Erich Gnaiger

ANAEROBIC METABOLISM IN AEROBIC MAMMALIAN CELLS : INFORMATION FROM THE RATIO OF CALORIMETRIC HEAT FLUX AND RESPIROMETRIC OXYGEN FLUX

Calorimetric and respirometric studies of cultured cells show that both neoplastic and non-neoplastic cell types maintain an anaerobic contribution to their total heat flux. In many mammalian cells this can be explained quantitatively by lactate production observed under fully aerobic conditions. Uncoupling and enhanced futile substrate cycling increase the ratio of heat flux to oxygen flux, the calorimetric-respirometric (CR) ratio. The interpretation of calorimetric and respirometric measurements requires an energy balance approach in which experimentally measured CR ratios are compared with thermochemically derived oxycaloric equivalents. The oxycaloric equivalent is the enthalpy change per mole of oxygen consumed, and equals -470 kJ/mol O2 in the aerobic catabolism of glucose, assuming that catabolism is 100% dissipative (the net efficiency of metabolic heat transformation is zero). CR ratios more negative than -470 kJ/mol O2 have been reported in well-oxygenated cell cultures and are discussed in terms of integrated aerobic and anaerobic metabolism.

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.

Proposals for a standardized sample handling procedure for the determination of elemental composition and enthalpy of combustion of biological material

A procedure has been developed for the prepn. of microbial biomass of std., defined quality suitable for the detn. of elemental compn. and enthalpy of combustion. Furthermore methods for the detn. of residual moisture and ash content of biomass samples have been established. The results indicate that samples should be prepd. in a freeze-dried (lyophilized) state and that residual moisture content should be detd. immediately prior to sample prepn. for combustion calorimetry and elemental anal. Results from such anal. should then be related to material which is first freeze-dried and subsequently oven dried (100 Deg for 24 h) as ref. state. The method outlined here for microbial biomass should prove suitable for biol. samples from a wide variety of sources including both pure proteins, fats, etc. as well as cells and tissues. [on SciFinder (R)]

Concepts on efficiency in biological calorimetry and metabolic flux control

Abstract Accurate definitions of efficiency are required to resolve controversies on the significance and comparability of measures of efficiency in biological energetics. This review on concepts of efficiency is arranged into 4 parts. First, some fundamental energy relations of equilibrium and nonequilibrium thermodynamics are defined and placed into a coherent context as relevant for efficiency in biology. The classical expression of the Carnot efficiency of a heat engine obtains a new meaning in terms of flux-force relations of nonequilibrium thermodynamics. Second, within this general thermodynamic frame, the specific treatment of energy transformations of chemical reactions is introduced, with particular emphasis on open systems with internal transformation and external transfer of matter. Third, the chemical transformations in ATP turnover and internal efficiencies of coupled reactions are analyzed in two parts. On the one hand, the enthalpy efficiency is relevant in the context of biological calorimetry in relation to uncoupling and the integration of aerobic and anaerobic metabolism. On the other hand, the molar Gibbs energy efficiency relates to the driving force of coupled reactions and to the control of flux. High metabolic power and maximum efficiency are mutually exclusive. Finally, the discussion of various expressions of efficiency in biological growth requires a careful distinction between energy conservation in transformations (chemical reactions) and energy acquisition in coupled transformation and transfer of energy in the form of externally supplied matter. Better understanding and management of biological resource utilization requires this combined analysis of efficiency in biological energetics.

Assessment of endothelial preservation in human cell cultures

BACKGROUND Impairment of microcirculation due to endothelial cell damage must be considered a limiting factor in organ preservation. The present study aims at a quantitative assessment of preservation-induced injury in cultured human endothelial cells. METHODS Monolayer cultures of human umbilical vein endothelial cells were exposed to cold (40 degrees C) hypoxic storage in University of Wisconsin solution, histidine-tryptophane-ketoglutarate solution, Euro-Collins solution, and saline solution. Cellular integrity was evaluated by viable cell count, ultrastructural analysis, and prostacyclin release after 24, 48, and 72 hours of storage and subsequent 6 hours of reincubation in culture medium at 37 degrees C. Expression of intercellular adhesion molecule-1 was investigated after 6, 12, and 24 hours of cold preservation and after 6 hours of rewarming. RESULTS Cellular viability was best maintained with University of Wisconsin and histidine-tryptophane-ketoglutarate solutions with no significant reduction of cell count up to 72 hours; Euro-Collins solution and saline solution caused a significant decline in cell numbers after 24 hours (p < 0.05). Morphology was best preserved by University of Wisconsin solution. Prostacyclin values were elevated after 24 hours in Euro-Collins solution and saline solution, after 48 hours in histidine-tryptophane-ketoglutarate, Euro-Collins, and saline solutions, and after 72 hours in Euro-Collins solution (p < 0.05, compared with University of Wisconsin solution). ICAM expression was weak after cold storage (24 hours) in University of Wisconsin solution, moderate after incubation in histidine-tryptophane-ketoglutarate and Euro-Collins solutions and intensive after storage in saline solution. In contrast, rewarming caused intensive expression of intercellular adhesion molecule-1 in all experimental groups as compared with controls, which showed baseline expression at any time. CONCLUSIONS From our results we conclude that in this model cellular integrity is best protected by University of Wisconsin solution, increased prostacyclin release is consistent with morphologic alterations and intercellular adhesion molecule-1 expression is clearly up-regulated in endothelial cells under reperfusion conditions after cold hypoxic storage.

Physiological calorimetry: heat flux, metabolic flux, entropy and power

Abstract Physiological calorimetry is concerned with the measurement of heat flux in living systems where heat flux is associated with the chemical flux of metabolic reactions. Calorimetry can be related to nonequilibrium thermodynamics if information on both the enthalpy of metabolic reactions and the molar Gibbs energy is available. The molar Gibbs energy of reaction (Gibbs force) is the scalar force conjugated to metabolic flux. The force conjugated to heat flux of an irreversible process is the Gibbs energy/enthalpy ratio. Metabolic power and heat flux of irreversible processes are distinguished as the time rate of Gibbs energy and enthalpy changes, respectively. Power is the product of fluxes and forces, related to the internal entropy production by the absolute temperature. In contrast, T Δ r S is the “bound energy” change which equals the heat change of a reversible process in a closed system and is not available for work. Heat flux in general is the sum of the dissipated power and the bound energy change per unit of time. This concept can be extended to vectorial heat flux along a temperature gradient. The temperature difference relative to the temperature of the heat source, traditionally viewed as the “efficiency of a reversible machine”, is in fact the thermal force for heat flux between heat source and sink. The thermal force times heat flux is the thermal power which can be maximally converted into work or can be irreversibly dissipated. A clear distinction between heat flux and power is conceptually revealing, despite the fact that both quantities have the same dimension with units [W per volume, or per mass or per defined system] when describing scalar and discontinuous processes.

Mitochondrial ischemia-reoxygenation injury and plasma membrane integrity in human endothelial cells

0 RGAN PRESERVATION under hypothermic ischemia is enhanced by storage solutions that protect the vascular endothelium from ischemia-reperfusion injury. Ischemia-reperfusion injury leads to primary graft failure and chronic rejection, and is commonly assessed by measuring endothelial activation and damage of the endothelial plasma membrane. However, corresponding primary intracellular events are little understood compared with the secondary cytokine/adhesion molecule cascade and inflammatory responses.‘-* Because protection of intracellular and cell membrane function is fundamental for further improvement of organ preservation, we developed highresolution respirometry as a sensitive diagnostic test for mitochondrial and plasma membrane competence.3 Whereas the plasma membrane remained impermeable after clinically relevant cold storage times of 8 hours and 20

Calorespirometry and spectrophotometry of the ciliated gill of the marine mussel mytilus Edulis (L.)

Abstract The energy metabolism of the ciliated gill of the marine mussel Mytilus edulis (L.) was studied under different conditions of energy supply and demand, using calorespirometry and spectrophotometry. When ciliary beat frequency was increased approximately two-fold at 30–155 Torr pO2 (4–20.7 kPa), metabolic rate nearly doubled. Metabolism remained aerobic and dissipative, with very little anaerobic capacity. Mitochondrial cytochrome reduction showed the same sensitivity to pO2 as metabolic rate, whether gills had basally active or stimulated cilia. These methods will be useful to determine the effects of natural stresses and aquatic pollutants on the control of energy flux in working ciliated tissues.