Clinical and Experimental Aspects of Bile Acid Metabolism

Clinical and Experimental Aspects of Bile Acid Metabolism From the Department of Medicine, St. Luke's Hospital, New York, N. Y.

THEODORE B. VAN ITALLIE, M.D. Associate Clinical Professor of Medicine, Columbia University, College of Physicians and Surgeons,. Director of M edicine and Attending Physician, St. Luke's Hospital

SAMI A. HASHIM, M.D. Assistant Professor of Nutrition, Columbia University, Assistant Attending Physician, St. Luke' 8 Hospital

FOR MANY years the bile acids were thought of almost entirely in terms of their role in fat digestion and absorption. Since 1943, when Bloch, Berg and Rittenberg10 demonstrated a direct metabolic relationship between cholesterol and cholic acid, the emphasis has changed remarkably. It is now recognized that the bile acids comprise the major products of cholesterol oxidation and that the enterohepatic cycle of bile acids exerts an important regulatory influence on cholesterol metabolism. Thus, investigators in the field of lipid biochemistry have increasingly turned to the sequence of reactions involved in the transformation of cholesterol to bile acids and to the enterohepatic bile acid cycle when attempting to explain the influence of certain diets, hormones and drugs on serum cholesterol levels. Students of liver disease have sought to find information of diagnostic and prognostic value in measurements of 'serum bile acid levels and partitions. Attempts have been made to influence bile acid metabolism by administration of nonabsorbable resins capable of sequestering bile acids in the intestinal lumen, thereby promoting their excretion in the feces. Such agents appear to have promise in the management of the pruritus that can occur in primary biliary cirrhosis and certain other forms of cholestasis. They may also prove to be useful in the treatment of a variety of hypercholesteremic states. 629

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CHEMISTRY

The bile acids are steroidal monocarboxylic acids which derive from cholesterol. 5, 10,22,52 In human bile, the predominant bile acids are cholic, chenodeoxycholic and deoxycholic acid, all in conjugated form. Bergstrom 7 has referred to cholic acid and chenodeoxycholic acid as "primary" bile acids since they are manufactured as end products by the human liver. Deoxycholic acid is a bacterial metabolite of cholic acid that is reabsorbed from the intestinal tract and re-excreted by the liver. The "primary" bile acids and some of their bacterial products, the "secondary" bile acids, are shown in Figure 1. In bile and in the upper gastrointestinal tract the bile acids are found conjugated with glycine or taurine. Sj6va1l54 , 55 reported that the ratio between glycine and taurine conjugates in the gallbladder bile of human subjects averaged 3.2: 1, with marked variation. However, all three acids were conjugated with glycine and taurine in approximately the same proportions. The ratios between glycocholic, glycochenodeoxycholic and glycodeoxycholic acids in gallbladder bile were 1.1: 1.0: 0.6. Conjugation of the bile acids appears to be carried out by liver microsomes and involves "activation" of the acid with coenzyme A to form cholyl-CoA, followed by condensation with the NH 2 group of either glycine or taurine to form an amide linkage,13, 20 for example: (1) R-COOH

+ ATP + CoASH ~ R-CO-SCoA + AMP + PP + H N-CH -COOH ~ R-CO-NH-CH -COOH +

(2) R-CO-SCoA

2

2

2

CoASH

In solution, the conjugated bile acids are present as anions or, depending upon the amino acid moiety and pH, associated with hydrogen. The term "bile salt" refers to the dry matter obtained from various biological sources. Bile acids are freely reabsorbed in conjugated form and the liver apparently does not alter the peptide linkage during the recycling process. 40 Normally, bacteria in the large intestine hydrolyze the bile acid conjugates and hence the bile acids appear in the feces only in the free form. 41 Three-dimensional models of the bile acids show that these molecules are constructed with their hydrophobic hydrocarbon groups predominantly on one side and the more polar hydrophilic groups on the other. It is this orientation (with a "foot in each camp") that appears to account for the surface-active properties of the bile acids, properties useful in promoting emulsification of fats and formation of micellar complexes with other lipids. 30 According to one source,57 hepatic bile contains 200 to 1830 mg./IOO m!. of bile acids, while gallbladder bile contains 1500 to 10,000 mg./lOO ml. The cholesterol content of hepatic bile is 80 to 170 mg./IOO ml., and of gallbladder 350 to 930 mg./IOO ml.

Clinical and Experimental Aspects of Bile Acid Metabolism

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27 26

CHOLESTEROL

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DEOXYCHOLIC ACID

CO-NH·CH 2 -COOH (GLYCINE CONJUGATES) \I ) -NH.CH 2 -CH 2-S0 2 0H (TAURINE

CONJUGATION IN LIVER Fig. 1. Conversion in the liver of cholesterol to "primary" bile acids (cholate and chenodeoxycholate). Intestinal bacteria modify primary bile acids further, with formation of such "secondary" acids as deoxycholate and lithocholate. Conjugation of bile acids with glycine or taurine takes place in the liver.

Pharmacologically, the bile acids are potent choleretic agents. Like the saponins, they irritate mucous membranes and have a digitalis-like effect on the heart. Administered by mouth, the bile acids appear to be nontoxic; when injected into the circulation they can produce severe nervous and cardiac depression and hemolysis. 56 Recently, Palmef et a1. 44 have reported that lithocholic acid, either in the free or conjugated

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forms, is capable of producing intense local inflammation when injected intramuscularly into normal human subjects. SYNTHESIS

The steps which must take place in order to transform cholesterol to cholic acid can be inferred from the structures of the two molecules (Fig. 1); however, the order in which these events occur has not been determined with finality. Apparently, both liver mitochondria and supernatant components are responsible for the catabolism of cholesterol to bile acids. There is evidence that in the formation of cholic acid the steroid nucleus is hydroxylated before oxidation of the side chain is completed. According to Bergstrom, 9 introduction of the 7a and 12a-hydroxyl groups precedes inversion of the 311-hydroxyl group and saturation of the Li5-double bond. Subsequently, the side chain isopropyl group is removed with oxidation of C-24 to a carboxyl (Fig. 1). Suld et a1. 58 have reported in vitro experiments strongly suggesting that the last three carbons of the side chain are split off as a unit by thiolytic cleavage of the bond between carbon atoms 24 and 25. In this way, propionyl coenzyme A and cholyl coenzyme A are formed from a postulated 27 carbon intermediate 3a, 7a, 12a-trihydroxy-24-keto-511-cholenstan-26-oyl coenzyme A. Although an active enzyme system capable of introducing a 7ahydroxyl group into deoxycholic acid has been found in rat liver, the human liver does not appear to be capable of converting deoxycholic to cholic acid. 9 This fact, perhaps, affords additional evidence that in man, at least, further hydroxylation cannot take place once the side chain has been oxidized. The existence of a close metabolic relationship between cholesterol and the bile acids was suspected by Lifschutz36 as early as 1914, and proven by Bloch et al. lO in 1943. Subsequent investigators have worked out the quantitative aspects of this relationship and have shown in rats with bile fistulas that as much as 80 to 90 per cent of administered cholesterol can be metabolized to bile acids, the remainder being excreted in the feces as neutral sterols. 18 • 52 In the intact animal, the nature of the diet may influence the proportion in which bile acids and neutral sterols are formed from cholesterol. A small, undetermined fraction of cholesterol may be used in the synthesis of steroid hormones. 51 The possible existence of some bile acid precursor other than cholesterol has been considered. Bergstrom et al. 9 have concluded that as yet there is no evidence to warrant postulation of another bile acid precursor. Under normal metabolic circumstances this may prove to be the case; however, during triparanol administration the desmosterol that accumulates apparently can be converted directly to bile acid. 24 • 33

Clinical and Experimental Aspects of Bile Acid Metabolism

633

CHOLESTEROL

~ approx. 0.8 gm/day

BILE ACIDS

\

GUT

approx.O.8gm/day in feces

Fig. 2. Enterohepatic cycle of bile acids. (Adapted from Bergstrom et a1. 9 )

ENTEROHEPATIC CYCLE

Unlike bile pigment, the bile acids are more than metabolic waste products: they play an important role in digestion and absorption. There exists an efficient mechanism that permits salvage and reuse of all but a small fraction of the bile acids that pass through the common bile duct into the duodenum each day. On the basis of figures for half-life and pool of cholic and chenodeoxycholic acids, Bergstrom 6 has estimated that approximately 0.8 gm. of bile acids is synthesized from cholesterol per day. This quantity enters a pool of perhaps 3 to 5 gm., which circulates and recirculates through the biliary tract, small bowel, cecum and portal vein (Fig. 2). It has been suggested that the traffic of bile acids through this enterohepatic cycle totals 20 to 30 gm. per day. An amount approximately equal to that manufactured (0.8 gm.) is lost each day in the feces. (It must be kept in mind that these figures are based on limited data and may very well require revision when more extensive studies have been made.) At the same time, roughly 0.4 gm. per day of neutral sterols is excreted in the stool. It is believed that, taken together, the fecal sterols and bile acids (correcting for dietary sterols) can account for all but a very small fraction of the cholesterol lost each day from the body. A number of reports have emphasized the important influence the enterohepatic cycle of bile acids can have upon cholesterol metab-

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olism. 7 • 46, 59 A classical study to illustrate this fact was performed in rats by Bergstrom and Danielsson. 8 They showed that when the enterohepatic circulation of the bile acids is interrupted by cannulation of the bile duct with formation of an external fistula, there follows a markedly increased synthesis of bile acids (12 times the normal rate). When 5 to 10 mg. of taurochenodeoxycholate was infused into the distal part of the severed bile duct, cholic acid synthesis was reduced to the range found in the norma] intact rat. These results indicate a homeostatic regulation of bile acid formation. Apparently when the concentration of bile acids in portal vein blood is reduced, the rate of bile acid formation will increase. Since the bile acids are derived from cholesterol, it is clear that an increased rate of synthesis of bile acids must involve the simultaneous degradation of cholesterol. Thus, the rates of synthesis and catabolism of cholesterol may be responsive in some way to the concentration of bile acids re-entering the liver from the portal vein. It has been reported 9 that in the rat cannulation of the bile duct, with the markedly increased bile acid synthesis that follows, does not result in any change in blood cholesterol levels. On the other hand, Byers and Friedman14 have reported that drainage of bile acids from the intestine in rats by ileal I\eimplantation of the bile ducts causes a fall in blood cholesterol. The ability of the liver to make up a bile acid deficit by synthesis of new cholesterol appears to vary among and within species. In any event, there is convincing evidence that the rate of cholesterol oxidation in the liver can influence profoundly the level of cholesterol in the serum. 59 Thus, the feeding of bile acids, which should overload the enterohepatic cycle and repress cholesterol oxidation, induces an increase in serum cholesterol levels in hypothyroid rats. 43 Beher and Baker3 have demonstrated an inhibitory effect of injected cholic acid on the incorporation of acetate-1-C 14 into liver cholesterol"in the rat, while Whitehouse and Staple 63 have found that conjugated bile acids added to the medium inhibit cholesterol oxidation to bile acids in liver slices. Conversely, a variety of procedures and agents that induce an increased loss of bile acids in the feces have been found capable of lowering serum cholesterol as well. 59 ROLE OF THE BILE ACIDS IN DIGESTION

The role of the bile acid conjugates in digestion and absorption has been extensively reviewed. 31 , 62 The surface-active properties of the bile acids have been referred to already. During digestion, the triglycerides, long-chain fatty acids and cholesterol are mixed with the bile and with other substances possessing both polar and nonpolar structures, such as lecithin, partial glycerides and ionized fatty acids. As a result, emulsions

Clinical and Experimental Aspects of Bile Acid Metabolism

635

and association colloids or micelles are formed which permit solubilization of otherwise nonpolar substances. Apparently, the bile acids together with the products of partial lipolysis play an important role in promoting hydrolysis of fats and absorption of cholesterol, conferring on certain products of digestion physical characteristics that facilitate absorption. It has been assumed by many observers that the bile acid conjugates promote digestion primarily by emulsifying substrate and thereby providing more surface for enzyme action. More recently, Borgstrom ll has suggested that the bile acids may not act primarily by emulsifying substrate but by keeping the reaction products in micellar form. Dawson and Isselbacher19 have studied the influence of bile acids upon the absorption and metabolism of long-chain fatty acids by the small bowel mucosa. Using an in vitro method, these investigators found that esterification of palmitate-l-C 14 was facilitated by adding taurocholate to the medium. In their studies, conjugated bile acids also stimulated incorporation of radioactivity of C 14-glucose into mucosal lipid. These results indicated that the conjugated bile acids promote fatty acid esterification, perhaps by directly influencing mucosal cell metabolism, in addition to any possible effect on absorption. INFLUENCE OF INTESTINAL FLORA ON ENTEROHEPATIC CYCLE

In the normal rat the half-life of the common bile acids has been calculated to be approximately two to three days.37 In rats treated with antibiotics that profoundly altered intestinal flora, Lindstedt and Norman38 found the half-life for cholic acid to be prolonged to about ten days. Similar prolongation of half-life of 24-C 14-cholic acid was observed by Gustafsson et a1. 27 in rats reared under germ-free conditions. Apparently, the intestinal microorganisms split the conjugated bile acids in the cecum and distally since germ-free rats and rats treated with appropriate antibiotics excrete only conjugated bile acids in the feces. 9 Another characteristic of certain intestinal bacteria is their ability to remove the 7a-hydroxyl group from cholic acid and chenodeoxycholic acid with the formation, respectively, of deoxycholic acid and lithocholic acid. While these latter acids appear to be the chief bacterial metabolites of the primary bile acids, a number of other bile acid derivatives presumably are also formed in the intestine. Only a few of these metabolites have been identified to date. Bergstrom 7 has emphasized that the intestinal microorganisms may actually incorporate certain bile acids such as lithocholate, or bind them so tightly that they are unavailable for reabsorption. It is also possible that deconjugation and dehydroxylation can reduce the polarity of the bile acids to a point where they are far less readily reabsorbed. Whatever the precise explanation, it seems likely that the intestinal

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flora are responsible for the attrition of a large proportion of the bile acids that "normally" escape the enterohepatic cycle and are lost in the feces. Goldsmith23 has reported that the administration to patients of neomycin in amounts of 0.5 gm. four times daily caused a three- to fivefold increase in fecal excretion of bile acids along with the previously demonstrated decrease in serum cholesterol levels. 50 Excretion of total sterols was not increased but during neomycin administration there was a change in the type of sterols excreted, with only cholestane compounds being present in the feces. (Normally, in the presence of intestinal flora, coprostane compounds predominate in relation to cholestane-type sterols.) Since neomycin is known to have a profound effect on the intestinal flora the change in character of the fecal sterols was not surprising. However, the lack of effect in man of a number of other antibiotics such as oxytetracycline50 on serum cholesterol does not seem consistent with the results obtained with neomycin and suggests that the cholesterol response to neomycin may be more complex than was first believed. Neomycin can have a damaging effect on the mucosa of the small intestine; moreover, the studies on germ-free animals suggest that other antibiotics destructive to the fecal flora would tend to reduce the rate of cholesterol turnover in the liver, not increase it. INFLUENCE OF DIET

In 1952, Groen et a1. 26 and Kinsell and co-workers34 showed in human subjects that substitution of vegetable for animal fats in the diet resulted in appreciable lowering of serum cholesterol levels. As these results ,vere refined and extended, it became increasingly apparent that the linoleic acid content of the vegetable oils was probably the positive factor responsible for the hypocholesteremic response. In subsequent studies, evidence has been presented suggesting that other polyunsaturated fatty acids substituted in the diet, essential and nonessential, also can exert a cholesterol-lowering effect.! Since cholesteryl linoleate is a predominant cholesterol ester in the serum, there has been speculation that an increased intake of linoleate might somehow inhibit cholesterol synthesis or make this sterol more available for oxidation and excretion. In this regard, Gordon et a1. 25 have noted in short-term experiments that when a linoleate-rich diet was fed to human subjects and cholesterol levels fell, a twofold increase in fecal bile acids occurred at the same time. Lewis35 studied patients with complete bile fistulas and found that oral or intravenous administration of fats rich in polyunsaturated fatty acids caused an increase in the rate of cholic acid excretion which preceded the drop in serum

Clinical and Experimental Aspects of Bile Acid Metabolism 300

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cholesterol. Long-term studies relating linoleate-rich diets to an increased rate of fecal bile acid excretion have yet to be reported. Fecal excretion of individual bile acids has been studied by Intengan et al. 32 in patients maintained on formula diets. It was demonstrated that the output of bile acids in the feces was greater during corn oil feeding than when coconut oil (a saturated fat) was substituted in the formula (Fig. 3). The predominant fecal bile acids were deoxycholic and chenodeoxycholic acids. Small amounts of lithocholic acid also were present. These studies suggested that the 7a-hydroxyl group of chenodeoxycholate was far less susceptible to bacterial removal than the 7a-hydroxyl group of cholic acid. It would seem that there are at least three mechanisms whereby dietary fatty acids may influence serum cholesterol: (1) Certain fats in the diet may influence the rate of cholesterol absorption and reabsorption from the intestinal tract. Wilkens 64 has shown by in vitro studies that cholesterol is more soluble in the more saturated fats and leES soluble in the highly unsaturated ones. His range of solubility percentages tends to parallel the serum cholesterol raising or lowering effects of the common fats and oils that have been studied. The efficiency of absorption of cholesterol from the gut also may depend on the composition of the fatty acid mixture available for esterification. In the intestinal

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mucosa of the rat, cholesterol apparently esterifies with different fatty acids such as oleate and linoleate at widely varying rates. (2) Dietary fatty acids may influence cholesterol metabolism by affecting the pattern of cholesterol esters in the serum and liver. Boyd 12 has suggested that the linkage of linoleic acid with cholesterol renders the steroid nucleus more subject to oxidation. (3) Dietary fatty acids may alter the balance of flora in the intestine and thereby influence the rate at which different catabolic products of cholesterol and the primary bile acids are formed by the intestinal bacteria. Also, Portman and Stare46 have reported studies indicating that diets containing an appreciable quantity of indigestible material (bulk) may cause more unmodified conjugated bile acids to reach the large intestine before they can be reabsorbed. INFLUENCE OF THYROID HORMONE

It is well known that in man the serum cholesterol levels tend to be high in hypothyroidism and low in hyperthyroidism. Accordingly, it is not surprising that thyroid hormone exerts a significant effect not only on cholesterol synthesis and excretion by the liver, but on bile acid metabolism as well. Bile-fistula rats excrete more cholesterol in the bile when treated with thyroxine. 47 Moreover, in the hyperthyroid rat an enhanced rate of excretion of chenodeoxycholic acid occurs, with a decrease in cholic acid output. 21 Under such circumstances, the increased output of chenodeoxycholate more than compensates for the decline in cholate excretion. (Normally, the rat puts out cholic acid and chenodeoxycholic acid in a ratio of 4: 1.) In hypothyroid rats excretion of both bile acids falls off, with the output of chenodeoxycholate showing marked diminution. These results have led to speculation that the thyroid hormone may inhibit the 12a-hydroxylating system, and, at the same time, promote oxidation of the cholesterol side chain. Recently, data have been presented showing that hypothyroid patients have a lower daily fecal output of bile acids and a longer bile acid half-life than euthyroid control subjeGts. 7 Treatment with thyroid hormone resulted in a return of these values toward normal. BILE ACID SEQUESTRANTS

From information concerning the dynamics of the enterohepatic bile acid cycle, it may be inferred that the oral administration of a substance capable of binding bile acids in the intestinal lumen, and thereby promoting their excretion in the feces, should result in an increased rate of cholesterol oxidation. Such a sequestration procedure would create,

Clinical and Experimental Aspects of Bile Acid Metabolism

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in effect, an "internal biliary fistula" with metabolic consequences somewhat similar to those obtained with external drainage of the bile. Bile acids are precipitated by a number of agents including iron salts and certain alkaloids. Indeed, Siperstein et a1. 53 have been able to lower serum cholesterol in cholesterol-fed cockerels by administering ferric chloride. However, such substances are toxic and cannot be used clinically. On the other hand, Tennent et al. 60 have reported that cholestyramine, an insoluble anion exchange resin with a polystyrene skeleton, is capable of binding bile acids in vitro and in the intestinal tract. This resin and a related polymer have been shown to inhibit serum cholesterol rise in cholesterol-fed cockerels. They also have a cholesterol-lowering effect in normocholesteremic dogs. In these animals, resin feeding for periods lasting up to a year induced a sustained drop to about 25 per cent below control values. In 26 patients with pretreatment cholesterol levels ranging from 133 to 400 mg. per 100 ml. oral doses of cholestyramine resin, usually 13 to 15 gm. per day produced an average decrease in serum cholesterol of 20 per cent. 4 Individual responses ranged between 6 and 38 per cent. The average decrease in three patients with familial hypercholesteremia was 25 per cent; in 15 patients with coronary heart disease it was 25 per cent; in four mild diabetics 15 per cent; and in four normocholesteremic subjects 18 per cent. Figure 4 shows the course of serum cholesterol concentration in a representative individual from each group. Return to pretreatment levels was rapid when cholestyramine (MK-135) was stopped. The most dramatic decreases in cholesterol level usually occurred in the individuals \vith the highest initial values. No toxic effects were observed in these and subsequent studies. Actually, the molecular weight of cholestyramine is over one million and therefore one would not expect this material to be absorbed from the intestinal tract. Mild gastrointestinal distress and constipation were experienced occasionally by some of the patients. Tennent et aI.,60 in studies on a dog, have shown that cholestyramine administration at a dose of 25 gm. per day tripled the fecal bile acid output and doubled the fecal sterol output. During the same time the cholesterol level fell by approximately 30 per cent. The increase in neutral sterol output suggested that the bile acid sequestrant also had interfered with cholesterol absorption. Carey and Williams17 fed 10 gm. of the resin per day to a normal volunteer subject and found that the fecal bile acid output was increased eightfold by this treatment. In amounts adequate to lower cholesterol (15 gm. per day), cholestyramine increases fecal fat output only slightly, if at all, in most subjects. However, at a level of 30 gm. per day (in divided doses), this material induces gross steatorrhea (Fig. 5).28,59 With this dose, output of fecal

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fat may exceed 15 gm. per day, with marked inhibition of uptake of 1131-triolein but not 113coleic acid. The mechanism whereby cholestyramine interferes with fat absorption is not clearly defined; however, the available evidence suggests that by reducing the quantity of bile acids available for digestive purposes the resin inhibits hydrolysis of dietary glyceride. During cholestyramine steatorrhea, xylose absorption is not impaired and the small intestine remains radiographically normal. When cholestyramine resin and triparanol (a cholesterol synthesis inhibitor)" were administered together to dogs, total cholesterol levels below 15' mg. per 100 m!. of serum were obtained. 59

Clinical and Experimental Aspects of Bile Acid Metabolism PLACEBO MK-135

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SERUM BILE ACID CHANGES IN LIVER DISEASE

The effect of liver disease upon bile acid metabolism and excretion depends to some extent upon whether the disorder primarily involves excretory or parenchymaI function. In practice, such a division usually represents a gross oversimplification; nevertheless, the distinction makes it easier to understand the changes in bile acid metabolism that can take place in liver disease. In disorders affecting biliary excretion, whether at the level of the smaller bile passages (primary biliary cirrhosis) or, because of extrahepatic obstruction (carcinoma of the head of the pancreas), retention of bile acids in the liver and blood ("hypercholanemia") invariably occurs early. In so-called primary biliary cirrhosis, obstruction in the mechanical sense cannot be discerned and it has been suggested that there is a regurgitation of bile by leakage through injured bile channels. 45 In obstructive jaundice, the process appears to be more straightforward with backing of bile proximal to the obstruction, dilatation of the bile passages, secondary inflammation and, ultimately, fibrosis. In either case, total bile acid levels in the blood are elevated, with values reported as high as 40 mg. per 100 ml. 42 In normal individuals serum bile acid values probably do not exceed 1 to 2 mg. per 100 ml., and at such low levels most of the analytical methods for bile acids are even more unreliable than usua1. 39 Osborn et a1. 42 studied 18 patients with obstructive jaundice and found

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a reasonably good correlation between serum bile acid and serum bilirubin levels (r = 0.71). TRIHYDROXY-DIHYDROXY BILE ACID RATIOS

Several investigators16 , 42, 48 have suggested that in jaundiced states information of clinical value can be obtained from the ratio of trihydroxy (cholate) to dihydroxy (chenodeoxycholate and deoxycholate) bile acids in the blood (TBA/DBA). It is difficult to identify a "normal" ratio of these moieties in the circulation since the total amount of bile acids in normal serum is negligible. In human bile, the TBA/DBA ratio has been variously reported as 1: 1 and 2.3: 1. 16 • 42 In any case, Osborn et al. 42 measured TBA/DBA ratios in 51 patients with various types of liver disease and reported the results in terms of three major groups: obstructive jaundice, portal cirrhosis and virus hepatitis. They found that in almost all cases of obstructive jaundice the total serum bile acid levels were raised more than 4 mg. per 100 m!. with the increase affecting both the trihydroxy and dihydroxy acids. In the acutely obstructed group the TBA/DBA ratio was invariably higher than 0.8. Similar ratios were obtained in patients with primary biliary cirrhosis, but in one of two patients with secondary biliary cirrhosis the TBA/DBA ratio was low. In the individuals with portal cirrhosis, values were highly variable. Only 13 of 24 patients had serum bile acid levels above 4 mg. per 100 m!., and in this group the TBA/DBA ratio was usually low, exceeding 0.8 in only three patients. In none of the patients was it possible to associate the prognosis with the total bile acid level or the TBA/DBA ratio. In the virus hepatitis patients total serum bile acid values also were very variable, correlating best with serum bilirubin. The TBA/DBA ratio was usually above 0.7. Most observers appear to agree that the finding of a low TBA/DBA ratio excludes the diagnosis of acute obstructive jaundice. On the other hand a high TBA/DBA ratio sometimes can occur in deeply jaundiced patients with portal cirrhosis but without biliary tract obstruction, and occasionally in patients with acute hepatitis. The mechanism for lowered TBA/DBA ratios is not readily explained. One reasonable possibility is that in hepatocellular disease there is failure of the 12a-hydroxylating mechanism. In obstructive jaundice interruption of the enterohepatic cycle of bile acids occurs, and in such a situation one would expect a reduced production of deoxycholate by intestinal flora and therefore a decreased rate of return of this dihydroxy acid to the liver. According to Rudman and Kendall,48 patients with "obstructive" jaundice display a rise in both DBA and TBA, largely in conjugated form. In such individuals TBA/DBA ratios ranged from 1 to 4 with

Clinical and Experimental Aspects of Bile Acid Metabolism

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appearance of conjugated bile acids in the urine. In contrast, individuals with hepatocellular injury showed predominantly DBA in their sera with bile acid detected in the urine only rarely. In their series, serum DBA concentration showed a correlation with serum bilirubin, while serum TBA concentration was proportional to the hypercholesteremia. Apparently in hepatocellular disease there is impairment not only of hydroxylation but of conjugation of the bile acids as well. It is believed that almost all of the circulating non-conjugated dihydroxy bile acids are bound to serum albumin and hence are not filtered by the glomeruli. 48 In contrast, a significant fraction of the conjugated trihydroxy bile acids can remain unbound, undergo glomerular filtration, and escape reabsorption by the renal tubules. Rudman and Kenda1l49 have found that t,he affinity for albumin of bile acids is reduced by the presence of hydroxyl groups in the steroid nucleus. Thus, DBA is bound to albumin to a greater extent than TBA. Some of the inconsistencies in the literature concerning serum bile acid levels and ratios in liver disease must result from the different analytical methods used, and the fact that the number of patients studied has been relatively small. Although measurement of the TBA/DBA ratio may have some clinical value in the differential diagnosis of jaundice, the information gained does not appear to justify the laboratory effort involved. The situation may change as simpler methods for bile acid analysis become available. PRURITUS AND HYPERCHOLESTEREMIA

It is well known that in acute impairment of biliary excretion there may be a concurrent rise in serum free cholesterol and phospholipid. Such hyperlipidemia has been observed both in experimental animals with artificially induced biliary obstruction 15 and in patients with such disorders as primary biliary cirrhosis and extrahepatic biliary obstruction. 2 Since the bile acids are the major catabolic product of cholesterol, it may be argued that accumulation of bile acids in the liver will lead to suppression of cholesterol oxidation. In addition, Byers et al. 15 have obtained evidence that in rats with ligated common bile ducts, the rate of cholesterol synthesis may increase, at least temporarily. Such a change in cholesterol balance could account for the hypercholesteremia that sometimes accompanies cholestatic disease. The rise in serum phospholipid and the relatively low proportion of serum esterified cholesterol that are often found in cholestasis are less readily explained. One of the most aggravating features of biliary cirrhosis is pruritus which, in some instances, can become so intense and unremitting that patients seriously contemplate suicide. 2 Patients with severe pruritus

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suffer from chronic insomnia and irritability, and their bodies may become covered with excoriations from uncontrollable scratching. Considerable evidence that the pruritus of patients with biliary cirrhosis is attributable to bile acid retention has accumulated. Support for this notion derives from the following facts: itching is common in patients with all types of biliary obstruction and, indeed, this symptom often may precede occurrence of jaundice by long intervals ;61 serum bile acids tend to be more elevated in patients with obstructive jaundice and pruritus than in patients with obstructive jaundice alone 42 (in one series, administration of whole ox bile to two patients with primary biliary cirrhosis greatly aggravated their pruritus2 ); and surgical drainage of bile in patients with intrahepatic or extrahepatic biliary obstruction induces temporary relief of pruritus. On the other hand, Osborn and associates42 have attempted to correlate changes in serum bile acid levels with the relief of pruritus induced by norethandrolone. In these patients, changes in serum bile acids bore no consistent relation to changes in pruritus. They concluded that it was" ... unlikely that bile acid retention in the blood was responsible for the pruritus." Other workers16 also have failed to demonstrate a consistent correlation between elevation of serum bile acid levels and itching. Because of its bile acid sequestering properties, cholestyramine has been used in treatment of the itching of primary biliary cirrhosis and various kinds of incomplete biliary obstruction. 17 , 29, 61 Administration of 6 to 12 gm. per day of the resin (in divided doses) has been found effective in management of the itching, with either complete or partial relief occurring in a majority of cases. Itching usually disappears within one to three weeks after treatment is started, with a concurrent fall in serum total bile acids. In some (but by no means all) patients serum lipids may return toward normal. It is believed that the relief of pruritus in patients treated with cholestyramine may be related to mobilization and fecal excretion of bile acids deposited in the skin. In Figure 6 are shown changes in serum bile acids and serum lipids in a patient treated with cholestyramine. Withdrawal of medication resulted in a return of pruritus and an exacerbation of hyperlipidemia and bile acid retention. Diets rich in linoleic acid also have been reported effective in the treatment of pruritus in several patients with biliary cirrhosis,61 and it would seem that at present the treatment of choice of the pruritus and hypercholesteremia associated with primary biliary cirrhosis or with incomplete biliary obstruction is cholestyramine resin, 6 to 12 gm. per day in four divided doses, and a diet in which the ratio of polyunsaturated to saturated fatty acids is 2: 1 or higher. It must be emphasized that treatment with cholestyramine is symptomatic, and that it is too early to know whether the course of primary

Clinical and Experimental Aspects of Bile Acid Metabolism

645

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Fig. 6. Effect of cholestyramine on serum lipids, serum bile acids and pruritus in a 49 year old woman with primary biliary cirrhosis (Van Itallie et a1. 61 )

Fig. 7. Finger tufts and palmar creases in a female patient with primary biliary cirrhosis prior to (left) and after one year of treatment with cholestyramine (right). Note disappearance of cholesterol deposits.

biliary cirrhosis can be altered by chronic administration of the sequestrant. However, patients experience an enhanced sense of well-being when relieved of pruritus, and if the hyperlipidemia also can be reduced, the skin xanthomata will improve. In one patient with primary biliary cirrhosis whose serum lipids fell with cholestyramine treatment the palms and soles, formerly rigid with lipid infiltrations and cracked with painful fissures, softened and healed (Fig. 7) allowing her to pursue sewing and other fine handwork tasks, and to wear shoes discarded a year previously. 61

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REFERENCES 1. Ahrens, E. H., Jr., Insull, W., Jr., Hirsch, J., Stoffel, W., Peterson, M. L.,

2. 3. 4.

5. 6. 7.

8.

9. 10. 11.

12. 1:3.

l-t. 15.

16. 17. 18.

19. 20. 21. 22.

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421 West 113th Street New York 25, N.Y. (Dr. Van Itallie)