Serum Bile Acids in Companion Animal - Medicine

GASTROENTEROLOGY: THE 1990s

0195-5616/93 $0.00 + .20

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE Sharon A. Center, DVM

The measurement of serum bile acids in veterinary clinical practice has emerged as a routinely used diagnostic test for liver function in the 1990s. Clinical use has evolved from initial interest in bile acid metabolism as a research tool in the 1960s and 1970s. Using a variety of animal species, basic research has clarified the biochemistry and physiology of bile acids. Elucidation of the physiologic regulation of serum bile acid concentrations in health has led the way for their use as clinical diagnostic tests, as research probes, and in the treatment of cholelithiasis and chronic liver inflammation. This article provides background on the investigative work involving bile acids in dogs and cats, bile acid physiology and metabolism in health and illness, clinical application of serum bile acids as a diagnostic test, and the use of bile acids as a therapeutic modality in modern veterinary practice. HISTORICAL BACKGROUND

As early as 1956, dogs were used to explore the physiology and enterohepatic circulation of bile acids. 9• 69• 7° Characterization of specific bile acid moieties in dogs and cats was accomplished in the 1960s and 1970s and was further refined in later years. 40• 77• 89• 90• 96• 99• 106- 107 Numerous studies of the physiologic variables influencing bile acid homeostasis have been done using dogs, cats, and rodent species. Areas of investigation have included characterization of membrane transport mechaFrom the Department of Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 23 • NUMBER 3 • MAY 1993

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nisms for bile acid uptake in enteric epithelium, hepatocyte and biliary structures; physicochemical characterization of bile under a variety of conditions; study of the choleretic effects of natural and synthetic bile acids and of numerous drugs; investigations into the influence of gallbladder function, cholecystokinin, regional bowel absorption, and hepatic uptake, storage, and excretion on bile acid enterohepatic circulation; determination of the influence of the portal circulation and total hepatic blood flow on hepatocellular bile acid extraction; characterization of the variables regulating bile acid synthesis; and studies of the influence of cholestasis on the biliary epithelium and hepatocytes and on the biliary, hepatocellular and circulating systemic bile acid milieu.

BILE ACID PHYSIOLOGY

Bile is a digestive secretion that provides a route for cholesterol and bilirubin elimination. Its major lipid components are cholesterol, bile acids, and phospholipids. All three components can be synthesized de novo by the hepatocyte and also can be imported by hepatic circulation. Bile acids are the predominant component of bile, composing approximately 85% of the biliary solids. Their continuous transhepatocellular secretion from blood into bile serves an important role in the generation of canalicular bile flow (bile acid-dependent bile flow). Bile acids act as ionic detergents and in that way provide a pivotal role in the intestinal absorption, transport, and secretion of lipids. They have many different biologic functions and pathologic effects (Table 1). Bile acids are organic acids derived from cholesterol, the obligatory precursor in all species studied. Synthesis from cholesterol occurs exclusively in the liver in a series of sequential steps involving numerous intermediates and several different hepatocellular organelles. Bile acid synthesis in the adult human has been well defined. Conversion of the C27 neutral sterol cholesterol to the primary bile acids occurs in nine steps, with each reaction being catalyzed by distinct enzymes. In the adult, these reactions are believed to occur in an orderly sequence with modifications to the steroid nucleus preceeding side-chain oxidation. The conversion of cholesterol to bile acids involves the addition of either one or two hydroxyl groups, epimerization of the 313-hydroxyl group, and degradation of the C27 side chain of cholesterol into a C24 bile acid and propionic acid. A simplified metabolic scheme is shown in Figure 1. The term "bile acid" refers to the molecular configuration in which the carboxylic acid side chain is nonionized; the term "bile salt" refers to the ionized form. Although the acid and salt forms coexist in aqueous solution, at a physiologic pH of 7.4, the salts predominate. 103 The biological properties of bile salts depend on the number and position of their hydroxyl groups and the type of conjugation they have undergone at the carboxyl group. In humans, dogs, and cats, the

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

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Table 1. BIOLOGIC ACTIONS AND PATHOLOGIC EFFECTS OF BILE ACIDS Bile flow: Bile salts generate a portion of the biliary secretions (bile salt-dependent bile flow) . Bile is an important route for solubilization and excretion of organic compounds, endogenous metabolites, and a variety of drug and mineral metabolites and xenobiotics. Solubilization of biliary lipids: Combined with lecithin, bile salts solubilize biliary cholesterol in the form of mixed micelles and vesicles. This is beneficial in avoidance of cholesterol choleliths.

Cholesterol elimination: (1) Bile acid synthesis from cholesterol is a principal pathway for cholesterol elimination. (2) Secretion of bile salts into bile is coupled with the secretion and elimination of phosphatidylcholine (lecithin) and cholesterol. Regulation of cholesterol synthesis: Bile salts influence the regulation of cholesterol synthesis, either by acting directly on the hydroxymethylglutaryl-coenzyme Q (HMG-CoA) reductase, or indirectly by modulating intestinal cholesterol absorption . Control of bile acid synthesis: The enterohepatic circulation of bile salts is thought to regulate bile acid synthesis through feedback on the rate limiting enzyme (?a-hydroxylase) in bile acid synthesis.

Regulation of hepatic lipoprotein receptors: Bile acids may modulate the rate of uptake of lipoprotein cholesterol by the liver.

Alimentary fat digestion and absorption: Bile salts form mixed micelles in the intestines and participate in the intraluminal solubilization, transport, and absorption of cholesterol, fat-soluble vitamins, and other lipids. Bile salts also optimize certain lipases. Alimentary transport of calcium and iron: Bile salts may be involved in the transport of calcium and iron from the intestinal lumen to the brush border. Perpetuation of hepatobiliary membrane injury: Retention of hepatotoxic (hydrophobic, membranocytolytic) bile acids in serum, bile, and liver tissue in patients with serious hepatobiliary disease are thought to promote the self-perpetuation of a "smouldering" chronic inflammatory process. The membranocytolytic effects of certain bile acids have been well established.

Modification of blood brain barrier in hepatic encephalopathy: High concentrations of circulating bile acids may promote increased permeability of the blood-brain barrier, thereby providing central nervous system access for noxious hepatoencephalopathic substances. Generation of gastroesophageal and duodenal ulcers: Because of their membrane damaging capabilities, reflux of bile into duodenum, stomach, and/or esophagus, may contribute to the formation and perpetuation of ulcerative lesions.

Diarrhea in bile acid malabsorption: Passage of unconjugated bile acids into the distal bowel can result in morphologic injury to enterocytes and colonic villi and development of diarrhea.

Immunosuppression: Certain bile salts inhibit lymphocyte response to mitogens. This may occur in patients with cholestatic liver disease.

Abnormal platelet function: Bile salts have been shown to reduce normal platelet aggregation when they are exposed to ADP.

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SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

629

major bile acids synthesized in the liver are cholic acid (CA) and chenodeoxycholic acid (COCA). In healthy humans, twice as much CA is synthesized compared to COCA, but because COCA undergoes more efficient enterohepatic circulation, more of it is preserved. The term "primary bile acid" refers to the common moieties synthesized de novo by the liver: CA and COCA. Dehydroxylation of these bile acids by anaerobic intestinal microorganisms produces the more hydrophobic "secondary bile acids"; CA is dehydroxylated to deoxycholic acid (DCA), and COCA is dehydroxylated to lithocholic acid (LCA) (Fig. 2). In health, a relatively small amount of secondary bile acids are contained within the bile acid pool; only about one third to one half of DCA and one fifth of LCA formed in the intestines is absorbed. The rate-limiting enzyme for bile acid synthesis is cholesterol 7ahydroxylase, which catalyzes the conversion of cholesterol to 7ahydroxycholesterol. 53 Bile acid wasting syndromes, such as occurs with ileal disease, biliary fistula, certain biliary diversions, or the administration of a bile acid binding resin (cholestyrarri.ine), increases hepatic 7ahydroxylase aCtivity and bile acid synthesis severalfold. 35 A list of variables that influence the rate of bile acid synthesis through 7ahydroxylase is shown in Table 2. In most mammals studied, the biosynthesis of bile acids appears to be regulated primarily by the amount of bile acids returning to the liver, and this is a function of the size of the bile acid pool and the number of enterohepatic circulations of the pool each day. 8• 11 • 22• 27 In humans, approximately six to eight cycles occur per day; two at each meal interval. LIVER

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Figure 2. Demonstration of bile acid degradation in the alimentary canal. Primary bile acids are dehydroxylated to form secondary bile acids, and conjugated bile acids are deconjugated by enteric bacterial enzymes.

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Table 2. VARIABLES INFLUENCING THE RATE OF BILE ACID SYNTHESIS Variables Bile fistula Bile acid malabsorption Cholestyramine Ax Lymphatic drainage (bile acid wasting) Portocaval anastomosis Glucocorticoids Adrenalectomy Thyroxin Thyroidectomy Glucose after starvation Fasting Oral administration bile acids (hydrophobic bile acids) Bile duct ligation/obstruction Diurnal rhythm (feeding time) Hepatic failure

Effect on Cholesterol 7a-hydroxylase

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(12 a-hydroxylase activity)

After synthesis, bile acids are conjugated in the liver. The normal liver is believed to conjugate almost all bile acids before their secretion into bile. 88 Conjugation results in the attachment of a second organic substituent, most often glycine or taurine, to the side chain carboxyl group or to one of the ring hydroxyl groups via an ester, ether, or amide linkage. The predominant conjugates in humans, dogs, and cats are C24 amides formed with the amino acids taurine or glycine. The relative proportions of glycine and taurine conjugates vary markedly among species. 2• 23 Humans conjugate with glycine and taurine, depending on the availability of tatirine. 39 Dogs and cats conjugate primarily to taurine. The dog has the ability to convert to glycine conjugation, but the cat seems to undergo obligate conjugation with taurine. Even taurine-depleted.cats seem to produce only small amounts of glycine-conjugated bile acidsY· 77• 78 In illness, alternative conjugation reactions may occur involving glucuronate, sulfate, and other moieties, but under normal circumstances, these reactions are minor. It is believed that both glucuronidation and sulfation occur in the liver, although it is possible that some sulfation also occurs in the kidney. 7 Sulfation and gluctironidation occur at the hydroxyl groups of the bile salt molecule.30• 91 • 112 By introduction of these polar groups, bil~ salts become more water-soluble and, as a result, undergo altered metabolism, excretion, and toxicity. For example, the renal clearance of sulfated bile acids is 20 to 200 times greater than the clearance of nonsulfated bile acids. 91 Of the common bile salts, only the most hydrophobic (lithocholic acid [LCA]) normally is found as a sulfate in liver tissue and bile. LCA is highly hepatotoxic and accumulates in the presence of cholestasis. Sulfated LCA is poorly resorbed from the intestine and .because it is rapidly lost from the enterohepatic circulation, .it normally constitutes only a small portion of the bile acid pool. Sulfation of LCA is a protective mechanism that prevents its accumulation. It has recently been suggested that bile acid sulfation may play a protective role in

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

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cholestasis by stimulation of bile flow and/or by reduction of biliary phospholipid and cholesterol secretion. By stimulating bile flow, sulfation may augment bile salt excretion and protect cellular membranes against the detergent properties of high bile salt concentrations. 112 Conjugation impedes passive intestinal aborption and thus enables bile acids to participate in intestinal digestion of dietary fats. 45 Conjugation with glycine or taurine increases the acidic strength of the side chain without altering its amphipathic properties. The pKa of glycine conjugates approximate 3.9, and the pKa of taurine conjugates is less than 1.0. Consequently, the most ubiquitous bile acid conjugates are ionized at the pH in the small intestine. Conjugated bile acids are actively absorbed in the distal ileum via high-affinity receptors. Approximately 95% of the bile acids are absorbed in the terminal ileum and are subsequently returned to the liver via the portal circulation. Conjugated bile acids may be deconjugated by enteric bacterial enzymes; this is the first step in bile acid degradation. 44 Detection of an increased proportion of unconjugated bile acids in an individual can be used as an index of intestinal bacterial activity. 31 In healthy humans, unconjugated bile acids are present in concentrations of up to 1 J..Lmol/ mL in the lumen of the distal small intestines. 68 Rapid nonionic passive absorption of unconjugated bile acids, particularly dihydroxy bile acids, occurs at all levels of the small intestine. This is possible because the pKa of unconjugated bile acids is approximately 5 to 6 at the pH of the intestinal contents. 82 Many different fecal bile acid metabolities formed by microbial enzymes have been identified. Bile acids may be subjected to oxidoreductions, and 5j3-bile acids may be converted into Sa-bile acids to some extent. Free or nonconjugated bile acids reabsorbed from the intestines are reconjugated efficiently in the liver. Rehydroxylation seems to be minimal in humans and has not been fully investigated in the dog or cat-7

THE ENTEROHEPATIC CIRCULATION OF BILE ACIDS

The enterohepatic circulation of bile acids is illustrated in Figure 3. After hepatic synthesis and conjugation, the primary bile acids are excreted through the biliary tract to be stored and concentrated in the gallbladder. Emptying of the gallbladder is usually, but not exclusively, initiated after meals following release of cholecystokinin. Cholecystokinesis can occur in the middle of the night in the dog and at random times during a prolonged fast. 25• 46• 51 • 65• 66• 84• 85• 101 After bile enters the alimentary canal, bile salts facilitate intraluminal solubilization and absorption of ingested lipids. They are absorbed throughout the entire intestines, although most prominently in the distal ileum, 73• 82 and are returned to the liver via the portal circulation. This cycling of bile acids between the liver-intestines-portal circulation is referred to as the bile acid enterohepatic circulation. This cycle is highly efficient, providing more than 95% of the bile salts ultimately secreted in bile. The intestinal

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Figure 3. Schematic representation of the bile acid enterohepatic circulation.

conservation of bile acids is very efficient; approximately 95% of luminal bile acids are absorbed through each pass in the intestines. The very small amount of bile acids lost in feces each day are replenished by hepatic synthesis. In health, the rate of intestinal absorption is the major factor determining serum bile acid concentrations in both the fasting and postprandial state.5• 56• 75 Feeding induces a small postprandial increase in the total serum bile acid concentration as a result of gallbladder contraction, efficient intestinal absorption, and portal transport of bile acids to the liver. Bile salts returning to the liver are bound to albumin and, to a lesser extent, to plasma lipoproteins, and uptake into the liver occurs mainly in the periportal areas. The increased load of bile acids in portal blood during the postprandial interval exceeds the clearing capacity of the liver. The hepatic extraction of bile acids is reported to be 90%. First-pass hepatic extraction of taurocholic, glycocholic, deoxycholic, and chenodeoxycholic acids is 80, 65, 55, and 40%, respectively. 103 The trihydroxy bile acids are more efficiently cleared than are the dihydroxy bile acids. Bile acids may exist within the hepatocyte as single molecules or may be bound to carrier proteins. The present concept of hepatic bile acid transport is that it is carrier-mediated against concentration and electrical gradients. Both sodium-independent and sodium-dependent carrier systems have been characterized.52• 105• 115 Cytosolic binding

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

633

proteins seem to facilitate the movement of bile acids within the hepatocyte to the canalicular apparatus. 92 This may occur by simple diffusion or by transport within vesicles via microtubule-dependent pathway associated with the Golgi apparatus. Secretion into the canaliculi may occur by simple exocytosis or may be facilitated by a carrier at the canalicular membrane. 28 Bile acid concentration within the canaliculus can achieve three to four times that within the hepatocellular cytosol. Recent studies suggest that hepatocytes may alter the destination of bile acid transport in cholestasis by "pumping" bile acids into the basolateral region of the hepatocyte across the sinusoidal membranes rather than into the canaliculus. This is consistent with the topographic shift in canalicular marker enzyme locations observed in cholestasis. This can explain the mechanism for the so-called "regurgitation" of bile acids into plasma in cholestasis.60 An overview of the enterohepatic bile acid circulation details four discrete circuits that contribute to the load of bile acids ultimately presented to the liver and that can be realized in the systemic circulation. A cholehepatic circuit has been defined between biliary radicles (canaliculi, bile ductules) and the liver. Regurgitation of bile acids in this circuit is important in the elevation of serum bile acids in cholestasis. A jejunohepatic circuit exists in which bile salts are absorbed from the proximal small intestine by passive absorption. This circuit may become important in the delivery of increased quantities of unconjugated bile acids to the portal circulation in the case of bacterial overgrowth of the bowel, a condition that increases bile acid deconjugation. An ileal-hepatic circuit is the major component of the enterohepatic bile acid circulation in health, as previously detailed. A colohepatic circuit allows for the passive absorption of bile acids that escape absorption by the small bowel or that are formed in the distal portion of the alimentary canal. This circuit can become important when bile acid malabsorption occurs as a result of ileal disease or resection. In summary, maintenance of an adequate concentration of bile acids in the alimentary canal and of the enterohepatic circulation depends on efficient bile acid absorption in the intestines. Intestinal reabsorption is highly efficient, and under normal conditions, bile acid synthesis is maintained at a relatively constant level sufficient to replace fecal losses. Conjugation of bile acids with taurine or glycine lowers their pKa values, abolishes bile salt precipitation, and increases their water solubility. Free bile acids have a significantly higher pKa and thus precipitate or are passively absorbed in the proximal intestines. They are thus relegated to an unimportant role as far as lipid miscibility and absorption are concerned. About 30% of the COCA is believed to be absorbed by nonionic diffusion in the jejunum. Absorption of bile acids in the colon is greatly influenced by the rate of bacterial dehydroxylation. The secondary bile acids tend to precipitate and to bind to bacteria in the colon and hence have low absorption.

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Factors Affecting the Enterohepatic Circulation

Bile acid pool size and kinetics are influenced by many variables including (1) the rate of bile acid synthesis, (2) meal-synchronization and completeness of gallbladder emptying, (3) gastric emptying and intestinal transit rate, (4) intestinal bile acid absorption efficiency, and (5) the enterohepatic cycling frequency. These variables and others that influence the use of an endogenous bile acid challenge test for evaluation of hepatobiliary function are summarized in Table 3. The size of the bile acid pool and the kinetics of bile acid circulation are of biomedical interest because of their relationship to gallstone formation. Considerable research has been done in this and related areas because of the importance of cholelithiasis in humans. Fortunately for the dog and cat, they are not as susceptible to cholelith formation as are humans. The dynamics of the enterohepatic circulation can influence the maximal bile acid concentrations attained in the systemic circulation and thus influence the sensitivity of bile acid quantification as a test of hepatobiliary function. Reduced intestinal absorption, incomplete gallbladder contraction, and an altered intestinal transit rate can each influence the optimal interval for postprandial sample collection. Although bile acid synthesis can be markedly reduced in patients with serious liver disease,

Table 3. VARIABLES AFFECTING THE ENDOGENOUS SERUM BILE ACID TOLERANCE TEST Dietary constituents: inadequate fat or amino acid content for optimal cholecystokinin release, gallbladder contraction, and postprandial challenge of the enterohepatic circulation. Inadequate meal consumption: food not consumed or insufficient ingested volume to optimally invoke gastric emptying, gallbladder contraction, and ingesta/bile transport to the distal ileum at the 2-hour postprandial interval. Delayed gastric emptying: results in a failure to stimulate gallbladder contraction and subsequently inadequate challenge of the enterohepatic circulation at the 2-hciur postprandial interval. Delayed Intestinal transit: lack of optimal timing to evaluate the postprandial challenge of the enterohepatic circulation. Too rapid Intestinal transit: reduced intestinal absorption of bile acids and subsequently inadequate challenge of the enterohepatic circulation at the 2-hour postprandial interval. Ileal disease or resection: reduced bile acid absorption resulting in an inadequate challenge of the enterohepatic circulation. Small intestine malabsorptlon/maldlgestion/steatorrhea: altered bile acid absorption resulting in an inadequate challenge of the enterohepatic circulation. lnterdigestlve gallbladder contraction: normal physiological variable; fasting bile acids exceed postprandial values.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

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a more efficient enterohepatic cycling, the presence of portosystemic shunting, and reduced hepatic extraction results in elevated systemic bile acid concentrations. The health and function of the gallbladder can have a major influence on the enterohepatic circulation of bile acids. The physiologic functions of the gallbladder include bile storage, concentration, and acidification, and release of bile into the intestines when stimulated by cholecystokinin and other neuropeptides. The gallbladder mucosa has a unique ability to withstand the detergent properties of bile acids as their concentration is continuously increased by fluid absorption. During storage, hepatic bile is concentrated to approxiinately 10 to 20% of its original volume via active transport processes that mobilize sodium chloride and sodium bicarbonate and water (passively) from the gallbladder. As the concentration of bile increases, bile salts form micelles, which eliminates the osmolal stresses that would develop in a conventional aqueous solution. Contraction of the gallbladder does not occur only in the postprandial intervaL Gallbladder contraction during the interdigestive interval has been clearly demonstrated. zs, 46, 51 , 66, 84, 85, Ioi The gallbladder undergoes intermittent partial emtpying during phase 2 of the migrating myoelectric complex. Interdigestive contraction is also believed to be triggered by a variety of gastrointestinal peptides and neuropeptides including gastrin, motilin, vasoactive intestinal peptide, secretin, glucagon, and pancreatic polypeptide .110 Altered gallbladder kinesis is believed to play a role in the development of gallstones. Impaired gallbladder emptying has been shown in human patients receiving total parenteral nutrition; these individuals are particularly prone to the formation of .biliary sludge and gallstones.63, 80 It is important to remember that the storage and concentration of bile in the gallbladder is not necessary for lipid digestion. Surgical removal of the gallbladder or cholecysto-duodenostomy or -jejunostomy does not lead to fat maldigestion, bile acid malabsorption, or inability to use the serum bile acid test. THE ROLE OF BILE SALTS IN LIPID DIGESTION AND ABSORPTION

Initial digestion of triglycerides and formation of a lipid emulsion occurs in the stomach. As chyme passes into the intestines, fatty acids, amino acids, and a lowered pH stimulate the secretion of cholecystokinin, which invokes gallbladder contraction. Bile salts initially prepare dietary fats for lipolysis by production of a stable lipid emulsion and eventually enhance the absorption of digested fat by formation of mixed micelles. However, several different lipases are optimized by the presence of bile salts. For example, triglycerides are split into monoglycerides and fatty acids by pancreatic lipase; low concentrations of bile salts optimize pancreatic lipase activation. Carboxylic ester hydrolase, the enzyme that splits cholesterol esters, also requires bile salts for proper function. Digestion of long-chain phospholipids is similarly dependent on the presence of bile acids.

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The major function of bile acids in the small intestine is to solubilize dietary lipids into micelles. As lipids diffuse through the unstirred water layer, they are transformed by association with bile acids into mixed micelles. Micelles enhance the diffusion of fats to the enterocyte surface for further digestion and absorption. It is well known that bile acids are essential for the digestion and absorption of certain lipid moieties, because complete diversion of bile from the intestines results in steatorrhea, impaired absorption of t:holesterol, and malabsorption of fat-soluble vitamins. In the absence of bile, a bleeding diathesis due to vitamin K deficiency develops within 21 days. Parenteral administration of vitamin K1 can rectify this deficiency within 12 hours. 72 The absorption of triglycerides is not fully dependent on the presence of bile salts; approximately 60 to 70% of triglycerides can be absorbed in the absence of bile acids. Only 50% of the dietary and biliary free cholesterol can be absorbed in the absence of bile acids. Bile salts are also thought to enhance the intestinal absorption of iron and calcium. EFFECTS OF VARIOUS NONHEPATIC AND HEPATIC DISORDERS ON SERUM BILE ACID CONCENTRATIONS

Table 4 summarizes the effects of various disorders on serum bile acid concentrations. Bile Acid Metabolism in Intestinal Disease

It is important to consider the underlying mechanisms involved with abnormalities in fat digestion and absorption that may influence the enterohepatic circulation of bile acids. Altered intestinal flora can result in perturbed36 bile acid metabolism which can injure intestinal and colonic epilelium. Some small bowel disorders result in bacterial overgrowth, which can impair bile acid absorption. Intestinal inflammation, pancreatic exocrine insufficiency, abnormally slow bowel transit, or indiscriminant use of antibiotics can each be associated with bacterial overgrowth in the small bowel. This can result in increased deconjugation, rapid absorption of free bile acids in the proximal jejunum, and subsequently fat mal digestion and steatorrhea. 86 In this circumstance, steatorrhea may be alleviated following readjustment of the bowel flora via treatment with an appropriate antibiotic. Loss of a critical volume of the terminal ileum as a result of inflammation or resection can cause increased delivery of bile acids to the colon, resulting in diarrhea and/or steatorrhea. If the extent of ileal loss or dysfunction is not severe, patients may develop diarrhea that resolves with cholestyramine administration. A positive response to cholestyramine suggests that bile acid malabsorption was in some way associated with the patient's diarrhea. Diarrhea develops when increased amounts of DCA

Table 4. EFFECTS OF VARIOUS NONHEPATIC AND HEPATIC DISORDERS ON SERUM BILE ACIDS Total Serum Bile Acid Concentrations Fasting

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Expected Qualitative Profile

Postprandial

Normal subjects

Dog : < 5 f!..moi!L Cat: < 5 f!..moi/L

Dog : < 15 f!..moi/L Cat: < 10 f!..moi/L

Ileal dysfunction Ileal malabsorption

Subnormal

Subnormal,

t

postprandial rise

t

Unconjugated moieties

Fat malabsorption/ maldigestion

Subnormal

Subnormal,

t

postprandial rise

t

Unconjugated moieties

Standard for comparison: trihydroxy > dihydroxy bile acids

Bacterial overgrowth

Variable

Variable

ttt

Cholecystectomy Biliary diversion

Slightly

t

Reduced rise, early postprandial rise

t t

Delayed gastric emptying

Slightly

t , exceeds postprandial

Expected increase not realized

Normal

t t

Rapid ·intestinal transit

Subnormal, variable

Subnormal

Delayed intestinal transit

May be increased, exceeds postprandial

Postprandial less than fasting value

Portosystemic shunting (congenital and acquired)

Normal after prolonged fast or

Reduced hepatic mass

May be normal after prolonged fast or t

Hepatic failure

Usually

Cholestasis

Profoundly

t ttt

t

Unconjugated moieties

Unconjugated moieties Deoxycholic acid vs cholic acid

Unconjugated Un~onjugated

Profound t t t , may see a marked change between fasting and postprandial values

t

trihydroxy:dihydroxy ratio in surgically created shunts without cirrhosis

Profound t t t , may see a marked change between fasting and postprandial values

t t

trihydroxy:dihydroxy ratio .sulfated ·moieties

Profound t t t , may see a marked change between fasting and postprandial values

t t

trihydroxy:dihydroxy ratio sulfated moieties

Profoundly t t t , fasting and postprandial have similar values

May have t unconjugated, t t sulfated, t t glucuronidated t trihydroxy:dihydroxy ratio

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and COCA are delivered to the colon. Bile salts, particularly the dihydroxy bile salts, inhibit water, sodium chloride, and bicarbonate absorption; promote potassium secretion; and accelerate colonic motility.6· 32· 43· 61 · 62· 97 When ileal loss or dysfunction are severe, fat malabsorption and steatorrhea develop. Bile acid concentrations in the small intestines decline owing to the inability of hepatic synthesis to replace excessive intestinal losses. Diarrhea in this case is not responsive to cholestyramine treatment. In humans,· only prodigious fecal losses reduce the bile acid pool size enough to impair dietary fat digestion and absorption. 39 Effective treatment requires restriction of long-chain triglycerides from the diet and administration of medium-chain triglycerides as a fat source, which does not require micellar solubilization. Some of these patients respond partially to oral bile salt replacement. Enterohepatic Circulation of Bile Acids After Cholecystectomy Cholecystectomy does not abolish diurnal variations of serum CA and COCA concentrations. It does, however, increase the fractional turnover rate and the rate of intestinal degradation of certain bile acids. Cholecystectomy increases the enterohepatic cycling of bile acids in the fasting state. The cholecystectomized patient has slightly high fasting serum bile acid concentrations and slightly low, early, and a less acute, postprandial serum bile acid rise. Owing to continuous exposure of CA to anaerobic microflora in the distal ileum, more is dehydroxylated to DCA. Detection of a higher concentration of DCA substantiates increased exposure of CA to intestinal bacterial dehydroxylation. Newly formed DCA is efficiently absorbed by active transport in the intestines. Concurrently, synthesis of CA is reduced by feedbacl:< inhibition owing to the increased transhepatic flux of bile acids. This leads to a slight reduction in amount of CA and its proportion in the total bile acid pool. Turnover and size of the DCA and COCA pools remain unaltered/9 Changes in Bile Acid Profiles in Liver Disease The percentage of individual bile acids in serum and bile has been thoroughly studied in humans with various types of liver injury. 1· 10· 71 · 76· 79· 88• 93· 102· 104· 114 Only limited information is available regarding bile acid profiles in serum and bile from dogs and cats with liver disease. 19· 99• 100 Healthy humans have a trihydroxy:dihydroxy bile acid ratio of 0.5 to 1.0. In extrahepatic bile duct obstruction, this increases to more than 1.0; in cirrhosis this reduces to less than 0.5. The mechanisms for these changes are not well understood. Because there are considerable overlaps between values, the ratio cannot distinguish between intrahepatic and extrahepatic cholestasis. Because the analytic procedures are costly and labor intensive, this ratio does not have clinical application.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

639

Humans with cirrhosis develop several different functional and anatomical abnormalities. These include the development of portosystemic shunts, reduction of liver cell mass and function, various degrees of intrahepatic cholestasis, and abnormal cholesterol and lipoprotein metabolism. These factors contribute to the many observed abnormalities of bile acid metabolism. The presence of portosystemic shunts results in a maldistribution of bile acids to outside the normal enterohepatic circulation, thus enlarging the distribution of the bile acid pool. These patients develop high urinary bile acid excretion. Serum bile acid profiles in dogs with surgically created portosystemic shunts showed large increases in the free and conjugated CA, smaller increases in COCA, and a reduction in the DCA concentrations.100 In the presence of seriously reduced hepatic function, the total bile acid pool may be reduced by up to 50%. This is believed to be due to a depression of both CA and COCA synthesis, although a disproportionately greater reduction of CA synthesis has been shown. Although impaired 12ahydroxylation of the CA precursor has been documented, many other factors also influence the biosynthetic pathway. 71 • 104 The hepatic secretion of COCA and the fractional turnover of CA and COCA are reduced, and usually there is a marked decline in DCA production. Collectively these effects result in a reduced trihydroxy:dihydroxy ratio. The changes are more accentuated in serum compared to bile. 1 Mild to moderate steatorrhea is common in humans with cirrhosis, and this may contribute to reduced DCA formation and absorption. 37• 54· 94· 109 Alterated bacterial flora and/or altered enteric conditions for bacterial 7a-dehydroxylase enzyme activity have been postulated as underlying mechanisms.114 Bile acid metabolism is markedly altered in cholestasis. Usually, there is an increase in the sulfation of COCA, leading to increased excretion of this dihydroxy bile acids. This leads to an increased trihydroxy:dihydroxy ratio. Alternative forms of conjugation such as sulfation and glucuronidation become important for bile acid elimination; between 10 to 30% of the bile acids may be sulfated. Many unusual bile acids may be found in urine and bile.

HEPATIC TOXICITY OF BILE ACIDS

Certain bile acids administered in large doses induce acute hepatocellular injury accompanied by cholestasis. Infusions of taurochenodeoxycholate and taurocholate in the rat cause severe biochemical abnormalities. In bile, these include a 30-fold increase in biliary leakage of proteins such as alkaline phosphatase, lactate dehydrogenase, IgG, and albumin.83 Enzyme induction is thought to contribute to the increased enzyme activities; taurocholic acid has been shown to be a potent inducer of alkaline phosphatase.95 Interestingly, simultaneous infusion of the taurine conjugate of ursodoxycholate (UDCA) prevents injury caused by other bile acids. This is one of the reasons that UDCA

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is used therapeutically in patients with chronic inflammatory hepatobiliary disease. Hepatocytes in zone 1 are most severely affected by bile acid toxicity. Injured hepatocytes exhibit swollen mitochondria and endoplasmic reticulum and loss of surface membrane integrity with extrusion of cytoplasm and organelles. Altered lipid composition of membranes is thought to be involved, as is abnormal membrane transport. 50• 111 • 113 Bile acid cytotoxicity is inversely proportional to the degree of hydroxylation: the dihydroxy bile acids (COCA, DCA) are most injurious. Increased lipid solubility also seems to be associated with cytotoxicity. Conjugation does not result in significant differences in bile acid toxicity, with the exception of deoxycholate conjugates, which induce enzyme leakage more rapidly.

INTERACTIONS BETWEEN ORGANIC ANIONS: BROMOSULFOPHTHELIUM, INDOCVZNINE GREEN, BILIRUBIN, AND BILE ACIDS

Because bile acids are organic anions, the mechanisms for hepatic uptake, storage, transport, and ultimately, secretion into the canaliculus have been scrutinized for competition with other organic anions (bilirubin, bromosulfophthelium [BSP], indocyznine green [ICG]). Initial evidence for the existence of different transport systems for organic anions came from study of mutant Corriedale sheep that appeared to be unable to secrete conjugated bilirubin and a number of other organic anions but eliminated bile acids normally in bile. The Dubin-Johnson syndrome represents a similar secretion defect in humans. A number of studies exploring organic anion interactions have been completed in vivo and in vitro and have provided divergent findings. Some studies have demonstrated the presence of shared competitive uptake and excretion mechanisms, whereas others demonstrate facilitation of uptake and elimination. The discrepancies among the organic anion studies have been attributed to the different experimental designs and concentrations of substrates and inhibitors used. Collectively, the findings support the currently held belief that two types of uptake mechanisms are involved for bile acids: sodium and non-sodium coupled transport. They also reflect that there is more than one route by which intracellular bile acids gain access to the canaliculus. The importance of the organic anion studies for the clinician are in the interpretation of serum bile acids when other organic anions are coexistent. Most notably, the problem occurs in the animal jaundiced because of hemolytic disease but for whom important hepatobiliary disease cannot be discounted. Clinical experience and experimental findings suggest that serum bile acid values are normal when hemolysis is the single cause of jaundice. It is important, however, to remember that severe anemia can lead to hepatocellular dysfunction and subsequently, mildly to moderately increased serum bile acid values.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

641

METHODS OF BILE ACID DETERMINATION

Initial methods used to measure bile acids required laborious extraction procedures to first isolate them from blood or bile. This involved the use of organic solvents, which resulted in incomplete quantification because some bile acids were lost in the extraction procedure. After extraction, bile acids were identified and quantified using thin-layer chromatography or techniques using spectrophotometric fluorescence. Improved methods providing better qualitative and quantitative determinations were developed that involved gas-layer chromatography and high-performance liquid chromatography. Unfortunately, none of these techniques lent themselves to routine clinical application because of their complexities. In the 1970s, it was discovered that bile acids could be measured directly in serum, thereby avoiding the laborious extraction procedures. Soon, methods were developed that had high sensitivity and acceptible interassay and intraassay variation for clinical application. Radioimmunoassays (RIA) were developed to measure specific common bile acid moieties in human sera. Most of these procedures are highly specific for conjugated primary bile acids. These have been used mainly for research purposes in humans. One commercial kit has been validated for use in the dog and cat but has only been studied in detail in sera collected after a 12-hour fast from clinically ill dogs with biopsy-confirmed liver disease. 12• 49• 81 Enzymatic procedures reliant on the activity of 3a-hydroxysteroid dehydrogenase (3a HSD) that required linkage to a detector system had been concurrently developed. None of the methods were practical for routine clinical application in veterinary patients until the direct enzymatic spectrometric method was refined for endpoint detection with a diformazan dye. The enzymatic procedure initially reported by Mashige57• 58 was optimized and validated for use in dogs and cats in 1984. 17 Subsequently, a series of papers describing the diagnostic efficacy of fasting and 2-hour postprandial serum bile acids determined by the direct enzymatic spectrometric method were reported. 3• 4• 13- 16• 18• 41. 48 Today, the most commonly used RIA procedure for the determination of serum bile acids in dogs and cats measures nonsulfated conjugated primary bile acids. 17• 21 It requires a very small volume of serum or plasma (10 ~J..l) and is linear to 50 j.Lmol/L. Values greater than 50 j.Lmol/L can only be quantified after dilution of the sample and reanalysis. The method is more expensive than the enzymatic procedure and must be hatched with other samples for analysis. The ranges reported for fasting and postprandial values for healthy dogs and cats are provided in Table 5. Most clinical laboratories are now using the enzymatic procedure for estimation of total serum bile acids in veterinary patients. The details of this method are shown in Figure 4. This technique measures all3a-hydroxylated bile acids regardless of whether they are conjugated. This method measures more bile acids than the RIA procedure; it provides a better estimate of serum bile acid concentrations because

z: N

Table 5. SERUM BILE ACID CONCENTRATIONS IN DOGS AND CATS WITHOUT HEPATOBILIARY DISEASE Dogs Assay Method

Liver Biopsy*

Number

no yes no no no no

25 40 15 26 13 8

2.46 1.7

yes no no no

40 15 26 13

9.26 6.8

yes yes no no

12 6 22 60

1.8 0.8 4.2 0.4

no

37

3.7

Enzymatic 12-hour fasting

2-hour postprandial

Mean/Median

-

1.07

2-hour postprandial

1.0 -

3.0

-

6.0

-

7.08

Radioimmunoassay 12-hour fasting

Cats

-

-

-

Sd/Sem

0.73 -

Range

-

0-2.141 0-17.118

-

0-8.821 0-14.848

1.3 0.281

-

0-5.049

-

0.712

3.9 3.9

yes

26

-

2.5

0-16104

yes

26

-

5.0

0-15104

no

34

1.6

Mean/Median

Sd/Sem

Range

0.91107

0-30.621 0-43.048

-

Number

1.31 4

-

2.024 1.9

Liver Biopsy

-

0- 21 .9 18

-

0.3 12

0.2-4.621 0.5-25.521

·11 a liver biopsy specimen was not obtained, the health status was determined as healthy on the basis of routine physical, hematologic, biochemical, and urinalysis evaluations. Some studies were completed with clinical patients suspected of having liver disease; the controls were animals that did not have liver disease on the basis of liver biopsy. sd = standard deviation; sem = standard error of the mean.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

~0

..... OH

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c::OH

3a-hydroxy bile acid

Plasma Pyruvate

~

NADH+H+

~ ~

Diformazan (end point color)

f~"l +3etHSO

~~ NAD

''oH

- 3aHSO

643

Nitrotetrazolium Blue (NTB)

1.33 M

Phosphoric Acid

37 10 minute

c incubation

I= I ~

Absorbance

Figure 4. Schematic representation of the direct enzymatic method for estimation of 3a-

hydroxylated bile acids. Spectrometric end-point is determined by a color change .in the reagent solution induced by the presence of 3a HSD·transferring a H+ to NAD+ and then to a diformazan dye. The amount of bile acid in solution is calculated from the difference in absorbance between the two tubes.

most of the common bile acids contain an hydroxyl group at the 3a position. Test methodology requires that other dehydrogenases, such as lactic dehydrogenase (LDH), be inactivated before bile acids are quantified. This is because other dehydrogenases can react with NADH in the test system and cause the diformazan color change that is used in the system to estimate the amount of bile acids. In most cases, pyruvate is successful in the inactivation of LDH. Unfortunately, LDH has several different isoenzymes, one of which is liberated from the liver during severe hepatocellular necrosis. This particular LDH is not inhibited by pyruvate. Nevertheless, its presence is obvious, because the induced color change is grossly apparent when LDH activity is present. The way the assay is done protects against false-positive test results due to interference with nonspecific dehydrogenases. Consulting Figure 4, each sample is evaluated with two tubes; one containing sera, 3a HSD, pyruvate, and buffer, and the other tube containing sera, and reagents but lacking the 3a-HSD. The tube lacking the enzyme is used as a blank. Any nonspecific color change caused by dehydrogenase activity appears in each tube equivalently and is negated by the "blanking" procedwe. Other methods to remove the LDH activities, such as heat inactivation or 1,6 hexanediol, are not suitable. 3 • 17 Heat inactivation takes too long and results in protein precipitation. The presence of 1,6 hexanediol adversely influences the 3a-HSD activity. The sensitivity of the direct spectrometric technique is 2.0 IJ..mol/L when 200 ~J..l of sera are used in the test system. Better test performance can

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be achieved by use of larger quantities of sera. Unfortunately, such volumes would require that 2.5 to 3.5 mL of blood be collected for each fasting and postprandial specimen. This is impractical for the petite veterinary patient. Because the absorption spectra of the endpoint diformazan dye is similar to that of hemoglobin, hemolyzed samples cannot be analyzed accurately. Lipemia must be eliminated by highspeed centrifugation and refrigeration of the sera so that accurate endpoint detection can be made. The eRzymatic spectrometric method can be adapted to an autoanalyzer if precautions are taken to ascertain the presence of lipemia and hemolysis, which can induce serious analytic errors. Laboratory quality control is important in visually inspecting each sample to avoid false-positive test results. The direct enzymatic spectrometric method has acceptible linearity up to 250 j.LmoV L when standard curves are prepared in canine or feline sera. 17 In our laboratory, we do not dilute the serum for further quantification, because there is no evidence in the experimental or clinical literature that refinement of the degree of bile acid elevation beyond 250 j.Lmol/L provides useful information. The reported ranges for fasting and postprandial values for healthy dogs and cats are provided in Table 5. DIAGNOSTIC APPLICATION OF SERUM BILE ACIDS

The diagnostic efficacy of fasting and 2-hour postprandial serum bile acid values in the dog and cat have established that they are useful in the diagnosis of hepatobiliary abnormalities associated with histologic lesions or portosystemic shunting. Overall, bile acid values perform better than routinely used screening tests to correctly identify these patients. They also improve the diagnostic performance of routine tests when used adjunctively. Serum bile acids have gained more favor in the veterinary profession than in human medicine because of several factors. Humans tend to present earlier to a clinician in the course of their disease, whereas animals often are presented in the more advanced stage of a disorder. In humans, the acute stage of hepatobiliary disease is usually associated with enzyme activity or serologic markers of viral infection or immune-mediated disturbances. If these are found, noninvasive imaging studies and the propriety of liver biopsy is immediately considered. In animals, the later stages of a chronic disorder may be associated with trivial or absent abnormal enzyme activity. In addition, the propensity of the dog for liver enzyme induction, particularly of ALP activity, consistently interferes with the detection of clinically significant liver disorders. Therefore, the less than abrupt presentation of our patients, the confusion created by liver enzyme induction in the dog, and the limited availability of imaging capabilities has supported the clinical use of a noninvasive and simple method for detection of hepatobiliary dysfunction. If reduced function is detected, the veterinary clinician can rapidly proceed to definitive diagnosis with confidence that a morphologic lesion will allow disease characterization or that a shunting defect will be identified.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

645

The following test performance statistics are relevant only for the direct enzymatic method for determination of total serum bile acid values. 14• 18 Specificity of fasting bile acids equal or exceed 95% in the dog and cat at values greater than 15 j.Lmol/L. Specificity of postprandial bile acid values achieve 100% in the dog at greater than 25 j.Lmol/L and in the cat at greater than 20 j.Lmol/L. At these bile acid concentrations, the positive and negative predictive values are greater than 92% in the dog and are greater than 97% in the cat. In· these studies, the overall test efficacy of serum bile acids was calculated using the following formula: (true-negatives plus true-positives)/total number of tests conducted. The overall test efficacy for fasting bile acids in the dog (n = 170) was 82.4% and in the cat (n = 108) was 66% The overall test efficacy for postprandial bile acids in the dog was 82.3% and in the cat was 81%. In the dog, we have estimated a false-positive test result rate of 2%. The issue of whether fasting or postprandial serum bile acid values perform better remains controversial in human medicine. Examination of the diagnostic efficacy of postprandial bile acid values in the dog has shown that they are nearly identical to that of the fasting values in most disorders. Evaluation of the diagnostic efficacy of postprandial serum bile acids in the cat has shown that the postprandial value performs better than the fasting values. Postprandial values perform better in animals with portosystemic vascular anomalies or acquired shunts. Use of both bile acid values provides the most reliable information in clinical patients. Serum bile acid values cannot differentiate liver diseases, because abnormal values overlap widely. Fasting and postprandial serum bile acid values from dogs with a variety of liver conditions are shown in Figure 5. Particular patterns frequently appear in the presence of shunting lesions, extrahepatic cholestasis, and intrahepatic cholestasis; these are illustrated in Figure 6. A normal liver rapidly clears bile acids so that only minor transient increases are realized in a postprandial serum sample. Deviation of the portal circulation, such as in portosystemic vascular anomalies or in some animals with cirrhosis, can seriously impair the enterohepatic bile acid cycle. Even though demonstrable portosystemic shunts may not exist, intrahepatic "shunting lesions" occur in association with collaginization of the hepatic sinusoids and disruption in sinusoidal blood flow caused by regenerative nodule formation. Minor changes in hepatic perfusion associated with dehydration, hypovolemia, or passive congestion are not believed to appreciably alter the SBA concentration as they do plasma bromosulfophthalein (BSP) or indocyanine green (ICG) clearance. Diseases associated with cholestasis result in high SBA concentrations owing to decreased biliary bile acids excretion and regurgitation of bile acids into the systemic circulation. Combination of serum bile acid values with results of other liver tests may reveal some useful diagnostic patterns. Detection of abnormal SBA concentrations in the absence of jaundice and abnormal liver enzyme activity indicates metabolically quiet liver disease associated with hepatoportal perfusion abnormalities or severely reduced hepatic

~

250 ...J

'0E

200

Vl

150

"

0

u < ~ iii

100

:::;; ::>

"'"'Vl

50 0 CIRRHOSIS

CHRONIC HEPATITIS

HEPATIC NECROSIS

CHOLESTASIS

NEOPLASIA

PORTOSYSTEMIC VASCULAR ANOMALY

GLUCOCORTICOID HEPATOPATHY

.

PASSIVE CONC:F:STION

MISCELLANEOUS

Figure 5. Fasting .and postprandial bile acid values in dogs with a variety of liver disorders. Dogs of the miscellaneous group were initially suspected of having liver disease on the basis of history, clinical signs, and increased liver enzymes, but ultimately did not have histologic lesions on light microscopic examination of liver tissue. (From Center SA, ManWarren R, Slater MR, et al: Evaluation of twelve-hour preprandial and two-hour postprandial serum bile acid concentrations for diagnosis of hepatobiliary disease in dogs. JAm Vet Med Assoc 199:217-226, 1991; with permission.)

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

....

e

0 A •

~ 250

~

647

PORTOSYSTEMIC SHUNT CIRRHOSIS INTRAHEPATIC CHOLESTASIS BILE DUCT OCCLUSION

200

~ 150

m

:::; 100 :::;) a: w

(/)

~

25

LS222blli2±£±slli222Bd FASTING

POSTPRANDIAL

Figure 6. Common patterns of fasting and 2-hour postprandial serum bile acids in patients with shunting lesions (portosystemic vascular anomaly and cirrhosis), extrahepatic bile duct occlusion, and intrahepatic cholestasis. Hatched area indicates the normal range.

mass. These findings typify occult cirrhosis or congenital portosystemic vascular anomaly, especially when the postprandial SBA concentration is greater than or equal to 250 j.Lmol/L. A pattern typified by normal SBA values in the presence of hyperbilirubinemia indicates prehepatic or hemolytic jaundice. Despite this ability to differentiate hemolytic from hepatocellular or cholestatic jaundice, such use of SBA values is seldom necessary. Usually, history and hemogram make the distinction clear. A pattern suggestive of glucocorticoid hepatopathy in the dog includes marked increase in alkaline phosphatase activity, normal serum bilirubin, and normal to mildly increased SBA values, coupled with the historical information or physical features associated with glucocorticoid excess. A pattern consistent with cholestasis includes marked enzyme abnormalities, especially increased ALP activity in the dog and increased ALP and -y-glutamyltransferase activities in the cat, moderate to marked hyperbilirubinemia, and marked increases in fasting and postprandial serum bile acid values. Animals with extrahepatic bile duct occlusion usually have SBA concentrations greater than 200 j.Lmol/L, and little if any difference exists between fasting and postprandial values. Animals with intrahepatic cholestasis usually have increased fasting and postprandial SBA values, but these values are usually lower than those associated with major bile duct occlusion. The pattern of intrahepatic cholestasis is extremely common in cats with liver diseases including cholangitis, cholangiohepatitis, hepatic lipidosis, and neoplasia. A wide range of enzyme, bilirubin, and SBA values are found in animals with primary and secondary hepatic neoplasia, seemingly dependent on the extent and distribution of the neoplastic involvement with the hepatic parenchyma and biliary structures. An attempt to correlate the severity of histologic lesion and the SBA concentration is fraught with complications. Most spontaneous hepatobiliary disorders have many different histopathologic abnormalities, which complicates any attempt to isolate the influence of individual factors on SBA concentrations.

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FASTING BILE ACIDS GREATER THAN POSTPRANDIAL BILE ACIDS: WHY?

Occasionally, clinically normal animals and animals with liver disease will have a fasting SBA value that exceeds the postprandial value. This can be attributed to spontaneous gallbladder contraction during fasting or to differences in gastric emptying rates, cholecystokinin release, alimentary response to cholecystokinin, altered intestinal transit time, or gastrointestinal tract flora. In consideration of these variables, it is noteworthy that differences in the optimal time of postprandial sample collection have been shown in individual human patients with liver disease and in clinically normal dogs and cats.16• 17• 18• 47• 48 Postprandial SBA values may therefore be underestimated in some animals because the sample is not acquired at that individuals most optimal sampling interval (when their SBA are at their highest level). This individual variation can only be avoided by obtaining blood samples frequently over a postprandial interval spanning 1 to 8 hours, making the test unsuitable for routine use.

LOW SERUM BILE ACID VALUES: FALSE-NEGATIVE TEST RESULTS

A variety of uncommon factors may promote low SBA values in the presence of hepatobiliary disease. Reduced flow of bile into the duodenum has been documented in some humans with early or transient obstruction of the biliary tree. Intestinal malabsoption of bile acids will invalidate the test, but this usually is obvious because the patient will have grossly evident diarrhea. In the author's experience, this is an uncommon complicating factor. Failure of a patient to eat the test meal will result in lack of enterohepatic bile acid cycling. This is avoided by visual confirmation that food has been consumed. Use of an inappropriate diet to invoke enterohepatic bile acid cycling can also complicate test interpretation. Maintenance canine and feline diets for healthy patients usually contain sufficient protein and fat to induce normal enterohepatic bile acid circulation. The use of canned diets seems more reliable. Ingestion of a small amount of food may not provide adequate mechanical stimulation for gastric emptying or enough fat and protein to initiate the enterohepatic bile acid cycle. These factors may result in an inadequate 2-hour endogenous postprandial bile acid challenge. Minimal amounts of food for the SBA test in clinical practice are 2 teaspoons for petite patients under 10 lbs, and 2 tablespoons for larger patients. The clinician, however, should aim for a larger ingested volume. The author usually aims for a meal of typical size for that patient and will blenderize food and feed it with a syringe if the patient is reluctant to eat. This helps to standardize the test conditions.

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

649

INBORN ERRORS OF BILE ACID METABOLISM

So far, there have been no recognized inborn errors in bile acid synthesis in domestic animals that would invalidate their use as a diagnostic test. In humans, inborn errors of bile acid metabolism are uncommon. Earliest recognized defects included a lipid-storage disease (cerebrotendinous xanthomatosis) and the cerebrohepatorenal syndrome of Zellwegers. Both of these defects involve impaired oxidation of the cholesterol side-chain. Since then, several other disorders of bile acid synthesis from cholesterol have been characterized. Generally, these defects lead to an early death. ATTEMPTS AT REFINEMENT OF THE BILE ACID TEST PROCEDURE

Determination of total 3a-hydroxylated serum bile acid concentrations provides a sensitive and specific indicator of hepatobiliary function compared to routinely used screening tests. Unfortunately, the endogenous bile acid tolerance test is influenced by many nonhepatic variables. Because of these modifying influences, consideration has been given to the development of an exogenous bile acid tolerance test that could circumvent many of these variables. Use of an intravenous tolerance test has been explored in humans with serious liver disease.55 Intravenous injection of glycocholic acid (GCA) and determination of its plasma clearance rate using a specific RIA showed increased sensitivity for the detection of liver dysfunction as compared to the endogenous bile acid concentrations. The GCA for this test required millipore filtration, autoclaving, and separate packaging into multiple dose vials with benzyl alcohol as a preservative. Intravenous injection required that a dilute bile acid solution be mixed with an equivalent volume of patient blood in order to avoid phlebitis. The blood mixed with bile acids underwent hemolysis. Samples were collected before and at eleven timed intervals over 45 minutes in anicteric patients. Overall, this procedure would not be convenient or acceptable in most small animal practices. To avoid some of the problems encountered with the GCA intravenous tolerance test, methods using radiolabelled bile acids for estimation of plasma disappearance have been explored. Results have shown that the plasma residence time of bile acids is too short in most patients to provide information better than that provided through the endogenous bile acid test.33, 34 The use of an intravenously administered tracer dose of radiolabelled bile acids has been studied in an attempt to obviate the need for an exacting RIA and to investigate the possibility of measuring one single spot plasma sample as an indicator of tracer clearance. 26' 98 Results have shown that fasting and 2-hour postprandial bile acid concentrations and BSP plasma clearance have better sensitivity. It was concluded that tracer dose clearance is more dependent on liver blood flow than on

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CENTER

liver function. This factor complicates the use of any test using an intravenously injected bile acid, because liver blood flow has considerable variation between individuals and may be increased in patients with significant liver disease. The efficacy of an oral bile acid challenge test using radiolabelled bile acids has been compared to an intravenous tolerance test using the same labelled moiety.33 Results showed that the oral tolerance test superceded the intravenous tolerance test but that the endogenous fasting bile acid con~ntration was a superior test. Measurement of the clearance of a bile acid administered by the oral route is more sensitive for the detection of impaired liver function than when a bile acid is given by the intravenous route. This is analogous to the blood concentration of a drug achieved after oral and intravenous administration. If a substance with high hepatic extraction of 80% is given intravenously, a 10% reduction in liver extraction due to disease will cause a proportionate 10% reduction in the plasma clearance. If the same substance is administered orally (and is completely absorbed), the fraction of the dose reaching peripheral blood from portal blood will be increased from 20% to 30% (by the 10% reduction in hepatic extraction). The apparent plasma clearance will fall by greater than 50%. Use of an intravenously administered bile acid also would reduce the high sensitivity of the endogenous or oral bile acid challenge to detect portal systemic shunting. 59 Because it seemed apparent that the oral challenge test held more promise, investigations using cholecystokinin to improve the reliability of enterohepatic bile acid cycling have been explored. 87 Following a fast, cholecystokinin infusion given to human patients does not improve the diagnostic accuracy of serum bile acids compared to fasting values. Oral loading with COCA has also been explored as a bile acid challenge test. Serum bile acids measured before and in the 3 hours after oral ingestion of COCA showed that fasting bile acid values had better diagnostic accuracy.23• 29 It is apparent that at least so far, the endogenous bile acid tolerance test provides better diagnostic accuracy than any of the exogenous oral bile acid, intravenous tracer bile acid, or cholecystokinin associated procedures. BILE ACIDS AS THERAPEUTIC AGENTS Dehydrocholic Acid

Dehydrocholic acid is a synthetic bile acid derived by oxidation of cholic acid; it has a keto group in the 3-position. It is not detected using the conventional enzymatic assay for bile acids. This bile acid has been used as a potent choleretic in physiologic studies of the biliary system. Dehydrocholate is a potent choleretic because its osmotic activity in bile is not diminished by micelle formation . It increases the total volume of bile by stimulating a watery biliary secretion without increasing biliary solids. It does not increase the elimination of bilirubin pigments and,

SERUM BILE ACIDS IN COMPANION ANIMAL MEDICINE

651

in fact, has been shown to decrease bilirubin excretion. Dehydrocholate does not increase the elimination of bile acids, cholesterol, or phospholipids. During passage through the liver, 50% of the dehydrocholate is believed to become 3a-hydroxylated-forming cholic acid. 38 One study has shown that extensive metabolism to other bile acid moieties also occurs. 24 For short-term use, dehydrocholate appears to be minimally hepatotoxic. Studies of patients under chronic therapy have not been reported to the author's knowledge. Dehydrocholate is used as a hydrocholeretic when a "thinning" of biliary secretions is desired. It has been used in dogs and cats with biliary sludge or preciptiation confirmed during laparotomy. It should always be used in this context with antibiotics and fluid therapy. Sludged bile or biliary concretions can be associated with biliary tract infection in a causal or secondary role. Fluid therapy is advised for concurrent treatment, because the patient must remain well hydrated if hydrocholeresis is to be induced. Observation of biliary sludge on ultrasonographic evaluation of a patient, in the author's opinion, is not an indication for use of dehydrocholate. Sludge or biliary precipitates are commonly observed in anorectic animals. Currently, UDCA (see following section) is being used in most animals that require hydrocholeresis. Ursodeoxycholic Acid UDCA has been promoted as a treatment for liver disease for the past 40 years in Asia. It is a powerful choleretic agent and can be used to treat sludged bile and cholelithiasis. It had recently received global attention in the treatment of chronic liver disease after it was marketed as a choleretic for medical dissolution of gallstones. During use for cholelith dissolution, it was discovered that among patients with coexistent serious liver disease, many underwent improvement in serum biochemical tests (enzymes, total bilirubin, increased albumin) and that some had a resolution of jaundice and ascites. 74 Many conferences, scientific papers, and investigative studies have now been directed to the use of UDCA as a therapeutic modality for chronic inflammatory cholestatic liver disease. UDCA has been shown to be beneficial for the treatment of chronic active hepatitis, primary biliary cirrhosis, sclerosing cholangitis, chronic persistent hepatitis, cirrhosis, biliary atresia in infants (improving their candidacy for liver transplantation), and the hepatic lesions associated with cystic fibrosis. Its precise mechanism of action remains controversial. It is believed that oral administration reduces small intestinal absorption of CA and probably also of COCA. The beneficial effect of UDCA in chronic cholestatic liver disease was initially attributed to displacement of hydrophobic bile acids from the endogenous bile acid pool. This is now contested. Confusion as to its mechanism of action has arisen because many of the initial reports concerning the efficacy of UDCA were generated from study of serum bile acid profiles in normal individuals. It is argued that serum bile acid

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profiles are not reflective of the overall metabolism of bile acids in an individual because they represent less than 1% of the total bile acid pool. Study of an individual pool size is essential for characterization of metabolic change in the management of a particular bile acid. This can be accomplished by use of a radiolabelled tracer dose of a bile acid, and such studies are now in progress. In healthy controls, these studies have shown that the DCA pool declines following treatment with UDCA. In patients with cholestasis, the- DCA pool is already reduced in size. Although the specific mechanism of action remains undetermined, it is clear that UDCA modifies the cytotoxic effects of the most noxious bile acids. Studies conducted on liposomes, erythrocytes, esophageal and gastric mucosa, and in vitro incubation of hepatocytes have shown that UDCA has minimal adverse effects on biological membranes. It has been postulated that UDCA reduces the expression of HLA antigens in the liver and biliary cells in patients with cholestasis. This might suppress the target of cytotoxic T cells, block the destruction of bile ducts, and prevent periportal or lobular necrosis, thereby slowing the progression of disease. Studies in humans with chronic liver disease have shown that UDCA treatment results in significant qualitative and quantitative alterations in serum and urinary bile acids; it becomes the major serum and urinary bile acid, and the total endogenous bile acids become markedly reduced. It is unknown if the choleretic effect induces these changes or if these changes are related to regulation of bile acid synthesis and enterohepatic cycling of endogenous bile acids. Extensive toxicity studies were conducted in dogs when this product was being evaluated for dispersal in the United States. It is not toxic to the dog. A recent study in healthy cats indicates that it is not toxic to this species. 67 Studies in rhesus monkeys, baboons, and rabbits have shown that UDCA is metabolized to lithocholic acid, a strongly hepatotoxic bile acid that is not adequately sulfated in these species. UDCA use is therefore contraindicated in these animals. The author has been using UDCA in the medical management of chronic cholestatic liver diseases in dogs for the past 3 years. The clinical impression is that it is beneficial, but without a rigorous controlled study, this remains only an impression. It is important to remember that use of any choleretic agent is contraindicated in patients with major bile duct occlusion. The dose used in dogs and cats with chronic liver disorders has ranged between 4 to 15 mg/kg per day. This is extrapolated from use in humans with liver disease.

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Address reprint requests to Sharon A. Center, DVM Department of Clinical Sciences Cornell University College of Veterinary Medicine Ithaca, NY 14853