Metabolic pathways of bile acid synthesis

Metabolic Pathways of Bile Acid Synthesis

WILLIAM

The major pathway for the metabolism and excretion of cholesterol in mammals is the formation of acidic steroids in the liver. Although in principle the theme is the same, there are variations that make different species quite distinctive. Man synthesizes only two primary bile acids and conjugates each of these with either glycine or taurine to yield primary bile salts. The rat and mouse, which have served as the prime experimental model for man, can produce several additional bile acids. Although the end products of cholesterol metabolism are not the same, certain generalizations seem valid at present. In particular, considerable evidence has been amassed to implicate the 7cr-hydroxylation of cholesterol as the initial rate-controlling step in bile acid biosynthesis. The sequence of events following this step and possible alternate pathways are less well delineated. Because of the number of enzymes committed to the formation of bile acids from cholesterol, it is possible that genetic or acquired defects in their synthesis may result in clinically apparent liver disease.

H. ELLIOT-T, Ph.D.

PAUL M. HYDE, Ph.D.* St. Louis, Missouri

From the Department of Biochemistry, Louis University School of Medicine,

St. St.

Louis, Missouri 63104. This paper represents Bile Acids XXXIV in the series from this laboratory. The literature has been reviewed through March 1971. We are pleased to acknowledge financial support from the National Institutes of Health, Grant HE 07878. Requests for reprints should be addressed to Dr. William H. Elliott, Department of Biochemistry, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104. * Present address: Department of Biochemistry, Louisiana State University School of Medicine, 1542 Tulane Avenue, New Orleans, Louisiana 70112.

568

The pioneering papers of Schoenheimer and Rittenberg [1,2] involving the use of deuterium as an “indicator” in the study of human metabolism generated an entirely new era of investigation. The biosynthesis of deuterated cholic acid from deuterated cholesterol was first demonstrated by Bloch, Berg and Rittenberg in 1943 [3]. In the succeeding years, cholesterol has been shown to be converted into bile acids and alcohols in all vertebrates investigated [4]. This conversion takes place in the liver, and the bile acids thus formed are designated primary bile acids. In most mammalian species, cholic and chenodeoxycholic acids are the primary bile acids; in a few species of birds, fish and reptiles allocholic and allochenodeoxycholic acid are also produced in appreciable amounts [4]. The bile acids are conjugated with glycine and taurine, which are then designated as bile salts, and are excreted into the bile. In the intestines, microorganisms transform some of the bile salts into secondary bile acids, such as deoxycholic and lithocholic acids. The major portions of all bile acids in the intestines are absorbed and returned to the liver via portal blood: only a small fraction is excreted

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daily In the feces. The bile acids returned to the liver via this route of enterohepatic circulation may be further metabolized before reconjugation and excretion into the bile. One of the tools employed early by Rittenberg and Schoenheimer for the quantitation of stable isotopes, the mass spectrometer, has found a new and more extensive use in recent metabolic studies. Coupled now with the gas chromatograph with its ability to separate small amounts of structurally similar components, the gas chromatograph-mass spectrometer [5] is currently utilized not solely for the quantitation of an isotope, although it can do this elegantly, but also rather to identify microgram or smaller quantities of metabolites by their characteristic fragmentation patterns in the rapid scanning instrument [6,7]. The foresight of Bergstr’bm to envisage the value of the instrumentation of Ryhage and Stenhagen resulted in a valuable tool for modern biochemistry. Mass spectrums of bile acids and sterols emanated from the Karolinska Institute as early as 1960 [8,9]. More detailed reviews of the application of this important tool in studies on bile acids and sterols are available [lo-121. NOMENCLATURE Substituents attached to the nucleus of sterols and bile acids are located by the same numbering system.‘:’ Reduction of the double bond in ring B of cholesterol provides either 5P-cholestan3~-01 (5p-cholestanol, coprostanol or “coprosterol,” a reduction product of intestinal microorganisms found in the feces) or %cholestan38-01 (5cr-cholestanol, cholestanol or “dihydrocholesterol,” a ubiquitous companion of cholesterol). Derivatives of 5/j-cholanic acid (Figure 1) con-

II 3 &

tfO0I-i

4,

I 7

5g - Cholrnic Acid Figure

1.

‘> Epimers at a given position are designated by the Greek letters, 01.,p-, or E-; i.e., the configuration indicated by a dotted line and read as extending into the paper is designated as a-, whereas the solid line which is read as projecting from the paper is designated as @-. For the few remaining derivatives in which the configuration is unknown, the Greek letter < is used, and the bond is represented as a wiggly line.

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stitute the largest group of naturaliy occurring bile acids; thus, cholic acid is 30(,7(x,120-trihydroxy-5/3cholanic acid. Precursors of this group are several CL; acids, derivatives of 5,p-cholestanoic acid (or “coprostanoic acid”). A few 5n.cholanic acids have been identified [4,13], and these have been designated with the prefix allo (Greek, other) attached to the trivial name; thus allocholic acid is 3a!,7a,12oc-trihydroxy-5a-cholanic acid BIOSYNTHESIS EXPERIMENTAL

OF BILE ACIDSAPPROACH

The principal approach to elucidation of the pathway of synthesis of bile acids from cholesterol has involved a study of the metabolism of hypothetical intermediates (1) in the ani,mal (rat) with a cannulated bile duct, or (2) in subcellular liver fractions. Although other experimental animals including man have been used, the general pathway outlined in Figure 2 summarizes our knowledge based upon these two approaches. Danielsson [14] has commented on the danger of invoking intermediates because of their formation in in vitro systems. This is exemplified most recently with the identification of a sequence of 23-, 24- and 25-hydroxylated derivatives of the sterol side chain, most of which are undoubtedly not involved in the systematic degradation of cholesterol to the bile acids [15]. The primary bile acids are produced From cholesterol by the rat with a bile fistula, but the rate of synthesis of bile acids in this animal is some seven to fifteen times greater than in the normal rat. The sequence of nuclear hydroxylations, dehydrogenations and reductions is generally associated with enzymes of the microsomal and supernatant fractions of liver homogenates, whereas the oxidative steps in the side chain appear to be catalyzed by the mitochondrial fraction. By 1955 Bergstrijm and colleagues [16] had acquired sufficient data to suggest that modifications of the sterol nucleus preceded degradation of the side chain. In general the results of experimentation over the last sixteen years have supported this hypothesis. Recent reviews on this topic are those of Lindstedt [17], Danielsson and Einarsson [18], Danielsson [19], Danielsson and Tchen [20], and Haslewood [4]. CHOLIC ACID FROM CHOLESTEROL Present information indicates that major transformations of cholesterol in the pathway to cholic and chenodeoxycholic acids (Figure 2) involve the intermediate formation of 5p-cholestane-3a,7a,-

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? (D

l

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12a-trio1 (Vi) and 5p-cholestane-3a,7a-diol(Xli), respectively. To form these derivatives cholesterol is hydroxylated at the 7clc-position (step 1) as shown by Lindstedt [21] and Bergstrijm [22,23]. The specific microsomal enzyme [24,25] which catalyzes the reaction is stimulated by feeding the ion exchange resin, cholestyramine [26,27], and increased many fold by cannulation of the bile duct [28-301. (See Berseus et al. [31] for details of preparation of compound Il.) The dehydrogenation of the diol (II) to the conjugated ketone (III) (step 2) is catalyzed by the microsomal fraction [32,33] and requires nicotinamide adenine dinucleotide (NAD) [31,34]. The rate-limiting step in the reaction is the dehydrogenation of the 3p-hydroxyl group [35]. 7a-Hydroxycholest-5-en-3-one may be an intermediate in the process, but the formation of this substance has not been demonstrated. The unsaturated derivative (Ill) is a key intermediate in the formation of either cholic or chenodeoxycholic acid. Step 3 in the pathway to cholic acid involves hydroxylation of compound III at the 12cx-position [36] to form 7cu,l&~-dihydroxycholest-4-en-3-one (IV). The reaction is catalyzed by the microsomal fraction, requires reduced nicotinamide adenine dinucleotide phosphate (NADPH), but appears to be inhibited by phenobarbital. 7a-Hydroxycholesterol (II) can also be hydroxylated by the enzyme system to cholest-5-ene-3p,7a,l2&riol, but the reaction proceeds at a much slower rate. The formation of 5P-cholestane-3a,7a,12a-triol (VI) from compound (IV) involves saturation of the nuclear double bond (step 4) and reduction of the 3-ketone (step 5). These reactions are catalyzed by two soluble enzymes and require NADPH [31,37]. Tritium from [4A-“H] NADPH (but not from the 4BJH derivative) appeared in the sterol (V) at the 5P-position (Figure 3); the hydrogen added at C-4 is derived from the medium [38]. A 3cr-hydroxysteroid dehydrogenase which catalyzes step 5 has been purified. The tritium from [4A-“H] NADPH (but not the 4BJH derivative) appeared almost entirely at the 3p-position [38,39]. This trio1 (VI) was obtained earlier by Mendelsohn and Staple [40] with the 20,000 x g supernatant fraction from liver homogenates; the studies have been extended to human liver homogenates with similar results

r411. The mechanism of degradation of the side chain to complete the formation of bile acids is not well understood. The initial reaction (step 6) involving w-oxidation at position 26 is stereospecific [42-441,

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1971

ELLIOTT,

Mechanism of enzymatic reduction oxosteroid to a 3.0x0-5p- or a 3.oxo&-steroid Figure 3.

nicotinamide

moiety

of NADPH

HYDE

of a A43with

the

[140].

and is catalyzed by the mitochondrial fraction of rat liver homogenates. Danielsson [45] identified the tetrol (VII) and the acid (VIII) as metabolites of the trio1 (VI) after incubation with mouse liver homogenates. Staple and colleagues [46] showed that NAD was necessary for the formation of the acid (VIII) from the tetrol (VII) (step 7) with a dehydrogenase from the supernatant fraction. The acid (VIII), 3a,7ol,12a-trihydroxy-5p-cholestan-26oic acid, has been identified in the bile of the alligator and in human bile [44,47], and after administration of radioactive cholesterol [48-501. Step 8 probably involves P-oxidation of the side chain at position 24 to form 301,7a,12a,24.&tetrahydroxy-5p-cholestan-26-oic acid. Hydroxylation at C-24 is catalyzed by enzymes in the mitochondrial fraction supplemented with the supernatant fluid, but subsequent reactions are catalyzed by enzymes in the supernatant fluid [44]. Masui and Staple [51,52] showed that a synthetic tetrahydroxy acid (IX) can be converted to cholic acid with the 105,000 x g supernatant fraction fortified with NAD or NADP. Tanaka [53] found similar conversion of the synthetic acid (IX) to cholic acid in the guinea pig with a cannulated bile duct. The final step 9 requires adenosine triphosphate (ATP) and coenzyme A (CoA) to provide propionyl CoA and cholyl CoA [44,46], and the latter is then conjugated with glycine or taurine to provide the bile salt. CHENODEOXYCHOLIC

ACID FROM CHOLESTEROL

The formation of chenodeoxycholic less well understood, but the central

acid (XIII) is role of 7a-hy-

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droxycholest-4-en-3-one (III) in the pathway to cholic or chenodeoxycholic acids is established. The determination whether this intermediate undergoes 12cr-hydroxylation or reduction of the nuclear double bond may well be the controlling factor toward formation of either of these two acids [54]. The evidence at hand suggests that the nuclear double bond and the 3-keto groups are reduced in that order to 5P-cholestane-3a,7a!-diol (XII) 7311. Partially purified preparations of the soluble enzymes, 3-oxo-A4-steroid 5p-reductase and 3a-hydroxysteroid dehydrogenase catalyze the conversion of 7a-hydroxycholest-4-en-3-one into the diol (XII) [39], supporting earlier observations of Mendelsohn et al. [55,56]. The saturated ketone, 7a!-hydroxy-5p-cholestan-3-one, was identified as an intermediate in the conversion of the unsaturated ketone (III) to the diol XII by the supernatant fraction from guinea pig liver homogenates [57]. This diol (XII) has been isolated from human bile after administration of a tracer dose of cholesterol [58]; the major biliary acid derived from this diol is chenodeoxycholic acid [59]. A sequence of steps comparable to 6 through 9 results in degradation of the side chain with formation of the substituted cholanic acid and propionic acid, presumably each in the form of a CoA derivative. A substance with chromatographic properties corresponding to 5p-cholestane-3a,7a,26-triol was detected after incubation of the diol (XII) with 20,000 g supernatant fluid [57], but the details of degradation to chenodeoxycholic acid are not presently known. Chenodeoxycholic acid is not hydroxylated in mammals to cholic acid [14], but the python [60], the eel [61] and the chicken [62] have been found to form some cholic acid from chenodeoxycholic acid. On the other hand, this acid (XIII) is metabolized by the rat and mouse to a-muricholic acid (XV) and p-muricholic acid (XVI) [63,64], the 3(~,6~,70!-trihydroxyand 3cr,6P,7p-trihydroxy-5,$ cholanic acids, respectively. p-Muricholate is formed from cY-muricholate [65] presumably through a 7-keto intermediate [66]. In the germfree rat 60 per cent of the fecal bile acid fraction is p-muricholate conjugated with taurine [67]. The 6,&hydroxylating system of the rat can be induced to provide detectable amounts of 3a,6p,12a-trihydroxy-5pcholanic acid as a metabolite of deoxycholic acid in the surgically jaundiced rat [68,69]. In the pig, which makes no cholic acid, chenodeoxycholic acid undergoes Ga-hydroxylation to form hyocholic acid (3a,Ga,7a-trihydroxy-5pcholanic acid) [4].

572

An alternate pathway to bile acids was proposed by Mitropoulos and Myant [70] to explain the identification of each of the following derivatives after incubation of cholesterol with mitochondrial and supernatant fractions. Cholesterol is converted via 26-hydroxycholesterol, 3p-hydroxy-cholest-tien-26-oic acid [71] and 3P-hydroxychol-5-enoic acid to lithocholic acid and thence to its metabolites 3a,6p-dihydroxy-5pcholanic acid, chenodeoxycholic acid and iy- and p-muricholic acids [72]. Little [73,74] or no cholic acid [75,76] has been found after administration of 26-hydroxycholesterol to rats, but the importance of the pathway is not known. Enteral microorganisms remove the 7a-hydroxy group from cholic and chenodeoxycholic acids to form deoxycholic acid (Xl) and small amounts of lithocholic acid (XIV), respectively [77]. In the pig similar 7a-dehydroxylation of hyocholic acid affords hyodeoxycholic acid (3a,6a-dihydroxy-5Pcholanic acid) [78]. A study of the intracellular localization of bile acids in rat liver [79] clearly indicated an important role for the cytoplasmic compartment. More than 70 per cent of the bile acids were found in this portion of the cell; the conjugated acids would be expected in this fraction because of their greater water solubility prior to release into the bile ducts. ALL0 BILE ACIDS The recent discovery of allo-bile acids, particularly allocholic acid [4,13], has stimulated an interest in these new derivatives. Although allocholanic acid and a few of its derivatives appeared first from the productive studies of Heinrich Wieland and Adolph Windaus prior to 1920, renewed interest in these 5acholanic acids must be attributed to Haslewood and colleagues in England, and Kazuno and Yamasaki and their colleagues in Japan [13]. With identification and synthesis of allocholic acid from the bile of the Gigi fish [80], this acid has now been recognized in the bile of the leopard seal, chicken, several fish, reptiles and birds [4,81,82], and has been obtained from mammals including man [4,83]. Allocholic acid is the major acidic biliary metabolite of cholestanol in the rat [84] and in the gerbil [85]. Glycoallodeoxycholic acid is a constituent of gallstones in rabbits fed a diet containing 1 per cent cholestanol [86]. Allochenodeoxycholic acid and its 3P-isomer were subsequently identified as biliary metabolites of cholestanol in the rat [87]. Current information suggests three related path-

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ways of biosynthesis of these new acids: via cholesterol and cholestanol. via 5pcholanic acids and via other 50.sterols. 5a-CHOLANIC

ACIDS

FROM

CHOLESTANOL

The relatronship of cholesterol to cholestanol stems from early experiments of Schoenheimer, Rittenberg and Graff [2] and Rosenfeld and Webster [88] who suggested that Al-cholestenone was the intermediate between these sterols. Subsequently this conversion was demonstrated in vitro and in vivo; a soluble enzyme from the supernatant fraction of rat liver homogenate fortified with NADP or NAD catalyzed the formation of AQholestenone [89]. The 5a-reductase necessary to provide 5crcholestanone was shown to require NADPH and was localized in the microsomal fraction as is the 3phydroxy reductase [90,91]. The location of these enzymes should be contrasted with the 5P-reductase of the supernatant fraction in step 4; the equatorial alcohol is the major product from the action of either the 50(- or 5p-reductase. Shefer et al. [26] showed that cholestanol (XVII) can be hydroxylated at the 7a-position at a rate comparable to that of cholesterol by microsomal enzyme systems fortified with NADPH and oxygen. Since the rate of hydroxylation of compounds I and XVIII can be enhanced by prior treatment of the animal with cholestyramine or phenobarbital, additional studies are necessary to ascertain whether the same enzyme system is active on both substrates I and XVII. 7a-Hydroxycholestanol (XVIII) can also be derived from 7a!-hydroxycholest4-en-3-one (Ill) by a microsomal fraction of rat liver homogenate via cis addition [92]; the tritium of [4B-“HI-NADPH is found only at the 5or-position, and the 4cu-hydrogen is derived from the medium (compare step 4). A comparison of the stereochemistry of the reduction of 3-oxo-A4-steroids to 3-0x05p- or 5custeroids with NADPH is shown in Figure 3 [92]. In the rat with a cannulated bile duct 5acholestane-3p,7a-diol (XVIII) is converted to allocholic and allochenodeoxycholic acids and their 3p-isomers [93]. Bjijrkhem and Gustafsson [94] concluded that 7a-hydroxylation precedes epimerization of the 3p-hydroxy group and the 12a-hydroxylation to provide 5cu-cholestane-3cy,7or,l2a-triol (XXI). Most efficient hydroxylation of the 3a,7a-diol (XX) occurred with a microsomal fraction fortified with NADPH. Epimerization of the 3p-hydroxyl group (XVIII) was catalyzed by a microsomal NAD-dependent 3p-hydroxysteroid dehydrogenase and a soluble NADP-dependent 3a-hydroxysteroid dehy-

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drogenase to provrde the 3au,7a.diol (XX). Allocholic acid (XXII) was formed in rats with cannulated bile ducts after administration of compounds XIX or XX (Figure 2). If this suggested pathway is shown to be correct, the question of identity of the enzymes in this pathway with those in the formation of cholic acid will warrant fruitful investigation. Presumably, a mechanism for the formation of allochenodeoxycholic acid is comparable to that af chenodeoxycholic acid. Data in Table I show the percentage conversion of several derivatives of cholestanol to allocholic and allochenodeoxycholic acids and their 38-epimers in the bile fistula rat. Clearly, cholestanol is a superior precursor of the trihydroxy acids in this species compared to its 26-hydroxy derivative. A comparable ratio of trihydroxy to dihydroxy acids appeared for the 3~, 7a-diol and the 3p,7a,26-triol. On the other hand, the total quantity of dihydroxy acids greatly exceeded the trihydroxy acids derived from 3P,7or-di-hydroxy-5or-cholestan-26-oic acid, suggesting that this acid is not on the pathway to the 3,7,12_trihydroxy acids [95]. More information is necessary to delineate fully the pathway of synthesis of these acids and to ascertain a relationship to the synthesis of the 5pcholanic acids. Since the rat metabolizes chenodeoxycholic acid to measurable quantities of CY-and ,#-muricholic acids, the question of similar metabolism in the allo-acid series is pertinent. Allochenodeoxycholic acid was recovered largely unchanged in the rat with a cannulated bile duct [96,97], but allocholic acid (-4 per cent) was identified as a biliary metabolite [97]; a companion acid showed mobility comparable to 3cu,6a,7a-trihydroxy-5a-cholanic acid, suggesting a-attack on the underside of the planar molecule at positions 12a- and 6a- rather than Gp-hydroxylation [97]. These observations indicate a significant difference in the metabolism of the planar 5crsteroid compared to the 5pTABLE

I

Per Cent Conversion of Substituted 5a-Cholestanes to Allocholanic Acids in the Bile Fistula Rat Allochenodeoxvcholic Acid

Derivative of 5a-cholestane 3p-01 3&7a-diol 3-0x0-7wol 3-oxo-701,1201-diol 3p-26.diol 3&7a-26-triol 3p,7a-diol-26-oic

301

3p

11 26

8 5 Minor

acid

. 20 28 64

... 16 4 16

Allocholic Acid 3a 70 49

38 7 3

18%931 WI I1041 [IO41

1.8 5 l-3

D381 I1391 WI

Major Major 16.5 44 8-12

Refer. ence

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OF BILE ACID SYNTHESIS-ELLIOTT,

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derivative. The significance in terms of the quantity of allocholic acid derived from allochenodeoxycholic acid warrants further study. From investigation on the metabolism of a series of 12-deoxybile acids (cholanic #acid [98], lithocholic acid [74], chenodeoxycholic acid [64], hyodeoxycholic acid [99], 3a,6P-dihydroxy-5p-cholanic acid [loo] and 7-ketokthocholic acid [66]) allochenodeoxycholic acid is the only known example of permissive 12~ hydroxylation in the rat. Like the 5P-epimer, allocholic acid undergoes loss of the 7cr-hydroxyl group in the intestine due to microbial metabolism to form allodeoxycholic acid [96,101]. Glycoallodeoxycholic acid is a poor detergent, causing crystallization of salts of fatty acids and deposition of gallstones in the rabbit on a diet of 1 per cent cholestanol [86]. In the rat allolithocholic acid and allodeoxycholic acid undergo 7a-hydroxylation in the liver to provide allochenodeoxycholic and allocholic acids, respectively [96,102-j. Finally, it should be noted that the dihydroxycY,p-unsaturated ketone (IV) may also enter into the formation of allocholic acid. An NADPHdependent microsomal fraction catalyzed the formation of 7&,12a-dihydroxy-5a-cholestan-3-one [103,104]; as was the case with reduction of 7’a-hydroxycholest-4-en-3-one, liver microsomes from female rats were about four times as active as those from male rats. Incubation of this ketone with the microsomal fraction and NADPH or NADH provided 5~-cholestane-3p,7~,12a-triol. Enzymes in the soluble fraction afford 5a-cholestane-3cr,7’a,l2a-triol (XXI). 5aCHOLANIC

ACIDS

FROM

5@HOLANIC

ACIDS

The formation of allo-bile acids from 5p-cholanic acids has now been satisfactorily explained. The identification of allodeoxycholic acid in the feces of rabbit as a major metabolite following intraperitoneal administration of deoxycholic acid [105] was clarified by Kallner’s observation [102] that this transformation occurred during enterohepatic circulation. In subsequent experiments Kallner established the conversion of deoxycholic acid in the cecum to lillclc-hydroxy-3-oxo-chol-4-enoic acid which could be reduced by the microsomal fraction of rat liver homogenate fortified with NADPH to the 3-oxo-5ct+acid, whereas the supernatant fraction provided deoxycholic acid. Thus, the intracellular localization of the Sp- and 5areductases which catalyze the reduction of the nuclear double bond in these unsaturated bile acids is the same as

574

that found for the Ca7 steroids. Following intraperitoneal administration of 12a-hydroxy-3-oxo-chol-4enoic acid, both deoxycholic and allodeoxycholic acids were isolated from bile [106]. On the other hand, administration of allodeoxycholic acid to the rat either intraperitoneally or intracecally provided allocholic and allodeoxycholic acids, demonstrating the conversion of the dihydroxy acid to the trihydroxy acid via liver enzymes, analogous to a similar conversion in the 5pseries. Similarly, the four isomers of 3-hydroxycholanic acid of the Spand 5crseries were identified in bile of rats after administration of 3-oxo-chol-4-enoic acid [107]; a mixture of 3,6-dihydroxy-licr-cholanic acids was reported following administration of 3p-acetoxychol5-enoic-24J4C [108]. 5aCHOLANIC

ACIDS

FROM

OTHER

5aSTEROLS

Allocholic acid occurs as a high proportion of the bile acids in certain lizards and to a lesser extent in various fish and birds [4]. A mechanism for the formation of 5orcholanic acids from the 5~ alcohols indigenous to these species has been studied by Hoshita [log] and Amimoto [l lo]. Thus, 5a-cyprinol (5a-cholestane-3a,7cr, 12@,26,27-pentol) was metabolized by the giant salamander to allocholic acid and an acid tentatively identified [ill] as 3a!,7a,12a!,264etrahydroxy-5wcholestatv26-oic acid. Okuda et al. [112] reported the isolation of a metabolite from bile of the iguana which appears to be 3a,7a,12a-trihydroxy-5,c-u~cholestan-26-oic acid. From these and earlier studies it appears that a pathway for biosynthesis of the allo-bile acids from preformed 5asterols may be similar to that outlined in Figure 2 from cholestanol. More detailed reviews of this subject and of the bile alcohols are available [13,113]. FORMATION MICROBIAL

OF SECONDARY METABOLISM

BILE

ACIDS;

Enteral mitcrobial metabolism of the bile salts is initiated by deconjugation to free the bile acid and proceeds via dehydroxylation at the 7a-position to provide deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. Although many of the strains of bacteria and fungi isolated from the human gastrointestinal tract possess the deconjugating enzyme(s) [114], only a few species of Streptococcus faecalis, bacteroides, Clostridia and Veillonella have the ability to remove the 7a-hydroxyl group. Mechanistically, it has been shown [23] that cholic acid labeled with

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::H specifically at positions 6a,6p,7a, or Sp undergoes an elimination of the Gp-hydrogen and 7cyhydroxy group to form the double bond at position 6-7. This is further reduced by transhydrogenation [70] to saturate positions 6 and 7. The same reaction sequence has been shown for the conversion of hyocholic acid to hyodeoxycholic acids [71]. Other enzymatic reactions taking place in the intestinal tract attributable to microorganisms include the oxidation of alcohols to ketones at positions 3, 7, and 12 of the bile acids [6,115]. This enzymatic activity may sufficiently alter some of the bile acids so that they will pass into the large intestine for further degradation [116,117]; others are reabsorbed as acids, primarily in the ileum in man [118], and undergo enterohepatic circulation. After absorption across the intestinal mucosa, the hemodynamics of portal blood flow, more than 500 times that of intestinal lymph, and the greater availability of binding sites for acids on the proteins in blood than in lymph, show that the portal venous system is virtually the exclusive carrier of bile acids to the liver [119]. CONTROL

OF BIOSYNTHESIS

Bile acid synthesis is believed to be controlled by at least two feedback mechanisms [120]: the amount of bile acids reaching the liver via the portal vein in enterohepatic circulation and the amount of cholesterol absorbed from the diet. The half-life of bile acids has been estimated to be from two to three days in man with a daily synthesis of about 500 to 600 mg of bile acids [121], of which cholic acid accounts for about 350 mg [122]. The evidence for bile acid feedback control of bile acid synthesis is strong. Diversion of bile acids from the intestine by bile duct cannulation increases the bile acid excretion rate seven- to fifteenfold as shown initially in rats by Eriksson [29] and later by others [123-1251. Sequestration of the bile acids by cholestyramine increases bile acid excretion and cholesterol catabolism when studied in the steady state [126]. In addition, Grundy and co-workers [127] showed that the administration of bile acids suppresses endogenous bile acid production in man. Removal of the ileum in man [127] has been shown to prevent reabsorption of bile acids and to increase bile acid production and cholesterol turnover. Hofmann and Poley [128] studied the efficacy of cholestyramine for treatment of diarrhea in patients with ileal resection.

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A major locus for this feedback regulation of biosynthesis of bile acids was established as step 1, the 7w-hydroxylation of cholesterol. Mosbach et al. [129] reasoned as follows: If animals with intact or interrupted enterohepatic circulation convert a given substrate to bile acids at the same or comparable rates, a rate-limiting process must not have occurred. Alternately, if a rate-limiting process had occurred, the rate of conversion of the substrate into bile acids will be smaller for the animal with intact enterohepatic circulation than in the animal with biliary diversion. They determined that intraduodenal infusion of sodium taurocholate at a rate of 11 to 14 mg/lOO gm of rat/ hour inhibited bile acid production in the rat with a cannulated bile duct and showed that this inhibition was approximately 90 per cent from either of three substrates, acetate-l-l%, mevalonolactone-2-J% or cholesterol-4-l+C. Similar experiments with 7a-hydroxycholesterol-4-l% showed an inhibition of less than 10 per cent. The mitochondrial system involved in shortening the side chain (steps 6 through 9) and the 12a-hydroxylation of the 7cr-hydroxycholest-4-en-3-one key intermediate, (III) have also been suggested as secondary sites for regulation. Thus, the quantity of bile acid formed is no longer regulated by the circulating bile acid once 7a-hydroxycholesterol (II) is formed, but the regulation of the amount of dihydroxy or trihydroxy primary acid formed may be determined by the enzymes involved in step 3. The formation of more chenodeoxycholic acid at the expense of cholic acid in the hyperthyroid animal [130] is apparently related to this problem. The manner in which bile salts control or affect the activity of the 7au-hydroxylase is not presently known nor is the identity of the bile acid or bile salt responsible for this feedback control [131]. A half-life time of two to three hours for the breakdown of the 7a-hydroxylase in the bile fistula rat has been reported [132]. Strong evidence exists for the relation of control of bile acid production and absorption of cholesterol. Abell, Mosbach and Kendall [133] first showed that increasing the dietary cholesterol of a dog could increase bile acid synthesis up to fourfold. Similar results were obtained in the rat [134,135] but not in the rabbit [136]. Whether or not man can increase his bile acid production after cholesterol ingestion is not clear [137] because of hepatic compensation by decreasing hepatic cholesterol synthesis. Clearly, the complex regulatory control of bile

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acid production must be linked to cholesterol synthesis in liver and extrahepatically, as well as to cholesterol catabolism into bile acids and neutral steroids. In summary, many of the intermediates in the main pathway from cholesterol to the primary bile acids are now identified, particularly those in the sequence of transformation of the steroid nucleus. A mechanism for formation of secondary bile acids in the intestine offers an explanation for the occurrence of these acids. A pathway to the newer allo bile acids is indicated. Feedback control of

biosynthesis of bile acids is now established at step 1, 7a-hydroxylation of cholesterol. On the other hand, the details of formation of the acidic moiety from the branched alkyl side chain are not completely delineated, the quantitative importance of alternate pathways of biosynthesis and the biologic significance of chenodeoxycholic acid and the allo acids are not clear, and the importance of other suggested sites of control of biosynthesis has not been established. These represent but a few of the areas in which renewed effort in investigation can be made.

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