment and in the and at 4 h These results that ent-cholesterol was rapidly the membrane of whereas of it may have been into the intestinal lumen. is likely that the of ent-cholesterol was in its only a small was found to be in the mucosa Because cholesterol is by as efflux mediated by or J. K. of cholesterol the intestinal Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), these may be for the rapid absorption of ent-cholesterol from the to the systemic circulation J. K. of cholesterol the intestinal Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K.K. Farese Jr., R.V. Turley deficiency cholesterol absorption in the on cholesterol 2004; PubMed Scopus Google Scholar). other may be rapid uptake and to the systemic circulation, the absorption of ent-cholesterol is incomplete. the given by intracardiac of this tracer was in the stool at 4 and this increased to at h In contrast, of cholesterol-d7 for of the intracardiac tracer at both time with the selective of ent-cholesterol its enrichment in the intracardiac injection was similar to that of cholesterol tracer at 4 h but was that of cholesterol at h The intracardiac injection a accumulation of over cholesterol-d7 in liver and bile at 4 h The enrichment of both tracers in liver and bile at is with over at ent-cholesterol tracer was still and that of cholesterol tracer in liver and bile, These results that the liver to the specificity of cholesterol absorption by selectively into bile for Lumenal levels of both ent-cholesterol and cholesterol in the increased with down the small intestine. a enrichment of ent-cholesterol with cholesterol in 4 at 4 Because only amounts of ent-cholesterol were found in the bile within 4 h of tracer be biliary ent-cholesterol to its enrichment within this time in the ent-cholesterol enrichment in the lumen increased at both 4 and h intracardiac injection In the ratio of ent-cholesterol to cholesterol tracers increased from 1 to 4 at 4 h and at In contrast, the ratio of ent-cholesterol to cholesterol tracers in the mucosa was at h with 4 The of ratio in the lumen is with the of Therefore, the may specificity in the enrichment of efflux mediated by von Bergmann K. Lutjohann D. H.H. sterol accumulation in Lipid Res. 2004; Full Text Full Text PDF PubMed Scopus Google or other in the intestinal and biliary secretion of ent-cholesterol suggest that ent-cholesterol may have enterohepatic a of this tracer was found in the circulation at 4 h time-course of biliary tracer excretion using bile hamsters the enterohepatic of but are by tracer ACAT-2 a role in cholesterol absorption when intestinal cholesterol levels are K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr., R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice..Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (300) Google Scholar). In only a small fraction of tracer was by 1 that the mucosal of cholesterol was in the is with the of a metabolic of cholesterol in the mucosa described Jr., Jr., H. of cholesterol in and cholesterol of mucosa Biol. Chem. Full Text PDF PubMed Google Scholar). In cholesterol was selectively into this and its absorption into the systemic circulation was with that of is of the role of this cholesterol in cholesterol absorption, but it that it may a of the of absorbed cholesterol that the proteins in the absorption can have on cholesterol of to be critical for the of cholesterol C. W. H.H. of cholesterol by 2004; PubMed Scopus Google Scholar). The results of this that cholesterol absorption is that ent-cholesterol is efficiently recognized and by both the mucosa and the An of the of this may a for at the intestinal absorption of both cholesterol and was by of and as as by to the Washington University the Washington University and the Washington University

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