Intact Liver Cells Research Articles

Development of binuclearity and DNA-polyploidization in the growing mouse liver.

Suspensions of intact liver cells were prepared from 36 male NMRI mice of different age after perfusion of the liver with ice-cold calcium- and magnesium-free phosphate buffer (CMF). The suspensions of the isolated hepatocytes were smeared on slides, fixed, hydrolized and stained by fluorescent acriflavine-Schiff-Feulgen reaction. The number of nuclei per cell was determined in a phase-contrast microscope. Quantitative fluorescent cytophotometric measurements of nuclear Feulgen-DNA were performed on individual nuclei. At the age of 0.5 month, 55% of the hepatocytes were found to be mononuclear, 45% binuclear. In the animal groups aged 1 month, 1.5 months, 3 months, 6 months and 12 months, the percentage of binuclear hepatocytes remained constant at about 80%. Very few liver cells with 3 or 4 nuclei were detected. Feulgen-DNA-measurements revealed a predominance of 2c and 4c nuclei at ages 1 month and 1.5 months with logarithmic increase of 8c nuclei and a decrease of the 2c nuclei. From 1.5 months on 16c nuclei were found, which never exceeded 8%. When total DNA-ploidy of the hepatocytes was calculated similar kinetics at a higher ploidy level were observed. 2c hepatocytes existed in small percentages at very young ages up to 1.5 months, but were also occasionally found in older animals. With increasing age the number of 16c hepatocytes increased logarithmically with a concomitant decrease of the 4c hepatocytes. The percentage of 8c liver cells remained more or less constant. Few hepatocytes with a 32c total DNA content were found in mice aged 3 months and older. In one-year-old mice the mean DNA-ploidy was calculated to be 5.8c per liver nucleus and 10.0c per whole hepatocyte.

Effects of Alanine on Gluconeogenesis in Isolated Rat Hepatocytes

The effect of alanine on pyruvate kinase was investigated in isolated rat hepatocytes. Alanine at concentrations of 2, 5 or 10 mM increased glucose production by 73% during the first 30 minutes of incubation of hepatocytes with 9 mM lactate and 1 mM pyruvate. After 3 minutes, the rate was not affected by the addition of alanine. A dose-response study showed that maximal stimulation of gluconeogenesis was achieved with 0.5 mM alanine. Using a method that measured recycling of phosphoenolpyruvate to pyruvate, it was found that in either fed or starved rats, alanine significantly decreased the percentage of phosphoenolpyruvate recycling when lactate was the substrate. However, no significant change in recycling was noted when either pyruvate or lactate-pyruvate was the glucose precursor. This study suggests that in intact liver cells, alanine has an inhibitory effect on pyruvate kinase. However, the inhibition is not of sufficient magnitude to completely account for the increase in glucose production when lactate is the substrate. It is hypothesized that alanine may have other effects on gluconeogenesis in addition to that of inhibiting pyruvate kinase.

Ethanol oxidation by isolated hepatocytes from fed and starved rats and from rats exposed to ethanol, phenobarbitone or 3-amino-triazole: No evidence for a physiological role of a microsomal ethanol oxidation system

In isolated hepatocytes from fed and starved rats, basal rates of ethanol oxidation were 1.15 and 0.71 μmoles/g wet wt, respectively, and were unchanged over the ethanol concentration range 8–96 mM. The addition of 4-methyl pyrazole (4 mM), a competitive inhibitor of alcohol dehydrogenase, largely abolished ethanol oxidation from 8mM ethanol, while at an ethanol concentration of 96 mM, the oxidation rate was inhibited by 87 per cent. Pyrazole was a less effective inhibitor of alcohol oxidation than 4-methyl pyrazole. In hepatocytes isolated from rats treated with ethanol, phenoharbitone or 3-amino-triazole, basal rates of ethanol oxidation were the same at ethanol concentrations of 8–96 mM and the rates were similar to, and never exceeded, the rate found in hepatocytes from normal fed rats. 4-Methyl pyrazole inhibited ethanol oxidation to the same extent in all liver cell preparations. regardless of the treatment the donor animal had received. Pyruvate stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation, together with the ethanol-induced accumulation of lactate, was abolished by 4-methyl pyrazole. This suggests that the capacity for alcohol oxidation in isolated liver cells is generally limited by the lack of suitable acceptors for the hydrogen generated in the cytoplasm by the alcohol dehydrogenase-catalysed oxidation of ethanol to acetaldehyde. Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration, irrespective of the prior treatment of the donor animal. This enhancement of ethanol oxidation and respiration was prevented by 4-methyl pyrazole. These artificial electron acceptors appear to act by circumventing normal pathways for the oxidation of cytoplasmic NADH generated in the conversion of ethanol to acetaldehyde. In cells from each treatment group, antimycin was more effective than rotenone as an inhibitor of ethanol oxidation; inhibition of ATP formation by oligomycin had least effect on alcohol oxidation. Ethanol oxidation by cells from alcohol-treated rats was most affected by these inhibitors of mitochondrial respiration. These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidizing system to ethanol oxidation in isolated, intact liver cells appears minimal. Thus they cast doubt on a physiological role for this system in vivo.

Interactions of glucagon with isolated rat-liver cells fate and subcellular localization of cell-associated hormone

The interaction of glucagon with isolated rat-liver cells was studied, and some results were compared with those obtained in parallel experiments with insulin. Attempts were made, in particular, to examine the functional relationships between receptor-binding and degradation, to assess the subcellular localization of cell-associated glucagon, and to compare the glucagonbinding properties of intact liver cells and whole-liver tissue. Binding determinations at the steady state are compatible with the existence of about 1.3 × 10 −5 sites per cell with a dissociation constant of 1.5 nM. Unlike that of liver membranes, the dissociation of bound glucagon is little affected by guanyl nucleotides. Glucagon in the incubation medium undergoes rapid inactivation and physical changes that suggest cleavage into iodotyrosyl peptides and free iodotyrosines. Most of the degrading activity is unrelated to the receptors as indicated by its low affinity (apparent K m 4 μM), its inhibition by polypeptides chemically unrelated to glucagon, and its partial association (about 50% of the total) with degradative components released extracellularly. In the absence of inhibitors, cell-associated glucagon also undergoes a progressive degradation, with a concomitant release of degraded products in the medium; this process may be, at least in part, receptor-mediated. However, in contrast with insulin, it represents only a minor fraction of the total degrading activity. About 80% of the glucagon, and 90% of the insulin associated to liver cells is recovered in the particulate fraction. On differential centrifugation, this material is almost exclusively recovered in the microsomal fraction; on sucrose density-gradient centrifugation, it is present to the extent of 70% in unidentified membranes of density 1.11–1.17. This pattern of distribution is unaffected by the time of incubation, indicating that if labeled hormone undergoes translocation, the latter is not detectable under the conditions used here. The subcellular distribution of the glucagon and insulinbinding activities is nearly the same whether the binding reaction is performed on intact cells or on isolated fractions, and, at saturation, intact cells bind about the same amount of hormone as does the particulate fraction derived from the cells. Thus, cell disruption does not affect the apparent accessibility of the binding sites. Crude particles and plasma membranes prepared from liver cells show a decrease in their ability to bind insulin and glucagon as compared with the same subcellular fractions obtained from whole liver; this is most pronounced with plasma membranes. Evidence that the collagenase preparation may be involved in these changes is presented.

Mouse pre-B cells synthesize and secrete μ heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.

The stereoselective enzymic reduction of the synthetic 9-ketocannabinoid, nabilone, in vivo, in isolated liver cells and in liver homogenate.

1. The enzymic reduction of the two optical isomers of nabilone, 6aR, 10aR and 6aS, 10aS, has been studied separately in the rat, in isolated hepatocytes, and in the 9000 g supernatant fraction of rat liver homogenate. The SS-nabilone was the more active substrate in all three cases. 2. In all three systems, the only carbinol formed from SS-nabilone was the S-(equatorial)-carbinol. In contrast, the reduction of RR-nabilone gave only S-(axial)-carbinol in the intact rat and in the isolated hepatocyte, but gave in the liver homogenate fraction a mixture of S-(axial)-carbinol and R-(equatorial)-carbinol. 3. The results show that the reduction of nabilone in vivo in the rat proceeds with rigid stereochemical control and that only the S-carbinols are formed. 4. The results also suggest that observations made with the intact liver cell may be more predictive of events in vivo than are those made with broken cell preparations.

Ethanol oxidation in isolated hepatocytes.

In isolated hepatocytes from fed and starved rats, rates of ethanol oxidation were 1.15 and 0.71 μmol x min−1 x (g wet wt)−1 respectively and were unchanged over the ethanol concentration range 8-96mM. The addition of the competitive inhibitors of alcohol dehydrogenase (ADH), pyrazole and particularly 4-methyl pyrazole (4-MP) abolished the oxidation of 8 mM ethanol and subsequently inhibited oxidation of 96 mM ethanol.In hepatocytes isolated from rats treated with ethanol, phenobarbitone or 3-amino-triazole, rates of ethanol oxidation were the same at ethanol concentrations of 8 and 96mM and the rates were similar to, and never exceeded the rate found in hepatocytes from normal fed rats. Pyrazole inhibited ethanol oxidation to the same extent in all preparations; 4-methyl pyrazole did likewise, but was more inhibitory than pyrazole.Pyruvate greatly stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation together with the excess accumulation of lactate was totally abolished by 4-methyl pyrazole.Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration irrespective of prior treatment of the donor animal, but again the enhancement of ethanol oxidation and respiration was totally abolished by 4-methyl pyrazole. It is probable that these agents, like pyruvate, act by stimulating the oxidation of NADH generated in the cytoplasm by the alcohol dehydrogenase catalysed conversion of ethanol to acetaldehyde.These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidising system (MEOS) in isolated intact liver cells appears minimal. Thus they cast considerable doubt on its physiological role in vivo.KeywordsMethylene BlueEthanol OxidationDonor AnimalIsolate Liver CellArtificial Electron AcceptorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Affinity labelling of α-adrenoceptors in intact liver cells by [ 3H]phenoxybenzamine

[ 3H]phenoxybenzamine of high specific activity (5.3 Ci/mmol) was synthesized and its binding to isolated, viable rat liver cells was studied. Phentolamine suppressible binding of [ 3H]phenoxybenzamine was irreversible and saturable (EC50: 10 nM, b max: 200 fmol/mg wet cell weight). Competition-inhibition studies showed structural and stereoselectivity compatible with α-receptors. The IC50 of unlabelled phenoxybenzamine to reduce specific binding (9 nM) or to block adrenaline-induced phosphorylase activation in the same cells (2 nM) was similar, whereas the IC50 of agonists to suppress binding was higher than their EC50's for phosphorylase activation. The results represent the first example of labelling α-adrenoceptors in intact liver cells. The sites labelled by [ 3H]phenoxybenzamine mediate the block of phosphorylase activation by α-adrenoceptor antagonists. However, the relationship of these sites to receptors that mediate responses to physiological, low concentrations of catecholamines remains to be clarified.

Effect of various metabolic inhibitors on biphenyl metabolism in isolated rat hepatocytes

The effect of three inhibitors of mitochondrial function (menadione, rotenone and 2,4-dinitrophenol) on drug metabolism in isolated rat hepatocytes has been studied. Menadione (at 1.25 × 10 −4 M) caused almost complete inhibition of biphenyl Phase I metabolism whereas rotenone (2 × 10 −5 M) inhibited the same reaction only by 25 per cent although the subsequent conjugation of the Phase I metabolite was markedly depressed. Qualitatively similar findings were observed with hepatocytes isolated from phenobarbital-pretreated rats, and with liver microsomes isolated from control rats. 2,4-Dinitrophenol (2 × 10 −4 M) caused a marked enhancement of biphenyl Phase I metabolism but a marked inhibition of subsequent conjugation. This enhancement of Phase I metabolism was not observed in control cells with other substrates (benzo[a]pyrene, 7-ethoxycoumarin), nor in biphenyl metabolism in “induced” cells or in liver microsomes isolated from control rats. It is tentatively suggested that products of 2,4-dinitrophenol metabolism may “activate” biphenyl metabolism in intact liver cells. Furthermore, it is suggested for all three inhibitors that direct effects on the drug metabolizing enzyme systems (Phase I and Phase II) are as important as their effects on mitochondrial function in explaining their inhibition of drug metabolism. It appears that Phase II metabolism of xenobiotics is more susceptible to inhibition by metabolic inhibitors than is Phase I metabolism, probably due to depletion of the cellular ATP levels.

Mutagenicity testing on Chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[ a]pyrene

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[ a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN — both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasised that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

Oxidation-reduction state of free NADP + during mixed-function oxidation in perfused rat livers—Evaluation of the assumptions of near equilibrium by comparisons of surface fluorescence changes and calculated NADP +:NADPH ratios

Changes in cellular pyridine nucleotide and flavoprotein oxidation-reduction states associated with mixed-function oxidation of p-nitroanisole, hexobarbital and aminopyrine by perfused rat livers were studied. Surface fluorescence techniques were compared with NAD(P) +/NAD(P)H ratios calculated from substrates assumed to be in near equilibrium with various dehydrogenases in freeze-clamped liver samples. p-Nitroanisole and p-nitrophenol caused a large decrease in pyridine nucleotide (366 → 450 nm) fluorescence as a result of fluorescence quenching. This decrease, therefore, did not reflect oxidation of pyridine nucleotides. Moreover, p-nitroanisole infusion decreased the free NADP +:NADPH ratios calculated from malic enzyme and isocitrate dehydrogenase. Hexobarbital, which did not produce fluorescence quenching, caused an oxidation in pyridine nucleotides as indicated by both a decrease in surface fluorescence and an increase in the calculated NADP +/NADPH ratio. These data indicate that free NADP +/NADPH ratios calculated from substrates, which are assumed to be in near equilibrium with NADPH-generating enzymes, indeed reflect the NADPH redox state in intact liver cells.

Cellular applications of 31P and 13C nuclear magnetic resonance.

High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously.

The effect of pargyline on the metabolism of ethanol and acetaldehyde by isolated rat liver cells

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 m m pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 m m, than of higher concentrations (1.0 m m) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.

Acetyl-CoA carboxylase. Evidence for polymeric filament to protomer transition in the intact avian liver cell.

Digitonin treatment of chick liver cells in monolayer culture perforates the plasma membrane, causing release of acetyl-CoA carboxylase and other cytosolic enzymes. The rate of carboxylase release is affected by conditions known to alter the position of the protomer-polymer (filament) equilibrium of the enzyme. Citrate, an allosteric activator of the carboxylase, induces polymerization of the protomeric avidin-sensitive form giving rise to the avidin-insensitive polymeric filamentous form. When cells are exposed to N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate which lowers intracellular citrate levels, the rate of carboxylase release from digitonin-treated cells is greatly accelerated. The presence of avidin, which rapidly enters the cell during digitonin treatment, inactivates carboxylase under conditions that promote depolymerization and rapid release, but not under conditions which promote polymerization and slow release. These findings indicate that carboxylase filaments exist in the intact chick liver cell when the cytoplasmic citrate level is high and undergo depolymerization when citrate levels fall.

Use of intact liver cells for metabolic activation in a simplified fluctuation test for detection of carcinogens and mutagens

Use of intact liver cells for metabolic activation in a simplified fluctuation test for detection of carcinogens and mutagens

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity ( K A = 3.8 · 10 7 M −1) and to a great number of acceptor sites ( N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA). These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [ 3H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.

Degradation of the Parathyroid Hormone in the Kidney and Liver

The cleavage of the parathyroid hormone has been reported to take place in various peripheral tissues, especially in the kidney and liver. In order to claify the mechanism of such a degradation, bovine PTH (bPTH 1--84) and its synthetic N-terminal peptide (bPTH 1--34) labelled with 125I by the chroramine-T method (125I-bPTH 1--84) and (125I-bPTH 1--34) or labelled with horseradish peroxidase Pox-125I-bPTH 1--84 and Pox 125I-bPTH 1 34) were used to study the disappearance from the blood stream and degradation and retension in the kidney and liver after intravenous injections in male Wiastar rats, weighing approximately 200g. Degradation of PTH was also studied in vitro, using isolated cells and a homogenate of the kidney and liver. PTH labeled with 125I and Pox was more readily degraded by the kidney than PTH labelled with 125I alone, but the opposite was found in the degradation by the liver. Isolated intact kidney cells degraded PTH less efficiently than the homogenate, but the isolated intact liver cells degraded PTH more efficiently than the homogenate, indicating the prominence of an intracellular mechanism of degradation in the kidney and an extracellular mechanism in the liver.

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices

The effect of glucagon on the phosphorylation of pyruvate kinase in 32P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10 −7 M glucagon, the incorporation of [ 32P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10 −9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.

Quantitation of pathways of ethanol metabolism

A method has been developed for estimating the sum of the contributions to ethanol oxidation by the microsomal ethanol-oxidizing system (MEOS) and catalase in the intact liver cell. It depends upon a comparison of the fate of the R hydrogen of ethanol and the hydrogen bound to carbon-2 of sorbitol under identical conditions. Limitations of the approach, particularly as regards isotopic effects, are defined. Under the condition of incubation of liver slices from rat and monkey at a concentration of ethanol of 3 mg/ml and from rat at 1 mg/ml, alcohol dehydrogenase catalysis is concluded to account, on the average, for 89% or more of the initial metabolism of ethanol. As by-products of this study, the stereospecificity of the sorbitol dehydrogenase-catalyzed reaction is shown to be of the A type in the rat, and evidence is obtained for the irreversibility of sorbitol oxidation in the intact liver cell.

Two methods for determining the activity of δ-aminolevulinate synthetase within intact liver cells in culture

Two methods are described by which δ-aminolevulinate synthetase activity is assayed within cultures of intact chick embryo liver cells. The methods depend on blocking further metabolism of δ-aminolevulinate with levulinate (25 m m). Under these conditions, the δ-aminolevulinate accumulates, much of it leaking out into the medium. The δ-aminolevulinate is measured colorimetrically in a 10-cm light-path cuvette. As little as 0.5 mg of cell protein/assay is required, and the lower limit of the spectrophotometric assay is 600 pmol/2 ml of culture medium. With the first method, the accumulation of δ-aminolevulinate can be followed during the induction, by chemicals, of δ-aminolevulinate synthetase within the intact cell. The second method serves as a quantitative assay for the amount of enzyme present at any one time point. Method 1 is sufficiently sensitive to measure the low activity of the uninduced enzyme as the accumulation of δ-aminolevulinate over a period of 16 hr. With cells in which δ-aminolevulinate synthetase has been chemically induced, method 1 yields a rate of δ-aminolevulinate production by these intact cells, 50–60% of the rate of δ-aminolevulinate produced by homogenates of the same cells incubated in an optimal assay medium. Glycine added to the culture medium at 5 m m increased by three-fold δ-aminolevulinate production by the induced intact cells. With method 2, the enzyme activity of intact cells which have been induced for a defined time, can be measured merely by replacing the culture medium with an “assay medium” containing levulinate (30 m m), glycine (50 m m), citrate (5 m m) and buffer and then incubating the cells for 5 hr. During this time, δ-aminolevulinate accumulates at a linear rate. The rate is equivalent to that of the homogenates of the same cells. The advantage of method 2 compared with the assay of homogenized cells is that the enzyme can be assayed in small amounts of cells without harvesting. One 3.5-cm dish containing 0.7 mg of cell protein is sufficient for one determination.