SEMINARS IN LIVER DISEASE-VOL.

12, NO. 4, 1992

Bile Acid Metabolism and Its Role in Human Cholesterol Balance

In intact humans, steady-state cholesterol homeostasis may be viewed as the balance between two inputs, the intestinal absorption of dietary cholesterol and cholesterol synthesis, and two outputs, bile acid synthesis and biliary secretion of cholesterol.',*The synthesis of steroid hormones and the sloughing of epithelial cells represent additional mechanisms of cholesterol removal. However, the contribution of the latter processes is small in comparison to the daily rates of bile acid synthesis and biliary cholesterol secretion. The liver regulates total body cholesterol metabolism. Preformed cholesterol is delivered to the liver, primarily from the uptake of plasma lipoproteins.'.'" In addition, more than 75% of the newly synthesized cholesterol in the whole body is produced by the liveP and it is the only organ capable of bile acid synthesis and biliary cholesterol secretion. Because all inputs and outputs to the total body cholesterol pool are focused in the liver, it is understandable why alterations in hepatic bile acid synthesis influence not just hepatic, but also total body metabolism of cholesterol. In this article I shall discuss the physiologic regulation of bile acid metabolism and describe how alterations in bile acid synthesis influence cholesterol balance in humans.

STEADY-STATE CHOLESTEROL HOMEOSTASIS Approximate values for steady-state inputs and outputs to the human total body cholesterol pool are given in Table I . There are two inputs to the pool: dietary cholesterol and newly synthesized cholesterol. The daily diet of the average American contains approximately 1 mmol of cholesterol and the fractional absorption of cholesterol from the intestine is 40 to 50%.'-la Thus, the net input to the cholesterol pool from dietary sources is 0.5 mmol. Although exact measurements of human total body cholesterol synthesis are lacking, results from both isotope and balance studies suggest that the major input to the body's cholesterol pool is from synthesis."-" The estimated hepatic input is approximately 1 mmol and the estimated extrahepatic input, 0.7 mmol. The total input to the body's cholesterol pool is therefore 2.2 mmollday. Routes of output are required to balance the input to the body's cholesterol pool from the sources just mentioned. One output pathway, the secretion of biliary cholesterol, is rela-

From the Section of Heparology and Hepatobiliary Researrh Ccnter, Division of Gastroenterology. University of Culorudo Heulrh Sciences Cenrer, Denver, Colorado. Reprint requests: Dr. Everson, Section of Hepatology and He-

patobiliary Research Center, Division of Gastrocntcrology, Univcrsity of Colorado Health Sciences Center, Denver, CO 80262. 420

tively constant, approximately 2 mmol each d a ~ . ' ~The . ' ~magnitude of this output alone is nearly sufficient to account for the total input of cholesterol. However, the fractional absorption of biliary cholesterol from the intestine is equal to or greater than the fractional absorption of dietary ch~lesterol.'~.~' Thus, only I mmol (50%) of the 2 mmol of daily biliary cholesterol is fecally eliminated. The daily rate of catabolism of cholesterol to bile acid, measured as bile acid synthesis, accounts for the output of the remaining excess cholesterol. Under most circumstances, output as newly synthesized bile acid exceeds the output as fecally eliminated biliary cholesterol. Small et a1 administered "C-cholesterol intravenously, measured the radioactivity recovered in fecally eliminated sterols, and deduced that bile acid synthesis was responsible for 70% of cholesterol eliminat~onin intact human^.'^

STEADY-STATE KINETICS OF BILE ACIDS The steady-state kinetics of bile acids are measured in hu' two major mans by the technique of isotope d i l ~ t i o n . ~The assumptions of this technique are: (1) the administered isotope mixes completely with the entire pool of bile acid, and (2) the size of the bile acid pool is constant during the period of the kinetic study. Although several variations the basic method is briefly described. Bile acids labeled with either radioactive or stable isotopes are administered to the study subject and a period of time is selected (more than 16 hours) to allow complete mixing of the isotope-labeled bile acid with the unlabeled bile acid pool. Bile or serum samples are obtained daily for 4 or 5 days to measure the enrichment and disappearance of isotope within the pool of bile acid. With enterohepatic cycling, a small portion of the pool (1.3% per cycle) is lost due to incomplete intestinal absorption (physiologic efficiency, 97 to 99%). As already defined, the size of the pool of bile acid does not change in the steady state. Synthesis of bile acid replaces that lost due to fecal elimination. In this way the isotopically laheled pool of bile acid becomes diluted with unlabeled newly synthesized bile acid (Fig. 1). The fractional turnover rate (FTR) of the pool is calculated from Lnllinear regression of isotopic enrichment versus time. The size of the pool is calculated from the intercept at time 0 and synthesis is the product of FI'R and pool. Numerous investigators have measured steady-state kinetics of bile acids in intact human subjects.30 The populations studied have been quite heterogeneous with regard to gender, age, degree of obesity, presence or absence of cholelithiasis, and coexistent disease states. The range of mean values for PTR, synthesis, and pool size in healthy individuals from these studies was considerable: FTR of chenodeoxycholic acid (CDCA): 0.14 to 0.40 d a y ' ; FTR of cholic acid (CA): 0.22 to 0.54 day-'; synthesis of CDCA: 106 to 264 mglday: syn-

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GREGORY T. EVERSON, M.D.

BILE ACID METABOLISM-EVERSON

42 1

TABLE 1. Approximate Values for Steady-State Cholesterol Balance in Humans mmolldav

FTR

Input

-

output

(1

-

AE) x (number in EHC).

FTR increases when AE decreases. The ileal transport of conjugated bile acid is the physiologic determinant of AD and is the product of the length of terminal ileum L,,, the number of enterocytes per unit length of ileum (E), and the absorptive capacity of each cell (CAC). Therefore:

1 .0 0.5

1.0 0.7

output

Biliary cholesterol Secreted Reahsorhed (50%) Bile dcld synthrsls

=

AE

2.0 10 12

=

thesis of CA: 180 to 409 mgiday; pool of CDCA: 526 to 1450 mg; pool of CA: 523 to 1514 mg. Estimates of the overall means for each value of hile acid kinetics are shown in Table 2. The size of the total pool IS approximately 6000 bmol (3 gm) and total synthesis is 1150 pmollday (575 mgiday).

Determinants of Fractional Turnover of Bile Acids Under physiologic conditions, the vast majority of the bile acid p w l is confined to the enterohepatic circulation (EHC). There is only one input to the pool, hepatic synthcsis, and one output, fecal elimination. FTR, which is a measure of disappearance of isotope from the pool, is an indirect measure of the rate of fecal elimination of hile acid. Two physiologic processes determine the rate of turnover. the absorptive efficiency

- b,

x E x CAC

Since the cellular transport of bile acids (CAC) by the terminal ileum operates at near-maximal capacity, AE is altered primarily by changes in length of terminal ileum (resection,'"' bypass3') or enterocyte density (Crohn's disease'>.'h). The degree of fecal elimination of bile acids after ileal resection or in patients with ileal disease is directly related to the length of the resection or the extent of disease, respectively. For these reasons, ileal resection has been used effectively in the treatment The degree of of nroderately severe hypercholesterole~nia.~' low density lipoprotein (LDL) lowering and stinlulation of the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, hepatic LDL receptor, and cholesterol-7a-hdyroxylase correlate directly with the length of ileal re~ection.'~ FTR increases when the number of EHCs increases. The number in the EHC (#EHC) is determined by: #EHC = (24 hour rate of hepatic secretion of bile acid) I pool In contrast to intestinal absorption, the hepatic secretion of bile acid operates Par below its maximal transport capacity. Virtually all con,jugated bile acid returning to the liver is rapidly

Serum Bile Acid Kinetics CA -

COCA

0.0,

FTR: .33/d

Pool: 2.8 mmol Syn: .92 mrnol/d

Time (d)

Time (d)

FIG. 1 . An example of a study of bile acid kinetics in a single individual. Stable isotopes ([i 1,12-2H]CDCA, [2,2,4,4] CA) were taken orally at time 0.The enrichment of the pools of bile acids with the isotopes is measured by mass spectrometry of bile acids isolated from serum. The technique is described in the text.

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Input Dietary cholesterol Intakc Absorhed (50%) Cholesterol synthesis Hepatic Extrahepatic

Ner Chanae

(AE) of the intestine for bile acid and the frequency of cycling of the bile acid pool within the EHC. Mathcmatically, FTR may be defined by:

SEMINARS IN LIVER DISEASE-VOLUME

12, NUMBER 4, 1992

Determinants of Bile Acid Kinetics Cholesterol

Genetic Elements Metabolism

Size

Intestinal Abspt Cell Function

# of EHC Gallbladder Storage S B Transit FIG. 2. Diagram of the numerous physiologic processes that influence t h e synthesis and FTR of bile acids. Changes in synthesis and FTR then determine the size of the bile acid pool. FTR may influence synthetic rate via effects on the magnitude of the hepatic flux of bile acids. Abspt: absorption; SB: small bowel. removed from portal blood and secreted into bile. For these reasons, the hepatic uptake, intracellular transport, and secretion of bile acids into bile are not rate-limiting for biliary secretion of bile acid. Under physiologic conditions, the hepatic secretion rate of bile acid will reflect the rate of delivery of bile acid to the liver from the intestine, which, in turn, is dependent on the rate of transit of bile acid from its site of entry into the bowel (duodenum) to its site of reabsorption (terminal ileum). Although the transport of bile acids from intestinal lumen to portal blood by ileal enterocytes and the flow of portal blood from the intestine to the liver are rapid, the transit of bile acid through the gallbladder and intestine are relatively slow.'841 For these reasons, the hepatic secretion of bile acids is reduced by either increased storage of bile acid in the gallbladder or prolongation of transit within the small b ~ w e l . ~ '

TABLE 2. Approximate Values for Steady-State Kinetics of Bile Acids in Humans Fractional turnover rates Chenodeoxycholate Cholate Synthesis (pmollday) Chenodeoxycholate Cholate Pool (prnol) Chenodeoxcholate Cholate Deonycholate

Determinants of Synthesis of Bile Acids The rate-limiting enzyme in the synthesis of bile acids is

~holesterol-7a-hydroxylase.~~."' Although this enzyme is under extensive investigation, the precise mechanisms regulating its synthesis and activity are incompletely defined. In man and intact animals, the activity of cholesterol-7a-hydroxylase is stimulated by treatments or conditions that increase the fecal elimination of bile acid^:""^ ileal resection, cholestyramine, psylli~m.~' These data support the traditional concept that bile acids regulate the activity of cholesterol-7a-hydroxylase via a negative feedback loop. Bile acid loss reduces the hepatic flux of bile acid, removes the inhibitory effect of bile acids on synthesis, and, as a result, the synthesis of bile acids increases. If the negative feedback of bile acid on bile acid synthesis were the sole regulatory element and hepatic free cholesterol, the substrate for cholesterol-7a-hydroxylase, was in abundance, then pool size would not change. Fecal loss would always be compensated for by enhanced synthesis. However, bile acid pool decreases under conditions- of excessive bile acid loss, such as after ileal resection or bypass. Studies of cultured hepatocytes suggest that bile acids may not directly regulate their own synthesis. Two separate groups have found that administration of high concentrations of bile acids to isolated rat hepatocytes does not alter rates of bile acid ~ynthesis.'~~' In contrast, studies comparing a wide variety of bile acids suggest that, although hydrophilic bile acids do not inhibit synthesis, hydrophilic bile acids In addition, other groups studied isolated pig h e p a t o c y t e ~ ~and ~ . ~observed ' inhibition of bile acid synthesis by bile acids. Despite these conflicting results, most investigators believe that bile acids are at least partially responsible for regulating their own synthesis.

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Bile Acids

The substrate for bilc acid synthesis is hepatic unesterified cholesterol. The two main inputs to this metabolically active pool of cholesterol are from the hepatic uptake of plasma lipoproteins (LDL, chylomicron remnants) and newly synthesized cholesterol. A role for substrate availability in the regulation of cholesterol-7a-hydmxylase is suggested by a number of observations. First, bile acid synthesis is the major catabolic pathway for cholesterol. If this pathway is analogous to other catabolic pathways, the need for stimulation of the pathway would be dependent on the need to eliminate cholesterol. If the pool of cholesterol was excessive, then stimulation of catabolism of cholesterol to bile acid would be necessary to restore cholesterol balance. Second, newly synthesized cholesterol may be a preferred substrate for synthesis of bile Thus, it is possible that changes in substrate pools of cholesterol might have been responsible, at least in part, for the stimulation of synthesis of bile acid observed after biliary diversion in whole animal studies. Third, synthesis of bile acids by isolated hepatocytes may not be inhibited by administration of bile but synthesis is decreased by removal of lipoprotein cholesterol584' or blockade of cholesterol ~ y n t h e s i s . ~Fourth. .~' although the gene for cholesterol-7a-hydroxylase has been sequenced and cloned since 1990,"'Mno genomic regions have been identified that could be classified as bile acid-dependent promoters or enhancers. Thus, the data presented support a role for the regulation of cholesterol-7a-hydroxylase by its substrate, hepatic free cholesterol.

Determinants of Pool Size Unlike FTR and synthesis, there is no physiologic mechanism for directly regulating the size of the pool of bile acid. For this reason, the size of the pool is best defined by: Pool

=

synthesis 1 FTR

The pool increases in direct proportion to increases in synthesis, as long as the FTR is constant. In contrast, the pool decreases as turnover increases, if the increase in turnover is not fully compensated by increases in synthesis. In this model, the size of the bile acid pool is a result of the numerous physiologic and regulatory processes that alter FTR and synthesis (Fig. 2). For example, during human pregnancy, the size of the pool of bile acids increases in direct proportion to expansion The presumed mechanism of the volume of the gallbladder.b5-0L of pool expansion is increased storage of the pool within the gallbladder, resulting in a reduction in the circulating bile acid pool (number in the EHC decreases), which, undcr conditions of unaltered absorptive efficiency by the intestine, leads to a decreased FTR.

METHODS USED TO MEASURE CHOLESTEROL BALANCE IN HUMANS Absolute Balance In this technique the actual inputs and outputs to the - ' ~ ~ ~ ~ - 'bal~ body's cholesterol pool are m e a s ~ r e d . ~ ~ Cholesterol ance is measured after subjects have been ingesting a diet containing a defined amount of cholesterol for a period of time (more than 4 weeks). Balance is defined as the difference between dietary intake and total steroid excretion. Negative balance occurs when fecal excretion of sterols exceeds the dietary intake of cholesterol and positive balance occurs when dietary intake of cholesterol exceeds fecal excretion of sterols. Negative balance implies removal of cholesterol from the body and

positive balance implies that cholesterol is accumulating in the body. The excretion of endogenous neutral sterols is determined by administering radiolabeled cholesterol intravenously and then, several days later, measuring the specific activity of cholesterol in plasma and the amount of radioactivity in fecal neutral sterols. Excretion of endogenous neutral sterols is calculated by dividing fecal radioactivity by plasma-specific activity. The fraction of dietary cholesterol that escaped absorption is calculated by subtracting the excretion of endogenous neutral sterols fmm total fecal neutral sterols. Conversely, the fraction of dietary cholesterol that is absorbed is dietary cholesterol intake minus fecally excreted dietary cholesterol. Daily cholesterol turnover is equivalent to the sum of fecally excreted endogenous neutral sterols and bile acids. The disposition of all of the dietary cholesterol is unknown in those subjects in positive cholesterol balance. Therefore, turnover can only be determined in those subjects with zero or negative cholesterol balance. The major advantage of the absolute balance method is that the actual inputs to and outputs from the body's pool of cholesterol are directly determined. In addition, confinement to a metabolic ward ensures accurate definition of cholesterol intake from the diet. However, the absolute balance method has several disadvantages. The validity of the results is dependent on several conditions that are difficult to achieve. Only individuals who are willing to conform to hospitalization in a metabolic unit and rigorously adhere to prescribed, often liquid formula, diets can serve as study subjects. Hospitalization of subjects on these metabolic units is commonly prolonged, from weeks to months. The accurate determination of balance is dependent on analysis of sterols from appropriately timed collections of feces. Subject compliance with multiple collections of fecal samples may be difficult at times and nursing staff commonly object to the unpleasantness inherent in collection and storage of feces. Finally, because of the above difficulties, the number of subjects reported in studies of absolute balance is characteristically small.

Relative Balance Because of the difficulties in performing absolute balance studies, a number of investigators have developed practical noninvasive techniques for assessing nearly all of the physiologic pathways that regulate human cholesterol homeostasis. These techniques asscss cholesterol metabolism with a minimum of discomfort and inconvenience for study subjects. However, absolute inputs and outputs to the cholesterol pool are not determined, the relative change or difference is used to define the effects of disease, treatment, or condition on cholesterol balance. The methods used in relative balance studies are described. Dietary intake of cholesterol is defined by validated techniques'' that utilize dietary records and computer-assisted analysis of records to calculate dietary composition. Typically, subjects record their diets on 3 separate days of the week for 2 consecutive weeks and commercially available programs, such as Food Processor 11, (ESHA Research, Salem, OR) are used to determine dietary cholesterol intake. The fractional absorption of cholesterol is determined by dual isotope technique' and absolute absorption is the product of fractional absorption and dietary cholesterol intake. The measurement of fractional absorption by dual isotope technique requires the administration of one radioisotope of cholesterol ('H) orally and a second (I4C), intravenously. Fractional absorption is equivalent to the ratio of radioactivity ('H/I4C) measured in plasma cholesterol 24 to 72 hours after isotope administration.

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BILE ACID METABOLISM-EVERSON

Currently, no technique exists for noninvasively and directly measuring total body or hepatic cholesterol synthesis in intact humans. However, it has been well-established that relative changes in cholesterol synthesis by peripheral blood mononuclear cells mirror the changes that occur in total body and hepatic cholesterol synthesis."-"-" Mononuclear cells are isolated from 30 ml of freshly obtained blood and sterol synthesis quantified by measuring the incorporation of ['"Clacetate into sterols produced by the cells. Relative changes in sterol synthesis by monocytes are used to indicate the relative changes in total body and hepatic cholesterol synthesis. Serum bile acid kinetics are equivalent to hiliary bile acid kinetics and are measured using stable isotopes and isotope dilution-mass Stable isotopes ("C or ?H) of bile acids are given orally and allowed to equilibrate with the entire pool of bile acid. Samples of serum are obtained daily for 4 to 5 days after isotope administration. bile acids are isolated from serum, and the enrichment of the pool in the stable isotope measured by mass spectrometry. Although the serum method is analytically rigorous, subject compliance is excellent. The discomfort to the subject of five venipunctures is trivial compared with the repeated nasoduodenal intubation that is necessary when measuring bile acid kinetics from bile samples The only other significant output of cholesterol, biliary cholesterol secretion, can only be measured by placement of a nasoduodenal tube. perfusion of nonabsorbable marker, such as beta-sitosterol, and collection of duodenal bile." The rate of cholesterol secretion is calculated from the ratio of the concentration of cholesterol to concentration of marker multiplied by the marker infusion rate. The 24-hour secretion rate of cholesterol is extrapolated from the mean hourly secretion rate achieved during intestinal perfusion. There arc several advantages of relative balance methods over absolute balance methods. First, nearly all of the techniques mentioned, except for the measurement of biliary cholesterol sccrction, are performed with a minimum of discomfort and inconvenience, ensuring maximum compliance of subjects with the study protocol. Second, studies may be perfbrmed in the outpatient setting, eliminating the need for prolonged stays on metabolic units. Third, the techniques assess specif~cpathways and yield more precise information regarding potential l o c ~of regulation. Fourth, the relative easc of conducting thcsc studics allows assessment of larger numbers o l subjects as is required when trying to determine effects of trcatmcnts, hormones, drugs, or other interventions. The main disadvantage of the "relative balance" method is that absolute values for inputs and outputs to the body's cholesterol pool are not determined. In particular, it is not known whether cholesterol synthesis by rnonocytes reflects rates of synthesis by the liver or whole body under all experili~ental conditions. The assessment of biliary lipid secretion requires nasoduodenal intubation, which is cumbersome, time-consuming. and uncomfortable for the study subject. Finally, rates uf cholesterol secretion may vary with the stin~ulusused (protein versus fat) in the perfusion forn~ula.~'

CHOLESTEROL BALANCE IN SPECIFIC HUMAN CONDITIONS Gallstone Formation in Women The risk of developing cholesterol gallstones is increased by pregnancy, contraceptive steroids, and conjugated estrogen~.''-'~Although female steroid hormones significantly alter hepatobiliary physiology, the exact mechanisms whereby these hormones induce gallstones are incompletely defined. For this reason, we and others have studied the effects of a variety of

12, NUMBER 4, 1992

exogenous female steroid hormones on hepatobiliary physiology. Relative balance techniques were used to follow the disposition of dietary cholesterol in subjects during use of conjugate estrogen" (bemarin, Ayerst Laboratories, New York). Subjects were first studied on a low cholesterol diet (443 + 119 mollda day) both off and on Premarin. They were then switched to a high cholesterol diet (2021 + 262, pmollday) and again studied both off and on Premarin. The following were measured at each study period: dietary intake of cholesterol, fractional absorption of cholesterol, cholesterol synthesis by peripheral blood mononuclear cells, serum bile acid kinetics, and hepatic rates of secretion of biliary lipids, including cholesterol. Premarin did not alter intestinal absorption of cholesterol or cholesterol synthesis by peripheral blood mononuculear cells. In contrast Premarin increased the lithogenic index of bile (p

Bile acid metabolism and its role in human cholesterol balance.

SEMINARS IN LIVER DISEASE-VOL. 12, NO. 4, 1992 Bile Acid Metabolism and Its Role in Human Cholesterol Balance In intact humans, steady-state choles...
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