Mitochondrial Respiration in Response to Iron Deficiency Anemia: Comparison of Peripheral Blood Mononuclear Cells and Liver.

Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue.

High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria.

Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O2 flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O2 concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O2 limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O2 flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.

Mitochondria: biological roles in platelet physiology and pathology.

Mitochondria are key regulators of cellular energy and redox metabolism, also playing a central role in cell signaling and death pathways. A number of processes occur within mitochondria, including redox-dependent ATP synthesis by oxidative phosphorylation and reactive oxygen species production. Mitochondrial permeability transition is a reversible process that may lead to cell death and is regulated by calcium and reactive oxygen species. Functional mitochondria are present in platelets, and evidence has demonstrated the direct involvement of these organelles in cellular ATP production, redox balance, as well as in platelet activation and apoptosis. Here, we review aspects of platelet physiology in which mitochondria are involved, as well as assess their function as new tools for studying a number of human diseases.

Bioenergetic failure of human peripheral blood monocytes in patients with septic shock is mediated by reduced F1Fo adenosine-5'-triphosphate synthase activity.

OBJECTIVE: Increasing evidence points to the role of mitochondrial dysfunction in the pathogenesis of sepsis. Previous data indicate that mitochondrial function is affected in monocytes from septic patients, but the underlying mechanisms and the impact of these changes on the patients' outcome are unknown. We aimed to determine the mechanisms involved in mitochondrial dysfunction in peripheral blood mononuclear cells from patients with septic shock. DESIGN: A cohort of patients with septic shock to study peripheral blood mononuclear cell mitochondrial respiration by high-resolution respirometry analyses and to compare with cells from control subjects. SETTING: Three intensive care units and an academic research laboratory. SUBJECTS: Twenty patients with septic shock and a control group composed of 18 postoperative patients without sepsis or shock. INTERVENTIONS: Ex vivo measurements of mitochondrial oxygen consumption were carried out in digitonin-permeabilized peripheral blood mononuclear cells from 20 patients with septic shock taken during the first 48 hrs after intensive care unit admission as well as in peripheral blood mononuclear cells from control subjects. Clinical parameters such as hospital outcome and sepsis severity were also analyzed and the relationship between these parameters and the oxygen consumption pattern was investigated. MEASUREMENTS AND MAIN RESULTS: We observed a significant reduction in the respiration specifically associated with adenosine-5'-triphosphate synthesis (state 3) compared with the control group (5.60 vs. 9.89 nmol O2/min/10(7) cells, respectively, p < .01). Reduction of state 3 respiration in patients with septic shock was seen with increased prevalence of organ failure (r = -0.46, p = .005). Nonsurviving patients with septic shock presented significantly lower adenosine diphosphate-stimulated respiration when compared with the control group (4.56 vs. 10.27 nmol O2/min/10(7) cells, respectively; p = .004). Finally, the presence of the functional F1Fo adenosine-5'-triphosphate synthase complex (0.51 vs. 1.00 ng oligo/mL/10(6) cells, p = .02), but not the adenine nucleotide translocator, was significantly lower in patients with septic shock compared with control cells. CONCLUSION: Mitochondrial dysfunction is present in immune cells from patients with septic shock and is characterized as a reduced respiration associated to adenosine-5'-triphosphate synthesis. The molecular basis of this phenotype involve a reduction of F1Fo adenosine-5'-triphosphate synthase activity, which may contribute to the energetic failure found in sepsis.