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Radioactive Carbon in the Study of Respiration in Heterotrophic Systems

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... The use of stable and radioactive isotopes to track metabolism of compounds of interest and their biosynthesis in many organisms and tissues began in the 1930 -1940s after Urey discovered deuterium and enriched nitrogen for 15 N, enabling their use by his colleagues at Columbia University (Rittenberg, Schoenheimer, Bloch, Ratner, Shemin, and others) to study intermediary metabolism. 11 CO 2 and [ 11 C]lactate, along with labeling with the stable isotope 13 C, were used to study carbohydrate metabolism (109,549,607). Later, 14 C labeling opened the door to studies of glucose metabolism in brain. ...
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The development of the use of carbon isotopes as metabolic tracers is briefly described. 13C-labelled precursors (13CO2, 13CH4) first became available in 1940 and were studied in microorganisms, but their use was limited by very low enrichments and lack of suitable analytical equipment. More success was achieved with 11C and especially 14C, as these radioactive tracers did not need to be highly enriched. Although the stable 13C isotope can be used at a low percentage enrichment in mass spectrometry, its application to magnetic resonance spectroscopy (MRS) requires very highly enriched precursors, due to its low natural abundance and low sensitivity. Despite such limitations, however, the great advantage of 13C-MRS lies in its exquisite chemical specificity, in that labelling of different carbon atoms can be distinguished within the same molecule. Effective exploitation became feasible in the early 1970s with the advent of stable instruments, Fourier transform 13C-MRS, and the availability of highly enriched precursors. Reports of its use in brain research began to appear in the mid-1980s. The applications of 13C isotopomer analysis to research on neuronal/glial relationships are reviewed. The presence of neighbouring 13C-labelled atoms affects the appearance of the resonances (splitting due to C-C coupling), and so allows for unique quantification of rates through different and possibly competing pathways. Isotopomer patterns in resonances labelled from a combination of [1-13C]glucose and [1, 2-13C2]acetate have revealed aspects of neuronal/glial metabolic trafficking on depolarization and under hypoxic conditions in vitro. This approach has now been applied to in vivo studies on inhibition of glial metabolism using fluoroacetate. The results confirm the glial specificity of the toxin and demonstrate that it does not affect entry of acetate. When the glial TCA cycle is inhibited, the ability of the glia to participate in the glutamate/glutamine cycle remains unimpaired, in that labelling of glutamine, which can only be derived from neuronal metabolism of glucose, persists. The results also confirmed earlier evidence that part of the GABA transmitter pool is derived from glial glutamine.
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Full-text available
The gluconeogenic pathway from 13C-labeled substrates, each of which contained the 14C-labeled counterpart at a tracer level, has been followed in isolated rat liver cells and in isolated perfused mouse liver. The gluconeogenic flux from glycerol, the synthesis of glycogen, the stimulation of glycogenolysis by glucagon, the recycling of triacylglycerol, and an increase in pentose cycle activity under the influence of phenazine methosulfate were all observed directly in the 13C NMR spectra of perfused liver or isolated hepatocytes. The relative concentrations of 13C label at specific carbons measured by the NMR spectra under these conditions agreed closely with 14C isotopic distributions measured in extracts of the same doubly labeled samples for specific activities of greater than or equal to 3%. The label distributions measured by both methods were the same to within the experimental errors, which ranged from +/- 2% to +/- 7% in these experiments.
Chapter
Die Anwendung von radioaktiven Indicatoren war zunächst beschränkt auf die Umwandlungsprodukte des Urans und Thoriums. Es kamen in dieser Epoche, die sich von 1913 bis 1933 erstreckte, hauptsächlich die radioaktiven Isotope von Blei und Wismut in Betracht. Die erste biologische Anwendung wurde 1923 veröffentlicht.
Chapter
Die Anwendung von radioaktiven Indicatoren war zunächst beschränkt auf die Umwandlungsprodukte des Urans und Thoriums. Es kamen in dieser Epoche, die sich von 1913 bis 1933 erstreckte, hauptsächlich die radioaktiven Isotope von Blei und Wismut in Betracht. Die erste biologische Anwendung wurde 1923 veröffentlicht.
Chapter
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During the last decade it has been recognized that the process of photosynthesis in green plants is unique, not because it involves a complicated photochemical decomposition of carbon dioxide for which there is no analogy in the organic world, but because it combines in a unique manner a number of processes each of which may be found in other living cells. If we turn from green plants to purple bacteria, for instance, we find that radiant energy is utilized for the reduction of carbon dioxide. These organisms, however, cannot use simply water as a hydrogen donor and hence are not able to liberate free oxygen. For the reduction of carbon dioxide they depend, in addition to light, upon energetically valuable hydrogen donors such as free hydrogen, hydrogen sulphide, or organic acids. The over-all energy balances of these photoreductions are, therefore, much less favourable than that of photosynthesis in plants. If we turn to organisms not sensitive to light, we find that carbon dioxide can be reduced in complete darkness by several species of bacteria and even by some animal tissues. In this case the mechanism is a coupled oxido-reduction in which an excess of hydrogen donors, either of inorganic or organic nature, has to be sacrificed to promote the ‘chemosynthesis’. It is clear that such dark reactions lead not to a gain but to an over-all loss of chemical energy.Recent advances in the field of respiration and fermentation have taught us that despite the infinite variety of metabolic reactions in living cells the principles governing them are few. Accordingly, it is conceivable that the different reactions involving a reduction of carbon dioxide have many important traits in common, and that the study of any one of them may lead to a better understanding of the process of photosynthesis.The present article is a report on the metabolism of certain unicellular chlorophyllous algae (several species of Scenedesmus, Ankistrodesmus, Rhaphidium) that are able to reduce carbon dioxide either in normal photosynthesis with the evolution of oxygen, or in photo-reduction with the absorption of an equivalent amount of hydrogen, or in chemosynthesis with the oxyhydrogen reaction as the driving force. The two latter reactions do not occur under normal aerobic conditions. They can be observed only after a few hours' incubation in hydrogen gas. The anaerobic treatment brings into play a hydrogenase which enables the algae to absorb or to release molecular hydrogen. This metabolic change we call adaptation. The adaptation consists in an enzymatic activation or rearrangement of some of the catalytic systems. It can be inhibited by traces of specific poisons like cyanide and hydroxylamine.Upon illumination in the adapted state, in presence of hydrogen and carbon dioxide, the algae reduce carbon dioxide with twice the volume of hydrogen, exactly akin to some purple bacteria. This we call photoreduction. The results of experiments with flashing light and with specific poisons show that in photosynthesis and photo-reduction the truly photochemical reactions are the same and remain unchanged. Hence, the difference appears to originate from the ways by which the oxidized products of the photochemical reaction are eliminated. In photosynthesis they are decomposed with the liberation of oxygen, in photoreduction they are reduced to water by hydrogen donors.The adapted state of the algae gives way to normal aerobic conditions not only under the influence of air, but also under the influence of higher light intensities. This we call reversion. It seems that reversion occurs whenever some intermediate oxidized products (which we call ‘peroxides’, because they must be the precursors to molecular oxygen) accumulate faster than they are reduced by the hydrogenase system. In absence of carbon dioxide no intermediate oxidized products (or ‘peroxides’) are formed and the adapted state is stable even at high light intensities. If not only carbon dioxide but also hydrogen is absent (i.e. in an atmosphere of pure nitrogen), light causes a release of hydrogen from the adapted algae. The reversion by light in presence of carbon dioxide can be prevented by the action of certain substances like hydroxylamine, or o-phenanthroline, In the presence of such inhibitors the adapted algae continue to metabolize like purple bacteria even in very strong light. The light saturation rate of carbon dioxide reduction with hydrogen remains, however, far smaller than the corresponding rate of photosynthesis.Very small amounts of oxygen prevent adaptation, but up to 10mm. Hg of this gas are tolerated by adapted algae in hydrogen. The reason is that at these low partial pressures the reduction of oxygen to water in the algae proceeds faster than the reversion. Under optimum conditions the formation of water from the elements, the oxyhydrogen reaction, is coupled with a simultaneous reduction of carbon dioxide, so that one-half molecule of carbon dioxide disappears for each molecule of reduced oxygen. The coupling between oxyhydrogen reaction and the reaction of carbon dioxide is such that in absence of carbon dioxide the first reaction remains incomplete; little more than one molecule of hydrogen is absorbed instead of the two necessary to form water. After a partial reduction of the oxygen leading to the ‘peroxide’ level, the reaction apparently continues in unknown directions with internal hydrogen donors. Again a different result is obtained after adding specific poisons. In presence of carbon dioxide and of poisons, the amount of hydrogen absorbed indicates the straightforward formation of water.A critical comparison of the results reported in this article leads to the assumption that the dark reduction of carbon dioxide can be represented very satisfactorily by reversing essential parts of a diagram drawn to describe various partial reactions in photosynthesis and photoreduction.My thanks are due to Drs J. Franck and D. Goddard, for helpful suggestions. I am particularly indebted to Dr E. Rabinowitch for a very thorough criticism of the manuscript.
Chapter
Quantum EfficiencySaturation PhenomenaInduction PeriodsPhoto-Oxidation Processes in PlantsThe Metabolism of the Purple Bacteria and van Niel's Theory of PhotosynthesisCarbon Dioxide Reduction in the Absence of Oxygen and the “Reduced State” of the Assimilating System in PlantsThe Reduction of Carbon Dioxide in the Dark
Article
1. Unicellular algae possessing a hydrogenase system (Scenedesmus and other species), and having been adapted by anaerobic incubation to the hydrogen metabolism, reduce oxygen to water according to the equation O(2) + 2H(2) --> 2H(2)O. 2. The oxyhydrogen reaction proceeds undisturbed only in the presence of carbon dioxide, which simultaneously is reduced according to the equation CO(2) + 2H(2) --> H(2)O + (CH(2)O) = (carbohydrate). 3. The maximum yield of the induced reduction is one-half molecule of carbon dioxide reduced for each molecule of oxygen absorbed. 4. Partial reactions are recognizable in the course of the formation of water and it is with the absorption of the second equivalent of hydrogen that the carbon dioxide reduction appears to be coupled. 5. The velocity of the reaction increases in proportion to the partial pressure of oxygen, but only up to a certain point where any excess of oxygen causes the inactivation of the hydrogenase system. The reaction then ends prematurely. 6. During the oxyhydrogen reaction little or no oxygen is consumed for normal respiratory processes. 7. Small concentrations of cyanide, affecting neither photosynthesis nor photoreduction in the same cells, first inhibit the induced reduction of carbon dioxide and then lead to a complete inactivation of the hydrogenase system. 8. Hydroxylamine, added after adaptation, has either no inhibitory effect at all, or prevents solely the induced reduction of carbon dioxide without inactivating the hydrogenase system. 9. Dinitrophenol prevents the dark reduction of carbon dioxide while the reduction of oxygen continues to the formation of water. 10. Glucose diminishes the absorption of hydrogen, probably in its capacity as a competing hydrogen donor. 11. The induced reduction of carbon dioxide can be described as an oxido-reduction similar to that produced photochemically in the same cells.