Isolated Hepatocyte Preparations Research Articles

Use of Isolated Hepatocyte Preparations for Cytochrome P450 Inhibition Studies: Comparison with Microsomes forKiDetermination

Predicting drug-drug interactions requires an assessment of the drug concentration available to the enzyme active site, both in vivo, and within an in vitro incubation. These predictions are confounded when the inhibitor accumulates within the liver, either as a result of active transport processes or intracellular binding (including lysosomal trapping). In theory, hepatocytes should provide a more accurate estimation of inhibitory potency compared with microsomes for those compounds that undergo hepatic accumulation. However, they are not routinely used for Ki determination and there is limited comparative information available. Therefore, the aims of this study were to compare Ki values determined in rat microsomes and freshly isolated hepatocytes using six cytochrome P450 inhibitors (miconazole, fluconazole, ketoconazole, quinine, fluoxetine, and fluvoxamine) with a range of uptake properties (cell-to-medium concentration ratios 4.2-6000). Inhibition studies were performed using four probe substrates for CYP2C, CYP2D, and CYP3A enzymes (tolbutamide and phenytoin, dextromethorphan and midazolam, respectively). Comparison of unbound Ki values (range 0.05-30 microM) showed good agreement between microsomes and hepatocytes for inhibition of 18 pathways of metabolism. In addition to this, there was no relationship between the cell-to-medium concentration ratios (covering over 3 orders of magnitude) and the microsomal to hepatocyte Ki ratio of these inhibitors. These data suggest that the hepatic accumulation of these inhibitors results from intracellular binding rather than the involvement of uptake transporters and indicate that microsomes and hepatocytes appear to be equivalent for determining the inhibitory potency of the six inhibitors investigated in the present study.

The use of flow cytometry to rapidly assess the cell number and viability of isolated hepatocyte preparations: Alan J Wigg, Department of Medical Biochemistry, Flinders University, Adelaide Australia; Loretta Wheatland, Department of Immunology, Flinders University, Adelaide Australia; John Phillips, Michael Berry, Department of Medical Biochemistry, Flinders University, Adelaide Australia

The use of flow cytometry to rapidly assess the cell number and viability of isolated hepatocyte preparations: Alan J Wigg, Department of Medical Biochemistry, Flinders University, Adelaide Australia; Loretta Wheatland, Department of Immunology, Flinders University, Adelaide Australia; John Phillips, Michael Berry, Department of Medical Biochemistry, Flinders University, Adelaide Australia

Preparation of equine isolated hepatocytes

In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0–2 °C, with continuous flow of 500–800 ml/min for 3–6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500–800 ml/min), and finally immersion of the liver lobe in UW solution (2 °C) during its transport to the laboratory. For equine isolated hepatocyte preparation a “three-step” perfusion procedure was elaborated: rewarming, chelating and collagenase perfusion. We found optimal cell yield and viability under the following conditions: rewarming with UW (38 °C) for 8–14 min, chelating with calcium free Hanks’ Balanced Salt Solution (HBSS, 38 °C) supplemented with 1 m m ethylene glycol-bis[β-aminoethyl esther]-N,N,N′N′-tetracetic acid at the flow rate of 450 ml/min for 6 min and enzymatic digestion with HBSS supplemented with 0.1% collagenase at 38 °C and 450 ml/min flow rate for 8–27 min. These conditions consistently generated cell harvests of 21×10 6±4.86 cells/g of perfused liver tissue with viability of 82.7%±10.2.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome P450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Peroxisome proliferators do not increase DNA synthesis in purified rat hepatocytes

There have been numerous reports that chemicals which induce peroxisomes in rodent liver increase DNA synthesis in isolated hepatic parenchymal cells, but not as well in vitro as in vivo. It is also known that tumour necrosis factor alpha (TNFalpha) is mitogenic in isolated hepatocytes. Since Kupffer cells are a major source of TNFalpha in the liver and have recently been shown to be activated by peroxisome proliferators, the possibility exists that the effect of peroxisome proliferators on DNA synthesis in parenchymal cells is via Kupffer cell contamination of isolated hepatocyte preparations. The purpose of this study was to evaluate this hypothesis by studying the effect of model peroxisome proliferators on purified hepatocyte preparations. Hepatocytes were prepared from rat liver by standard calcium-free and collagenase perfusion. Subsequently, cells were centrifuged through Percoll to remove contaminating non-parenchymal cells. Cells were at least 99.9% pure as assessed by cell counting using specific markers for hepatocytes (resorufin O-glucoside) and Kupffer cells (FITC-labelled latex beads). Hepatocytes were cultured in Williams medium + 10% fetal bovine serum for 24 h followed by culture for 48 h in Williams medium plus or minus drug or mitogen additions. Under these conditions epidermal growth factor stimulated DNA synthesis assessed by incorporation of [3H]thymidine approximately 5-fold over control levels. The peroxisome proliferators WY,14-643 and nafenopin, however, had no effect on DNA synthesis, although they did increase acyl-CoA oxidase as expected. In contrast, TNFalpha increased cell proliferation nearly 10-fold in purified hepatocytes, an effect nearly doubled by WY-14,643. Further, when conditioned medium from purified Kupffer cells incubated with WY-14,643 was added to pure hepatocytes, DNA synthesis was increased over 2-fold in a time-dependent manner. Collectively, these data support the hypothesis that peroxisome proliferators do not influence DNA synthesis in isolated hepatocytes per se. Rather, they stimulate cytokine production by Kupffer cells which in turn increases DNA synthesis in parenchymal cells. An increase in mitogenic cytokine production by Kupffer cells is necessary for stimulation of DNA synthesis in purified rat parenchymal cells.

The isolated hepatocyte preparation: 30 years on.

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.

A COMMENTARY ON THE USE OF HEPATOCYTES IN DRUG METABOLISM STUDIES DURING DRUG DISCOVERY AND DEVELOPMENT*

Isolated hepatocytes and liver slices, in short-term suspension or longer-term culture, offer the prospect of providing qualitative metabolic information and quantitative pharmacokinetic parameters from key animal species and man at early stages of the drug discovery–development continuum. The propensity for changes in the fidelity of drug metabolism after removal of hepatocytes from the organ has long been recognized. The many and varied approaches which have been undertaken in an attempt to compensate for physiological shortcomings of in vitro hepatocyte systems are reviewed. In this respect, short-term suspension culture may provide a baseline against which to measure the success of extended culture methods, but it should be remembered that even freshly isolated hepatocyte preparations have deficiencies and liabilities that may affect the nature of information gathered. This article discusses the current advances and shortcomings of hepatocyte suspensions and cultures, along with liver slice technology, at both quantitative and qualitative levels.

Isolated hepatocytes--past, present and future.

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca(2+)-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase. The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.

Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions

Since its introduction in 1969, the high-yield preparation of isolated hepatocytes has become a frequently used tool for the study of hepatic uptake, excretion, metabolism and toxicity of drugs and other xenobiotics. Basic preparative methods are now firmly established involving perfusion of the liver with a balanced-saline solution containing collagenase. Satisfactory procedures are available for determining cell yields, for expressing cellular activities and for establishing optimal incubation conditions. Gross cellular damage can be detected by means of trypan blue or by measuring enzyme leakage, and damaged cells can be removed from the preparation. Specialized techniques are available for preparing hepatocyte couplets and suspensions enriched with periportal or perivenous hepatocytes. The isolated hepatocyte preparation is particularly convenient for the study of the kinetics of hepatic drug uptake and excretion because the cells can be rapidly separated from the incubation medium. Isolated liver cells have also proved valuable for investigating drug metabolism since they show many of the features of the intact liver. However, they also show important differences such as losses of membrane specialization, some degree of cell polarity and the capacity to form bile. The many consequences of the hepatic toxicity of xenobiotics including lipid peroxidation, free radical formation, glutathione depletion, and covalent binding to macromolecules are also readily studied with the isolated liver cell preparation. A particular advantage is the ease with which morphological changes as a result of drug exposure can be observed in isolated hepatocytes. However, it must be remembered that the isolation procedure inevitably introduces changes that may make the cells more susceptible than the normal liver to damage by xenobiotic agents. Despite its limitations, the isolated hepatocyte preparation is now firmly established in the armamentarium of the investigator examining the interaction of the liver with xenobiotics.

Lipoproteins do not influence cholesterol synthesis in freshly isolated rat hepatocytes, cautionary note

1. 1. Suspensions of freshly isolated rat hepatocytes were used to study the effects of native and derivatized lipoproteins on the rate of cholesterogenesis. 2. 2. Short-term incubation of the hepatocytes with a variety of lipoproteins failed to modify the rate of cholesterol synthesis as determined by the incorporation of tritium from tritiated water into cholesterol after separation from other lipids by thin-layer chromatography. 3. 3. Neither an increase in the cholesterol content of the particles (β -very-low-density lipoproteins) nor derivatization of the lipoproteins (lactosylated-low-density lipoproteins or high-density lipoproteins associated with a tris-galactoside-terminated cholesterol derivative) nor cholesterol-containing liposomes were effective in this respect. 4. 4. Whether this behaviour represents an artefact of the isolated hepatocyte preparation is unknown yet.

Age-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats

The biliary excretion of i.v. injected ouabain was examined in male and female Wistarderived rats in relation to age. The hepatic uptake velocity for ouabain was also determined in isolated hepatocyte preparations obtained from male rats of various ages. Biliary recovery values of ouabain (percent of the dose) were fairly comparable for young male and female rats (3–4-month old). Recovery progressively decreased with age, the first 10-min recoveries at 24 months being about one-third those of respective young values in both sexes. A significant linear relation was demonstrated between the first 10-min recovery (Y, percent of the dose) and rat age (X, month), yielding the relations of Y = 17.75 − 0.43X for males and Y = 18.99 − 0.43X for females respectively. Similarly, the initial uptake velocity (Y, nmol/mg/min) for ouabain decreased in a linear fashion with age (X, month), yielding a significant negative correlation (Y = 0.704 − 0.0021X, r = −0.839, P < 0.005, N = 21) at an ouabain concentration of 8 μM. Kinetic studies using non-linear regression analysis revealed a significantly lower V max value (0.533 ± 0.041 nmol/mg/min) in old (24–29 months) rats compared to the young (4–4.5 months) value (1.193 ± 0.105 nmol per mg/min, P < 0.05), while the affinity constant ( K m , μM) did not differ significantly between young and old animals (203.12 ± 25.42 μM in young rats vs 283.68 ± 28.90 μM in old rats, mean ± SE, 0.05 < P < 0.1). The results of the present study suggest that the age-dependent decrease in the biliary recovery of i.v. injected ouabain in rats can be largely explained by the decrease with age in the hepatic uptake of ouabain. Furthermore, the results provide further support for our previous thesis that the decrease in the lateral mobility of hepatocyte plasma membrane proteins, as revealed by the fluorescence recovery after photobleaching technique, may play a signfiicant role in the age-dependent decrease in the physiological function(s) of the hepatocyte plasma membrane, such as the hepatobiliary transport of ouabain.

Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea.

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)

CCl4-induced toxicity in isolated hepatocytes: the importance of direct solvent injury.

CCl4 is proposed to induce cellular injury through its metabolites that are generated by a cytochrome P-450 dependent step. These free radical products can interact with membrane structures, thereby generating lipid peroxides. The latter process has been implicated as a major mechanism of CCl4 hepatoxicity, although this relationship has been difficult to demonstrate when using isolated hepatocyte preparations. This report demonstrates that there are at least two mechanisms by which CCl4 induces injury in isolated hepatocytes. One occurs within minutes of exposure to CCl4 and is characterized by modest malondialdehyde formation and no decline in cellular-reduced glutathione. SKF 525A, metyrapone and promethazine did not protect against this early damage. A second phase of damage, evident particularly after 3 hr, is characterized by a marked increase in malondialdehyde formation, a fall in cellular glutathione and substantial further cellular damage. These changes could be moderated by the cytochrome P-450 inhibitors and promethazine, and antioxidant. Further examination of the initial phase of damage reveals an immediate dose-related inhibition of O2 consumption. This could not be prevented by SKF 525A or metyrapone and was associated with loss of ability to stimulate mitochondrial respiration with dinitrophenol. Rapid recovery to initial O2 consumption rates occurred with time as CCl4 evaporated from the incubation system. This was associated with a partial return of dinitrophenol stimulation of mitochondrial O2 consumption despite significant glutamate dehydrogenase release. A portion of this recovery could be inhibited by SKF 525A, suggesting that some O2 consumption was due to CCl4 metabolism and ensuing lipid peroxidation. These data suggest that early CCl4 toxicity is a direct consequence of its solvent properties and is partially reversible; subsequent damage may be mediated by lipid peroxidation. This solvent injury has not been previously recognized and may have relevance not only to a reversible toxicity as demonstrated with isolated hepatocytes but also in vivo as well.

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations—I: Metabolite formation and irreversible binding to cellular macromolecules

The metabolism of endogenous estrogens, estradiol and estrone, and the irreversible binding of estrogens to cellular macromolecules have been examined and compared in subcellular microsomal and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol, an NADPH-generating system, and denatured DNA, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 3.26 nmoles/mg protein in 1 hr (S.D. 0.39; 7.9% of total steroid) while binding to DNA was found to be 0.288 nmole/mg DNA/mg protein (S.D. 0.025; 0.39% of total steroid). No significant difference was observed between microsomal preparations from untreated, phenobarbital-treated or 3-methylcholanthrene-treated rats. Irreversible binding to proteins was also demonstrated in the intact hepatocyte cell incubations. After 2-hr incubations of estradiol with hepatocytes, 5.9% (S.D. 1.4%) of the steroid(s) was irreversibly associated with cellular proteins (approximately 1.43 pmoles/mg/min). Analysis of the organic-soluble metabolites demonstrated the presence of the catechol estrogens and their metabolites, 2-hydroxyestradiol, 2-hydroxyestrone, 2-methoxyestradiol, and 2-methoxyestrone. Estrone and estriol were also identified. The aqueous-soluble materials isolated from hepatocyte incubations contained glucuronide, sulfate, and apparent thioether conjugates, as determined by liberation from estrogen metabolites by treatment with β-glucuronidase, sulfatase, and Raney nickel. Thus, extensive primary and secondary metabolism of estrogens occurs in intact hepatocyte incubations. Furthermore, irreversible binding of estrogens to cellular proteins occurs in these intact cells having demonstrated conjugative pathways of metabolism.

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations—II. Inhibition studies

The abilities of various inhibitors and metabolism modifiers to alter the metabolism of estradiol and the irreversible binding of estradiol to proteins were examined in subcellular microsomal incubations and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol and an NADPH-generating system, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 77.59pmol/mg/min (SD 6.1; 7.6% of total steroid). 2-Bromoestradiol and 4-bromoestradiol, inhibitors of estrogen 2-hydroxylase, effectively decreased this irreversible binding of radiolabeled estradiol metabolite(s) to microsomal proteins to 17 pmol mg −1 min −1 (2.1% of total estradiol). These haloestrogens were also effective inhibitors in the intact hepatocyte cells, decreasing the amounts of organic metabolites, aqueous-soluble conjugates, and protein-bound materials. The HPLC radiochromatograms of the organic-extracted fractions from the 2 h hepatocyte incubations demonstrate that the catechol estrogen products, i.e. 2-hydroxyestrogens and 2-methoxyestrogens, were present in lower amounts in the incubations containing the bromoestrogens than in control incubations containing no inhibitor. Ascorbic acid and cysteine, general modifiers of oxidative pathways of metabolism, also affected estradiol metabolism in microsomal and hepatocyte preparations. Both these agents were able to decrease the irreversible binding of estradiol to proteins in the microsomal assays. Ascorbic acid decreased the general metabolism of estradiol in the hepatocyte incubations but did not decrease irreversible binding to proteins. The addition of cysteine to the hepatocyte incubation resulted in an increased metabolism of estradiol and the production of more aqueous-soluble radiolabeled metabolites than the control incubations; however, cysteine did not decrease the amounts of estradiol metabolite(s) irreversibly bound to proteins. Investigations of steroid metabolism in the isolated hepatocytes thus provide an effective in vitro technique for examining the overall oxidative, reductive, and conjugative pathways that are functional in the liver and enables one to investigate the abilities of inhibitors, regulators, and modifiers to affect the metabolic processes. Also, these hepatocyte studies demonstrate that the inhibitors of estrogen 2-hydroxylase, 2-bromoestradiol and 4-bromoestradiol, can enter and act in the intact cells. Consequently, these agents may be useful pharmacological probes for examining the functions of catechol estrogens in other tissues.

The digitoxin metabolism in isolated hepatocytes from young and old male Wistar rats

The metabolism of digitoxin (Dt3) in isolated hepatocyte preparations was studied in young (3-mth-old) male and female rats, young castrated (3-mth-old) male rats and old (22- to 28-mth-old) male rats. When hepatocytes were incubated with [ 3H]Dt3, the predominant metabolite of Dt3 was digitoxigenin-bis-digitoxoside (Dt2) which was 70–90% of the total metabolites in the three male rat groups. Only in the young female rat group was the proportion of Dt2 (40%) slightly exceeded by that of digoxigenin-bis-digitoxoside (Dg2) (50%). A kinetic analysis for Dt3 degradation velocity obtained from studies using different Dt3 concentrations in old male rats yielded a V max of only 21% of the young value. In young castrated males, the V max also decreased to 13% of the young non-castrated males, which is approaching the young female value. The changes were primarily due to the decline of Dt2 formation velocity in these groups. Apparent K m values also decreased with both castration and aging. The plasma testosterone levels which were much higher in young male rats than in female rats became significantly lower in young castrated rats as well as in old male rats. The results suggest that apparent decreases in V max and K m values observed in old male rats for Dt3 metabolic degradation may be at least partly through the steroidal hormone control, presumably, the decline of androgenic induction during aging.

Barbiturate N-demethylase activity in isolated rat hepatocytes

The isolated hepatocyte preparation has become an increasingly popular system for studying xenobiotic metabolism, and there is a need for methods of determining metabolic activity. Described here is a simple and sensitive radiometric assay for barbiturate N-demethylase activity in the isolated hepatocyte suspension. The demethylation of 1,3-dimethylphenobarbital is determined by measuring the formation of its only significant metabolite, N-methylphenobarbital. The reaction is a typical P-450-mediated dealkylation.

Evidence for a lactate transporter in the plasma membrane of the rat hepatocyte.

1. The rate of uptake in 15 s of L- and D-lactate was studied in isolated hepatocyte preparations from starved rats. 2. Observations were also made of the effect of different concentrations of unlabelled L- and D-lactate on L-[14C]- and D-[14C]-lactate uptake. 3. Lactate uptake into hepatocytes appears to occur by two processes, one saturable and at least partially stereospecific, the other non-saturable, at least up to 28 mmol/l, and probably representing simple non-carrier assisted diffusion. 4. The 15 s uptake of lactate is inhibited by alpha-cyano-3-hydroxycinnamate and by rho-chloromercuriphenylsulphonate but not by the general anion transporter inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, ouabain or the structural analogue DL-rho-hydroxyphenyl-lactate. 5. Transporter-mediated lactate entry into liver cells is enhanced by lowered extracellular pH. 6. It is suggested that the contribution of the diffusion component of lactate entry into the cell may be exaggerated by the artifactual nature of the isolated hepatocyte preparation, and that under some circumstances in vivo, lactate transport into hepatocytes could determine the rate of lactate removal from blood.

11] Isolated perfused rat liver technique

The function of the liver in the metabolism of xenobiotics can be studied with a spectrum of liver preparations ranging from the intact organ in vivo, through perfusion systems, liver slices, isolated hepatocytes, homogenates, and membrane fractions, to purified enzymes.. The use of the isolated perfused liver is also attractive because of the intact architecture of the organ, the possibilities of controlling blood and bile flow, and the ease of the manipulation of the composition of the perfusion medium. Advantages include the large number of perfusate samples that may be collected from and compared with the intact animal and the smaller number of interactions with endogenous compounds. In addition, it is possible to use the concentrations of the substrates and metabolic inhibitors that would not be tolerated in vivo because of their toxicity. Compared with isolated hepatocyte preparations, the perfusion system does not require damaging treatment with Ca 2+ -free solutions and digesting enzymes and the normal functional polarity of cells and their localization in the liver lobule is maintained.

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.