Ruth Ann Welch, DVM; Heather L. Tarpley, DVM; Kenneth S. Latimer, DVM, PhD

Class of 2005 (Welch) and Department of Pathology (Tarpley, Latimer), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Overview

Determination of bile acids is a common diagnostic test for hepatic function in mammals. This test also is useful in avian medicine because elevated liver enzyme activity, such as increased aspartate transferase (AST), does not always correlate with the presence of liver disease. Quantitation of bile acids is sensitive and specific for liver function, because their synthesis, secretion, extraction from portal blood, and re-excretion depend on adequate liver function. A bile acid assay is easily performed and relatively inexpensive. Because increased bile acids can be observed with portosystemic shunts, liver failure, and cholestasis, other diagnostic procedures including surgical biopsy are usually required to differentiate these specific etiologies. Bile acid measurement can also be used to further evaluate elevated liver enzyme activity, monitor the progression of liver disease, and evaluate the effectiveness of treatment.

Bile Acid Formation and Secretion

Bile acids are the major constituent of bile, and in mammals, compose approximately 67% of this secretion. Bile acid synthesis in mammals and birds occurs in the liver and is considered a primary pathway of cholesterol metabolism. Cholesterol is the precursor molecule for bile acid synthesis (Fig. 1). This multistep process involves the conversion of cholesterol to cholic and chenodeoxycholic acids (Fig. 2). The rate limiting step of this process is catalyzed by the enzyme 7 alpha-hydroxylase. The activity of this enzyme is influenced by fasting, cholestasis, glucocorticoid exposure, liver failure, and lymphatic drainage (bile acid wasting) which can affect bile acid production. Within the liver, bile acids are conjugated by the addition of glycine or taurine, which are polar amino acids. Conjugation inhibits intestinal bile acid resorption and promotes lipid degradation. Bile acids are secreted via the bile duct into the small intestine and exert a detergent-like action such that ingested fats are emulsified and solubilized for digestion and absorption. In normal conditions, only conjugated bile acids are present in bile. In birds (regardless of the presence of a gall bladder), there is continuous secretion of bile acids. Presence of food in the duodenum stimulates the release of hormones such as secretin, avian vasoactive intestinal peptide (VIP), and/or cholecystokinin. In avian species that have a gall bladder, the effects of these hormones include relaxation of the sphincter of Oddi, gall bladder contraction, and expulsion of bile acids through the bile duct and into the small intestine. Some species of birds, such as psittacines, are similar to horses in that they lack a gall bladder.

Figure 1. Cholesterol is the precursor for bile acid formation in the liver.

Enterohepatic Circulation

Approximately 90% to 95% of bile acids are absorbed from the intestines into portal blood and delivered to the liver, creating enterohepatic circulation. In mammals, the vast majority of bile acids are absorbed in the ileum whereas 5-15% of bile acids absorbed into portal blood are derived from absorption of unconjugated bile acids from the large intestine. Bile acids are extracted from portal blood by hepatocytes. Bile acid production is stimulated primarily by bile acids returning to the liver and is also influenced by the size of the bile acid pool and number of enterohepatic circulation cycles. Two to five cycles of enterohepatic circulation occur during and immediately following meals. Normally, only a small quantity of bile acids enters systemic circulation and is eventually cleared during periods of fasting.

Methods of Measuring Bile Acids

Measurement of bile acids in peripheral blood indicates the amount of bile acids not extracted from portal blood that have subsequently entered systemic circulation. Bile acids can be measured in serum by direct enzymatic assay or radioimmunoassay (RIA). The enzymatic method measures total serum bile acid concentrations regardless of its conjugation status. RIA uses a labeled iodine tracker and antiserum against the amino acid (taurine or glycine) to which the bile acid is conjugated. As expected, values obtained with the enzymatic method are slightly higher than values ascertained by RIA (so results of tests using different methodologies cannot be compared). Both methods are effective for determining bile acid concentrations in birds.

Adverse effects on the enzymatic method of bile acid analysis may vary, depending upon the reagent kit. Kits marketed by Trinity Biotech, St. Louis, MO for use with the Hitachi 912 analyzer may provide inaccurate values in the presence of marked lipemia and icterus. Results of tests using RIA are not affected by hemolysis and lipemia. However, the use of radioisotopes requires special guidelines for procurement and disposal of the isotopes, laboratory monitoring for contamination, and safety precautions for personnel. The cost of the two procedures is equivalent. Sample volume (50 µ L) requirements are identical in both assays, which is beneficial in very small patients.

Protocol for bile acid testing in dogs and cats usually entails blood sampling after a set period of fasting and two hours after feeding a fatty meal. The postprandial release of a large quantity of bile acids challenges the efficiency of the enterohepatic cycle and hepatic function, so the postprandial bile acid measurement is a sensitive indicator of liver function in mammals. In contrast, usually only a single bile acid concentration after a 12-hour fast is measured in birds, though postprandial concentrations can also be determined. In sick birds, crop stasis may be present and can prolong postprandial stimulation of bile secretion. Postprandial bile acid concentrations have not been found to be significantly different in birds with (ducks, chickens, raptors) and without (psittacines, pigeons) gall bladders.

Pathophysiology of Elevated Bile Acids

Determination of bile acids provides a highly specific and sensitive indicator of hepatic disease in both birds and mammals since bile acid conjugation, excretion, and extraction requires normal hepatic function. Bile acid measurement alone cannot differentiate specific hepatic disease etiologies. Interpretation of bile acid concentrations in birds is similar to that in mammals, though postprandial responses are more variable in birds than in mammals. Elevated circulating bile acids may be detected if the degree of hepatic damage prevents adequate extraction of bile acids from the portal blood or if enterohepatic circulation is blocked or bypasses the liver. A diseased liver, however, will continue to function somewhat, so bile acids in the general circulation will eventually be cleared during periods of fasting.

Although reference ranges for bile acid concentrations have not been established for all avian species, bile acid concentrations are considered to be elevated in racing pigeons and most psittacine species if they are > 70 mmol/L in fasted (12-hour) samples. Postprandial bile acid concentrations vary between bird species and are more challenging to interpret.

Though the liver synthesizes bile acids, decreased bile acid concentration is typically not associated with decreased hepatic function since the majority of bile acids are recycled. Decreased bile acids are usually attributable to delayed gastric emptying or an ileal abnormality.

Conclusions

Bile acid determination is a sensitive and specific diagnostic test for liver function in mammals and birds. Increased bile acid concentrations can be used to document liver disease and monitor its progression or resolution over time. In birds, only a single 12-hour fasting sample is needed to accurately measure bile acid concentration in birds regardless of whether or not they possess a gall bladder. Although determination of bile acid concentration can confirm the presence of liver disease, a biopsy usually is required to determine disease etiology.

References

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2. Cray C, Anderopoulos A: Comparison of two methods to determine plasma bile acid concentrations in healthy birds. J Avian Med Surg 17:11-15. 2003.

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8. King MW: Cholesterol and bile acid metabolism. Indiana State University School of Medicine, http://web.indstate.edu/thcme/mwking/cholesterol.html. This excellent website provides a detailed overview of cholesterol and bile acid synthesis and metabolism.

9. Lumeij JT: Hepatology. In: Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application, Lake Worth, Wingers, 1994, pp. 522-537.

10. Lumeij JT: Avian clinical biochemistry. In: Kaneko JJ, Harvey JW, Bruss ML (eds): Clinical Biochemistry of Domestic Animals, Sand Diego, Academic Press, 1997, pp. 857-883.

11. Tennant BC: Hepatic function. In: Kaneko JJ, Harvey JW, Bruss ML (eds): Clinical Biochemistry of Domestic Animals, Sand Diego, Academic Press, 1997, pp. 327-352.

12. Willard MD, Twedt DC. In: Willard MD, Tvedten H, Turnwald GH (eds) Small Animal Clinical Diagnosis by Laboratory Methods 3rd edition, W.B. Saunders Company, 1999, pp.172-207.

Acknowledgment

"Hyacinthine Macaw" by Rita Thornton is from the Other Wildlife section of The Art of Rita Thornton website and is used with permission.

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