Value of the in vitro or in vivo monoethylglycinexylidide test for predicting liver graft function.

BACKGROUND: An adequate function test for donor livers is still lacking. The monoethylglycinexylidide (MEGX) test, performed in vivo in the donor to measure the metabolic rate of lidocaine conversion to MEGX, has been proposed as a function test for donor livers to predict postoperative organ function. METHODS: In the present study, we investigated whether the MEGX formation rate measured in needle biopsy specimens in vitro correlates with the rate of MEGX formation in vivo. The in vivo MEGX test was performed in the donors and in the recipients on days 1 and 2. The in vivo and in vitro MEGX tests were compared with posttransplant liver function in the recipients in order to investigate their possible relevance as predictors of graft function. RESULTS: The MEGX formation rate in needle biopsy specimens in vitro showed a significant correlation with the MEGX serum concentration found in the donor. A low rate of MEGX formation in the biopsy specimens tended to predict initial poor function of the grafts. In the donor, the MEGX test did not correlate with general liver function after transplantation. Only the MEGX serum concentration in the recipients on day 2 gave an indication of graft function. CONCLUSIONS: MEGX formation in liver biopsy specimens in vitro properly reflects metabolic function of the particular liver. Therefore, liver biopsies may be a valuable tool to help predict liver function in vivo. However, the MEGX test alone is not sufficient to provide the gold standard to determine liver function in donor and transplantation livers.

Comparison of five incubation systems for rat liver slices using functional and viability parameters.

Precision-cut liver slices are presently used for various research objects, e.g. to study metabolism, transport, and toxicity of xenobiotics. Various incubation systems are presently employed, but a systematic comparison between these incubation systems with respect to preservation of slice function has not been performed yet. Therefore, we started a comparative study to evaluate five of these systems: the shaken flask (an Erlenmeyer in a shaking water bath), the stirred-well (24-well culture plate equipped with grids and magnetic stirrers), rocker platform (6-well culture plate with Netwell insert rocked on a platform), the roller system (dynamic organ culture rolled on an insert in a glass vial), and the 6-well shaker (6-well culture plate in a shaking water bath). The liver slices were incubated in these incubation systems for 0.5, 1.5, and 24.5 h and subsequently subjected to viability and metabolic function tests. The viability of the incubated liver slices was evaluated by: potassium content, MTT assay, energy charge, histomorphology, and LDH leakage. Their metabolic functions were studied by determination of the metabolism of lidocaine, testosterone, and antipyrine. Up to 1.5 h of incubation all five incubation systems gave similar results with respect to viability and metabolic function of the liver slices. However, after 24 h, the shaken flask, the rocker platform, and the 6-well shaker incubation systems appeared to be superior to the stirred well and the roller incubation systems.

The applicability of rat and human liver slices to the study of mechanisms of hepatic drug uptake.

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 microm thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereoselectively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose(27)-Human Serum Albumin (Lact(27)-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver.

Effect of cold and warm ischaemia on drug metabolism in isolated hepatocytes and slices from human and monkey liver.

1. The influence of short-term cold storage in University of Wisconsin organ preservation solution (UW) on the ability to metabolize lidocaine, testosterone and 7-ethoxycoumarin in isolated human and cynomolgus monkey (Macaca fascicularis) hepatocytes and liver slices has been investigated. 2. The human liver tissue was obtained from two different sources, i.e. healthy liver tissue from patients undergoing partial hepatectomy because of metastases of colorectal carcinoma (PH livers) and donor tissue remaining as surgical waste after reduced size or split liver transplantation (Tx livers). Tx livers were perfused in situ with ice-cold UW avoiding warm ischaemia. This in contrast with PH livers, where the operation caused warm ischaemia for 5-90 min. 3. Liver slices and hepatocytes from cynomolgus monkey liver showed comparable metabolic rates for the substrates tested, indicating that all hepatocytes in the slice are participating in the biotransformation of the substrates. These monkey liver preparations can be stored up to 18 h with only a slight loss of their metabolic capacity. 4. Liver slices and isolated hepatocytes from the Tx livers as well as isolated cells from the PH livers could also be stored up to 18 h without losing metabolic capacity. However, for liver slices prepared from PH livers cold storage is not recommended, because metabolic function was reduced by approximately 40% after 18 h.

The capability of isolated hepatocytes and liver slices of donor livers to predict graft function after liver transplantation.

AIMS/BACKGROUND: In liver transplantation, adequate function tests for donor livers and transplanted livers are of utmost importance to provide an objective basis for decision-making. Isolated hepatocyte and/or slice preparations from human donor liver tissue may be suitable to test the quality of the organ to be transplanted. METHODS: Surgical waste material remaining after reduced size or split liver transplantation in children was used to prepare slices and isolated hepatocytes. The viability of these preparations as well as drug transport and metabolism functions were determined and related to graft function in 32 liver recipients. RESULTS: The in vitro tests used in the present study apparently did not select non-viable livers. In vitro preparations of the primary non-function grafts which occurred in the investigated group showed normal viability, metabolic and uptake function. CONCLUSION: These results indicate that either the presently used viability tests are not sensitive enough to detect potential organ failure or that other factors besides the hepatocyte viability at the time of transplantation are of paramount importance to the graft function of the recipient, such as complications during and after transplantation or the viability of the non-parenchymal cells.

Characterization of the uptake of rocuronium and digoxin in human hepatocytes: carrier specificity and comparison with in vivo data.

Mechanisms of drug transport in the liver have been investigated predominantly in rodents. Most of the in vitro drug research in the liver is performed in liver preparations of animals. The results of such experiments frequently are discussed in relation to anticipated metabolic profiles in man, but these extrapolations are often inappropriate because of large interspecies differences in drug metabolism. In the present study, the mechanisms and specificity of the uptake of the organic cation rocuronium and the cardiac glycoside digoxin were investigated in human hepatocytes and were compared with results obtained in rat hepatocytes. The extraction ratio for the intact liver was calculated from the measured uptake rates of the compounds in the human cells in vitro and compared with published in vivo data. The initial hepatic extraction ratio, calculated from the in vitro uptake data for digoxin and rocuronium, very well reflected the initial extraction ratio for distribution in the liver in vivo in man. Uptake of 100 microM rocuronium was inhibited by 40 microM K-strophantoside (80% inhibition), and although not significantly, by 160 microM procainamide ethobromide, whereas no inhibitory effect was found in the presence of 160 microM taurocholic acid. In a previous study in rat hepatocytes, marked inhibition of digoxin uptake by quinine and only minimal inhibition by the diastereomer quinidine was demonstrated, showing clear stereoselectivity in transport inhibition. Unexpectedly, the uptake of digoxin in human hepatocytes was not inhibited significantly by quinidine or quinine, which indicates clear species differences. This is the first study to investigate the uptake mechanisms of organic cations and cardiac glycosides in human hepatocytes in some detail. The results show that uptake characteristics of drugs found in rats can not be extrapolated directly to humans.

Effect of human liver source on the functionality of isolated hepatocytes and liver slices.

In vitro experiments using human liver tissue to study drug metabolism and transport are usually performed and interpreted without real consideration of the differences in procurement of the tissue, if it is obtained from different sources. Therefore, in this study the functionality of isolated hepatocytes and liver slices prepared either from healthy human liver tissue obtained from patients undergoing partial hepatectomy [livers from partial hepatectomy (PH-livers)] or from donor tissue remaining after reduced-size or split-liver transplantation [livers from transplantation (Tx-livers)] was compared. From each liver sample, both liver slices and hepatocytes were prepared and compared with respect to viability and drug disposition. The viability of hepatocytes was assessed by trypan blue exclusion, ATP content, and energy charge and that of liver slices by potassium retention. In both preparations phase I metabolism was studied using lidocaine and testosterone as substrates, whereas phase I and II metabolism was assessed with 7-ethoxycoumarin. The membrane transport capability of the hepatocytes was investigated by measuring the uptake of taurocholic acid. The hepatocytes from PH-livers and Tx-livers showed similar viabilities and functional capacities. Metabolism in cells and slices from Tx-livers was found to be quantitatively comparable. However, liver slices from PH-livers showed a significantly lower metabolic capacity, compared with cells from the same tissue. This may indicate that only some of the hepatocytes in the liver slices from PH-livers participate in the metabolism of the compounds studied and that a selection of healthy cells takes place during isolation of the hepatocytes. Our results imply that hepatocytes isolated from Tx-livers and PH-livers can be used in the same study without consideration of the procurement of the tissue. However, the procurement of the tissue may significantly influence the functions of liver slices; the liver slices prepared from PH-livers showed significantly lower metabolic function, compared with slices prepared from Tx-livers.