Hemoglobin-free Perfused Rat Liver Research Articles

Proton movement accompanying monocarboxylate permeation in hemoglobin-free perfused rat liver

While considerable information has accumulated recently from studies with isolated liver mitochondria on transport systems for diand tricarboxylates (cf. reviews [ 1,2] ), comparatively little is known on the properties of uptake and release by the liver cell of the major metabolic fuels which are either uncharged (e.g. glucose) or weak monocarboxylic acids (fatty acids, lactic and pyruvic acids). The experiments to be reported here represent attempts to follow the rate and the extent of monocarboxylic acid uptake and release by the liver cell. With the help of a ‘model monocarboxylic acid’, benzoic acid (pK = 4.2), which is considered metabolically inert when compared to the compounds named above, a simple and continuous method to follow monocarboxylic acid movements into and out of the hepatocyte will be outlined. The experimental system is that of the hemoglobin-free perfused rat liver [3,4] . The underlying idea is based on the concept of Overton [5] , elaborated and applied by Conway to the yeast cell [6,7] and by Hohorst, Kreutz and Bticher to the liver cell [8] , that permeant monocarboxylates are passively transported across biological membranes in the form of the uncharged acids. Therefore, net monocarboxylate transport should be coupled to net proton transport, no matter whether the mechanism is ‘p&on symport’ or ‘hydroxyl antiport’. In other words, the compartment from which monocarboxylic acid is leaving is expected to be rendered more alkaline, and vice uersa. On this basis, extracellular pH is explored as an indicator of monocarboxylate gradient equilibration in an open (non-recirculating) system of perfusion.

State of oxidation-reduction and state of binding in the cytosolic NADH-system as disclosed by equilibration with extracellular lactate-pyruvate in hemoglobin-free perfused rat liver.

1 The extracellular ratio [lactate]/[pyruvate] is used as a means to poise the cytosolic NADH/NAD+ system. This is achieved by stepwise changes of [lactate]/[pyruvate] in the perfusate in an open system. Fluorescence and absorbance at NADH-specific wavelengths is recorded simultaneously with single or dual wavelength methods. Fluorescence excitation and absorbance difference spectra are obtained with the help of an instrument computer. The readout is calibrated by infusion of internal standards, permitting an estimation of the enhancement of quantum yield of NADH-specific fluorescence. 2 Mathematical treatment on the basis of the mass action law permits extrapolation to fully reduced and fully oxidized cytosolic bound NADH. In addition, the apparent equilibrium constant for bound NADH/NAD+ and lactate/pyruvate is obtained (0.022 to 0.071), corresponding to a midpoint potential of bound NADH of – 250 to – 265 mV. Accordingly, the ratio of dissociation constants, KNAD+/KNADH, relevant for the cytosolic binding sites, is 200–700. Cytosolic NADH-binding capacity is estimated to be around 80 nmol/g liver wet weight. Under normoxic conditions at [lactate]/[pyruvate] of 10 in the perfusate, the ratio of contents of cytosolic bound NADH/free NADH is 100. 3 The existence of near-equilibrium conditions in the system is suggested by measurement of net H+ transport coupled to lactate import, as well as by determination of an intracellular dependent indicator metabolite couple, α-glycerophosphate/dihydroxyacetone phosphate. Problems regarding the validity of tissue contents of lactate/pyruvate as indicator for the cytosolic NADH/NAD+ potential are discussed.

Direct measurement of the state of the lactate dehydrogenase system in hemoglobin-free perfused rat liver

Appropriate correction of tissue contents of lactate and pyruvate shows that the cellular contents of these metabolites are of limited cell physiological use, especially in abnormal metabolic states. However, the ratio of contents of α-glycerophosphate/dihydroxyacetone phosphate and of extracellular concentrations of lactate/pyruvate are useful in this context. The extracellular ratio [lactate]/[pyruvate] provides a means to poise the redox potential of cytosolic NADH. This is achieved by stepwise changes of [lactate]/[pyruvate] in perfusate of rat liver perfused in an open system. Dependent intracellular changes are followed (a) by photometric and (b) by analytical methods. Mathematical treatment of observed increments on the basis of the mass action law allows extrapolation to fully reduced and fully oxidized cytosolic NADH. In addition, the apparent equilibrium constant for bound NADH/NAD and lactate/pyruvate is obtained. The ratio of dissociation constants, D NAD/ D NADH, relevant for the cytosolic sites, is calculated at an assumed pH of 7.0. The midpoint potential of bound NADH is around −260 mV.

The state of NADPH/NADP + during mixed-function oxidation of hexobarbital in hemoglobin-free perfused rat liver

The effect of dinitrophenol (52 μm), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms.In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).

The steady state level of catalase compound I in isolated hemoglobin-free perfused rat liver

Helmut SIES lnstitut ff~r Physiologische Chemie und Physikalische Biochemie, der Universittit Mftnchen, Germany and Britton CHANCE Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Received 25 September 1970 1. Introduction The long study of the catalytic and peroxidatic ac- tivities of catalase has led to little information on its function in vivo, particularly in mammalian systems [1, 2]. In bacteria, e.g. Micrococcus lysodeikticus, spectroscopic evidence indicated that catalase is large- ly saturated with H202 in the form of the primary intermediate, compound I [3]. In mammalian tissue, the correspondence of the order of reaction and the overall flux rates for methanol metabolism in the rab- bit were suggestive of catalase's role in methanol oxi- dation [ 1 ]. Similar conclusions were reached by Tephly, Parks and Mannering [4] for methanol metab- olism in the rat. Heppel and Porterfield [5] found coupled nitrite oxidation in rat liver homogenates, and Portwich and Aebi [6] demonstrated the coupled oxidation of formate in rat liver slices. In the present investigation, spectrophotometric measurements of absorbance in the 660 nm band re- gion of catalase compound I from isolated perfused rat liver have been performed. Experimental evidence which supports an interpretation of changes of the ob- served signal to be due to the catalase system will be * Postal address: Dr. H. Sies, Institut fllr Physiologische Chemie und Physikalische Biochemie, Goethestrasse 33, 8-Miinchen- 15, Germany. presented; it includes the effects of the above-men- tioned hydrogen donors, of hydrogen peroxide gener- ating substance, of oxygen tension, and of inhibitors of the catalase system. 2. Experimental Spectrophotometry of light transmitted through a lobe of perfused liver was performed with the Rapid- spektroskop of Howaldtswerke Deutsche Werf, Kiel, as adapted by Brauser [7]. Dual wavelength absor- bance photometry by sinusoidal wavelength modula- tion [7] was used for more sensitive study of absor- bance changes as a function of time (see also [8]). Bandwidth of monochromatic light was 4 nm. Com- pensation for spectral characteristics of the apparatus was afforded by a reference beam which was reflected from a magnesia surface. In preliminary experiments, the rotating filter ap- paratus of Chance [9] as adapted for absorbancy measurements [ 10] was used with a 660 nm mea- suring filter and a 720 nm reference filter, having bandwidths of + 30 and + 7 nm, respectively. Light transmitted through a liver lobe was collected by a light pipe and was detected by an infrared-guarded silicone diode. Oxygen uptake was followed with Ag-Pt-micro- electrodes inserted into the perfusion circuit before 172 North.Holland Publishing Company - Amsterdam

Positive increase of redox potential of the extramitochondrial NADP(H) system by mixed function oxidations in hemoglobin-free perfused rat liver

FEBS LettersVolume 9, Issue 4 p. 205-208 Full-length articleFree Access Positive increase of redox potential of the extramitochondrial NADP(H) system by mixed function oxidations in hemoglobin-free perfused rat liver Helmut Sies, Helmut Sies Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this authorMartina Kandel, Martina Kandel Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this author Helmut Sies, Helmut Sies Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this authorMartina Kandel, Martina Kandel Institut für Physiologische Chemie und Physikalische Biochemie der Universität München, GermanySearch for more papers by this author First published: August 17, 1970 https://doi.org/10.1016/0014-5793(70)80356-8Citations: 25AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Th. Bücher, H. Sund Pyridine Nucleotide Dependent Dehydrogenases (1970), Springer Berlin, Heidelberg, New York 439– 2 H.A. Krebs, R.L. Veech, S. Papa J.M. Tager E. Quagliariello E.C. Slater The Energy Level and Metabolic Control in Mitochondria (1969), Adriatica Editrice Bari 329– 3 H. Sies and B. Brauser, European J. Biochem., in press. 4 B. Brauser, H. Versmold, Th. Bücher, Z. Physiol. Chem., 349, (1968), 1589– 5 B. Brauser, H. Sies, Th. Bücher, FEBS Letters, 2, (1969), 170– 6 W.J. Marshall, A.E.M. McLean, Biochem. Pharmacol., 18, (1969), 153– 7 B. Chance, F. Jöbsis, Nature, 184, (1959), 195– 8 M. Klingenberg, in: Methoden der enzymatischen Analyse, ed. H.U. Bergmeyer, 2nd edition (Verlag Chemie, Weinheim) in press. 9 J.R. Williamson, B.E. Corkey, J.M. Lowenstein Methods in Enzymology 13, (1969), Academic Press New York, London 434– 10 H.J. Hohorst, F.H. Kreutz, Th. Bücher, Biochem. Z., 332, (1959), 18– 11 Th. Bücher, M. Klingenberg, Angew. Chem., 70, (1958), 552– 12 M. Klingenberg, 11 Coll. Ges. Physiol. Chem. (1961), Springer Berlin, Heidelberg, New York 82– 13 J.R. Williamson, S. Papa J.M. Tager E. Quagliariello E.C. Slater The Energy Level and Metabolic Control in Mitochondria (1969), Adriatica Editrice Bari 385– 14 R.G. Thurman, R. Scholz, Z. Physiol. Chem., 351, (1970), 294– 15 R. Scholz, R.G. Thurman, Federation Proc., 29, (1970), 925– Abstr. 3873 16 M.J. Waterman, H.S. Mason, Biochem. Biophys. Res. Commun., 39, (1970), 450– Citing Literature Volume9, Issue4August 17, 1970Pages 205-208 ReferencesRelatedInformation

Reduction kinetics and content of cytochrome P-450 by application of dual wavelength techniques to hemoglobin-free perfused rat liver

1. Introduction For a better understanding of the function of the CO-binding pigment, cytochrome P-450, more basic knowledge of its properties is required. The pigment has so far resisted purification attempts by conversion to a substance called P-420 [l] . We shall try here