The simultaneous determination of oxidized and reduced glutathiones in liver tissue by ion pairing reverse phase high performance liquid chromatography with a coulometric electrochemical detector.

The oxidized (GSSG) and reduced (GSH) forms of glutathione were quantified simultaneously in rat liver tissues by ion-pairing reverse phase high pressure liquid chromatography coupled to a coulometric electrochemical detector (HPLC-EC). Other biological thiols namely cysteine, cystathionine, homocysteine and methionine were shown to be well resolved from the glutathiones. Standard curves for GSH and GSSG were linear over the range of glutathione concentrations found in biological tissues with a correlation coefficient (r) greater than 0.999. Rat liver tissue content of GSH (16.96 +/- 4.29, n = 5) and GSSG (0.467 +/- 0.188 n = 5) found in this study are similar to results reported by other investigators. This method is also applicable to determine glutathione levels in rat bile samples. The advantages and disadvantages of employing coulometric over amperometric, as well as precautions in establishing HPLC-EC for the detection of oxidized and reduced glutathiones is discussed.

Total protein output during rapid reduction of bile salt secretion rates in man.

An investigation was undertaken to study the effect of bile salt secretion on total biliary protein secretion in man. Bile was collected in eight patients from a tube in the bile duct. Collection was started after a meal and continued for six hours, in order to obtain bile salt secretion rates over the entire physiological range. Total protein secretion rates did not vary with change in bile salt secretion or bile flow. The protein pattern assessed by SDS-PAGE did not vary with bile salt secretion. The results indicate that bile salt secretion has little influence on biliary protein secretion under these conditions in man. Changes in bile salt secretion were associated with linear change in bile flow, but there was no relationship between bile flow and protein secretion rates. This argues against convective sieving of plasma proteins into bile.

Adenine nucleotide tissue concentrations and liver allograft viability after cold preservation and warm ischemia.

The relation between adenine nucleotide liver concentrations and the viability of liver allografts after cold preservation and warm ischemia was studied. A rat model was used with storage times defined in terms of allograft viability. Livers were excised and stored for 4 hr at 4 degrees C or 1 hr at 37 degrees C (viable if transplanted) or for 8 hr at 4 degrees C or 2 hr at 37 degrees C (not viable if transplanted) in a solution containing 0.9% NaCl and 2 mM CaCl2. Adenine nucleotide, malondialdehyde, and glutathione concentrations were measured in liver biopsies at the end of the storage periods and in control livers. During cold preservation, ATP concentrations decline, but degradation is largely halted at AMP, and this is independent of the length of storage or viability of the allograft. Graft failure is not due to lack of availability of intramitochondrial substrate (AMP) for rephosphorylation to adenosine triphosphate (ATP), nor is it likely that provision of such substrate will be helpful. On the other hand, with warm ischemia, degradation to inosine, hypoxanthine and xanthine occurs and nonviable livers develop higher levels of xanthine than viable ones; in fact, xanthine concentrations provide 100% discrimination between viable and nonviable warm preserved livers. Malondialdehyde concentrations were also significantly greater in the warm preserved nonviable livers, indicating that some lipid peroxidation may occur even before reperfusion of allografts. Glutathione concentrations were similar in all experimental groups.

Markers of allograft viability in the rat. Relationship between transplantation viability and liver function in the isolated perfused liver.

The relationship between transplant viability and liver function has been examined. Wistar rat livers were preserved at 4 degrees C for increasing intervals and then transplanted into Wistar rat recipients. Two critical times were identified, the longest preservation period with 100% transplantation success (4 hr) and the shortest preservation period with 100% transplant failure (8 hr). The comparable critical times were also identified in livers preserved at 37 degrees C (1 hr and 2 hr). Liver functions were studied by the isolated perfused liver technique in other rat livers stored at 4 degrees C or 37 degrees C for the critical times. Two liver function tests, AST and LDH concentration in perfusate, discriminated between viable and nonviable livers across as well as within preservation groups. AST gave the best separation between viable and nonviable livers. Some functions such as ALT concentration in perfusate separated viable from non viable allografts only within preservation groups. Other liver functions were more sensitive to preservation temperature than allograft viability. Oxygen consumption after cold preservation for either critical time was about twice control levels. Urea production was far below control levels in warm-preserved livers but almost normal in cold-preserved livers. Our results indicate that AST release into perfusate can be used as a screening technique to optimize preservation methods, reserving transplantation for confirming the most promising results.

Biliary proteins and the nucleation defect in cholesterol cholelithiasis.

A study was performed to determine whether differences in gallbladder proteins might be present in patients with rapidly nucleating bile. Gallbladder and hepatic bile protein concentrations were measured using a fluorometric assay. The method was validated by an independent technique, i.e., hydrolysis and amino acid analysis. Persons with cholesterol gallstones had significantly higher gallbladder bile protein concentrations than patients without gallbladder disease or patients with pigment stones. The protein concentration correlated with the in vitro nucleation time in the cholesterol stone group. Gallbladder bile proteins were also purified by chromatography and gradient ultracentrifugation. Proteins from patients with cholesterol gallstones accelerated the nucleation time of control bile, whereas protein from controls had little effect. Hepatic bile protein concentrations were similar in persons with and without cholesterol gallstones. The gallbladder-to-hepatic bile ratios of a variety of solutes were examined. The ratio for protein in the cholesterol gallstone group can be explained straightforwardly by water reabsorption in the gallbladder, whereas the very low ratio in patients without cholesterol gallstones suggests that their gallbladders reduce protein mass by a process such as protein absorption or degradation during water absorption in the gallbladder.

Bile salt dependent bile flow in the rabbit: evidence for the importance of an amiloride inhibitable pathway.

Bile salt dependent flow and electrolyte secretion in response to two bile salts were studied in awake rabbits. It was found that sodium glycodeoxycholate had a much greater choleretic and cholioneretic efficiency than sodium taurocholate. The effect of the bile salts on flow and electrolyte secretion was not linear across the range of bile salt secretion rates studied. When amiloride was administered significant decreases in choleretic and cholioneretic efficiencies occurred, but furosemide had no effect. It is concluded that bile salts stimulate electrolyte transport via amiloride inhibitable cellular processes, and that this electrolyte transport is in part responsible for bile salt dependent bile flow.

Evidence for a potent nucleating factor in the gallbladder bile of patients with cholesterol gallstones.

A study was performed to determine whether the rapid nucleation time of gallbladder bile obtained from patients with cholesterol gallstones was due to the addition of a nucleating agent or the removal of an antinucleating agent by the gallbladder. Isotropic phases of gallbladder bile from normal controls (control bile) and from patients with gallstones (abnormal bile) were mixed 50:50 (vol/vol) and the nucleation times of the mixtures and parent biles were determined. The mixtures had rapid nucleation times, similar to those of the gallbladder bile from gallstone patients, indicating that a nucleating factor was present in the abnormal bile. Experiments were then performed using mixtures in which the proportion of abnormal bile was reduced. These studies showed that the nucleating agent was potent. The results were not due to changes in cholesterol saturation or total lipid concentration. The conclusions reached in the first study were supported in a second set of similar experiments in which hepatic bile from gallstone patients was mixed with their own gallbladder bile. It was also found that filtration of abnormal bile through an XM-300 Amicon filter did not eliminate its nucleating potency, indicating that the results could not be explained by the presence of residual microcrystals in the abnormal bile.

Nucleation of cholesterol monohydrate crystals from hepatic and gall-bladder bile of patients with cholesterol gall stones.

Nucleation time and cholesterol saturation index of hepatic and gall-bladder bile were measured in 16 patients with cholesterol gall stones to determine whether a gall bladder or liver defect was responsible for the rapid nucleation time of gall-bladder bile in such patients. Although hepatic bile was consistently more saturated than gall-bladder bile, the in vitro nucleation time of gall-bladder bile was more rapid. Dilution of gall-bladder bile to hepatic bile concentrations did not affect nucleation time. The results indicate that the gall bladder plays an important role in the production of the rapidly nucleating bile which is found in patients with cholesterol gall stones, and that this role is not simply concentration of bile by the gall bladder. Normal and abnormal gall-bladder biles were also compared in a larger group of patients. The view that there is a nucleation defect in cholesterol cholelithiasis which is independent of cholesterol saturation was confirmed. Subgroups of normal and gall-stone population were defined by the nucleation time and saturation index. Results suggested that solitary stones may be produced under different conditions than multiple stones. Some putative nucleating factors were examined but none was found to distinguish between normal and gall-stone bile.

Bile salt-associated electrolyte secretion and the effect of sodium taurocholate on bile flow.

Sodium taurocholate has a remarkably varied effect on bile flow. Preisig et al. showed that a portion of electrolyte secretion is related to bile salt secretion, and other studies have suggested that this fraction of electrolyte secretion is important in bile salt--dependent flow. In the present study in the baboon and the dog, it was found that the magnitude of bile salt-associated electrolyte secretion was closely correlated to the effect of sodium taurocholate on bile flow. Theophylline, an agent widely used to alter cellular electrolyte transport, simultaneously reduced the fraction of biliary electrolyte secretion related to bile salts and the effect of the bile salt on flow. The study suggests an important role for electrolyte secretion in the modulation of the effect of bile salts on bile flow.

[14C]erythritol clearance and canalicular bile acid-independent flow in the baboon.

The use of [14C]erythritol for the quantitative assessment of hepatic bile formation has been studied in baboons using sodium taurocholate to generate canalicular bile flow. It has been found that increments in [14C]erythritol clearance are equal to taurocholate-induced increments in bile flow, but there was no change in [14C]erythritol clearance when bile flow was increased by secretin. No evidence was found to support the view that bile acids affect bile acid-independent bile flow.

Unilateral hepatic duct obstruction in the primate.

The effects of obstruction of one hepatic duct were investigated in two studies in the rhesus monkey. A model to divide bile flow from the two sides of the liver was developed in the first study. Bile flow from one side of the liver was obstructed and backflow from the obstructed biliary tract was investigated in five animals. Test solutions containing sulfobromophthalein (BSP) and conjugated cholic acid-14C were introduced into the obstructed side of the liver at pressures below and above the maximum secretory pressure. These solutes were detected in the circulation and in the bile on the free side of the liver. BSP, secreted on the free side, was conjugated normally (52.7 percent) for this species. There was also backflow of water from the obstructed side and continuing secretion into the obstructed side. It was determined that there were no functionally significant biliary communications between the two separately cannulated portions of the liver. In the second study, graded unilateral obstruction and recovery were studied in five animals. Bile flow, eyythritol-14C clearance, and bile acid, bilirubin, and bicarbonate secretion rates were reduced during obstruction and recovery. The bile:plasma ratio of erythritol-14C fell significantly (1.13 to 0.97) and the bicarbonate concentration increased during partial obstruction. On the free side, bile flow, erythritol-14C clearance, and bile acid secretion rates increased during obstruction. The increase in bile flow was due largely to an increase in canalicular bile acid-dependent flow. The studies indicated that reduced bile flow during acute obstruction is due to both hydrostatic backflow and secretory failure. The latter occurs mainly at the canalicular level, whereas the bile ductules are spared at least relatively. During acute obstructions compensations in unobstructed portions of the liver are due to increased secretion of solute at the canaliculus.

Effect of bile acid synthesis rate on cholesterol secretion rate in the steady state.

In order to discover the effect of bile acid synthesis on cholesterol secretion into the bile, 7 rhesus monkeys were studied at various stable interruptions of the enterohepatic circulation of bile acids. In 4 animals, 2.5 and 7.5% interruptions were compared; 2.5 and 15% interruptions were compared in 5 animals. There was a significant decrease in the cholesterol secretion rate, 0.048 mmoles per 24 hr +/- 0.016 (SD), and a significant increase in bile acid synthesis rate, 0.62 mmoles per 24 hr +/- 0.03 (SD) at the 7.5% interruption. No significant changes in bile acid or phospholipid secretion rates occurred. Therefore, bile acid synthesis rate influences the cholesterol secretion rate in the chronic stable state. At 15% interruption there was a significant decrease in the bile acid secretion rate from 13.4 mmoles per 24 hr +/- 1.8 (SD) to 8.0 mmoles per 24 hr +/- 2.4 (SD), suggesting that the maximum percentage interruption of the enterohepatic circulation of bile acids, at which the bile acid synthesis rate is maintained in this species, has been overestimated. A large variation in cholesterol secretion rate was found in the 7 animals when the 2.5% interruption was examined alone. Bile acid secretion and synthesis rates were considerably more stable at this interruption. Additional determinants of cholesterol secretion into the bile which are independent of bile acid metabolism probably exist.

The contribution of the extrahepatic bile ducts to bile formation.

This study was performed to determine the contribution of the extrahepatic bile ducts to bile flow in the rhesus monkey. Bile flow from the two sides of the liver was divided. The major extrahepatic bile ducts remained connected to one side of the liver only. Bile flow, and the concentrations of [14C]erythritol, bicarbonate, bile acid, and bilirubin in bile samples from the two sides of the liver, in the fed state were measured and compared. An estimate of the net flow from the extrahepatic ducts was obtained from the [14C]-erythritol concentrations on the two sides of the liver and the bile flow rate on the side with the extrahepatic ducts. The [14C]erythritol bile-plasma ratio was significantly lower in bile collected from the side with the extrahepatic bile ducts, than in bile from the other side of the liver. About 10% of total hepatic bile flow originated in the extrahepatic bile ducts, in the fed state. The bicarbonate-[14C]erythritol concentration ratio was significantly higher in bile from the side with the extrahepatic bile ducts. Bicarbonate - bile acid, and bicarbonate-bilirubin concentration ratios were also significantly higher in bile from the side of the liver with the extrahepatic ducts. The extrahepatic bile ducts have a physiologically significant role in the secretion of bile water. Bicarbonate is secreted in association with water in the extrahepatic ducts.

The effect of bile acid synthesis on cholesterol secretion into the bile.

Cholesterol secretion into the bile has been shown to be related to the bile acid secretion rate. It has been suggested that the availability of bile acid micelles controls the entry of cholesterol into the bile. However, previous data could have been interpreted to indicate that bile acid synthesis controls cholesterol secretion into the bile. To discover whether bile acid synthesis has a significant influence on cholesterol secretion, Rhesus monkeys were studied during the period of increasing bile acid secretion and bile acid synthesis, which begins 6-10 hours following interruption of the enterohepatic circulation of bile acids. This is the only condition in which bile acid synthesis and bile acid secretion increase simultaneously. The cholesterol secretion rate fell signficantly during this period, and this effect was enhanced by phenobarbital administration. An increasing cholesterol secretion rate would have been expected if micellar attraction controlled cholesterol secretion under these conditions. Bile acid synthesis appears to have an important influence upon cholesterol secretion into the bile.

Analysis of the components of bile flow in the rhesus monkey.

Bile flow studies were performed in three groups of awake rhesus monkeys. In the first group, the increase in bile flow stimulated by secretin was not accompanied by an increase in erythritol-14C clearance. Resection of the gastric antrum and small intestine in the second group resulted in stable bile flow at fixed bile salt secretion rates. Linear regression lines for bile flow versus bile salt secretion rate and erythritol-14C clearance versus bile salt secretion rate were parallel, and the Y-axis intercept for the latter was consistently higher than for the former. We concluded that erythritol-14C clearance equals canalicular flow, and ductular reabsorption is constant at all bile salt secretion rates in this species. Bile flow was studied during fasting and feeding, over 6 days, in the third group. A model incorporating four bile flow components was developed and tested by multivariate regression analysis. The data fit the model quite well, explaining greater than 90% of the variation in bile flow. A method of measuring the contribution to bile flow of each of the four components is provided.

Bile production in fasted and fed primates.

Previous experiments have suggested differences in the bile salt independent flow rate between monkeys and dogs, and in the ability of bile salt secretion to influence bile flow. The present study was performed to determine whether the availability of food could have influenced these results. Bile flow and bile salt secretion rates were studied in fasted and fed primates. In the fasting experiments, sodium cholate, sodium taurocholate, and monkey bile were infused into the duodenum; only sodium cholate was infused in the feeding experiments. The results showed that bile flow was linearly related to the bile salt secretion rate in both fasting and feeding animals. Bile salt independent flow is extremely variable during fasting and is increased and stabilized by feeding. These studies indicate that the differences found in previous experiments are probably attributable to variations in experimental design.