Monitoring hepatic cholesterol 7α-hydroxylase activity by assay of the stable bile acid intermediate 7α-hydroxy-4-cholesten-3-one in peripheral blood

Abstract

We describe an accurate method for monitoring the enzymatic activity of hepatic cholesterol 7α-hydroxylase (C7αOH; CYP7A1), the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Assay of 7α-hydroxy-4-cholesten-3-one (C4), an intermediate in bile acid synthesis, revealed that the level of C4 in peripheral blood serum or plasma showed a strong correlation to the enzymatic activity of hepatic C7αOH, both at steady-state conditions (r = 0.929) as well as during the rapid changes that occur during the diurnal phases.This assay should be of value in clarifying the regulation of bile acid synthesis in vivo in laboratory animals and humans since it allows for the monitoring of hepatic C7αOH activity using peripheral blood samples. We describe an accurate method for monitoring the enzymatic activity of hepatic cholesterol 7α-hydroxylase (C7αOH; CYP7A1), the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Assay of 7α-hydroxy-4-cholesten-3-one (C4), an intermediate in bile acid synthesis, revealed that the level of C4 in peripheral blood serum or plasma showed a strong correlation to the enzymatic activity of hepatic C7αOH, both at steady-state conditions (r = 0.929) as well as during the rapid changes that occur during the diurnal phases. This assay should be of value in clarifying the regulation of bile acid synthesis in vivo in laboratory animals and humans since it allows for the monitoring of hepatic C7αOH activity using peripheral blood samples. The breakdown of cholesterol to bile acids in the liver is a major pathway for cholesterol elimination (1Russell D.W. Setchell K.D. Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Google Scholar, 2Angelin B. 1994 Mack-Forster Award Lecture. Review. Studies on the regulation of hepatic cholesterol metabolism in humans.Eur. J. Clin. Invest. 1995; 25: 215-224Google Scholar, 3Dietschy J.M. Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans.J. Lipid Res. 1993; 34: 1637-1659Google Scholar). Stimulation of this process results in adaptive responses that promote hepatic cholesterol synthesis and LDL receptor-mediated uptake. The ensuing lowering of plasma LDL cholesterol (LDL-C) has been shown to be beneficial in preventing coronary heart disease (4Levine G.N. Keaney Jr., J.F. Vita J.A. Cholesterol reduction in cardiovascular disease. Clinical benefits and possible mechanisms.N. Engl. J. Med. 1995; 332: 512-521Google Scholar). Bile acids are synthesized by two biochemical pathways (5Axelson M. Sjovall J. Potential bile acid precursors in plasma–possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man.J. Steroid Biochem. 1990; 36: 631-640Google Scholar): the classic (neutral) pathway, regulated by the enzyme cholesterol 7α-hydroxylase (C7αOH; CYP7A1) that directly converts cholesterol into 7α-hydroxycholesterol (6Shefer S. Hauser S. Bekersky I. Mosbach E.H. Biochemical site of regulation of bile acid biosynthesis in the rat.J. Lipid Res. 1970; 11: 404-411Google Scholar), and a second alternative (acidic) pathway where cholesterol is converted to oxysterols by sterol 27-hydroxylase prior to 7α-hydroxylation catalyzed by oxysterol 7α-hydroxylase (7Bjorkhem I. Eggertsen G. Genes involved in initial steps of bile acid synthesis.Curr. Opin. Lipidol. 2001; 12: 97-103Google Scholar). The importance of C7αOH as a regulator in whole-body cholesterol homeostasis is well established in both animals and humans (8Schwarz M. Russell D.W. Dietschy J.M. Turley S.D. Alternate pathways of bile acid synthesis in the cholesterol 7alpha- hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding.J. Lipid Res. 2001; 42: 1594-1603Google Scholar, 9Bjorkhem I. Araya Z. Rudling M. Angelin B. Einarsson C. Wikvall K. Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver. No coordinate regulation of CYP7A1 and CYP27A1.J. Biol. Chem. 2002; 277: 26804-26807Google Scholar, 10Rudling M. Angelin B. Stahle L. Reihner E. Sahlin S. Olivecrona H. Bjorkhem I. Einarsson C. Regulation of hepatic low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol 7alpha-hydroxylase mRNAs in human liver.J. Clin. Endocrinol. Metab. 2002; 87: 4307-4313Google Scholar). In addition to the classical ways of stimulating C7αOH activity by resin treatment or ileal resection (11Akerlund J.E. Reihner E. Angelin B. Rudling M. Ewerth S. Bjorkhem I. Einarsson K. Hepatic metabolism of cholesterol in Crohn’s disease. Effect of partial resection of ileum.Gastroenterology. 1991; 100: 1046-1053Abstract Full Text PDF Google Scholar, 12Reihner E. Angelin B. Rudling M. Ewerth S. Bjorkhem I. Einarsson K. Regulation of hepatic cholesterol metabolism in humans: stimulatory effects of cholestyramine on HMG-CoA reductase activity and low density lipoprotein receptor expression in gallstone patients.J. Lipid Res. 1990; 31: 2219-2226Google Scholar), new drugs are under development. Among those are inhibitors of the apical sodium-dependent bile acid transporter (SLC10A2) that blocks the active uptake of bile acids in the distal ileum (13Root C. Smith C.D. Sundseth S.S. Pink H.M. Wilson J.G. Lewis M.C. Ileal bile acid transporter inhibition, CYP7A1 induction, and antilipemic action of 264W94.J. Lipid Res. 2002; 43: 1320-1330Google Scholar, 14Ichihashi T. Izawa M. Miyata K. Mizui T. Hirano K. Takagishi Y. Mechanism of hypocholesterolemic action of S-8921 in rats: S-8921 inhibits ileal bile acid absorption.J. Pharmacol. Exp. Ther. 1998; 284: 43-50Google Scholar, 15Gälman C. Östlund-Lindqvist A-M. Björquist A. Schreyer S. Svensson L. Angelin B. Rudling M. Pharmacological interference with intestinal bile acid transport reduces plasma cholesterol in LDL receptor/apoE deficiency.FASEB J. 2003; 17: 265-267Google Scholar) and antagonists for the hepatocellular bile acid receptor, farnesoid X receptor (16Urizar N.L. Liverman A.B. Dodds D.T. Silva F.V. Ordentlich P. Yan Y. Gonzalez F.J. Heyman R.A. Mangelsdorf D.J. Moore D.D. A natural product that lowers cholesterol as an antagonist ligand for FXR.Science. 2002; 296: 1703-1706Google Scholar). Direct stimulation of C7αOH activity by, for example, hormonal treatment also holds some promise (17Rudling M. Angelin B. Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.FASEB J. 2001; 15: 1350-1356Google Scholar). The increased interest in this field makes it important to have more reliable and rapid methods to assay changes in bile acid synthesis. Presently, bile acid synthesis can be estimated from analysis of fecal excretion (18Kekki M. Miettinen T.A. Wahlstrom B. Measurement of cholesterol synthesis in kinetically defined pools using fecal steroid analysis and double labeling technique in man.J. Lipid Res. 1977; 18: 99-114Google Scholar) and from isotope dilution studies (19Lindstedt S. Turnover of cholic acid in man.Acta Physiol. Scand. 1957; 40: 1-7Google Scholar). Direct assay of the enzymatic activity of C7αOH or its mRNA requires liver biopsy. All these methods are laborious, costly, and do not allow for dynamic studies. It has previously been shown that an intermediary product in the synthesis of bile acids, 7α-hydroxy-4-cholesten-3-one (C4), is present in blood plasma, and that its plasma level appears to reflect bile acid synthesis (20Axelson M. Aly A. Sjovall J. Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man.FEBS Lett. 1988; 239: 324-328Google Scholar). Later studies have also shown that the plasma level of C4 correlates with the enzymatic activity of C7αOH assayed in human hepatic microsomes (21Axelson M. Bjorkhem I. Reihner E. Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man.FEBS Lett. 1991; 284: 216-218Google Scholar) as well as with bile acid synthesis in humans (22Sauter G. Berr F. Beuers U. Fischer S. Paumgartner G. Serum concentrations of 7alpha-hydroxy-4-cholesten-3-one reflect bile acid synthesis in humans.Hepatology. 1996; 24: 123-126Google Scholar). However, the originally described procedure is rather complex and, therefore, the study of larger series of samples has been difficult. Interestingly, to the best of our knowledge there are no available published data regarding the use of C4 to monitor bile acid synthesis in laboratory animals. In the current paper, we describe a method for assay of C4 in serum or plasma that takes advantage of commercially available columns for sample preparation. This allows the analysis of 50–75 samples per day compared with earlier described procedures where only 10–15 samples could be analyzed (20Axelson M. Aly A. Sjovall J. Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man.FEBS Lett. 1988; 239: 324-328Google Scholar). By studying rats at different established steady-state conditions known to modulate bile acid synthesis in this species, we have tested the relevance of the data generated. We have found that the C4 level in rat serum strongly correlates with the enzymatic activity of hepatic C7αOH not only during steady-state conditions but also during the diurnal changes that occur in this species. The described method should be of value for monitoring C7αOH activity and presumably bile acid synthesis, and will be of great use in further studies on the in vivo regulation of the dynamics of bile acid synthesis in both humans and laboratory animals. The HPLC system used was the Hewlett Packard 1100 series (Hewlett Packard GmbH, Waldbronn, Germany). It consisted of a autosampler (G1313A), a column compartment (G1316A), a diode array detector (G1315A), a degasser (G1322A), and a pump (G1312A). The software used was HP Chemstation® for LC. The column was a Nova-Pak® C18 steel column, 3.9 × 300 mm, 4 μm particle size, (part. No. WAT011695, Waters Corp., Milford, MA). Sample preparation columns were mounted in a Varian CEREX® SPE processor (Varian Inc. Palo Alto, CA), to which had been added a removable custom-made 5.5 × 21.4 × 8.4 cm stainless steel water-jacketed block heater with 24 positions, hole diameter 1.1 cm. The column heater was connected to a Lauda E100 water bath (Lauda-Königshofen, Germany) set to 66°C in order to obtain 64°C column temperature. The eluted samples were evaporated to dryness at 60°C under a stream of nitrogen using a Techne sample concentrator and a Techne Dry block, type DB3D (Techne, Cambridge, UK). Acetonitril, methanol, chloroform, and hexane were HPLC grade (LiChrosolv®, Merck, Darmstadt, Germany). Pyridin and chlorotrimethylsilane were purchased from Merck and hexamethyldisilazane was from Pierce (Rockford, IL). Cholesterol and cholic acid were from Sigma (St. Louis, MO). Human blood serum and plasma (Table 1) were obtained from the blood donor center and were aliquoted and stored at 4°C, −20°C, −85°C, and in liquid nitrogen. Human serum samples for the experiment shown in Fig. 5 were from five healthy males 26–33 years of age. The studies were approved by the Ethics Committee at Huddinge University Hospital.TABLE 1Effect of storage conditions and time on the 7α-hydroxy-4-cholesten-3-one concentrationStorage TimeStorage Temp.1 Day2 Days15 Days3 Months6 Months10 Months+4°C8.0 ± 0.27.5 ± 0.36.3 ± 0.13.7 ± 0.11.1 ± 0.10−20°C7.4 ± 0.26.8 ± 0.17.1 ± 0.17.4 ± 0.47.1 ± 0.3−85°C7.5 ± 0.26.9 ± 07.2 ± 0.17.2 ± 0.57.2 ± 0.1N2 (l)7.7 ± 0.36.7 ± 0.17.4 ± 0.26.7 ± 0.46.8 ± 0.3Plasma samples were aliquoted and stored at +4°C, −20°C, −85°C, and in liquid nitrogen. Samples were analyzed concomitantly after the indicated storage times. Mean and SEM are shown. Open table in a new samples were aliquoted and stored at +4°C, −20°C, −85°C, and in liquid nitrogen. Samples were analyzed concomitantly after the indicated storage times. Mean and SEM are shown. rats and 7 to and to 300 were All animals were under conditions with to water and of rats cholestyramine for 7 or cholic acid for was with rat and the cholic acid was by rat with into which cholic acid had been The rat All rats were the day and In the diurnal rats were at and All animals were under conditions with to water and The were and were by of or by and by was and serum was by and stored at The were in liquid nitrogen and stored at All animal studies were approved by the and plasma cholesterol and were using a using commercially available MA). of by and assay of cholesterol was on of plasma using a system H. Rudling M. Angelin B. B. hormone receptor of the cholesterol 7alpha-hydroxylase to hormone but to Endocrinol. Scholar). was from by enzymatic with cholesterol as described L. L. of and in rat liver and Scholar). The of the was from the at using as the of we tested different with and C18 as a a C4 columns with or in a and we an The for sample was tested from to and it was found that C4 had the at a column of using a concentrations of conditions were also tested using of hexane and and It was found that the level of was obtained using at the serum from rats and from was with of with of and in was In the in Fig. we used serum from for where we used SPE columns were with × and with × of were mounted in the Varian CEREX® SPE processor with a block heater and to 64°C The samples were in water for and at for prior to the The samples were to on the columns for 5 to were and samples were the which were with 64°C The block was and columns were with × at temperature. to with × of columns were by a stream of nitrogen for The eluted product was evaporated to dryness at 60°C under a stream of nitrogen and in of this was into the HPLC a was used at a of The and column were and C4 was from the of the using the known of were from rat liver by of liver as described earlier K. Angelin B. Ewerth S. K. Bjorkhem I. Bile acid synthesis in assay of hepatic cholesterol 7 alpha-hydroxylase activity by isotope Lipid Res. Scholar). The activity of C7αOH was as the of 7α-hydroxycholesterol from cholesterol by using isotope dilution in samples from K. Angelin B. Ewerth S. K. Bjorkhem I. Bile acid synthesis in assay of hepatic cholesterol 7 alpha-hydroxylase activity by isotope Lipid Res. Scholar). In animal experiment the enzyme assay was on in order to are as ± or ± were with was used of correlation was using the correlation We the procedure to obtain the best possible and for C4 as described in and a HPLC after of human serum as described in the for the The time for C4 was the had a time of The and C4 were from Fig. We the of the method by the addition of of C4 to of a human serum sample The of for the method was from analysis of C4 in a serum sample that was and analyzed in The assay was to of We set the of C4 to blood plasma results as we analyzed C4 in serum and plasma samples from It was found that the C4 was not different plasma was ± = as compared with ± = for We the of the method using serum and plasma samples from two The of were and for the serum and plasma The of the method was by C4 in plasma samples obtained from Sample were stored at +4°C, −20°C, and −85°C, and in liquid from day to (Table It was found that storage conditions were storage for more The as from data with an time of to was with a of of In a experiment using results were with a of of We to the of well plasma C4 could to the enzymatic activity of C7αOH in the liver at steady-state this we established of rats = with cholestyramine = or cholic acid = were and serum and liver were for assay of and lipoprotein in serum and for enzymatic activity of hepatic C7αOH as well as for HMG-CoA A strong was found the enzymatic activity of hepatic C7αOH was to serum C4 A was also present animals on and cholic acid Fig. the animals on were analyzed a was (r = of plasma showed that cholic acid treatment increased plasma cholesterol plasma were not (Table Serum lipoprotein cholesterol showed that the in cholesterol cholic acid was and LDL treatment not plasma plasma were increased The serum lipoprotein of animals showed larger and was of plasma and enzymatic activity of hepatic HMG-CoA = = = ± ± ± compared with ± ± compared with ± ± ± compared with ± = or cholestyramine = for 7 or cholic acid = for and are compared with Open table in a new = or cholestyramine = for 7 or cholic acid = for and are shown. The activity of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was strongly by cholestyramine cholic acid not the activity of the enzyme (Table it was that at steady-state assay of C4 in peripheral blood showed a strong correlation to the enzymatic activity of C7αOH in the liver. We C4 in peripheral blood also the enzymatic activity of C7αOH in conditions the activity of this enzyme is It is well known that the enzymatic activity of hepatic C7αOH has a strong diurnal with in the of the S.S. D.J. Hepatic cholesterol 7 Regulation in vivo at the and mRNA level in to diurnal and bile acid Biol. Chem. 1990; Full Text PDF Google Scholar). We of rats at different of the and assayed hepatic C7αOH activity and the C4 in blood It was found that the level of C4 in the blood serum the diurnal as the enzymatic activity of hepatic C7αOH in order to the C4 in humans with those in used laboratory and we plasma C4 in different species. can be in Fig. and rats had and plasma C4 compared with By the plasma C4 level was in a C4 level in plasma that was only of that in humans The present results that of C4 in peripheral blood samples can be used as an accurate of hepatic C7αOH activity in C4 in peripheral blood also the hepatic C7αOH enzyme activity the enzyme is regulated as during the diurnal in the The and HPLC method to C4 a system and as an the HPLC we a system previously described by that system can as L. liquid of 7 alpha-hydroxy-4-cholesten-3-one in human Scholar) and can be We found that the of C4 from the was the was used at of In the present we a sample procedure and used commercially available The during sample was and was using a water-jacketed steel described methods custom-made water-jacketed columns (20Axelson M. Aly A. Sjovall J. Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man.FEBS Lett. 1988; 239: 324-328Google Scholar, L. liquid of 7 alpha-hydroxy-4-cholesten-3-one in human Scholar) that are to The of the current sample preparation procedure with HPLC the of samples that can be The of the method showed a of of for both blood serum and conditions not the C4 C4 in samples stored at for more The that the C4 level not of and that storage to at not C4 that C4 is a intermediate that is a for the use of this method in larger studies. studies have shown a plasma C4 and the in with C. Paumgartner G. Bile acid as a of value of 7alpha-hydroxy-4-cholesten-3-one in Scholar, S. M. Angelin B. Einarsson K. Serum 7 alpha-hydroxy-4-cholesten-3-one concentrations in the of bile acid in with correlation to 1993; 34: Scholar, H. Serum 7 alpha-hydroxy-4-cholesten-3-one and in the of bile acid J. 1996; Scholar) and also plasma C4 and the enzymatic activity of C7αOH in the liver gallstone with gallstone with chenodeoxycholic or cholestyramine (21Axelson M. Bjorkhem I. Reihner E. Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man.FEBS Lett. 1991; 284: 216-218Google Scholar). the best of our there are no data available regarding the use and of C4 in laboratory animals. We found a plasma C4 and enzymatic activity of C7αOH in the liver of both at steady-state conditions and during the rapid regulation of the which during the diurnal S.S. D.J. Hepatic cholesterol 7 Regulation in vivo at the and mRNA level in to diurnal and bile acid Biol. Chem. 1990; Full Text PDF Google Scholar). the plasma C4 level the diurnal changes of hepatic C7αOH there to be a in the plasma C4 the enzymatic activity was the for C4 in rat blood is the data from the diurnal changes that it should be 5 the of of C4 into blood is It to be the into the is from the or C4 is from the bile the liver or after from the The of plasma of C4 be important for data are The different showed that the rat and the had the C4 five those in human plasma The mouse and the had two plasma C4 the C4 as those found in The C4 level was found in C4 of only that found in We obtained an plasma C4 level of using human samples from five This level is in with previously published data where from to have been (21Axelson M. Bjorkhem I. Reihner E. Einarsson K. The plasma level of 7 alpha-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man.FEBS Lett. 1991; 284: 216-218Google Scholar, L. liquid of 7 alpha-hydroxy-4-cholesten-3-one in human Scholar, C. Paumgartner G. Bile acid as a of value of 7alpha-hydroxy-4-cholesten-3-one in Scholar, C. M. J. Paumgartner G. Beuers U. Effect of cholestyramine on bile acid and synthesis during of acid in J. Clin. Invest. Scholar, Bjorkhem I. M. Einarsson K. for biosynthesis of cholesterol and bile acids are not in gallstone Scholar). data on the of the C4 in and humans in the current are in with previously published data on the enzymatic activity of C7αOH in these species. our data (17Rudling M. Angelin B. Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.FASEB J. 2001; 15: 1350-1356Google Scholar, P. Angelin B. Rudling M. Cholesterol and lipoprotein metabolism in of by hormone treatment in Biol. Scholar, M. Angelin B. Stimulation of rat hepatic low density lipoprotein by of a regulatory in Clin. Invest. 1993; Scholar, M. P. Angelin B. Growth hormone and bile acid synthesis. for the activity of hepatic cholesterol 7alpha-hydroxylase in the rat.J. Clin. Invest. Scholar, Reihner E. Rudling M. L. Einarsson K. Angelin B. of on hepatic cholesterol metabolism in gallstone activity of cholesterol 7 1995; Scholar, E. Angelin B. Bjorkhem I. Einarsson K. Hepatic cholesterol metabolism in cholesterol gallstone Lipid Res. 1991; Scholar, Rudling M. Angelin B. Bjorkhem I. P. K. Einarsson K. Hepatic cholesterol metabolism in human 25: Scholar), it can be found that rats have the C7αOH activity by humans have the activity a and reliable method that reflects hepatic C7αOH activity using a peripheral blood sample has been a method is of interest for since it the activity of the enzyme in it should important to the mRNA This is of since C7αOH enzymatic activity appears to also be regulated at the level M. P. Angelin B. Growth hormone and bile acid synthesis. for the activity of hepatic cholesterol 7alpha-hydroxylase in the rat.J. Clin. Invest. Scholar, S. G. M. Cholesterol 7alpha-hydroxylase from human and rat liver are in by 1996; 24: Scholar), the of regulation is it will allow the dynamic study of enzyme activity and bile acid synthesis in laboratory animals. it will the of in more the of the synthesis of bile acids in human as and gallstone and be to monitor the treatment with drugs that with bile acid This was by from the the for the and the the of and the 7α-hydroxy-4-cholesten-3-one cholesterol 7α-hydroxylase