Storage Of Liver Research Articles

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 °C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 °C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 ± 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 °C is near neutrality (7.0 to 7.4).

A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver

Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall's hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed “University of Wisconsin” (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38 °C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 ± 2.4 ml/2 hr/100 g) in livers stored in HC (8.8 ± 2 ml), PPF (9.9 ± 2.2 ml), and UW (10.3 ± 1.9 ml); it was slightly depressed in EC (6.7 ± 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 ± 0.8 ml, P < 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 ± 0.7 ml) but significantly depressed (3.5–6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers.

Differential effects of freezing on total hepatic peroxisomal fatty acid oxidation and on the rate-limiting enzyme, acyl-CoA oxidase

Fatty acid oxidation defects can be acutely fatal, leading to the collection of tissues which are frozen for future analysis. Since peroxisomes can also oxidize long-chain fatty acids, differentiation of the contributions from the peroxisome as opposed to the mitochondria is important. We studied the effects of freezing and storage of rat livers on peroxisomal and mitochondrial β-oxidation as measured by cyanide sensitivity of the oxidation of [1- 14C]oleoyl-CoA to 14CO 2 and acidsoluble labeled products. In addition, we examined the effects of freezing and storage on the rate-limiting enzyme for peroxisomal β-oxidation, acyl-CoA oxidase, by the H 2O 2 generation method. Marked reduction in the oxidation of [1- 14C]oleoyl-CoA was found for both peroxisomal and mitochondrial systems upon freezing at −18 or −70°C for 2 days which declined further on storage at these temperatures for 12 weeks. Loss of activity after freezing was greater for the mitochondrial than the peroxisomal β-oxidation system. By contrast, acyl-CoA oxidase activity was resistant to these changes, maintaining prefrozen activities despite storage for 12 weeks. The contribution of the peroxisomal system to β-oxidation was 32% of the total rate of oxidation of [1- 14C]oleoyl-CoA in the rat liver. These findings indicate that the contributions of the peroxisomal system to total fatty acid oxidation may be considerable, that freezing of the liver results in drastic reduction in enzyme activities of both peroxisomal as well as mitochondrial β-oxidation, but that the rate-limiting enzyme of the peroxisomal system, acyl-CoA oxidase, retains full activity despite freezing and storage.

Effect of cold storage and freezing of bovine and porcine liver on activity and subcellular distribution of mitochondrial enzymes

The subcellular distribution of the mitochondrial enzymes lipoamide dehydrogenase (LIPDH), citrate synthase (CS), and beta-hydroxyacyl-CoA-dehydrogenase (HADH) in bovine and porcine liver tissue was studied by measuring the enzyme activities in a phosphate buffer extract of tissue (total activity) and in liver press-juice (cell plasma). In slaughter-fresh liver most of the activity was located in the mitochondria. During storage of liver under refrigeration (+2 degrees C) for several days a large decrease in total LIPDH activity and a lesser decrease in HADH activity, but no change in CS activity were observed. There was no or only little release of the three enzymes into the cell plasma during storage; this indicates that storage of liver at +2 degrees C was not accompanied by a marked damage of mitochondria. Freezing (-20 degrees C) and thawing of bovine and porcine liver caused some losses of the total activity of HADH and particularly of LIPDH but not changes in CS activity. There was a considerable increase in the activities of LIPDH, CS, and HADH in the press juice after freezing and thawing of liver tissue. Apparently freezing of liver results in damage to the mitochondria and, therefore, in a partial release of the three enzymes from the inner membrane of the mitochondrion into the cell plasma. By storage of liver under refrigeration the mitochondria became more sensitive to freezing and thawing. Prolonged frozen-storage of liver resulted in an increased release of LIPDH, CS, and HADH into the cell plasma.

Comparison of cold storage and perfusion of dog livers on function of tissue slices

We compared how two methods of hypothermic preservation affect physiological functions of tissue slices of dog liver. Livers were preserved by either (i) cold storage (CS) in Collins' solution or (ii) continuous perfusion (P) with a perfusate, containing hydroxyethyl starch, sodium gluconate. adenosine, and potassium phosphate, recently developed in our laboratory. Livers were cold stored for 6 to 8, 24. or 48 hr, and perfused for 24 or 72 hr. Tissue slices of preserved livers were incubated at 30 °C and analyzed for volume control, electrolyte-pump activity (K and Na). and adenine nucleotide concentration. Also, mitochondria were isolated after preservation to quantify respiratory activity. Slice functions of livers preserved for short periods (6 to 8 hr by CS and 24 hr by P) were similar to those for control livers. After normothermic incubation, the mean (±SD) water content of tissue (expressed per unit dry mass of tissue) was 2.3 ± 0.3 kg/kg for control, 2.6 ± 0.4 kg/kg for 6- to 8-hr CS, and 2.5 ± 0.5 kg/kg for 24-hr P. Longer periods of preservation resulted in cell swelling, and water content was 3.3 ± 0.4 kg/kg for 24- to 48-hr CS and 2.8 ± 0.3 kg/kg for 72-hr P. The mean (± SD) K Na ratio was nearly normal for livers preserved for short periods: 3.7 ± 0.5 for control, 4.1 ± 0.2 for 6- to 8-hr CS, and 3.3 ± 0.4 for 24-hr P. Longer periods of preservation resulted in the loss of ion-pump activity and K Na ratios of 2.7 ± 0.2 for 24- to 48-hr CS and 1.7 ± 0.2 for 72-hr P. Energy metablism (ATP synthesis and mitochondrial activity) was well-maintained under most preservation conditons and did not appear to explain the loss of ion-pump activity. The metabolic function of tissue slices is better maintained in livers perfused for 24 hr than in livers cold stored for 24 hr. Perfusion, rather than cold storage, may therefore be necessary to maintain optimal function when livers are hypothermically preserved for 24 hr or more.

The Sampling and Analysis of Human Livers.

A comprehensive approach to the analysis of human livers has been developed in a pilot program for a National Environmental Specimen Bank (NESB). Since 1980, the pilot NESB program has examined the collection, processing, storage and analysis of human livers. Sampling protocols, handling procedures and analytical methods have been developed and implemented considering the requirements for valid analytical results. Sampling and handling included the use of cleanroom technology, specific clean implements and packing materials made from titanium and Teflon and flash-freezing and preservation at liquid nitrogen temperature. Neutron activation analysis played a major role in the implemented analytical scheme. The scheme combined up to four analytical techniques to determine the distribution of 29 trace elements in 66 human livers.

Adenine nucleotides and glycolysis during liver preservation by simple hypothermic storage.

Adenine nucleotides and glycolytic metabolites were examined during hypothermic storage of porcine livers by different preservation methods. The parameters ATP, ADP, AMP, G6P, F6P, FDP, GAP, 2PG, PEP, Pyr and Lact were investigated. The results indicate a dependence of adenine nucleotides but no dependence of glycolytic metabolites on preservation method. Storage by plasma protein fraction or Collins C2 solution causes a better result in TAN concentration after 6 h than albumin and Ringer storage or Ringer trickle perfusion. Conditioning the organ for a better outcome of preservation aimed at increasing the glycolytic rate is of questionable value.

Microbial Flora of Livers, Kidneys and Hearts from Beef, Pork and Lamb: Effects of Refrigeration, Freezing and Thawing

Aerobic plate counts (APC) of livers, kidneys and hearts obtained from beef, pork and lamb soon after slaughter were nearly always <104 and often <103 per cm2. Differences in APC of different sites of the same liver, kidney or heart, within each species, were not significant (P>0.05). APC of livers, kidneys and hearts from pork and lamb after storage for 1, 3 or 5 days at 2 C were not significantly different (P>0.05) from those at day 0. APC of beef livers, kidneys and hearts after 5 days at 2 C differed significantly (P<0.05) from those at day 0, 1 and 3. Temperature abuse of fresh organs for 6–12 h at 30 C before freezing caused major increases in count. Frozen storage of livers, kidneys and hearts (4 days at −20 C) did not cause significant changes in APC. The initial microbial flora of fresh livers, kidneys and hearts was varied with coryneform bacteria and Micrococcus sp. often constituting a major part (>25%) of the microbial flora. After storage for 5 days at 2 C, Pseudomonas sp. more often became a major part of the microbial flora of liver samples. Frozen storage for 4 days at −20 C did not change the microbial flora of beef samples greatly; in pork and lamb, coryneform bacteria more frequently became a major part of the microbial flora after freezing. Changes in pH of livers, kidneys and hearts during storage for 5 days at 2 C were small.

Histamine storage and metabolism of human liver in disorders of the biliary tract.

In three clinical-biochemical trials (all prospective, two balanced but only the last one randomized) histamine methyltransferase (HMT) activity and histamine content were determined in liver tissue of patients being operated for an epigastric tumour or a chronic duodenal ulcer (control group) and of those suffering from disorders of the biliary tract (test group). With median HMT-activities between 361 and 427 pmol/(min×mg protein) in the control and the test groups the human liver showed the highest histamine methylation capacity of all organs of all species hitherto investigated. This explained the well-known high elimination rate from the portal plasma by a single passage through the liver.

Effects of storage conditionsin vitroon the assay of chick liver glycogen

1. The reproducibility of an enzymic method for assaying chick liver glycogen was found to be +/- 1.6%. 2. Storage of liver at either -21 or -70 degrees C for up to 4 weeks, after freezing in liquid nitrogen, had no significant effect on glycogen concentration. However, the concentration of free glucose increased by about 50% during this time. 3. Storage of liver in ice-cold saline for up to 3 h proved unsatisfactory because of a significant loss of glycogen. 4. There was no significant difference in immediate glycogen concentration in liver tissue that was freeze-clamped, frozen in liquid nitrogen or collected into ice-cold saline. The free glucose concentration was higher, when estimated immediately after, with the latter two methods of collection of liver tissue.

Histidine metabolism in mackerel (Scomber scombrus). Studies on histidine decarboxylase activity and histamine formation during storage of flesh and liver under sterile and non‐sterile conditions

SummaryThe concentrations of histidine and histamine in the flesh and liver of mackerel have been determined during storage at 0, 2, 10 and 23°C. Under sterile and non‐sterile conditions little histamine was produced during storage of muscle even after 18 days at 0°C. At 10°C the levels of histamine exceeded 100 mg/100 g tissue in both liver and muscle after 5 days’ storage. The concentration of histidine in muscle remained virtually constant throughout all storage periods but in liver it increased markedly. Histamine showed greatest increase in liver samples. Histidine decarboxylase activity as measured by release of 14CO2 from 14C‐L‐Histidine in crude extracts showed a decrease in muscle and an increase in liver. The significance of bacterial and tissue enzymes on the production of histamine is discussed.

Liver adenine nucleotide metabolism during hypothermic anoxia and a recovery period in perfusion

Reduction of the level of ATP and of the total adenine nucleotide pool has been studied during cold storage of livers initially flushed with Ringer's lactate (R-L), Collins' solution (CO), modified Collins' solution (CMg), and a new hyperosmolar “intracellular” solution containing sucrose (KMgS). The reduction of ATP and nucleotides after 24 hr of anoxia is not influenced by the composition of the flushing solution. When the preserved livers are reperfused with oxygenated blood, the remaining adenine nucleotides are rapidly rephosphorylated, attesting to the persistence of mitochondrial functions irrespective of the solution used. In contrast, a synthesis of adenine nucleotides during the first hours following the anoxic period can only be obtained with the two solutions which have the highest osmolarity and magnesium sulfate content, the CMg and KMgS solutions.

EFFECT OF LYOPHILIZATION AND STORAGE OF LIVER 9,000 χ g SUPERNATANT FRACTION ON THE METABOLISM OF IMIPRAMINE IN SEVERAL SPECIES OF ANIMALS

Effects of lyophilization and storage of liver 9,000 × g supernatant fraction on the activity of drug metabolizing enzymes in some species of animals were studied. Imipramine metabolizing enzyme activity and the content of cytochrome P-450 were stable for, at least, one month without any loss of the activity when lyophilized liver 9,000 × g supernatant fraction of some animal species, i.e., rat, mouse, guinea pig, rabbit and dog, was stored under reduced pressure in a deep freezer (—20 °C). There were no differences of Imipramine metabolism between lyophilized liver 9,000 × g supernatant fraction which was kept in a deep freezer for one month and a fresh fraction which was prepared immediately before determinations of the enzyme activity and cytochrome P-450. The data presented here indicated that the lyophilized preparation of the liver 9,000 × g supernatant as well as the fresh preparation can be used for the determination of the drug metabolizing enzyme activity.

Effects of lyophilization and storage of rat liver microsomes on activity of aniline hydroxylase, contents of cytochrome b5 and cytochrome p-450 and aniline-induced p-450 difference spectrum.

Effects of lyophilization and storage of rat liver 9, 000×g supernatant fraction or microsomes on activity of aniline hydroxylase, contents of cyt. P-450 and cyt. b5 and aniline-induced P-450 difference spectrum were studied. The lyophilization of rat liver 9, 000×g supernatant fraction or microsomes produced about 20% decrease in the activity of aniline hydroxylase. Aniline hydroxylase was stably stored for 6 months if lyophilized 9, 000×g supernatant fraction of rat liver had been stored in an atmosphere of nitrogen or in a low pressure of air. Activity of aniline hydroxylase and contents of cyt. P-450 and cyt. b5 were not reduced during aerobical storage for a week. When lyophilized microsomes were stored for 6 months anaerobically, aniline hydroxylase, cyt. P-450 and cyt. b5 were more stable as compared to aerobical storage. However, activity of aniline hydroxylase was reduced approx. 40% by the storage. The reduction of aniline hydroxylase activity was more significant than that of contents of cyt. P-450 and cyt. b5. Reduction of either activity of aniline hydroxylase or contents of cyt. P-450 and cyt. b5 was more marked when lyophilized microsomes were stored with glycerol. Aniline hydroxylase in lyophilized 9, 000×g supernatant fraction from phenobarbital treated rabbit was stably stored, and aerobically little activity was lost during storage for 7 months.

Effects of lyophilization and storage of rat liver microsomes on activity of aniline hydroxylase, contents of cytochrome b5 and cytochrome P-450 and aniline-induced P-450 difference spectrum

Effects of lyophilization and storage of rat liver 9,000 × g supernatant fraction or microsomes on activity of aniline hydroxylase, contents of cyt. P-450 and cyt. b5 and aniline-induced P-450 difference spectrum were studied. The lyophilization of rat liver 9,000 × g supernatant fraction or microsomes produced about 20% decrease in the activity of aniline hydroxylase. Aniline hydroxylase was stably stored for 6 months if lyophilized 9,000 × g supernatant fraction of rat liver had been stored in an atmosphere of nitrogen or in a low pressure of air. Activity of aniline hydroxylase and contents of cyt. P-450 and cyt. b5 were not reduced during aerobical storage for a week. When lyophilized microsomes were stored for 6 months anaerobically, aniline hydroxylase, cyt. P-450 and cyt. b5 were more stable as compared to aerobical storage. However, activity of aniline hydroxylase was reduced approx. 40% by the storage. The reduction of aniline hydroxylase activity was more significant than that of contents of cyt. P-450 and cyt. b5. Reduction of either activity of aniline hydroxylase or contents of cyt. P-450 and cyt. b5 was more marked when lyophilized microsomes were stored with glycerol. Aniline hydroxylase in lyophilized 9,000 × g supernatant fraction from phenobarbital treated rabbit was stably stored, and aerobically little activity was lost during storage for 7 months.

Twelve-hour liver preservation in the pig using hypothermia and hyperbaric oxygen

Abstract In an attempt to prolong successful storage of the pig liver beyond the 6–8 hours previously described from this laboratory, the addition of cell-membranestabilizing drugs to the preserving solution and the use of a potassium-rich solution were investigated. Using simple hypothermia alone, 10- and 12-hour storage was unsuccessful. Using the previously described hypothermic technique, but with the addition of hyperbaric oxygen at 3 atmospheres absolute (A.T.A.), consistently successful 12-hour preservation was achieved.

Successful 20-hour storage of the canine liver by continuous hypothermic perfusion

Summary Fifteen canine livers were stored by means of continuous hypothermic (10°C) perfusion using homologous serum as the perfusate. The organs were transplanted as allografts. Succinate and glucose were added as substrates and other additives included hydrocortisone, heparin, insulin, and Keflin. Flow rate was adjusted to give a portal vein pressure of 6 to 8 cm of H 2 O and hepatic artery pressure of 80 mm Hg. Of the initial seven attempts to store the liver for 20 hrs only one organ sustained life for 1 week and it exhibited severe damage. The remaining six died within 12 to 48 hrs from a hemorrhagic diathesis. Two of two organs were held successfully for 7 brs using an identical storage procedure; one of these animals survived 8.5 months. Storage conditions were then altered. The perfusate pH was adjusted to 7.0 (37°C) instead of 7.25 (37°C) which was used in the initial nine attempts. Also, substrate level was supplemented after 6 to 10 hrs of perfusion. The six animals receiving these latter organs after 20-hr storage were walking 2 to 4 hrs after surgery and they exhibited no bleeding. Four of the six organs were considered successfully stored for the 20-hr period. A reusable all-Silastic perfusion system was developed for the above studies. The perfusion circuit consists of a series of Silastic tubings and incorporates a simple reusable membrane oxygenator.

Kinetische untersuchungen über die enzymatische hydroxylierung von phenazon, oxydative o-demethylierung von kodein und N-demethylierung von aminophenazon durch rattenlebermikrosomen

Hydroxylation of phenazone and maminophenazone- N-, and codeine- O-demethylation was determined in liver supernatants (9000 g for 20 min) of adult male rat in relation to enzyme and substrate concentrations, incubation times and temperatures, concentrations of glucose-6-phosphate, DPN and nicotinamide, type of buffer and its concentration and pH, and also storage times at room temperature. The activities were proportional to the volume of supernatant used. On exceeding the optimum substrate concentration of phenazone hydroxylation was inhibited. This inhibition was slight for degradation of aminophenazone and not detected for codeine. Addition of glucose-6-phosphate and DPN had no effect, nicotinamide increased only the breakdown of aminophenazone. Pre-treatment with nicotinamide in vivo was also effective. The three reactions studied also differed in response to different buffers and storage of whole liver or supernatant at room temperature. The best incubation conditions differed somewhat for the different substrates and a “standard incubation system” for several enzymatic reactions is not easy to formulate. The results presented support the view that several DPNH 2-dependent oxidases with mixed functions are present in liver microsomes. Generalizations of the results obtained for one substrate must be qualified even for the same type of reaction.

Observations on the effects of glycerol on the cold storage of the canine liver

Observations on the effects of glycerol on the cold storage of the canine liver

The effects of hydrocortisone on glycogen storage and phosphorylase activity of frog liver.

The effects of hydrocortisone on glycogen storage and phosphorylase activity of frog liver.