Protein binding capacity in vitro changes metabolism of substrates and influences the predictability of metabolic pathways in vivo.

UNLABELLED: Large numbers of lipophilic molecules are attached to fractions of serum protein, e.g. albumin, in vivo. Cell culture medium of most in vitro hepatocyte models for the prediction of metabolism does not contain albumin. Consequently, in vitro availability and metabolism of substrates could differ significantly from the in vivo situation. The influence of albumin on the in vitro metabolism was tested on a new lipophilic compound. METHODS: Primary human and rat hepatocytes were cultured in a collagen sandwich configuration and incubated with (14)C-labeled compound X127 that is known to exhibit a high propensity to bind to plastic surfaces. Groups contained either 1% (w/v) BSA or none. Substrates as well as metabolism products were determined with radio-HPLC and radioactivity levels in the medium were recorded. RESULTS: Quantitative differences were seen in the distribution of the compound in BSA and non BSA containing groups, thus indicating a substantial binding of the compound to polystyrol surfaces of cell culture dishes. Metabolic radio-HPLC profiles showed different patterns after 24 h of incubation between the two species as well as between the BSA- and non-BSA groups within the species. CONCLUSIONS: With addition of albumin the adherance of lipophilic substrates and metabolites to cell culture dish surfaces can be neutralized and in vitro systems can more closely mimic the in vivo situation.

A new bioassay including a small scale hepatocyte bioreactor for hepato-mediated toxicity testing in a target cell line.

New approaches for in vitro testing of hepato-mediated toxicity are undertaken to offer alternatives to in vivo animal testing. The described bioassay for hepato-mediated toxicity testing is based on a small scale hepatocyte-bioreactor with pig hepatocytes connected to a silicon sensor based microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells and the microphysiometer alone. EAR represents the metabolic activity of tested cells (hepatocytes and ZR 751 cells) under the influence of perfused media, compared to controls, which were set to 100%. Cyclophosphamide (CYCL), whose cytostatic effect is dependent on CYP 450 biotransformation was used as a model substrate. CYCL showed decrease of EAR in hepatocytes, but not in ZR 751 cells. Bioreactor supernatant including CYCL was pumped into the microphysiometer and EARs of the target ZR 751 cell line were recorded. After 7 h of bioreactor supernatant perfusion the ZR 751 cell line showed an EAR decrease of 18.68% +/- 10.18, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of CYCL by the bioreactor. This new bioassay serves as an example of future applications for hepatocyte bioreactors in automated toxicity testing devices, e.g. in preclinical drug studies or evaluation of hepato-mediated toxicity, not depending on cell destruction or further assays.

Evidence-based medicine: lung volume reduction surgery (LVRS).

Lung volume reduction surgery (LVRS) was developed as a means of surgical treatment for severe pulmonary emphysema. To date, various studies have been designed to explain the mechanisms involved in pathophysiological changes after treatment, to define criteria for patient selection, to identify the surgical technique of choice and to propose appropriate follow-up care. Preliminary results of follow-up studies (up to five years) have already been published, indicating improved pulmonary function and quality of life after surgical treatment. However, the alarming results from the National Emphysema Treatment Trial (NETT) Research Group indicated a considerable risk for death in patients with homogenous emphysema and low forced expiratory volume in one second (FEV1) undergoing LVRS. This brief review summarizes the results of currently published studies to supply evidence for selection criteria in order to better define the subset of patients for which LVRS offers an effective and safe means of palliation from the symptoms of advanced COPD. Due to acceptable morbidity and mortality rates, stapler device wedge excision and closure has become the standard procedure for removing non-functioning, hyperinflated lung areas in heterogeneously affected organs. LVRS is carried out in two ways - using video-assisted thoracoscopic surgery (VATS) as well as thoracotomy/sternotomy-and performed in unilateral and bilateral procedures. In contrast, most clinics have found laser resection of emphysematous parenchyma to be unsuccessful. In some patients, LVRS was carried out as an alternative to lung transplantation, whereas in others, it served as a bridge-to-transplant procedure. LVRS has proven effective in the reduction of dyspnea, especially in patients with recovery options in both the circulatory and pulmonary system. In responders, recovery from labored breathing and O(2) dependency and increased physical capacity are usually accompanied by improved spirometric data. These results are mainly explained by a more regular breathing pattern and an increase in the maximum volume of ventilation in the affected lung. In most cases, functional improvement is maximized during the first six months postoperatively and decreases steadily thereafter indicating the need for a systematic postoperative patient care after surgical treatment. After indicating at-risk patients who should not be considered for LVRS, long-term results from the multicenter NETT research group will hopefully help clarify the impact of this treatment on survival of patients further.

Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices.

OBJECTIVE: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). METHODS: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. RESULTS: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/microg DNA and of 1.8 +/- 0.14 microg/h/microg DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/microg DNA (28.7, 42.8) and 32.7 ng/h/microg DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively. CONCLUSION: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems.

In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates.

In this article we introduce an in vitro model for hepato-mediated toxicity testing consisting of a Hepatocyte-Bioreactor connected to a microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells. The EAR in this system represented the metabolic activity of a tested cell line under the influence of bioreactor supernatant. Cyclophosphamide (CYCL), a well-known hepato-activated cytostatic drug was used as a model substrate because of its widespread clinical use. The predrug CYCL needed CYP 450 dependent activation to its active cytotoxic metabolite 4-OH cyclophosphamide. Primary pig hepatocytes from slaughterhouse organs were cultured in a collagen sandwich configuration in specially designed flasks and after 3 days introduced into a 50 ml recirculating perfusion system including 30 microg/ml CYCL. In a parallel open circuit, this bioreactor was connected to three perfusion chambers of a microphysiometer system housing 1.5 x 10(5) ZR 751 cells (breast tumor cell line). Bioreactor supernatant including CYCL was pumped at 150 microl/min into the microphysiometer. The recorded EARs under CYCL influence were correlated to controls, which were set to be 100%. After 1 and 7 h of bioreactor supernatant perfusion, including activated CYCL, the ZR 751 cell line showed an EAR of 98.99%+/-3.15 (mean+/-SD) and 81. 32%+/-10.18 (P<0.05), respectively, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). The inactivated predrug CYCL showed no effect on the EAR: Perfusion of medium with 30 microg/ml CYCL alone, excluding the bioreactor activation, resulted in an EAR of 100. 11%+/-4.74 (mean+/-SD) after 7 h. Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of the predrug by the bioreactor.

New non-woven polyurethane-based biomaterials for the cultivation of hepatocytes: expression of differentiated functions.

A new non-woven polyetherurethane support suitable to host cultured hepatocytes has been developed. Prior to its use in bioreactors and artificial liver devices, the biocompatibility of this new material was investigated. The experiments have shown that the survival and functionality of hepatocytes entrapped in the non-woven polymer were longer than that of monolayer cultured hepatocytes, under serum-free culture conditions. Hepatic specific metabolic functions, namely, synthesis of urea and synthesis and secretion of plasma proteins, were well maintained by hepatocytes entrapped in non-woven polyetherurethane sheets. Cells also retained the expression of biotransformation activities of 7-ethoxycoumarin-O-deethylase as well as CYP2A1, CYP2B1 and CYP3A1. The results presented in this paper point to non-woven polyetherurethane sheets as a suitable biocompatible support for functional, three-dimensional hepatocyte cultures.

Cryopreserved porcine hepatocyte cultures.

Cryopreservation of freshly isolated hepatocytes is regarded the standard technique for long term storage of liver cells. Frankly, we were not successful in reproducing viability rates of about 70% of that which have been reported by most authors as results of various freezing protocols for hepatocyte suspensions. In fact, we saw mostly devastating results. We assume that intracellular ice crystal formation as well as osmotic changes during freezing and thawing of liver cells cause hazardous effects, especially on membranes of cells after enzymatic isolation, and, thus, generally result in a severe loss in number and impaired specific hepatocyte functions in subsequent culture. We tried to improve results by freezing cell cultures instead. We allowed hepatocytes to regain a more stable condition prior to storage and placed them in tissue flasks in a uniform configuration, rather than to pack cell suspensions in vials or bags under rather indefinable conditions. Porcine hepatocytes (viability 92+/-2%) were isolated from slaughterhouse organs and cultured in a double gel (sandwich) configuration. At day 3, cultures were rate controlled frozen (RCF) and stored in a cell bank for three hours (Group A) or 14 days at -80 degrees C (Group B), respectively. Non-frozen cells (NF) and cultures subjected to a linear freezing rate of -10 degrees C/min (LFR, Group C) with 3 h of storage served as controls from identical cell batches. Upon thawing, at day 2 of subsequent culture, fluorescence microscopy studies revealed a survival rate of 75+/-10% (mean+/-S.D.) in the RCF groups. Time of storage (3 h, 14 d) did not influence results. Survival in Group C was 10+/-5%. Cell integrity was measured by LDH-release, which indicated a larger damage of cells in the LFR group, and thereby resembled the morphological findings. Functional parameters, such as albumin synthesis and CYT P 450-activity were comparable to non-frozen liver cells at 48 h after thawing in the RCF groups (A + B), and showed significantly higher levels in these groups as compared to the LFR Group (C). We recommend to freeze hepatocytes in culture in a rate controlled fashion rather than cell suspensions. This way a cell bank of cryopreserved hepatocyte cultures for batch controlled investigations can be easily obtained. This could ameliorate the availability of rare (human) cell material and might also improve the quality of data generated from in vitro experiments in hepatology or pharmacology/toxicology with liver cells from identical sources. It remains to be seen whether this technique might also be of value in hybrid bioartificial liver devices to make these systems become readily available upon clinical demand.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

[The artificial liver--an interim report]. / Die künstliche Leber--ein Zwischenbericht.

A Bioartificial Liver (BAL) is not available to date. Human therapeutic applications of different systems of BAL as of 1998 are presented and discussed. It is concluded that--as for now--no artificial liver device has gained any importance for the treatment of liver failure, and that some critical issues in this field of research have not been sufficiently investigated and/or are not resolved. This review analyzes reports of clinical applications of BAL from the following research groups (numbers in brackets indicate patients treated with an artificial liver device): Demetriou/Rozga, Los Angeles (31 patients), Williams, London (1 patient); Gerlach, Berlin (1 patient); Strom, Virginia (5 patients). The BAL systems used in these studies cannot be directly compared because there are considerable differences in the quality and in quantity of the functional unit employed in bioreactors, the experimental design, patient selection, to mention just a few points. None of the systems investigated so far could convincingly prove its effectivity in replacing impaired liver function neither in animal models nor in a clinical application. It remains to be shown, whether liver cells cultured in bioreactors remain stable, i.e. viable and functionally active, for a sufficient period of treatment. Selected metabolic or detoxifying functions of the bioreactor are difficult to assess, since these functions do not necessarily serve as pars pro toto for the complex clinical presentation of liver failure and therefore cannot sufficiently validate any organ replacement system. Furthermore, since some applications combine biological units with other components, such as active charcoal, it be-comes even more difficult to assess the role of hepatocytes in these settings. Case reports of patients treated with BAL usually refer to BAL as a "successful bridge to transplant", thereby demonstrating the positive effect of orthotopic/auxiliary liver transplants in the treatment of acute liver failure rather than the potential benefit derived from an artificial liver device. Randomised studies have been proposed and urged for years in order to prove the effectivity of these systems which, in part, are already available for clinical use. Because of the heterogeneous patient group in question the design of such protocols will be a difficult task. It must be asked, whether currently used artificial liver systems have left basic science research too early; the use of "black box" applications in humans cannot draw its legitimation merely from the fact that an effective conservative treatment of liver failure is not available so far.

[Technique and rationale of lymph node dissection in bronchial carcinoma]. / Technik und Rationale der Lymphknotendissektion bei Bronchialcarcinom.

Any operation for lung cancer that is planned as curative includes dissection of the lymph nodes in the mediastinum in order to allow correct staging of the disease. It is well known that ipsilateral lymph node metastases (N2) mean a poor prognosis. Since most patients with this finding die from metastatic tumor progression within 3 years after the operation, it can be assumed that positive findings in the mediastinal lymph nodes represent generalization of the cancer. Therefore, the probability of a therapeutic effect from more radical removal of lymph nodes in the mediastinum is rather low. On the other hand, it is justified to apply and evaluate additional systemic treatment in these tumor stages.

Cell morphology, albumin secretion and glutathione S-Transferase expression in collagen gel sandwich and immobilization cultures of rat hepatocytes.

In order to investigate whether collagen gel sandwich and immobilization cultures of rat hepatocytes are suitable in vitro models for long-term pharmaco-toxicological studies, the expression of the key phase II biotransformation enzyme, glutathione S-transferase (GST, EC 2.5.1.18), has been studied in the presence or absence of l-proline (60mug/ml) in the culture medium. Additionally, hepatocytes morphology was followed and albumin secretion into the medium measured. As judged by inverse phase light microscopy and transmission electron microscopy, cells cultured in both organotypical models remained viable and well differentiated for at least 14 days. Albumin secretion increased 2.5-fold after 7 days of culture, in comparison with the values found after 2 days, and remained thereafter relatively constant. When l-proline was added to the medium of sandwich and immobilization gel cultures, steady-state secretion levels of 7.1 and 5.1 mug albumin/hr, respectively, were already obtained after 4 days of culture. Total, Mu, Alpha and Pi class GST activities were determined using a general substrate and isoenzyme specific substrates, respectively. After 7 days of culture, total GST activities were decreased as compared with the values obtained for freshly isolated cells. On the contrary, Mu class GST activities were kept at a constant level. Alpha class GSTs were maintained at a 50% activity level and GST 7-7 activity was shown to be slightly induced. l-proline prevented an initial decline in total and Mu class GST activities in both culture models. The GST subunit pattern, measured after affinity chromatography by reversed phase HPLC, reflected the GST activity results.

Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities.

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 degrees C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of l-proline (final concentration 60 mug/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. l-Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.

Porcine hepatocytes for biohybrid artificial liver devices: a comparison of hypothermic storage techniques.

Two hypothermic preservation techniques were investigated to assess their possible role in on-demand cell supply for bioartificial liver support devices. Porcine hepatocytes from slaughterhouse organs were isolated and either cold stored in a modified University of Wisconsin solution for up to 72 h or directly cultured in a sandwich configuration, frozen at Day 3 of culture, and stored for up to 30 days with subsequent long-term culture (14 days) in both groups. Cold storage for 72 h resulted in a decreased viability of cells (58.7 +/- 7.9%) with well preserved ultrastructures in the remainder of cells. In subsequent culture, albumin secretion was slightly increased, and cytochrome P450 IA1 dependent 7-ethoxy-coumarine deethylation activity was reduced to about 40% of control values. After cryopreservation, hepatocyte cultures revealed no severe damage to ultrastructures of cells, and functional parameters (albumin, 7-ethoxycoumarine deethylation) were comparable with controls after an initial drop in activity directly after thawing. Length of storage time did not influence results. Both hypothermic preservation protocols might eventually play an important role for bioartificial liver processing and on-demand cell supply, dependent on the individual reactor design.

Hypothernic storage of pig hepatocytes: influence of different storage solutions and cell density.

For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.

Extracorporeal artificial liver: the influence of a second cell layer on the morphology and function of immobilized human hepatocytes.

Hepatocytes in long-term cultures represent a promising approach to preserve liver function under standard culture conditions. Hepatocyte cultures as the key components in an extracorporeal artificial liver (EAL) in the treatment of hepatic insufficiency, would be a great advantage. However, one of the numerous unsolved problems is the limitation of the surface area of a future EAL. To decrease the dimensions of same, we modified the cell immobilization technique by placing a second layer of immobilized human hepatocytes onto a layer of pre-immobilized hepatocytes creating a "sandwich immobilization" (SI) system. Immobilization and sandwich immobilization were compared over an investigation period of 30 days: functional performance mirrored by cholinesterase (CHE) and albumin secretion showed remarkable differences only in the course of the first week, whereas we found almost no differences from day 8 on. The total DNA-values on days 0, 1, 7, 14, 21 and 30 varied strongly after the first week but were very similar up to day 30. Finally, it appears disadvantageous to enlarge number/cm2 of (human) hepatocytes in long-term cultures or for application in an EAL by means of sandwich immobilization.

Hepatic levels of bile acids in end-stage chronic cholestatic liver disease.

In chronic cholestatic liver disease hydrophobic and potentially cytotoxic bile acids are assumed to accumulate in the liver. To test this hypothesis we investigated bile acid levels and pattern in livers and serum of patients with, (A) end-stage chronic cholestatic liver disease, and with (B) end-stage cirrhosis of alcoholic/chronic hepatitic origin who underwent liver transplantation. Bile acids were also analyzed in (C) normal liver tissue. Levels of bile acids were 215 +/- 39.1 nmol/g liver (wet weight) in chronic cholestasis and 120 +/- 32.7 and 56.1 +/- 24.2 nmol/g liver in group B and group C (P < 0.01 and P < 0.005), respectively. Cholic acid was the prevailing bile acid in chronic cholestasis (51%) and was elevated eight-fold as compared to group C (P < 0.005). Chenodeoxycholic acid contributed 41% to total bile acids and was elevated four-fold (P < 0.005). Deoxycholic acid contributed only 1.5% to bile acids in chronic cholestasis as compared to 27% in group C (P < 0.01) and was absent in group B. Levels of lithocholic acid tended to be increased in chronic cholestasis as compared to group C and its sulfation was impaired (P < 0.05). The pattern of serum bile acids in chronic cholestasis agreed well with the bile acid pattern in the explanted livers. We conclude that hepatic accumulation of hydrophobic chenodeoxycholic acid and impaired sulfation of lithocholic acid might contribute to tissue degeneration in chronic cholestatic liver disease due to the detergent effects of these bile acids.

Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes.

BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. UDCA is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2+ agonist and stimulates biliary exocytosis and hepatocellular Ca2+ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. METHODS: The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accummulation of sn-1,2-diacylglycerol (DAG), the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. RESULTS: Immunoblotting studies showed the presence of four isoenzymes (alpha, delta, epsilon, and zeta). The phorbol ester phorbol 12-myristate 13-acetate (1 mumol/L) induced translocation of alpha-PKC, delta-PKC, and epsilon-PKC from cytosol to a particulate membrane fraction, a key step for activation of PKC. TUDCA, but not taurocholic acid, selectively induced translocation of the alpha-PKC isoenzyme from cytosol to the membranes. In addition, TUDCA induced a significant increase in hepatocellular DAG mass and stimulated membrane-associated PKC activity. CONCLUSIONS: TUDCA might stimulate Ca(2+)-dependent hepatocellular exocytosis into bile in part by activation of alpha-PKC.

Formation of cholic acid and chenodeoxycholic acid from 7 alpha-hydroxycholesterol and 27-hydroxycholesterol by primary cultures of human hepatocytes.

It has been suggested that chenodeoxycholic acid is preferentially formed by the alternative or 'acidic' pathway of bile acid biosynthesis starting with 27-hydroxylation of cholesterol, while cholic acid is derived from 7 alpha-hydroxycholesterol which initiates the 'neutral' pathway. We have studied bile acid formation from each of these precursors using human hepatocytes cultured in a novel sandwich collagen configuration. Culture supernatants were analyzed using capillary gas chromatography and gas chromatography-mass spectrometry. 27-Hydroxycholesterol and 7 alpha-hydroxycholesterol were both found to be efficiently converted to cholic acid as well as chenodeoxycholic acid. Analysis of acidic intermediates after addition of 7 alpha-hydroxycholesterol to the cultures revealed a significant increase of side-chain oxygenated C24- and C27-steroids with a 3-oxo-7 alpha-hydroxy-delta 4-ring structure. These data indicate that (i) the 'neutral' pathway is connected to the 'acidic' pathway by side-chain oxidation of C27-steroids with a 3-oxo-7 alpha-hydroxy-delta 4-ring structure and that (ii) the relative formation of cholic acid and chenodeoxycholic acid is regulated by metabolic events distal to the initial hydroxylation at either position 7 or position 27 of the cholesterol molecule.

Analysis of laser Doppler flux motion in man: comparison of autoregressive modelling and fast Fourier transformation.

In order to investigate laser Doppler (LD) flux motion in healthy subjects and patients with peripheral arterial occlusive disease (PAOD), spectrum analysis of LD signals is needed. Autoregressive analysis (AR) is presented as an alternative method of power spectrum estimation. This procedure is compared to the commonly used fast Fourier transform algorithm (FFT) by describing the analytical power of both spectra in the analysis of flux motion waves. LD signals were recorded from the forefoot of 8 healthy volunteers and 11 patients with different degrees of PAOD. The flux, concentration of moving blood cells and velocity signal was digitized and stored for off-line analysis. Special software was designed to calculate AR and FFT spectra of the LD signals and to compare the suitability of both methods for the spectral analysis of LD recordings. Additionally, three-dimensional spectrum diagrams were calculated to demonstrate time-dependent flux changes during standardized provocation maneuvers. AR facilitates the determination of frequency and amplitude of flux motion waves as compared to the FFT. Low frequency-large amplitude waves (LF waves) were detected in both groups. High frequency-small amplitude waves (HF waves), which predominantly appear in severe ischemia, were observed in 7 of the 11 patients and in 2 of the 8 controls. The spectra revealed pulsatile waves in all healthy controls, but only in 1 of the 11 patients. AR modelling allows a reliable description of important flux motion components and has considerable advantages in spectral estimation of LD signals as compared to the FFT.