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.

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.

Sinusoidal lining cell damage: the critical injury in cold preservation of liver allografts in the rat.

We have previously defined viability limits in a rat transplantation model. All liver allografts stored in a simple preservation solution (NaCl 0.9%, CaCl2 2 mM) at 4 degrees C for 4 hr or at 37 degrees C for 1 hr were viable upon transplantation, but all those stored at 4 degrees C for 8 hr or at 37 degrees C for 2 hr were nonviable. Only cold-preserved, nonviable livers showed increased vascular resistance, platelet trapping and an initially low, but then high, rise in aspartate transaminase (AST) upon reperfusion, all suggesting injury to the microcirculation, with secondary injury to the hepatocyte. In the present study, we investigated the morphological changes that occur in livers stored for the defined critical times, using light and electron microscopy after perfusion-fixation. Accurate and reproducible identification of specimens as belonging to viable or nonviable and warm- or cold-preserved could be made in this way. Preservation in the cold first resulted in reversible changes consisting of cellular swelling, alterations of intracellular organelles, and partial denudation of the sinusoidal lining (cold-preserved viable group). Later, under conditions of nonviable cold preservation, detachment of cell bodies of sinusoidal lining cells with nuclear changes and almost complete denudation of the sinusoidal lining was observed. Endothelial cells of larger vessels were only injured mildly. In contrast, under conditions of warm preservation, changes involving mitochondria and later nuclei were found in hepatocytes, and blebbing was more extensive. Endothelial cells were spared relatively. We also examined livers stored in isotonic citrate solution at 4 degrees C for 8 hr and 16 hr, the critical times determined for this solution in another model of rat liver transplantation. The findings were very similar to storage in saline with respect to the changes in the sinusoidal lining cells after cold preservation for the two critical times. The results provide convincing evidence of a qualitative difference between warm and cold preservation injury, with relatively selective damage to hepatocytes or sinusoidal lining cells, respectively. Endothelial damage represents the primary event, resulting in the loss of organ viability following hypothermic storage. Thus morphology may serve as a useful viability marker after preservation.

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.

Evidence for a channel for the electrogenic transport of chloride ion in the rat hepatocyte.

Chloride is the major inorganic anion in bile but its mechanism of passage from blood to bile is uncertain. Specific membrane channels account for most net inorganic anion flux in other cell types such as the proximal tubular cell and red blood cell; disulfonic stilbenes inhibit anion movement through these channels. Therefore, we have sought the presence of similar channels in the hepatocyte. Net inorganic anion flux or conductance was initiated in isolated rat hepatocytes by valinomycin in the presence of an outward potassium gradient. Potassium concentration in the extracellular medium increased from 2.75 +/- 0.02 in control cell suspensions to 3.15 +/- 0.04 in valinomycin-treated cell suspensions. Membrane potential difference (Em) (mV), determined as the distribution of [14C]tetraphenyl phosphonium ion was -28 mV in control cells and -42 mV in valinomycin-treated cells (p less than 0.05). Intracellular chloride concentration (36Cl-) (mEq per liter of cell water) decreased significantly from 38.6 in control cells to 32.0 in valinomycin-treated cells. The observed intracellular concentrations (36Cl-) in both control and valinomycin-treated cell suspensions closely approximates values predicted on the basis of the Nernst equation: 41 and 29 (mEq per liter of cell water), respectively, suggesting that the chloride ion is passively distributed on the basis of the membrane potential difference. Furthermore, net rate-limited cell water loss of approximately 15% of control values was associated with the above valinomycin-stimulated changes in ion distribution, as assessed using three methods of cell water volume determination.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

Effect of sodium taurocholate on amiloride-inhibitable sodium uptake and on intracellular pH of isolated rat hepatocytes.

Evidence is accumulating that bile salts affect cellular mechanisms for the transport of inorganic electrolytes. We investigated the effect of amiloride on 22Na+ uptake rates in the presence and absence of sodium taurocholate (NaTC) and the effect of NaTC on intracellular pH (pHi) in isolated rat hepatocytes. Initial 22Na+ uptake rates (nanomoles per milligram of protein per minute) were significantly suppressed by amiloride (0.6 mmol/L), although the effect was small. NaTC significantly increased 22Na+ uptake rates. The amiloride-sensitive portion of 22Na+ uptake was significantly increased in the presence of NaTC (0.36 +/- 0.07 SEM nmol/mg protein/min vs. 0.20 +/- 0.07 nmol/mg protein/min, P less than 0.02). The pHi was estimated by using the pH probes carbon 14-labeled 5,5'-dimethyloxazolidinedione and acridine orange. NaTC caused intracellular alkalinization. Amiloride caused intracellular acidification, and the reduction of pHi by amiloride was enhanced in the presence of NaTC, although this enhancement is difficult to interpret because of the large effects of amiloride on pHi relative to those of NaTC. Our results indicate that NaTC affects sodium uptake by isolated hepatocytes probably by stimulating the Na+-H+ antiport.

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.

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.

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.

Ileal excretion and bacterial modification of bile acids and cholesterol in patients with continent ileostomy.

Bile acid (acidic sterol) and neutral steroid excretion were determined in 15 patients, five with conventional ileostomy, five with continent ileostomy, and five with continent ileostomy and an ileal resection. Acidic sterol losses were normal in conventional ileostomy patients and not significantly increased in those with continent ileostomy alone. Bile acid excretion rates were significantly increased in patients with a continent ileostomy and an ileal resection. Neutral steroid excretion was similar in all groups and not different from normal. Deoxycholic acid was not detected in ileal effluent of patients with conventional ileostomy and less than 2% of neutral steroid excreted was in the form of bacterial metabolites of cholesterol. The same was true of six of the 10 patients with continent ileostomies; in the other four patients at least 10% of acidic or neutral steroids were excreted as secondary bile acids or as a coprostanol. Modification of steroids was not related to ileal resection. Continent ileostomy was associated with a significant increase in percentage water content and a reduction in the pH of ileal effluent.

Effect of binding of ionised calcium on the in vitro nucleation of cholesterol and calcium bilirubinate in human gall bladder bile.

Biliary calcium may be a nucleating agent in cholesterol cholelithiasis. A study was designed to determine the effect of binding of ionised calcium on in vitro nucleation time. Ultracentrifuged and microscopically clear gall bladder bile from cholesterol gall stone patients was divided into two aliquots. One aliquot served as control and ionised calcium was bound in the second aliquot by addition of EDTA. Nucleation time was observed for the two groups. Addition of EDTA had no effect on lipid composition of the biles. EDTA bound all ionised calcium. Calcium bilirubinate precipitated from all controls on day 1 but was absent in all samples with EDTA. Addition of EDTA had no effect on cholesterol crystal nucleation time; nucleation time was rapid in both the controls and samples with EDTA. Ionised calcium is essential for calcium bilirubinate precipitation but is not responsible for the rapid nucleation time of bile from cholesterol gall stone patients.

Quantitative analysis of major, minor and trace elements in gallbladder bile of patients with and without gallstones.

The concentration of 25 major, minor and trace elements in human bile was determined by inductively coupled plasma atomic emission spectrometry. Gallbladder bile was obtained during surgery from patients with cholesterol gallstones, pigment stones and with no biliary tract abnormalities (controls). Comparison of the concentration of elements (microgram per gram of solids) did not reveal any significant differences among the three patient groups. The in vitro nucleation time was not related to the concentration of any element measured.

Effect of mucous glycoprotein on nucleation time of human bile.

The purpose of this study was to determine whether mucous glycoprotein is the nucleating factor responsible for the rapid in vitro nucleation time of gallbladder bile from persons with cholesterol gallstones. Ultracentrifugation and ultrafiltration of abnormal bile removed all detectable mucous glycoprotein, yet bile that had been filtered exhibited as rapid a nucleation time as unfiltered bile. When abnormal bile was heated to 95 degrees C for 60 min, nucleation time was significantly prolonged. Rapid nucleation time could be restored to heated abnormal bile by addition of small volumes of unheated bile. Purified human mucous glycoprotein accelerated nucleation time of human bile, but mucous glycoprotein from control patients was as effective as that from gallstone patients. There was a direct relationship between mucous glycoprotein concentration and effect on nucleation time. Mucous glycoprotein may be important in the early stages of stone formation, but it is probably not the agent responsible for the sharp discrimination between control bile and gallbladder bile from patients with cholesterol stones found in the in vitro nucleation time test. The markedly prolonged nucleation time of heated abnormal bile is preliminary evidence that the nucleating factor may be a heat-labile protein other than mucous glycoprotein.

Quantitative and qualitative comparison of gall bladder mucus glycoprotein from patients with and without gall stones.

Human gall bladder mucus glycoprotein was isolated by Sepharose 4B gel filtration followed by caesium chloride density gradient ultracentrifugation from four groups: patients with cholesterol gall stones, patients with pigmented stones, patients with complete obstruction of the cystic duct and patients with no biliary tract abnormalities (controls). Mucus glycoprotein concentrations in cholesterol gall stone bile (203 micrograms/ml +/- 199 SD, n = 17), pigment gall stone bile (110 micrograms/ml +/- 77 SD, n = 6) and control gall bladder bile (96 micrograms/ml +/- 98 SD, n = 11) were not significantly different. While bile from patients with complete obstruction of the cystic duct contained significantly higher concentrations of mucus glycoprotein (6220 micrograms/ml +/- 4130, n = 4). In vitro cholesterol nucleation time was not correlated to gall bladder mucus glycoprotein concentrations. Qualitative analysis of the carbohydrate and amino acid composition showed a basic structure typical of mucus glycoproteins in general. It is unlikely that either quantitative or qualitative differences in mucus glycoproteins are responsible for the rapid in vitro nucleation time characteristic of cholesterol gall stone patients.

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.