C.N. Petrunka

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°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°C (1 hr and 2 hr). Liver functions were studied by the isolated perfused liver technique in other rat livers stored at 4°C or 37°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.

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.

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.

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°C or 1 hr at 37°C (viable if transplanted) or for 8 hr at 4°C or 2 hr at 37°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

Nucleation of cholesterol from vesicles isolated from bile of patients with and without cholesterol gallstones

Bile was obtained from patients with and without cholesterol gallstones at surgery. Biliary vesicles were separated from micelles by gel filtration. The cholesterol/phospholipid ratio in vesicles was much higher than in micelles. Cholesterol crystals nucleated from vesicular fractions, but nucleation from the micellar fractions was slow or did not occur at all. Cholesterol nucleated from vesicles obtained from bile of control patients as rapidly (2.4 days +/- 0.7) as from patients with stones (2.4 days +/- 0.9) and there was no difference in the vesicular cholesterol/phospholipid ratio. The effect of alteration of the bile salt environment was studied by changing the concentration of sodium cholate in the eluting buffer. At low concentrations (5 mM) only vesicles were eluted from the column. These vesicles had a relatively low cholesterol/phospholipid ratio and cholesterol nucleated slowly from these vesicles. At higher concentrations the proportion of micelles increased. The proportion of vesicles decreased progressively but their cholesterol/phospholipid ratio increased and the nucleation time fell. These studies demonstrate that cholesterol nucleates from vesicles in the absence of micelles, that control vesicles are not protected by tightly bound antinucleating substances and that exposure of vesicles to micelles strips relatively more phospholipid than cholesterol from the vesicular fraction, resulting in vesicles with higher cholesterol/phospholipid ratios and shorter nucleation times.

Effect of cold preservation on lymphocyte adherence in the perfused rat liver.

A study was designed to determine if cold preservation induces an increase in lymphocyte adherence to liver sinusoids on reperfusion. Rat livers were stored at 1°C in University of Wisconsin solution for 45 min, 8 hr, or 30 hr, and then reperfused for 90 min at 37°C in an isolated perfused rat liver apparatus. Just prior to reperfusion, isogeneic rat lymphocytes prepared on a Fi-coll-Paque gradient were added to the perfusate. In some studies lymphocytes were labeled with a fluorescent lipophilic membrane marker. There was no change in the number of circulating lymphocytes in an anhepatic circuit. When livers were present in the circuit, lymphocytes were lost from the perfusate into the liver in all studies, with the most rapid decrease occurring within 10 min of reperfusion. The length of preservation had a marked and statistically significant effect on the rate of disappearance of lymphocytes from the perfusate. Reduction by 50% of the number of lymphocytes infused did not affect the results when expressed as percent lymphocytes remaining in perfusate. To exclude the possibility that the loss of lymphocytes into the liver was due to a damaged subpopulation of lymphocytes, two livers stored 3for 45 min were put into the circuit in sequence. The percent reduction in cells due to exposure to a second liver was not significantly different from that observed when cells were exposed only to a single liver. Histological studies showed fluorescence-labeled lymphocytes adherent in sinusoids, and the number of labeled cells was directly related to the length of preservation. Cold preservation induces an increase in lymphocyte adherence in the reperfused liver, which might be important in graft malfunction and rejection.

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.

OXIDATIVE STRESS PLAYS A ROLE IN THE PATHOGENESIS OF FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of upper and lower motorneurons. The disease affects 0.6–2.6:100,000 individuals1, with a mean age at onset of approximately 55 and a duration of two to three years on average2. Motorneuron death results in muscle weakness and paralysis with eventual ventilatory failure and death.