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In dogs with biliary obstruction, hemoperfusion through an anion exchange resin (Dowex 1, Bio-Rad Laboratories, Richmond, Calif.) or an uncharged resin (XAD-2, Rohm & Haas Co., Philadelphia, Pa.) resin was carried out to determine whether selected protein-bound anions, normally extracted by the liver and excreted in bile, could be removed. Sulfobromophthalein, chenodeoxycholic acid, and total bilirubin were continuously removed by the resins during the 2-hr perfusion period. Indocyanine green was not removed. Blood levels of chenodeoxycholic acid and bilirubin in the dogs remained relatively constant, but adsorption of anions to the column continued throughout the perfusion, suggesting mobilization of tissue stores. Bile acids were adsorbed to a greater extent by XAD-2 than by Dowex 1, in agreement with in vitro equilibrium binding studies. Similarly, sulfobromophthalein was removed more effectively by Dowex 1 during hemoperfusion, and the charged resin had a greater affinity for sulfobromophthalein in vitro. Platelets and leukocytes were adsorbed during hemoperfusion and in vitro studies. Hemolysis was minimal. Electrolytes remained unchanged. Serum protein concentrations remained constant, but XAD-2 adsorbed some prealbumin and caused some denaturation of ceruloplasmin. Hemoperfusion through charged or uncharged resins appears to be a feasible procedure for removal of protein-bound anions in an animal model designed to simulate the excretory defect present in hepatic failure, and provides an experimental method for assessing the toxicity of protein-bound metabolites.

When clinical studies of potential gallstone-dissolving agents began, it was thought that all the natural bile components might be suitable and that the goal of pharmacologic dissolution might be achieved through expansion of the bile acid pool. But the experience described here showed that only chenodeoxycholic acid dissolved stones, that it dissolved only cholesterol gallstones, and that it acts by rendering bile unsaturated in cholesterol.

A multicompartment model is described for the enterohepatic circulation of conjugated bile acids in man which encompasses bile acid synthesis, amino acid conjugation, bacterial deconjugation, hepatic reconjugation, and bacterial dehydroxylation. When the model was used to predict the fate of administered ring-labeled bile acid, there was good agreement between observed and calculated values. The model was also used to assess the validity of the customary single-compartment analysis of bile acid specific activity decay curves. The errors in estimates of pool size and synthesis rate were found to be small if the Lindstedt procedure was used (tracer administered as free bile acid and specific activity measurements made on combined conjugates of that acid). However, if tracer was given as a conjugated bile acid and the specific activity was determined on only that conjugate, the errors potentially were unacceptably large, especially if cholyltaurine was used. Several novel functions were defined for the enterohepatic circulation of conjugated bile acids, and these were used to derive equations describing biliary bile acid composition for both steroid and amino acid moiety in terms of hepatic and intestinal factors. The model should be of value for quantitative characterization of bile acid metabolism in health and disease.

Measurement of bile acid synthesis rate and pool size by isotope dilution is a reliable technique if the proper isotope is used, the isotope is injected in the chemical form of the free acid, and the pool is sampled validly and for a sufficient time period. However, the accuracy of the method remains uncertain. By defining the relative synthesis rates of the two primary bile acids, one obtains the proportion of each synthesized from cholesterol—a fundamental hepatic property that is significantly altered in several disease conditions. Deoxycholic acid input from the intestine may also be defined by the isotope dilution technique, but the method gives no information on deoxycholic acid formation. Pool size represents the interaction of synthesis and intestinal conservation and may not be used to predict secretion. Specific recommendations are made, for isotope dilution studies in man, regarding chemical form and location of label for the tracer, route and time of administration of tracer, measurement of specific activity in biliary bile acids, and expression of data.

Bile acid metabolism was studied in 10 patients previously cholecystectomized for cholesterol cholelithiasis, in 12 healthy subjects, and in 13 patients with cholesterol cholelithiasis (disease control subjects). The rate of bacterial deconjugation and 7-dehydroxylation was measured by using [2, 4-3H ]cholyl- [1-14C]glycine; measurements of synthesis and pool size were made simultaneously. Total bile acid pool size and biliary bile acid composition were determined by gas-liquid chromatography. Compared with healthy subjects, patients with cholecystectomy showed significantly increased deconjugation and dehydroxylation of the labeled cholylglycine. However, the pool of cholylglycine was considerably smaller, so that the absolute rate of deconjugation was similar. The pool of deoxycholylglycine was twice as large as that in the healthy control and the disease control subjects. In addition, ketohydroxy bile acids (resulting from bacterial dehydrogenation) composed 7 to 15% of the bile acid pool. The 7-keto and 12-keto derivatives of cholic acid were identified by mass spectrometry. The total bile acid pool in cholecystectomized patients did not differ from that in healthy control subjects but was larger than that in the disease control subjects. The size of the total bile acid pool in cholecystectomized patients correlated highly with the size of the pool of secondary bile acids, suggesting that its apparent return to a normal size in these patients after cholecystectomy could be explained in part by the increased input of secondary bile acids from the intestine.

A systemic schedule, in the format of an algorithm, is proposed for the monitoring of transaminase levels in patients ingesting a potentially hepatotoxic drug, and for the subsequent management according to the value found. The scheme is based on the assumption that hepatotoxicity will be signaled by an increased transaminase level and that this test with subsequent determination of serum alkaline phosphatase and liver biopsy provides an adequate data base for the decision to discontinue drug therapy or to continue it at the same or at decreased dosage.

A saturated mixed-chain glycerol triether, 1-hexadecyl-2,3-didodecyl glycerol (1-hexadecoxy-2,3-didodecoxypropane), was synthesized with (3)H at positions 9 and 10 or (14)C at position 1 of the hexadecyl moiety. In acute feeding experiments in rats, less than 0.2% of the triether was absorbed, based on lymph and fecal recoveries. Radioactivity was present exclusively as triether in feces, indicating that it was not degraded by digestive or bacterial enzymes. Chronic feeding experiments in rats confirmed the nonabsorbability of the triether and further indicated that it was nontoxic, did not influence the absorption of dietary fat, and mixed intimately with the fat present in colonic contents and feces. The triether that was absorbed was deposited as triether in adipose tissue, liver, and spleen. When administered intraperitoneally to mice, the triether was stored in the tissues and was not metabolized. When the triether was partitioned between an oil phase of triolein or fatty acid and monoglyceride, and an aqueous micellar phase, the triether remained exclusively in the oil phase. The triether appears to be an ideal nonabsorbable oil-phase marker for use in lipid absorption studies.

A tritium-labeled glycerol triether was tested as a nonabsorbable oil-phase marker in studies of fat absorp- tion in normal rats and in rats with steatorrhea induced by various doses of cholestyramine. Animals were fed a test meal containing 3H-labeled triether and l4C-labeled trilinolein. Fat absorption was estimated in the following three ways: (a) by isotope ratios (the change in 3H/14C in the test meal and in feces); (b) by isotope recovery (the total fecal excretion of 14C radioactivity) ; and (c) by chemical recovery (the total fecal fat excretion). Absorption calculated from isotope ratios agreed well with that calculated from isotope recovery over a range of fat absorption of SCrlOO%, thus validating the use of this lipid marker under these conditions of fat malabsorption. Absorption calculated from chemical recovery was consistently poorer than that calculated from isotope ratios or isotope recovery, thus suggesting that cholestyramine increased the excretion of nondietary (endogenous) fat. Triether may be of value for studying the absorption of compounds present predominantly in the oil phase during digestion and may have significant advantages over other proposed lipid markers.

Seven women with gallstones were given 0.75 to 4.5 g per day of chenodeoxycholic acid, a primary bile acid, to promote micellar solubilization of cholesterol in bile. In Case 1 three stones that had remained unchanged in size during six years of observations disappeared after six months of treatment. A single stone in Case 2 and multiple calculi in Cases 3 and 4 progressively grew smaller during 14 to 22 months of chenodeoxycholic acid administration. Gallstone size did not change in the other three. Chenodeoxycholic acid and total bile acid pool sizes (measured in two patients), which were reduced before therapy, were markedly expanded by chenodeoxycholic acid, and the ratio of bile acids and lecithin to cholesterol in bile increased in all patients. Liver function and morphology remained normal; moderate dose-related diarrhea occurred. Chenodeoxycholic acid may offer medical treatment for cholesterol cholelithiasis in man.

The disorders of fat digestion caused by low intrajejunal pH were studied prior to operation in a patient with a gastrin-producing islet cell tumor (proved by bioassay), gastric hypersecretion, steatorrhea, and normal histological appearance of the jejunum. Samples of small intestinal contents were analyzed for pH, digestive enzymes, lipolytic products, and bile acids after administration of test meals; in vitro pH manipulation was carried out to define the reversibility of the observed changes. Irreversible inactivation of pancreatic lipase attributable solely to an acidic intrajejunal pH produced by gastric hypersecretion was the major defect. Consequent failure to form dispersible lipolytic products resulted in a low concentration of lipid in the micellar phase. In addition, the concentration of bile acids was low in acidic samples because of extensive dilution by gastric secretion as well as precipitation of glycine dihydroxy conjugated bile acids. In vitro neutralization caused the precipitated bile acid to enter solution but did not increase micellar lipid. When neutral samples, in which lipolysis proceeded normally, were acidified in vitro, the lipolytic products passed from the aqueous to the oil phase and bile acid precipitation also occurred. However, since lipolysis was greatly reduced in all acidic samples, inactivation of pancreatic lipase appeared to be the initial and dominant digestive abnormality.

When a 3-keto bile acid methyl ester was chromatographed on basic alumina inactivated with tritiated water, the enolic hydrogen atoms at C-2 and C-4 exchanged with tritium atoms. The (3)H-labeled keto ester was reduced with borohydride, and the resultant mixture of 3alpha- and 3-hydroxy epimers was resolved by preparative thin-layer chromatography to yield a pure 2,4-(3)H-labeled bile acid ester. Lithocholic, chenodeoxycholic, deoxycholic, and cholic acids having a specific activity of 1-10 micro c/ micromole were prepared from their 3-keto derivatives. The tritium label remained intact during alkaline saponification in vitro and enterohepatic cycling in vivo in human subjects.

Bile acid kinetics and biliary lipid composition were characterized in six women with gallstones before and after 6 mo of oral therapy with chenodeoxycholic acid, an agent that induces dissolution of cholesterol gallstones in man. Over a dosage range of 1-4 g/day, absorption varied from 0.8 to 2.3 g/day. The chenodeoxycholic acid pool expanded two-to sixfold, and bile became composed predominantly (> 90%) of chenodeoxycholic acid conjugated chiefly with glycine. Cholic acid and deoxycholic acid pools decreased markedly, so that the total bile acid pool expanded much less, about twofold on the average. Cholic acid synthesis decreased in five of the six patients, consistent with negative feedback inhibition of cholic acid synthesis by chenodeoxycholic acid. In four patients whose bile was above or close to saturation with cholesterol, the bile became unsaturated; in two patients, whose bile was unsaturated, it remained so. In five patients with radiolucent gallstones, chenodeoxycholic acid therapy was continued after completion of kinetic and composition measurements; the stones decreased in size or dissolved entirely during the subsequent 6 to 18 mo. Similar measurements of bile acid kinetics and biliary lipid composition were made before and after a 6-mo period without medication in a control group of six healthy women; no changes occurred.

After oral administration of [2,4-(3)H]-cholyl[(35)S]taurine to eight healthy subjects with indwelling nasoduodenal tubes, the specific activity of the cholyl and taurine moieties and the distribution of radioactivity in biliary bile acid and urinary metabolites, as well as total urinary and fecal (35)S and (3)H, were measured at intervals for 4-8 days. Similar measurements were made after [(35)S]taurine was given orally or intravenously or instilled into the distal intestine. The daily fractional turnover rate of the taurine moiety of cholyltaurine was low and similar to that of the cholyl moiety, indicating that deconjugation occurring during enterohepatic cycling was less than half that previously observed for glycine-conjugated bile acids. Some of the cholyl moiety was absorbed but, since reconjugation occurred predominantly with glycine, little reincorporation into the cholyltaurine pool was observed. Some of the taurine moiety was also absorbed intact but entered large taurine pools, and little reincorporation into the cholyltaurine pool was seen. Oral administration of taurine expanded the cholyltaurine pool and induced a decrease in the fractional turnover rate of the cholyl moiety of cholyltaurine, interpreted to indicate a greater reincorporation of the cholyl moiety because of increased reconjugation with taurine. Taurine moiety not absorbed as taurine appeared to be absorbed largely as sulfate which, like taurine, entered large endogenous pools. Little fecal excretion of (35)S occurred. (35)S was excreted in urine as taurine and sulfate, and excretion in the first 24 h (as percentage of administered dose) correlated highly (r = 0.93) with the daily fractional turnover rate of the taurine moiety. When taurine was instilled into the distal intestine, it appeared as such in plasma, but the more distal the site of instillation, the greater the fraction of urinary (35)S present as sulfate. The [(35)S]sulfate appeared to have come from bacterial degradation of [(35)S]taurine because, when [(35)S]taurine was given intravenously, (35)S was excreted in urine chiefly as [(35)S]taurine with little SO(4)=(-)[(35)S] being present.

The concentration of bile acids in the stomach was measured, both in the fasting state and after a test meal, in 10 patients with gastric ulceration of the proximal lesser curvature and in 10 matched controls. A simple method was developed for measuring total bile acid concentration in serial samples of gastric content, which involves labeling of the bile acid pool, determination of total radioactivity in serial samples, and calculation of bile acid concentration from selected determinations of specific activity. After eating food, patients with ulceration of the proximal lesser curvature had significantly higher concentrations of bile acids in the stomach than did normal subjects, and this difference was due to increased reflux of bile. Acidification of the meal favored precipitation of bile acids. However, at the onset of reflux and at its peak, a large proportion was still in solution. The proportion of labeled bile acid in solution could be significantly reduced by the addition of cholestyramine to the test meal. It is suggested that reflux of bile into the stomach may be a causative factor in gastric ulcer or, if secondary to the presence of an ulcer, may delay healing.

[24-(14)C]Dehydrocholic acid (triketo-5-beta-cholanoic acid) was synthesized from [24-(14)C]cholic acid, mixed with 200 mg of carrier, and administered intravenously to two patients with indwelling T tubes designed to permit bile sampling without interruption of the enterohepatic circulation. More than 80% of infused radioactivity was excreted rapidly in bile as glycine- and taurine-conjugated bile acids. Radioactive products were identified, after deconjugation, as partially or completely reduced derivatives of dehydrocholic acid. By mass spectrometry, as well as chromatography, the major metabolite (about 70%) was a dihydroxy monoketo bile acid (3alpha,7alpha-dihydroxy-12-keto-5beta-cholanoic acid); a second metabolite (about 20%) was a monohydroxy diketo acid (3alpha-hydroxy-7,12-di-keto-5beta-cholanoic acid); and about 10% of radioactivity was present as cholic acid. Reduction appeared to have been sequential (3 position, then 7 position, and then 12 position) and stereospecific (only alpha epimers were recovered). Bile flow, expressed as the ratio of bile flow to bile acid excretion, was increased after dehydrocholic acid administration. It was speculated that the hydroxy keto metabolites are hydrocholeretics. The proportion of cholesterol to lecithin and bile acids did not change significantly after dehydrocholic acid administration. In vitro studies showed that the hydroxy keto metabolites dispersed lecithin poorly compared to cholate; however, mixtures of cholate and either metabolite had dispersant properties similar to those of cholate alone, provided the ratio of metabolite to cholate remained below a value characteristic for each metabolite. These experiments disclose a new metabolic pathway in man, provide further insight into the hydrocholeresis induced by keto bile acids, and indicate the striking change in pharmacologic and physical properties caused by replacement of hydroxyl by a keto substituent in the bile acid molecule.

Cholyl-2,4-(3)H-glycine-1-(14)C was administered orally to eight healthy subjects with indwelling nasoduodenal tubes. The distribution of radioactivity among bile acids and the specific activity of cholylglycine were determined in bile collected at intervals for 7 days. (3)H and (14)C were measured in stool. (14)C in breath was calculated from interval (14)CO(2) specific activity determinations. The daily fractional turnover of the glycine moiety (mean +/-SE, 106+/-17%) was three times greater than that of the cholyl moiety (38+/-7%). On the basis of certain assumptions, it was calculated that about 18% of the cholylglycine pool was deconjugated per enterohepatic cycle. The extent of deconjugation appeared to be unrelated to the efficiency of absorption of the cholyl moiety, which averaged 90-95% per enterohepatic cycle. (14)C was recovered predominantly in breath (52+/-5% of administered dose), and 24 hr (14)CO(2) excretion correlated highly (r = 0.95) with daily fractional turnover of the glycine moiety. (3)H excretion occurred predominantly in feces, and the rate correlated highly (r = 0.92) with the daily fractional turnover of the cholyl moiety. Deoxycholylglycine became labeled with (3)H rapidly, indicating the occurrence of bacterial 7-dehydroxylation of the cholyl moiety and absorption of deoxycholic acid. This biotransformation occurred in all eight subjects but varied in degree and was unrelated to the degree of deconjugation. Since ingested glycine-1-(14)C was not incorporated into bile acid glycine, appearance of (14)C in deoxycholylglycine (observed in three of eight subjects) indicated that 7-dehydroxylation of cholylglycine can occur without deconjugation. Dehydroxylation was also observed in vitro when fecal homogenates were incubated with cholylglycine.

We have examined in vitro some requirements for an artificial support system for use in hepatic failure. These experiments compared methods of removing from plasma (1) certain unbound substances implicated in the pathogenesis of hepatic coma, and (2) some model protein-bound substances of variable toxic potential known to be extracted by the liver and excreted in the bile. A Craig thin film countercurrent dialyzer was used, and dialysis rates were related to protein binding by determining concentrations of unbound species by ultrafiltration. Compounds not bound to protein (methionine, glutamine, butyrate, ammonia, and urea) were readily dialyzed (>90%). Dialysis of protein-bound anions (unconjugated bilirubin, chenodeoxycholate, sulfobromophthalein, and methyl orange) was negligible and was enhanced little by addition of a competitive anion (salicylate) or an acceptor (plasma or a water-soluble polycation) to the dialysate. The major rate-limiting factor was the low concentration of unbound species, rather than the membrane. When plasma was passed over columns containing anion exchange or uncharged resins, protein-bound material was removed efficiently; anion exchange resins were superior to uncharged resins. Both hemoperfusion through anion exchange columns and hemodialysis may therefore be essential excretory components of an artificial liver.

Ingested triglyceride is hydrolyzed by pancreatic lipase to yield monoglycerides and fatty acids, which are dispersed by bile salts into aggregates of micellar size. To determine the molecular arrangement of these aggregates, the surface interaction of appropriate fatty acids, glycerides, and their analogs with synthetically prepared, pure bile salts varying in conjugation, number, and position of hydroxyl groups and type of A/B ring juncture has been characterized. Monoolein was readily penetrated by bile salts. The degree of penetration was proportional to the intrinsic surface activity of the bile salt, and this correlated with the number and position of hydroxyl substituents: taurocholate π A and Δ V A indicated that at high areas the bile salt nucleus lies parallel to the interface; as the area was reduced, the nucleus became perpendicular and the ionic head of the bile salt was forced into the aqueous phase. With further compression, dihydroxy and trihydroxy bile salts separated from the monolayer and passed into the bulk phase; monohydroxy bile salts remained associated with the film, reflecting their greater surface activity. Penetration of monoglycerides, monoglyceride analogs, diglycerides, and triglycerides of oleyl and stearyl homologs indicated that the interaction of bile salts with monoglycerides was much stronger than that with higher glycerides, fatty acids, or propylene glycol monoesters. At 25°C, penetration did not occur unless spacing between the polar heads of monoglycerides was present. The mechanism of penetration was considered to be hydrogen bonding between the hydroxyl groups of the bile salt nucleus and those of the glycerol moiety of the monoglyceride. Monoglycerides form a mesomophic phase in water. Bile salts adsorb to the presumably lamellar or cylindrical arrays and round them off into smaller aggregates; a spherical model of the bile salt-monoglyceride-fatty acid aggregate is proposed. In this model, bile salts function as wetting agents, rather than as detergents, since they adsorb to the interface and do not penetrate the hydrocarbon chains. The ability of bile salts to penetrate films of monoglycerides plus the existence of a mesomorphic phase in the water-poor region of the ternary composition phase diagram of the bile salt-water monoglyceride system suggests that a mesomorphic phase of bile salt-water-monoglyceride and fatty acid may occur during pancreatic lipolysis at the oil/water interface. Thus the mechanism proposed by Lawrence for spontaneous detergence may well occur in biological systems.

The aphorism, "To study the phenomena of disease without books is to sail an uncharted sea, while to study books without patients is not to go to sea at all" (210), applies aptly to bile salts:6 for to consider the function of bile salts in ignorance of their physical properties is to be deprived of many helpful concepts; conversely, studies of the physical properties of bile salts which have been carried out in ignorance of their physiological functions have overlooked many of their distinctive properties. l �.This review summarizes the authors' views of information presently available o n the physical properties of bile salts in solution and at interfaces, both of which are relevant to their physiological function. The detergent

A rapid, sensitive, specific, and valid radioimmunoassay for conjugated cholyl bile acids has been developed. It uses antiserum, produced in rabbits, to cholylglycine coupled by amide linkage to bovine serum albumin. In a radioimmunoassay system, [3H]cholylglycine and antibody reached equilibrium in 1 hr at 42 C. Bound antigen was precipitated by polyethylene glycol, and the free antigen in the supernate was determined by liquid scintillation spectrometry. The displacement curve was linear when the percentage binding of tracer was plotted against logarithmic increase of unlabeled cholylglycine from 5 to 80 pmoles. The antibody bound cholylglycine and cholyltaurine similarly but showed much less affinity for conjugated dihydroxy bile acids or unconjugated bile acids. Cholesterol and hormonal steroids were not bound. The final assay method used less than 0.1 ml of serum and was validated by showing close agreement between radioimmunoassay values and those determined by gas liquid chromatography. In 40 fasting normal subjects, serum levels of immunoreactive conjugated cholyl bile acids were less than 1 μΜ (0.54 ± 0.04 μΜ, mean ± se ). This method should be useful for assessing hepatic function in health and disease.

Thin-layer adsorption chromatography on silicic acid has been scaled down by applying the adsorbent to microscope slides. Rapid, qualitative separations of mixtures of bile acids, bile acid conjugates, bile acid derivatives, and lipid classes are shown.

Chylomicrons isolated from rat chyle were iodinated with iodine131 and the protein specific activity determined before and after incubation with normal rat serum. A marked decrease in chylomicron protein specific activity occurred during incubation because of adsorption of nonradioactive soluble serum protein as well as exchange of part of the labeled chylomicron protein with lipoprotein protein. Ultracentrifugal fractionation of serum after such incubation revealed significant radioactivity in all of the lipoprotein fractions, but with low specific activity reflecting dilution by larger protein pools. Serum proteins with density greater than 1.21 contained much less radioactivity.

A case of idiopathic hypoparathyroidism with a history of repeated grand mal seizures is reported. The patient exhibited papilledema and retinal hemorrhages despite normal cerebrospinal fluid pressure. The fundal findings, unilateral neurological signs and electroencephalographic abnormalities simulated a brain tumor, but cleared after treatment with dihydrotachysterol and calcium gluconate. Lead mobilization from skeletal stores occurred during therapy, and mechanisms involved in interrelationships between lead, calcium and phosphorus metabolism are discussed.

The presence of lipase in gastric aspirates containing less than 3 % contamination with duodenal reflux (as determined by tests for bile acid) was demonstrated in all samples of apparently normal gastric juice from 73 subjects when assayed with a trioctanoin substrate emulsified in sodium taurodeoxycholate and buffered at p H 6. This enzyme, in contrast to lipase(s) in duodenal aspirate, was stable at p H 2, had a lower p H optimum, was rapidly inactivated by trypsin even in the presence of bile acid, and was moderately inhibited by added fatty acid. Like duodenal lipase(s), gastric lipase had greater activity against short chain than long chain triglycerides and was more active against the fatty acids in the 1 than in the 2 position of triglyceride. It had a much smaller apparent molecular weight (40,000 to 50,000) than duodenal lipase (>500,000) by gel filtration chromatography and had only moderate esterifying properties compared with duodenal lipase(s). Synthetic triglycerides were cleaved more slowly by gastric lipase than by pancreatic lipase in pancreatic fistula juice or duodenal content. Triglycerides of human milk particles were cleaved by gastric lipase and lipases present in duodenal content, but not by pancreatic fistula juice which lacked bile acid, suggesting that milk particle triglyceride is resistant to pancreatic lipase unless bile acid is present. In healthy adults, the concentration of gastric lipase in gastric contents was much less than that of pancreatic lipase in duodenal contents, and gastric lipase did not contribute significantly to the lipolytic activity of duodenal content after a test meal. Gastric lipase was significantly decreased in the gastric contents of 3 achlorhydric patients. Medium chain triglyceride was not cleaved by 16 hr of incubation at p H 1.8 or 1 hr of incubation at p H 1, suggesting that "acid hydrolysis" does not occur. Gastric lipase probably contributes to digestion of milk triglyceride in infants, as well as to hydrolysis of administered medium chain triglyceride, especially in children with decreased pancreatic lipase concentrations. Its limited activity against long chain triglyceride suggests that gastric lipase has little role in normal fat digestion in adults.

1. 1. Certain long-chain monoglycerides have a high solubility in bile-salt solutions forming mixed monoglyceride-bile-salt micelles. Monoglycerides in such an optically clear, micellar solution are readily hydrolyzed by pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3). Using a rat-pancreatic-juice preparation containing negligible esterase activity, the hydrolysis of a number of pure monoglycerides in micellar solution has been studied. 2. 2. The rate of hydrolysis was essentially linear with time and enzyme concentration up to a level of around 60–70% hydrolysis of the substrate. The extent of hydrolysis fell when the concentration of micelles of identical composition was increased. There was a broad pH optimum 5.5–7.5. Bile salts greatly enhanced the rate of hydrolysis at low concentrations, but this effect was possibly chiefly due to their dispersing effect; at higher concentrations, they caused a marked inhibition of hydrolysis. Typical anionic detergents influenced hydrolysis in the same manner as bile salts. With a cationic detergent the enzyme concentration had to be considerably increased to obtain the same rate of hydrolysis. 3. 3. 2-Monoolein was not attacked to any appreciable extent by pancreatic lipase; thus the specificity of lipase for the 1-ester position could be directly shown. Glycol monooleate was readily hydrolyzed; 1-α,α-dimethyl-monodecanoin was not split. 4. 4. Under experimental conditions where 1-monoolein was extensively hydrolyzed, middle-chain saturated 1-monoglycerides were hydrolized to a very limited extent. Extensive hydrolysis could be obtained by increasing the micellar amphiphile to micellar bile-salt ratio, by adding other amphiphiles such as fatty acid, or by using a two-phase heptane-buffer system where the monoglyceride was concentrated at the interface. The results of these and other experiments were interpreted as showing the tightness of packing of the micelle to be a critical factor influencing the extent of hydrolysis. 5. 5. Titration of the fatty acid of a mixed bile salt-monoglyceride-fatty acid micelle indicated a pK a of 6.9 with the experimental conditions present.

Fed cholestanol is converted by the rabbit to 5α-bile acids which coprecipitate with the normally occurring 5β-bile acids to form gallstones composed of calcium and sodium glycoallodeoxycholate and glycodeoxycholate. The present study shows that oral administration of large doses of neomycin prevents gallstone formation in the cholestanol-fed rabbit and reduces the elevated concentration of allodeoxycholic acid in bile, with a reciprocal increase in allocholic acid concentration. The reduction in the concentration of allodeoxycholic acid and in the incidence of gallstones is proportional to the dose of neomycin; at a concentration of allodeoxycholic acid below about 20% of total bile acids, gallstone formation does not occur. Neomycin probably exerts its action by modifying the anerobic intestinal flora which dehydroxylate allocholic acid to allodeoxycholic acid; if so, this suggests that both hepatic and bacterial transformations are essential steps in the pathogenesis of cholestanol-induced cholelithiasis. The bile of rabbits on a normal diet contains allodeoxycholic acid (5% of total bile acids). A similar decrease in allodeoxycholic acid concentration and reciprocal increase in allocholic acid concentration is observed when neomycin is administered to rabbits on a normal diet.

A method, featuring perfusion of both gastric and duodenal markers with collections from the stomach and duodenum, was developed and validated for simultaneous measurements of total duodenal and gastric secretory outputs in man. Calculations of duodenal reflux into the stomach, of contamination of duodenal contents by gastric contents, and of the amounts -recycled between the two organs were made. The values so obtained were used to establish application of duodenal perfusion (with simultaneous gastric aspiration) for precise measurements of total secretions into the duodenum by simple calculations. The technique was used to show that total pancreatic enzyme output after intraduodenal stimulation by perfused amino acids was identical with that obtained after a maximally tolerated dose of pancreozymin given by vein. However, simultaneous intraduodenal and intravenous stimulation caused a pancreatic enzyme output significantly exceeding either method of stimulation alone, or any previously reported, thereby suggesting that conventional stimuli cannot attain maximal secretory capacity for enzymes. When applied to determination of total bilirubin output in the duodenum, the results approximated those reported by other methods.

The absorption of cholesterol has been studied in man by perfusing the upper jejunum with a micellar solution of bile salt, 1-monoglyceride, and cholesterol-14C, with a triple lumen tube with collection sites 50 cm apart. The absorption of micellar components between the collection sites was calculated from their concentration changes relative to those of the watersoluble marker, polyethylene glycol. Control experiments were performed with cholesterol-free perfusions of saline or bile salt-monoglyceride solutions. Steady state conditions were obtained. Each of the components of the micelle was absorbed to a different extent during passage through the test segment of jejunum. Bile salt was not absorbed (mean, -3%), but micellar monoglyceride was rapidly hydrolyzed and absorbed almost completely (mean, 98%). Cholesterol radioactivity was absorbed to an intermediate extent (mean, 73%), and the absorption of chemically determined cholesterol (mean, 46%) indicated that much of the disappearance of radioactivity represented true absorption and not simple exchange. The specific activity of the perfused cholesterol fell during passage through the loop. This fall was interpreted as signifying the continuous addition of nonradioactive endogenous cholesterol by the test segment. However, the decrease in specific activity may also be considered to signify exchange, in that nonradioactive molecules entered the lumen as radioactive molecules were absorbed. Plant sterols appeared in the intestinal contents during the perfusion and must have been contributed by the perfused segment. The perfusate and samples taken from the upper and lower collection sites were examined by ultracentrifugation to define the physical state of cholesterol. It was found that cholesterol in the perfusate or upper collection site samples did not sediment, but that 23% of the cholesterol in the lower collection site samples was sedimentable (mean of three experiments); bile salt, as control, was not sedimentable. Solubility experiments in model systems showed that cholesterol possessed low solubility in bile salt solution; its solubility increased markedly and in linear proportion to the amount of fatty acid or monoglyceride or both that was added to the bile salt solution. These findings suggest that polar lipid such as fatty acid or monoglyceride as well as bile salt is essential for normal micellar solubilization of cholesterol in intestinal content. They suggest the necessity of considering an insoluble sedimentable phase of particulate sterol in intestinal content as well as an oil and micellar phase for a complete description of sterol absorption. The marked difference in the rates of absorption of individual micellar components suggests that micellar lipid is not absorbed as an intact aggregate and is consistent with the view that polar lipid such as fatty acid is absorbed in molecular form by diffusion from a micellar solution. The experiments confirm previous findings demonstrating that fat absorption without bile salt absorption occurs in the upper small intestine in man.

Balance studies, which focused on bile acid metabolism, were carried out on 9 patients with ileal resection in order to define the importance of bile acid malabsorption in the pathogenesis of the diarrhea and steatorrhea. The influence of cholestyramine (Q) or type of dietary fat—long chain triglyceride (LCT) or equicalorically substituted medium chain triglyceride (MCT)—on bile acid synthesis, diarrhea, steatorrhea, and fecal electrolyte excretion was measured during steady state intervals of four randomized periods (LCT, LCT + Q, MCT, and MCT + Q). During control (LCT) periods, all patients had bile acid malabsorption, excreting >75% of administered taurocholate-14C within 24 hr, and this malabsorption was accompanied by a 10- to 20-fold increase in bile acid synthesis. Patients with ileal resection of less than 100 cm had greatly increased concentrations of bile acids in fecal water compared with normal control subjects. Administration of cholestyramine significantly decreased fecal weight, frequency, and sodium ion excretion as well as the concentration of bile acid in fecal water. These observations, together with our previous demonstration that bile acids induce water and electrolyte secretion by the human colon, provide evidence that a major cause of diarrhea in these patients was the increased passage of bile acids into the colon. Cholestyramine caused the already increased bile acid synthesis to increase still more in some of these patients; maximal synthesis was 0.1 to 0.3 mmole per kg of body weight per day, based on gas chromatographic measurements of fecal bile acids. All patients had mild steatorrhea ( 20 g per day); fat malabsorption appeared to be a major cause of diarrhea, since replacement of LCT by MCT caused a decrease in fecal weight, sodium, and potassium as well as a striking decrease in steatorrhea. Unabsorbed fatty acids were converted in part to hydroxy fatty acids by intestinal bacteria and it is proposed that unabsorbed fatty acid or its bacterial product, hydroxy fatty acid, or both, contributed to diarrhea. Bile acid malabsorption appeared to play no direct role in the diarrhea in 2 of 3 patients, since there ~ was no response to cholestyramine and the concentration of bile acids in fecal water was low. Malabsorption of bile acids was nonetheless important in the syndrome, since jejunal bile acid concentrations were decreased (causing fat maldigestion) in 2 of 3 patients, and fat maldigestion, together with decreased mucosal surface, was the probable explanation for the severe steatorrhea. These studies define two syndromes of bile acid malabsorption, present evidence for different mechanisms of diarrhea in each, and describe a therapeutic program.

Bile acid and vitamin B 12 absorption were compared prospectively in 22 patients with distal ileal resection, 7 with similar diarrhea but apparently normal ileal function, 5 with the stagnant loop syndrome, and 3 with steatorrhea due to digestive abnormalities. Also studied were 18 healthy control subjects. A test meal containing glycine- 14 C-labeled glycocholate, 3 H-ring-labeled taurocholate, vitamin B 12 - 57 Co with intrinsic factor, and 51 CrCl 3 as nonabsorbable marker was administered to fasting patients. Bile acid absorption was assessed by appearance of 14 CO 2 in breath and by fecal excretion of 14 C or 3 H; B 12 absorption was measured by excretion of 57 Co in urine (Schilling test). Based on fecal excretion of 3 H, nearly all patients with ileal resection had unequivocal bile acid malabsorption. Measurement of 14 CO 2 in breath was less sensitive since four false negatives were identified. Measurement of both 14 CO 2 and fecal 14 C was as sensitive as measurement of fecal 3 H. Of the 14 patients with resections less than 100 cm, 4 had normal Schilling tests, despite nearly all having bile acid malabsorption; both tests were equivocal or abnormal in patients with larger resections. In patients with similar diarrhea but no evidence of ileal dysfunction, bile acid malabsorption was not present, since the ratio of fecal bile acid radioactivity to marker radioactivity was significantly smaller than in patients with ileal resection; B 12 absorption was normal. The breath test appears to be a useful screening test for the detection of the stagnant loop syndrome. Its interpretation is uncertain in patients with ileal dysfunction, since such patients may also have bile acid malabsorption. Thus, the clinical value of the breath test or measurement of bile acid absorption in such patients probably can only be determined by future studies which relate test results to therapeutic response.

Serum conjugates of cholic acid were determined by radioimmunoassay and compared with conventional tests in 38 patients during the course of chronic active liver disease that responded to treatment; 16 patients subsequently relapsed when treatment was discontinued. At the time of diagnosis, values for these bile acids were always significantly elevated. At biochemical resolution of conventional liver tests, the values were still elevated in 33 of 38 patients, and this finding correlated well with evidence of continuing histologic activity. At histologic remission of disease activity, these values were less than twice the upper limit of normal in all 22 patients who subsequently remained in remission without treatment, but were significantly higher in nine of 16 who later relapsed. During relapse, serial determinations showed that elevation of serum conjugates of cholic acid preceded an increased glutamic oxalacetic transaminase. Serum concentrations of these bile acids in chronic active liver dis...