Toxic Bile Salts Research Articles

Tauro β-muricholate is as effective as tauroursodeoxycholate in preventing taurochenodeoxycholate-induced liver damage in the rat

Cholestasis and enhanced biliary leakage of proteins such as lactate dehydrogenase (LDH) and albumin are known to be induced by infusions of relatively toxic bile salts such as taurocholate (TC) and taurochenodeoxycholate (TCDC). Tauroursodeoxycholate (TUDC) was previously shown to prevent these bile abnormalities when simultaneously infused (1–5). In the present study, we examined whether tauro β-muricholate (T β-MC) has a similar effect. The enhanced biliary excretion of LDH and albumin induced by the infusion of TCDC at a rate of 0.4 umol/min/100 g was markedly prevented by the simultaneous infusion of T β-MC or TUDC at a rate one-fourth that of TCDC. Increased LDH level in plasma and hemolysis caused by the infusion of TCDC were also reduced by either T β-MC or TUDC. These results indicate that T β-MC has a preventive effect on TCDC-induced hepatobiliary changes, which is as efficient as that of TUDC as shown previously, suggesting that the 7 β-hydroxy group is important for this hepatoprotective effect. Furthermore, our results suggest that β-muricholic acid may also have clinical value since current reports demonstrate a beneficial effect of ursodeoxycholic acid on a variety of cholestatic conditions, including primary biliary cirrhosis.

Hepatobiliary Abnormalities Associated with Total Parenteral Nutrition

Evidence of hepatic dysfunction (clinically, chemically, and morphologically) and biliary-tract abnormalities is common in patients receiving TPN. The entity might present as hepatocellular injury (fatty liver, steatonecrosis), intrahepatic cholestasis, acalculous cholecystitis, or cholelithiasis. Infants manifest primarily intrahepatic cholestasis, whereas adults manifest fatty liver early and intrahepatic cholestasis later in the course of therapy. Both groups are at risk for the development of biliary-tract abnormalities. Although most of these changes in the adult are mild and reversible, a small number of patients have recently been reported to develop progressive liver disease. In infants, however, the changes may be more severe, and lead more frequently to progressive liver disease and death. Although this progression to chronic liver disease is worrisome, it is uncertain whether, in patients on long-term therapy, it is due to the TPN or to other conditions or therapies associated with their clinical condition. The pathogenesis of each of these lesions may be multifactorial, including carbohydrate overfeeding, essential-fatty-acid deficiency, amino-acid deficiencies or imbalances, carnitine deficiency, bile-salt toxicity, lipid emulsions, bile-flow reduction, and gallbladder stasis. Therapies in these patients are aimed at alterations in the preceding etiologies (decreased glucose loads, contraction of the gallbladder). Further work is necessary to delineate the exact mechanisms of this entity, especially with regard to the causal relationships of TPN and this entity and to the development of chronic liver disease.

Extrahepatic obstructive cholestasis reverses the bile salt secretory polarity of rat hepatocytes.

To elucidate the consequences of extrahepatic cholestasis on the structure and function of hepatocytes, we studied the effects of bile duct ligation on the turnover, surface distribution, and functional activity of the canalicular 100-kD bile salt transport protein (cBSTP). Basolateral (blLPM) and canalicular (cLPM) liver plasma membrane vesicles were purified to the same degree from normal and cholestatic rat livers and the membrane bound cBSTP identified and quantitated using polyclonal anti-cBSTP antibodies. Cholestasis of 50 h resulted in an increased release of cBSTP into bile, thereby decreasing its in vivo half-life from 65 to 25 h. Furthermore, a significant portion of cBSTP accumulated at the basolateral surface and in intracellular vesicles of cholestatic hepatocytes. This redistribution of cBSTP was functionally paralleled by decreased and increased electrogenic taurocholate anion transport in cLPM and blLPM vesicles, respectively. These results demonstrate that biliary obstruction causes a reversal of the bile salt secretory polarity of rat hepatocytes. The resulting increase in basolateral (sinusoidal) bile salt efflux might protect hepatocytes from too high an accumulation of toxic bile salts within the cell interior.

Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties--studies on isolated hepatocytes and lipid membrane vesicles.

To characterize the relative toxicity of different bile salts, isolated hepatocytes were incubated with different concentrations of one bile salt or with identical concentrations of different bile salts and their conjugates. Incubation lasted for 1 hr; samples were taken at intervals and studied for enzyme release, urea synthesis and stimulation by glucagon, and by electron microscopy. While the trihydroxylated bile salt, taurocholate, did not produce alterations at concentrations up to 1,500 microM, the dihydroxylated salts, chenodeoxy- and deoxycholate, caused enzyme release and membrane lysis, and inhibited urea synthesis at concentrations above 500 microM. In contrast, ursodeoxycholate was ineffective at concentrations up to 1,500 microM. Conjugation of these bile salts did not result in significant differences with the exception of deoxycholate conjugates which induced enzyme leakage more rapidly. Studies of lipid membrane vesicles revealed corresponding alterations. The monohydroxylated salt, taurolithocholate, caused cellular damage as indicated by enzyme loss and impairment of hormonal sensitivity of cells at low concentrations (30 to 100 microM). Dihydroxylated salts produced a different time course of membrane leakage, ultrastructural changes and release of volume marker and lipid in liposomes, suggesting a possible different mechanism of damage induced by this bile salt. Both systems can readily be used to study bile salt membrane interactions.

Studies on the mechanism of bile salt-induced liposomal membrane damage.

The damage of phosphatidylcholine membranes by bile salts such as cholate, deoxycholate (DC), chenodeoxycholate (CDC), ursodeoxycholate (UDC), as well as their glyco- and tauroconjugates, and lithocholate (LC) were studied. The permeabilities of liposomes differing in size (700 and 1,700 A in diameter) were determined at increasing bile salt concentrations. The release of entrapped raffinose (3H) (MW: 594) or inulin (3H) (MW: 5,000) was measured by pelleting of the liposomes and subsequent determination of the radioactivities in the supernatant. The release of these uncharged volume markers of different size points to a formation of membrane leaks increasing in size with increasing bile salt concentration. Determination of the membrane damaging threshold concentrations of bile salts demonstrated a higher stability of the smaller liposomes. Incubation of the smaller liposomes with increasing DC concentrations results in a successive substitution of lecithin by DC. The predominantly DC-containing vesicles are of remarkable stability against higher DC concentrations. The damaging properties of bile salts increase with decreasing number of hydroxy groups, with the exception of UDC and its conjugates which are much less membrane toxic than the other dihydroxy bile salts. Conjugation with glycine or taurine slightly enhances the membrane toxicity of bile salts with the exception of UDC. Sulfation of the 3-alpha-hydroxy group of LC reduces the damaging effect to about 10%.

Hepatic failure in infants on total parenteral nutrition (TPN): Clinical and histopathologic observations

Total parenteral nutrition (TPN) is an important adjunct in the care of neonates with surgical disorders. Although cholestasis complicates TPN in 30% of cases, it is usually transient in nature. This report, however, describes 9 instances (8 fatal) of progressive liver failure in surgical neonates who were maintained on prolonged TPN (6 wk-15 mo) and demonstrated distinct hepatic pathologic changes. Diagnosis included complicated gastroschisis, (2) bowel atresia with peritonitis (1), NEC (4), prune belly and antenatal peritonitis (1), and total colonic and ileal aganglionosis (1). Mean gestational age was 35 wk with sexes equally affected. The 8 fatal cases had persistant cholestasis that failed to respond to changes in parenteral protein or glucose concentrations. Enteral feedings were not tolerated. Clinical deterioration was manifested by increasing serum bilirubin (22–35 mg%), clotting dysfunction, enzyme elevations (SGOT), and hepatomegaly. Histologic examination of the liver showed significant distortion of hepatic architecture with hepatocytes arranged in pseudoacini rather than cords and trapped by fibrous tissue. Bile duct proliferation was noted, but ducts contained no bile. Bile plugs were seen in canaliculi only. Hepatocytes showed diffuse vacuolization (+for fat) and contained brown pigment (+for iron). Portal triads were free of inflammation. Ultrastructural examination showed hepatocytes in an acinar array surrounded by collagen. Cystic changes in the endoplasmic reticulum, osmophilic cytoplasmic pigment, lipid droplets, and severe glycogen depletion consisten with acytotoxic insult were seen. Prematurity, immature liver function, and amino acid imbalance with formation of a toxic bile salt are implicated in these findings. Peritonitis, sepsis and inability to tolerate enteral feedings may also play a role. These observations indicate a need to modify TPN application in selected prematures requiring long-term nutritional support.

Effects of Sulfation of Taurolithocholic and Glycolithocholic Acids on their Intestinal Transport

The intestinal transport of the 3-α-sulfate esters of glycolithocholic and taurolithocholic acids was studied in an animal model (guinea pig). Absorption of these compounds was by active transport and was limited to the ileum. This contrasts with the nonsulfated compounds which can be reabsorbed from all sections of the small bowel. Absorption of the nonsulfated compounds was greater in the distal region, probably reflecting two processes: (1) active absorption and (2) passive diffusion. In the proximal regions, only passive diffusion occurred. Ileal transport of sulfated taurolithocholate was depressed by the presence of sodium taurocholate. These results support the concept that sulfation of the potentially toxic lithocholic acid bile salts could curtail their intestinal absorption and enhance their fecal elimination rates.

Relative Toxicity of Different Bile Salts on the Normal Gall Bladder.

In a previous publication we have shown that an experimental increase of the bile-salt concentration in the bile produces changes in the gall bladder wall similar to “human cholecystitis” Sixteen experiments had been performed, using “commercial bile-salts” (Armour and Company), and so-called “purified bile-salts”, i. e., the commercial variety freed from non-alcohol soluble impurities by alcohol precipitation. The latter proved the more effective preparation of the two. The technique employed consisted of the retrograde introduction of a fine ureter catheter into dog's gall bladders through an opening in the common duct and introduction of the bile-salt solution through this catheter. To elucidate this action further, we have made a more thorough investigation using different bile-salt fractions. The following results were obtained from 55 additional experiments: The process of hydrolyzing increases rather than decreases the effectiveness of the bile salts. While a 20% solution causes a tremendous reaction on the gall bladder wall, a concentration as low as 8.7% still produces a marked effect. Two cases of bile peritonitis occurred in this group. In a series of 13 experiments, a special study was made of Na desoxycholate. This proved to be the most powerful fraction, a concentration of 15% causing gangrene of the gall bladder wall, bile peritonitis, or even death, while even a concentration of 7.5% gave a marked reaction. Cholic and apocholic acid are slightly weaker in their actions than desoxycholic acid, apocholic being the stronger of the two. Positive reactions were obtained with 7% apocholic acid, while higher concentrations of cholic acid were necessary to produce similar changes. Glycocholic acid, on the other hand, has much less effect on the gall bladder wall than the fractions described above. An 8% solution yielded a normal gall bladder regularly, a reaction being obtained only with a 13% solution. Injection of taurocholic acid, obtained by evaporation of dog bile to about one-third of its volume, however, gave strongly positive results, i. e., bile peritonitis and death accompanied by a marked change in the gall bladder wall. For the sake of completeness, we examined the action of Decholin (Dehydrocholic acid), which is used in the medical management of human cholecystitis. A 20% solution can be injected into the dog's gall bladder without producing a marked reaction in the gall bladder wall.

Effect of Human Blood Serum on the Toxicity of Bile Salts.

Approximately two dozen white mice were used in these experiments. The effect on the toxicity of bile salts when injected intraperitoneally was observed, using normal saline and blood serum as vehicles. The lethal dose of stock bile salts, dissolved in normal saline had previously been found to be 0.009 gm. This product contained proteins, etc., which were removed, since they might influence the results. The procedure consisted of cleaning with chloroform and ether and dissolving the solid matter in alcohol. The alcoholic solution cleared by standing was pipetted off, filtered, and evaporated and the residue dried in a hot air oven. The residue was then immediately weighed so as to obviate as much as possible the adsorption of water which factor would necessarily influence the weight. The solutions when made up contained 0.001 and 0.002 gm. of bile salts to the cc. Their slight variation in acidity was not sufficiently marked in the author's experience to influence the results. The surface tension estimated by the drop method for the serum and saline salt mixtures was virtually the same for like concentrations of the salt. When normal saline was used as a vehicle 0.009 gm. of bile salts were found lethal as in previous experiments. When fresh human serum (not older than 3 hours) was used in a like capacity, at least twice the amount was necessary to insure a fatal outcome. Thirty-six hours were considered a logical time over which to read results, especially since animals which did not die within that time survived. We observed that human serum is protective toward this toxic product. It is questioned that this is due to the presence of a developed resistance on the part of the body.

I. THE DIFFUSIBLE CALCIUM AND THE PROTEINS OF THE BLOOD SERUM IN JAUNDICE

Investigations into the state of the blood calcium in diseases accompanied by jaundice have been carried out mainly in three directions: the direct examination of the total calcium content of the whole blood, serum or plasma of jaundiced patients and experimental animals; the determination of the toxicity of bile and bile salts as modified by the action of calcium salts, and the injection of these into the blood stream of experimental animals; and by therapy with calcium salts, cod liver oil and ultraviolet radiation, and by the action of extract of parathyroid hormone in experimental animals and in man. The most consistently and most widely quoted works are those of King and Stewart,1Lee and Vincent,2Vines,3Groves and Vines,4Kirk and King5and Walters and Bowler.6It is largely on the basis of these investigations, in which the three methods of approach mentioned were employed, that the current opinion

Effects of Sodium Citrate on Bile Salt Hemolysis.

In a previous report1 we observed an increase in toxicity of bile salts∗ injected intraperitoneally into the white mouse when sodium citrate was added. In the present study the effects of varying dilutions of bile salts and sodium citrate on human blood cells in vitro have been observed.When bile salts are added to red blood cells of different individuals and allowed to stand at room temperature for 18 hours, hemolysis occurs through dilutions of 1:200 to 1:6400. The bloods of 100 undiagnosed individuals were examined in order to determine their susceptibility to bile salt hemolysis. The large variation in ability of bile salts to hemolyse different bloods is independent of changes due to age of cells, and independent of the temperature and humidity under which the cells are kept.When sodium citrate is added to bile salts, hemolysis of red cells takes place in higher dilutions of the bile salts than when the bile salts per se are employed and the dilution at which hemolysis takes place depends upon the co...