Bile Acid Metabolism and Fat Absorption in Newborn Infants

Symposium on Recent Clinical Advances

Bile Acid Metabolism and Fat Absorption in Newborn Infants

John B. Watkins, M.D.*

The absorption of nutrient lipids is essential for the growth and development of newborn infants. To perform this process efficiently, the infant in its transition from an in utero existence must possess or acquire, among other functions, the many complex hepatic and intestinal mechanisms required for the absorption of dietary lipid. For example, in order to have sufficient bile salts for completion of the intraluminal phase of fat absorption, the liver must be able to (1) synthesize bile salt from cholesterol; (2) conjugate bile salt with taurine or glycine; and (3) secrete the conjugates into bile. The gail bladder must be able to concentrate and then deliver the bile into the duodenum at concentrations sufficient to solubilize and disperse the products of lipolysis. Finally, the intestine, specifically the ileum, must possess the specialized mechanisms which permit reabsorption of the secreted bile salts, and thereby maintain the bile salt pool and establish an effective entero-hepatic circulation. Recent studies have established that during the neonatal period considerable inefficiency in fat absorption exists/ 5 and that the mechanisms which permit mature bile salt metabolism are incomplete in the fetus and newborn infant. 36 The purpose of this paper is to review recent information concerning the developmental aspects of bile acid metabolism, to discuss the physiochemical basis of lipid absorption, and to highlight some of the pathophysiologic processes encountered with abnormalities of bile acid metabolism.

MECHANISMS FOR FAT ABSORPTION Dietary lipids are composed principally of triglycerides. These biologically important lipids are largely water-insoluble, and as such are poorly *Instructor in Pediatrics, Harvard Medical School; Assistant in Medicine and Assistant in Clinical Nutrition, The Children's Hospital Medical Center, Boston, Massachusetts; Formerly Research Associate in Medicine; Fellow in Gastroenterology, Boston University School of Medicine, Boston, Massachusetts Supported in part by U.S. Public Health Service Grant No. 14523 and the Siegfried and Irma Ullmann Fund for Cystic Fibrosis Research.

Pediatric Clinics of North America- Vol. 21, No.2, May 1974

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available as a nutrient source. Chemically, triglycerides are composed of three fatty acids esterified to glycerol. When characterized according to their physical properties in an aqueous environment, they are among the group of compounds classified as polar, water-insoluble, non-swelling amphiphiles 54 (loving both), meaning in this case oil and water. Therefore, although they are water-insoluble, triglycerides do orient at the oilwater surface and spread to form a monolayer. The process of fat digestion is integrally related to these physical properties and proceeds through a series of steps to transform the triglycerides into lipids more capable of being solubilized within the luminal phase and then absorbed at the mucosal level. The first step is emulsification, a process by which large fat globules are converted into smaller stable droplets. This requires mechanical energy, which is supplied by the physical churning action of the stomach, and the slow release of the small lipid droplets through the pylorus into the duodenum. Pancreatic lipase then interacts with the emulsified triglyceride to hydrolyze the fatty acid ester linkages, specifically at the first and third positions of the triglyceride molecule. 11 The rate of this reaction is directly related to the surface area of substrate (triglyceride) rather than substrate concentration in bulk, 11 a phenomenon which is due to the fact that lipase, a water-soluble enzyme, interacts with the water-insoluble triglycerides only at their oil-water interface. 1 Thus the formation of small emulsion particles dramatically increases the surface area available for lipolytic activity. The end-products of this hydrolytic reaction are fatty acids and 2-monoglycerides, lipids in conjunction with lecithin which serve as emulsifiers to stabilize the particle size of the small lipid droplets. 16 The lipolytic products within the jejunum are now in potentially absorbable forms. However, for several reasons it can be predicted that emulsification and lipolysis acting alone without the action of bile salts would elaborate an end-product incompletely digested and suboptimally absorbed: (1) the pH optimum of pancreatic lipase is above the pH of the intestinal lumen, (2) fatty acids and monoglycerides exert endproduct inhibition on pancreatic lipase, and (3) being incompletely solubilized, the lipolytic products are suboptimally presented to the intestinal mucosa for absorption.

THE ROLE OF BILE SALTS Bile salts, by virtue of their unique "detergent" properties, 55 dramatically alter the intraluminal environment and are central to the completion of the intraluminal phase of lipid digestion and absorption. Their primary function in the intestine (see Fig. 1) is to solubilize the products of lipolysis and to create an environmental optimal for lipid absorption. 2 " Bile salts are amphiphilic molecules possessing both hydrophilic and hydrophobic centers. 29 At low concentrations they exist as unassociated molecules (monomers) but at higher concentrations, they form polymolecular aggregates termed micelles. 9 Transition from a monomeric to a micellar solution occurs at the critical micellar concentration. The criti-

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CHEMICAL

Figure 1. Schematic representation of the physical and chemical states of lipids in the intestinal content. The lipids of the oil phase are in equilibrium with the aqueous micellar phase. The emulsion droplets, micelles, and microvilli are drawn approximately to scale. (Reproduced from Hofmann, A. F., and Small, D. M.: Detergent properties of bile salts: Correlation with physiologic function. Ann. Rev. Med., 18:333, 1967, with permission.)

Oil phase Triglyceride Diglyceride Folly acid (unionized)

Emulsion droplets

Microvilli

tOOm)'

cal micellar concentration is fixed under laboratory conditions but under physiologic conditions in the intestine it represents a range which is infhienced by the intraluminal pH, the chemical nature of the substituent molecules, the electrolyte concentration, and the temperature. 10 At the jejunal pH during a meal, and in the presence of polar lipids (fatty acids and phospholipids), the critical micellar concentration is in the range of 1 to 2 mM. 26 The jejunal bile acid concentrations reported for most normal adults is 5 to 25 mM., 47 although for newborn infants values are reported to be considerably lower (1.2 to 3.0 mM). 43 The creation of the micellar phase together with solubilization of the lipolytic products accelerates the lipolytic reaction toward completion and vastly increases the absorption of lipid presented to the intestinal epithelium. It should be noted that recent evidence indicates that micelles are not absorbed intact by the mucosa, but that micellar lipid must diffuse as single molecules across the aqueous unstirred layer overlying the luminal surface of the mucosa in order to be absorbed. 66 The dynamics of this process are under investigation. In addition to lipid solubilization, bile salts combine with co-lipase, a polypeptide cofactor, to alter the kinetics of pancreatic lipase.50· 66 At concentrations above the critical micellar concentration of bile salts, and in the presence of co-lipase, the pH optimum of lipase is shifted from 8.0 to 6.5, close to the usual intraluminal pH of the intestine, and the enzyme activity is increased several fold. 6· 41 The mechanisms which maintain adequate intraluminal bile salt concentrations and thus insure continuation of these all important functions are two-fold: hepatic synthesis of bile salt and a selective, active reabsorption of the secreted bile salt conjugates in the distal ileum. In the adult, conjugated bile salts at the intraluminal pH are not passively reabsorbed to any appreciable degree in the proximal jejunum; hence, the majority of bile salts pass unabsorbed into the ileum. 56 By being absorbed distally, jejunal bile salt concentrations are maintained at levels suf-

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ficient for the continued formation of mixed micelles, thereby increasing the solubilization of lipid and permitting continued lipid absorption. The reabsorptive capacity of the ileum for bile salts is immense and it conserves 95 to 98 per cent of the bile salt secreted each day so that only 2 to 5 per cent is lost in the feces. The bile salt pool contained within the enterohepatic circulation remains nearly constant however, because of a delicate feedback mechanism whereby the amount of bile salt lost each day is replaced by the hepatic synthesis of new bile salt. 12

BILE SALT SYNTHESIS Bile salts are synthesized from cholesterol in the liver. The significant alterations which occur during this synthesis are the introduction of two hydroxyl functions at the 7 and/or 12 positions, transformation of C-3 hydroxyl group from the beta to the alpha position, and oxidation of the cholesterol side chain. Transformation of the cholesterol nucleus occurs so that the C-5-6 double bond of cholesterol is saturated stereospecifically to produce a cis configuration at the A and B rings, an alteration which causes all of the polar hydroxyl groups to orient at one side of the molecule (Fig. 2). Various intermediate metabolic sequences have been suggested and these pathways have been recently reviewed. However, in general, it appears that hydroxylation precedes side chain oxidation and that cholesterol-7 a-hydroxylase is the major regulatory enzyme for bile acid biosynthesis as well as the site at which feedback inhibition of bile acid synthesis occurs. Two main primary bile acids are synthesized, cholic acid and chenodeoxycholic acid, and together constitute about 80 per cent of the bile acids circulating within the enterohepatic circulation in normal adults. Free bile acids are not normally found in human bile but are conjugated with the amino acids glycine and taurine before secretion. The addition of these groups serves to increase bile acid solubility and to increase their resistance to precipitation at low pH. Cholic acid for ex-

BILE SALT

CHOLESTEROL

,.~

~·o1H

Figure 2. Formation of cholic acid from cholesterol. Upper panel depicts the conventional formulas. The lower panel represents the molecular shape and indicates the reorientation of the A + B rings of the steroid nucleus which permits the hydrophilic polar groups to lie on one side of the molecule and the hydrophobic groups to lie on the opposite side.

BILE ACID METABOLISM AND FAT ABSORPTION IN NEWBORN INFANTS

•

OH"

505

CONHCH,COO-

OH

TAUROCHENODEOXYCHOLATE

GLYCOCHOLATE

! ••

!

#00"

COOH

OH

OH

DEOXYCHOLIC ACID

LITHOCHOLIC ACID

Figure 3. The primary bile acids, cholic and chenodeoxycholic acid, are synthesized in the liver and secreted as the glycine or taurine conjugates. The secondary bile acids, deoxycholic and lithocholic acid, are formed by intestinal bacteria which deconjugate and dehydroxylate the primary bile acids.

ample precipitates at pH 6.5 and its glycine conjugate at pH 4.3, while the taurine conjugates remain soluble until pH 1.2. Thus, at the range of normal intraluminal pH, the conjugated bile acids are almost completely ionized. Conjugation with taurine is a pyridoxine-dependent step 2 and appears to be preferential to glycine conjugation in that the ratio of glycine to taurine conjugated bile acids may be increased by feeding taurine whereas the ingestion of glycine has little effectY The ratio of glycine to taurine conjugates is about 1 : 3 at birth and reverses to the adult ratio of 3 : 1 after 3 to 4 months. 46 In situations of rapid bile salt turnover or losses as occur with intestinal bacterial overgrowth or ileal resection this ratio may increase to 13 or 15 to 1. In such instances the rapid turnover of the glycine pool may be of both diagnostic and pathophysiologic significance.z;;, "9 Secondary bile acids deoxycholic and lithocholic acid are produced within the intestine by intestinal bacteria (Fig. 3). Bacterial alterations of bile salts generally involve first deconjugation and then 7 a dehydroxylation, although there is some evidence that the latter reaction may proceed without an initial deconjugation. 25 Deoxycholic acid is readily reabsorbed from the ileum and colon and is returned to the liver where it is reconjugated and secreted into bile, making up some 20 to 30 per cent of the total bile acid pool in adults. Lithocholic acid on the other hand is poorly soluble at body temperature and is absorbed only to a minor extent, with the majority being excreted in the feces. 45

FETAL AND NEWBORN BILE ACID METABOLISM Maintenance of the bile acid pool requires the precise integration and regulation of several organ systems. Immaturity of hepatic or intestinal functions would profoundly influence this balance and determine the ability of the newborn infant to absorb nutrient lipid. Several studies

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utilizing gall bladder bile from stillborn infants and cord blood have established that the primary bile acids, cholic acid and chenodeoxycholic acid, as well as the secondary bile acid, deoxycholic acid, are present in fetal gall bladder bile and serum. 5 • 51 In addition, lithocholic acid has been isolated from the meconium of newborn infants. 53 What then is the source of fetal bile acids? Does the fetus synthesize, conjugate, and secrete bile acid or do these findings merely reflect placental transfer? Is the fetus capable of responding to bile acid loss with bile acid synthesis and thus demonstrate the responses necessary for independent extrauterine functions? Smallwood, Lester, and Jackson, 30 utilizing elegant intrauterine surgical techniques, have sought to answer some of these important questions. Utilizing 14 C-labeled cholic acid, they have demonstrated that the fetal dog, near term, can take up, conjugate and secrete tracer amounts of bile acid. 31 In addition, the fetal dog will excrete a taurocholate load into the biliary system, 38 the fetal gall bladder can concentrate and deliver the bile into the intestine and the reappearance of the secreted bile acid into the fetal circulation establishes that an enterohepatic circulation for bile acids does exist in utero. The fetal bile salt pool is reduced in size, however, when compared to the adult on a weight basis but the fetus is capable of responding to biliary drainage and depletion of the pool with synthesis from cholesterol of new bile acid. 36 Interestingly, in the dog, cholic acid synthesis predominates with little incorporation of label into chenodeoxycholic acid and no evidence for the fetal synthesis of deoxycholic or lithocholic acid. In subsequent studies to determine the origin of the secondary bile acids detected in fetal bile and plasma, the maternal cholic and deoxycholic acid pools were specifically labeled. Placental transfer to the fetus of maternal deoxycholic acid was demonstrated, implying that fetal deoxycholic acid is derived from the mother rather than a unique fetal synthetic pathway.36 There is as yet no information on the magnitude or importance of this transfer or on the contribution of the fetus to the maternal bile acid pool. The placental transfer of organic anions such as bilirubin or bile salts varies considerably between species and seems in part to be inversely correlated with the relative hepatic maturity at birth. This has been well demonstrated for the placental transport of bilirubin in the dog3 and in the monkey. 5 7 The dog has a relatively mature fetal liver and little placental transport of bilirubin, whereas the monkey, which may be jaundiced at birth, has a less mature liver and a diminished ability to conjugate and to secrete bilirubin, and thus relies upon the placenta as an excretory organ. Preliminary information from similar studies on the placental transport of bile salt suggests a similar trend. 5 7 Accordingly, although the fetal liver may be well adapted for the intrauterine environment, the questions remain as to whether in man hepatic function is sufficiently mature at birth to permit the infant to maintain adequate intraluminal concentrations of bile acid and to respond to fecal losses with the synthesis of new bile salts, and/or whether intestinal function is sufficiently mature to conserve these important biological detergents and to establish an effective enterohepatic circulation for bile salts. Immature hepatic func-

507

BILE ACID METABOLISM AND FAT ABSORPTION IN NEWBORN INFANTS

Table 1.

Relation of Duodenal Bile Acid Concentration to Coefficient of Fat Absorption BILE ACID CONCENTRATION

(mM./L.)

COEFFICIENT OF FAT ABSORPTION

No. of Infants >80% No. of Infants <80% Total x 2 ~ 4.04

0-2.0

2.01-4.0

>4.0

Total

0

19 8 27

21 3 24

40 22 62

11 11

p 0.01

Data demonstrate a positive and significant relation between intraduodenal bile acid concentration and fat absorption in 62 newborn infants. Published with the kind permission of Dr. Uri Lavy, personal communication.

tion in newborn infants has been demonstrated in regards to bilirubin conjugation 19 and excretion 18 and for BSP excretion, 68 but there is little information concerning bile salt metabolism. Dietary lipid absorption in neonates is inefficient by adult standards and only begins to approach adult levels (95 per cent of ingested fat absorbed) by 6 to 9 months of age. 14 Evidence that intraluminal phase defects and altered bile acid metabolism contribute to this defect is for the most part indirect. Glycerides are excreted in feces during the first 3 to 11 days of life, 63 which implicates either incomplete lipid hydrolysis due to insufficient concentrations of pancreatic lipase and/or incomplete micellar solubilization of lipolytic products because of diminished bile acid concentrations. Lavy et al., in studies of low birth weight infants, reports normal lipase activity but low intraluminal bile acid concentrations after feedings.3 5 The intraluminal bile acid concentrations in these infants subsequently increased with age; an increase which correlated well with improved fat absorption (Table 1). Other investigators, using various perfusion techniques, have reported somewhat lower lipase values for the newborn infant but in all cases intraluminal bile acid concentrations were reduced below the amount estimated to be necessary to solubilize completely the products of lipolysis. 43 Attempts to isolate directly the aqueous phase and to quantitate the hydrolysis and micellar solubilization of lipid have not yet been performed in newborn infants; however, in one study of 10 older children, 6 to 13 months of age, micellar solubilization of lipid and not lipid hydrolysis was the rate-limiting intraluminal phenomenon. 49 Studies quantitating the total body pool of bile acid have been recently reported for newborn infants utilizing nonradioactive deuteriumlabeled bile acid. The bile acid pool size was determined by an isotope dilution technique 44 and found to be reduced in newborn infants to approximately one half the adult values, when compared on the basis of body surface area. 45 The values for premature infants between 32 and 36 weeks' gestation were further reduced to approximately one-half to one-

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CHOLATE POOL

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CHOLATE SYNTHESIS

me/ M 2 1 DAY

600

500

250

400

200

300

150

200

100

100

50

PREMATURE

FULL TERM

ADULT'f'

Figure 4. Comparison of cholate pool size and synthetic rate in premature infants, fullterm infants and adults, corrected for body surface area. The data on pool size and synthesis rate for adults ('Y) are extrapolated from Vlahcevic et al.62 while data for full term infants are from Watkins et al.65 The data for premature infants are from unpublished observations of Watkins eta!.

third that of the full term infants 64 (Fig. 4). Intraluminal bile acid concentrations were similarly reduced in conjunction with the pool size measurements to levels below that associated with efficient solubilization of the products of lipolysis. Bile acid synthesis rates when compared to adult values were increased relative to the pool size; however, they were still insufficient to maintain an expanded pool size, or adequate intraluminal concentrations. The newborn infant then is capable of responding to fecal losses through the synthesis of bile salt but demonstrates an immaturity of bile salt metabolism particularly in regard to the regulation of pool size. Further studies are needed to evaluate bile salt secretion rates, to establish the mechanisms for bile salt reabsorption and to assess the functional maturity of the enterohepatic circulation. The information gained should provide much needed help in understanding the developmental aspects of lipid absorption, and form the basis for nutritional therapy in infants at risk.

APPLICATION TO DISEASE STATES AND FUTURE DEVELOPMENTS Abnormalities of bile salt metabolism have been implicated as having pathophysiological importance in two principal areas, persistent intractable diarrhea of infancy and in cholestatic syndromes such as biliary atresia. The role of bile salt in association with diarrheal states stems in part from the observations of Burke and Anderson, 8 who initially reported a transient monosaccharide intolerance in neonates who exhibited bacte-

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rial overgrowth and increased intraluminal concentrations of free bile salts. Subsequent investigations directed towards delineating the effect of these unconjugated bile salts on intestinal function have demonstrated an interference with monosaccharide absorption associated with ultrastructural evidence for direct toxic injury to the mucosa. The intraluminal effects of bile salt deconjugation include alteration of the physical properties of bile salts leading to their precipitation and/or reabsorption from the intestine, reduced intraluminal bile salt concentrations, poor micellar solubilization of lipid and fat malabsorption. Thus, on two counts, situations leading to stasis and bacterial overgrowth may,potentiate continued malabsorption. 61 Regulation of intestinal fluid and electrolyte secretion is also affected by intraluminal factors. Hofmaim and Poley originally described a "choleretic diarrhea" in patients 27 with ileal resection or disease. Such patients have increased fecal loss of bile salts, which results in elevated colonic concentrations of bile acid. Utilizing elegant perfusion studies these and other investigators have been able to demonstrate that increased concentrations of (particularly) the dihydroxy bile acids produce actual secretion of water and electrolyte into the colon. 40 These patients often exhibit a dramatic cessation of diarrhea with the administration of cholestyramine, a resin which binds intraluminal bile salts. Similar effects have been demonstrated to occur with perfusion of conjugated bile salts in the small intestine as well as through a mechanism possibly mediated by cyclic AMP. 4 • 67 The importance of these mechanisms for the postoperative neonate or the infant with intractable diarrhea have not yet been determined but the interesting possibility is raised that intraluminal factors may potentiate some of the chronic diarrheas in infancy. Another major area of concern is the role of bile salts in the production of hepatic injury and cholestasis. There is increasing evidence, for example, that the biliary atresias are probably not developmental anomalies, as neither extrahepatic nor intrahepatic biliary atresia is seen in aborted fetuses, stillbirths, or premature infants. 7 Indeed some of the entities may be inherited/ 3 thus leading to interest in defects of bile acids synthesis or secretion. Lithocholic acid, a secondary bile acid produced from chenodeoxycholic by intestinal bacteria (Fig. 3), for example, produces cholestasis and bile duct proliferation in experimental animals. 33 In addition it has been isolated from meconium, and found in the urine of infants with biliary atresia. 38 Palmer has demonstrated that such bile acids are sulfated at the three hydroxyl group in adults, a modification which increases their solubility in aqueous solution 44 and enhances urinary excretion. This pathway for detoxification is established in utero 23 and has been demonstrated to exist in infancy. In fact one infant with biliary atresia, when administered labeled lithocholic acid, was found to excrete this bile salt principally as the conjugated sulfate derivative in the urine. 3 " The search for synthetic defects producing other possibly toxic monohydroxy bile acids has led to the identification of several unsaturated bile acids in infants with biliary atresia. 39 In addition, two infants with

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biliary atresia have been found who secrete large amounts of coprostanic acid, with an unusual C 27 bile acid in their urine. 13 The importance of this finding is highlighted by the work of Hanson et al. who have recently reported the same bile acid in a child with a familial type of cholestasis. 24 This child is unable to convert the C 27 bile acid by side-chain reduction to cholic acid, a c24 bile acid, thus demonstrating the first known defect in the synthesis of bile acids in a child with cholestasis. Other areas of investigation include analysis of the serum bile acid patterns in children with other forms of cholestasis. These studies have provided preliminary information which may enable one to differentiate between biliary atresia and cholestasis secondary to infection or other causes. Javitt et al. 34 have noted that the ratio of trihydroxy to dihydroxy bile acids is altered in biliary atresia and that this ratio does not revert to normal following administration of cholestyramine, in contradistinction to situations of incomplete biliary obstruction. These investigations, and others concerned with the effects of pharmacologic agents on bile flow and bile acid synthesis, are the beginning of an exciting period of investigation which can be expected to lead to a greater understanding of the mechanisms of bile acid synthesis and secretion and provide treatment for the cholestatic syndromes in infancy. ACKNOWLEDGMENT

The author wishes to thank Dr. Richard J. Grand for his comments and help in the preparation of this manuscript, Dr. Roger Lester whose thoughtful discussions and interest stimulated much of the work reported in this paper, and Mrs. Helen O'Meara for her secretarial assistance.

REFERENCES 1. Benzonana, G., and Desnuelle, P.: Etude Cinctique de !'action de la lipase pancreatique sur des triglycerides en emulsion: Essai d'une enzymologice en milieu heterogene. Biochim. Biophys. Acta, 105:121-136, 1965. 2. Bergeret, B., and Chatagner, F.: Desulfinication et decarboxylation enzymatiques de l'acide 1-cystine sulfinique: satrans formation en alanine et en hypotaurine. Biochim. Biophys. Acta (Arnst), 9:147, 1952. 3. Bernstein, R. B., Novy, M. J., Piasecki, G. J., et al: Bilirubin metabolism in the fetus. J. Clin. Invest., 48:1678, 1969. 4. Binder, H. J., and Rawlins, C. L.: Effect of conjugated dehydroxy bile salts on electrolyte transport in rat colon. J. Clin. Invest., 52:1460, 1973. 5. Bongiovanni, A.M.: Bile acid content of gall bladder in children and adults. J. Clin. Endocrin. Metab., 25:678, 1965. 6. Borgstrom, B., and Erlanson, C.: Pancreatic juice co-lipase: physiological importance. Biochim. Biophys. Acta, 242:509-513, 1971. 7. Brent, R. L.: Persistent jaundice in infancy. J. Pediat., 61:111, 1962. 8. Burke, V., and Anderson, C. M.: Sugar intolerance as a cause of protracted diarrhea following surgery of the gastrointestinal tract in neonates. Aust. Ped. J., 2:219, 1966. 9. Carey, M. C., and Small, D. M.: Micelle formation by bile salts. Arch. Intern. Med., 130:506-527, 1972. 10. Carey, M. C., and Small, D. M.: The characteristics of mixed micellar solutions with particular reference to bile. Amer. J. Med., 49:590-608, 1970. 11. Desnuelle, P.: Pancreatic lipase. Adv. Enzymol., 23:129, 1961. 12. Dowling, R. H., Mack, E., and Small, D. M.: Effect of controlled interruption of the enterohepatic circulation of bile salts by biliary diversion and by ileal resection on bile salt secretion, synthesis and pool size in the Rhesus monkey. J. Clin. Invest., 49:232-242, 1970.

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13. Eyssen, H., Parmentier, G., Compernolle, F., et al.: Trihydroxycoprostanic acid in the duodenal fluid of two children with intrahepatic bile duct anomalies. Biochem. Biophys. Acta, 273:212, 1972. 14. Filer, L. J.: Excretion of fat by normal full-term infants fed various milks and formulas. Amer. J. Clin. Nutrition, 10:1299, 1970. 15. Fomon, S. J.: Infant Nutrition. Philadelphia, W. B. Saunders Co., 1967. 16. Frazer, A. C., Schulman, j. H., and Stewart, H. C.: Emulsification of fat in the intestine of the rat and its relationship to absorption. J. Physiol., 103:306, 1944. 17. Garbutt, J. T., Heaton, K. W., Lack, L., et al.: Increased ratio of glycine- to taurine-conjugated bile salts in patients with ileal disorders. Gastroenterology, 56:711, 1969. 18. Gartner, L. M., and Arias, I. M.: Formation, transport, metabolism and excretion of bilirubin. New Eng. J. Med., 280:1339, 1969. 19. Gartner, L. M., and Arias, I. M.: Pharmacologic and genetic determinants of disordered bilirubin transport and metabolism in the liver. Ann. N. Y. Acad. Sci., 151 :833, 1968. 20. Gracey, M., Burke, V., and Oshin, A.: Influence of bile salts on intestinal sugar transport in vivo. Scand. J. Gastro., 6:273, 1971. 21. Gracey, M., Burke, V., Oshin, A., et al.: Bacteria, bile salts and intestinal monosaccharide malabsorption. Gut, 12:683, 1971. 22. Gracey, M., Papadimitriou, J., Burke, V., et al.: Effects on small intestine function and structure induced by feeding a deconjugated bile salt. Gut, 14:519, 1973. 23. Gustaffson, J. A., Shakleton, C. H. L., and Sjovall, J.: Identification of 22-, 24- and 26hydroxy cholesterol in the steroid sulfate fraction of feces from infants. Europ. J. Biochem., 8:467, 1967. 24. Hanson, R. F., Klein, P. D., Williams, G. C., et al.: Familial paucity of intrahepatic bile ducts with a defect in the metabolism of trihydroxyloprostanic acid (THCA). Clin. Res., 21 :867, 1973. 25. Hepner, G. H., Hofmann, A. F., and Thomas, P. J.: Metabolism of steroid and amino acid moieties of conjugated bile acids in man. I. Cholylglycine. J. Clin. Invest., 51:1809, 1972. 26. H6fmann, A. F.: The role of bile salts in fat absorption: the solvent properties of dilute, micellar solutions of conjugated bile salts. Biochem. J., 89:57, 1963. 27. Hofmann, A. F.: The syndrome of ileal disease and the broken enterohepatic circulation: Choleretic enteropathy. Gastroenterology, 52:752, 1967. 28. Hofmann, A. F., and Borgstrom, B. J.: The intraluminal phase of fat digestion in man: The lipid content of micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest., 43:247-257, 1964. 29. Hofmann, A. F., and Small, D. M.: Detergent properties of bile salts: Correlation with physiological function. Ann. Rev. Med., 18:333, 1967. 30. Jackson, B. T., and Egdahl, R. H.: The performance of complex fetal operations in utero without amniotic fluid loss or other disturbances of fetal maternal relationships. Surgery, 48:564, 1960. 31. Jackson, B. T., Smallwood, R. A., Piasecki, G. J., et al.: Fetal bile salt metabolism I. The metabolism of sodium cholate- 14C in the fetal dog. J. Clin. Invest., 50:1286-1294, 1971. 32. Javitt, N. B.: Cholestatic syndromes. Amer. J. Med., 51:637, 1971. 33. Javitt, N. B., and Emerman, S.: Effect of sodium taurolithocholate on bile flow and bile acip excretion. J. Clin. Invest., 47:1002, 1968. 34. Javitt, N. B., Morrissey, K. P., Siegel, E., et al.: Neonatal choleastatic syndromes: Diagnostic value of serum bile acid pattern and cholestyramine administration. Pediat. Res., 7:119, 1973. 35. Lavy, U ., Silverberg, M., and Davidson, M.: Role of bile acids in fat absorption in low birth weight infants. Pediat. Res., 5:387, 1971. 36. Lester, R., Little, J. M., Greco, R., etal.: Fetal bile salt formation. Pediat. Res., 6:375, 1972. 37. Lindstedt, S.: The turnover of cholic acid in man: bile acids and steroids. 51. Acta Physiol. Scand., 40:1-9, 1957. 38. Lyhovin, B. A., Norman, A., Strandvik, B.: Metabolism of lithocholic acid ••-••c in extrahepatic biliary atresia. Acta Ped. Scand., in press. 39. Makino, I., Sjovall, J., Norman, A., and Strandvik, B.: Excretion of 3B hydroxy-5-cholenoic and 3 hydroxy-5a-cholanoic acids in urine of infants with biliary atresia. FEBS Letters, 15:161, 1971. 40. Mekjjian, H. S., Phillips. S. F., and Hofmann, A. F.: Colonic secretion of water and electrolytes induced by bile acids: Perfusion studies in man. J. Clin. Invest., 50:1569, 1971. 41. Morgan, R. G. H., and Hoffman, N. E.: The interaction of lipase, lipase cofactor and bile salts in triglyceride hydrolysis. Biochim. Biophys. Acta, 248:143-148, 1971. 42. Mosbach, E. H.: Hepatic synthesis of bile acids. Arch. Intern. Med., 130:478, 1972. 43. Norman, A., Strandvik, B., Ojamae, 0.: Bile acids and pancreatic enzymes during absorption in the newborn. Acta Paediat. Scand., 61:71, 1972.

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WATKINS

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