Philip A. Kramer

A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers

The assessment of metabolic function in cells isolated from human blood for treatment and diagnosis of disease is a new and important area of translational research. It is now becoming clear that a broad range of pathologies which present clinically with symptoms predominantly in one organ, such as the brain or kidney, also modulate mitochondrial energetics in platelets and leukocytes allowing these cells to serve as “the canary in the coal mine” for bioenergetic dysfunction. This opens up the possibility that circulating platelets and leukocytes can sense metabolic stress in patients and serve as biomarkers of mitochondrial dysfunction in human pathologies such as diabetes, neurodegeneration and cardiovascular disease. In this overview we will describe how the utilization of glycolysis and oxidative phosphorylation differs in platelets and leukocytes and discuss how they can be used in patient populations. Since it is clear that the metabolic programs between leukocytes and platelets are fundamentally distinct the measurement of mitochondrial function in distinct cell populations is necessary for translational research.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the ‘canary in the coal mine’ by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value–the Bioenergetic Health Index (BHI)–can be calculated to represent the patients composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood

Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, including the oxidative burst from neutrophils and monocytes from individual donors. With the exception of neutrophils, all cell types tested exhibited oxygen consumption that could be ascribed to oxidative phosphorylation with each having a distinct bioenergetic profile and distribution of respiratory chain proteins. In marked contrast, neutrophils were essentially unresponsive to mitochondrial respiratory inhibitors indicating that they have a minimal requirement for oxidative phosphorylation. In monocytes and neutrophils, we demonstrate the stimulation of the oxidative burst using phorbol 12-myristate 13-acetate and its validation in normal human subjects. Taken together, these data suggest that selection of cell type from blood cells is critical for assessing bioenergetic dysfunction and redox biology in translational research.

Bioenergetics and the oxidative burst: protocols for the isolation and evaluation of human leukocytes and platelets.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.

Mitochondria in monocytes and macrophages-implications for translational and basic research.

The mitochondrion plays a crucial role in the immune system particularly in regulating the responses of monocytes and macrophages to tissue injury, pathogens, and inflammation. In systemic diseases such as atherosclerosis and chronic kidney disease (CKD), it has been established that disruption of monocyte and macrophage function can lead to chronic inflammation. Polarization of macrophages into the pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes results in distinct metabolic reprograming which corresponds to the progression and resolution of inflammation. In this review, we will discuss the role of the mitochondrion in monocyte and macrophage function and how these cells specifically influence the pathophysiology of atherosclerosis and CKD. We propose that assessing monocyte bioenergetics in different disease states could (1) enhance our understanding of the energetic perturbations occurring in systemic inflammatory conditions and (2) aid in identifying therapeutic interventions to mitigate these disorders in patients.

Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients.

Translational bioenergetics requires the measurement of mitochondrial function in clinically relevant samples and the integration of the data in a form that can be applied to personalized medicine. In the present study, we show the application of the measurement of the Bioenergetic Health Index (BHI) to cardiac surgery patients.

Hemoglobin-associated oxidative stress in the pericardial compartment of postoperative cardiac surgery patients

Atherosclerosis and valvular heart disease often require treatment with corrective surgery to prevent future myocardial infarction, ischemic heart disease, and heart failure. Mechanisms underlying the development of the associated complications of surgery are multifactorial and have been linked to inflammation and oxidative stress, classically as measured in the blood or plasma of patients. Postoperative pericardial fluid (PO-PCF) has not been investigated in depth with respect to the potential to induce oxidative stress. This is important because cardiac surgery disrupts the integrity of the pericardial membrane surrounding the heart and causes significant alterations in the composition of the pericardial fluid (PCF). This includes contamination with hemolyzed blood and high concentrations of oxidized hemoglobin, which suggests that cardiac surgery results in oxidative stress within the pericardial space. Accordingly, we tested the hypothesis that PO-PCF is highly pro-oxidant and that the potential interaction between inflammatory cell-derived hydrogen peroxide with hemoglobin is associated with oxidative stress. Blood and PCF were collected from 31 patients at the time of surgery and postoperatively from 4 to 48 h after coronary artery bypass grafting, valve replacement, or valve repair (mitral or aortic). PO-PCF contained high concentrations of neutrophils and monocytes, which are capable of generating elevated amounts of superoxide and hydrogen peroxide through the oxidative burst. In addition, PO-PCF primed naive neutrophils resulting in an enhanced oxidative burst upon stimulation. The PO-PCF also contained increased concentrations of cell-free oxidized hemoglobin that was associated with elevated levels of F2α isoprostanes and prostaglandins, consistent with both oxidative stress and activation of cyclooxygenase. Lastly, protein analysis of the PO-PCF revealed evidence of protein thiol oxidation and protein carbonylation. We conclude that PO-PCF is highly pro-oxidant and speculate that it may contribute to the risk of postoperative complications.

The emerging theme of redox bioenergetics in health and disease.

Mitochondrial function has long been recognized as central to normal physiology and a contributor to a broad range of pathologies. Much of the early research in mitochondrial biology focused on the mechanisms to generate ATP and characterization of mitochondria from highly energetic tissues such as the heart or liver. More recent studies emphasize the role of mitochondria in redox signaling and in less energetic cells such as those in the innate immune system and the vasculature. In this short overview, we discuss some of these recent developments in translational and basic research in mitochondrial pathophysiology. Advanced high throughput analytical techniques are now allowing the assessment of bioenergetic health in human populations and the emergence of the exciting new field of metabolotherapeutics. These have led to the emergence of the new field of redox bioenergetics which encompasses both the canonical aspects of mitochondrial energy production and the organelles′ role in cell signaling and disease.

Pleiotropic effects of 4-hydroxynonenal on oxidative burst and phagocytosis in neutrophils.

Metabolic control of cellular function is significant in the context of inflammation-induced metabolic dysregulation in immune cells. Generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are one of the critical events that modulate the immune response in neutrophils. When activated, neutrophil NADPH oxidases consume large quantities of oxygen to rapidly generate ROS, a process that is referred to as the oxidative burst. These ROS are required for the efficient removal of phagocytized cellular debris and pathogens. In chronic inflammatory diseases, neutrophils are exposed to increased levels of oxidants and pro-inflammatory cytokines that can further prime oxidative burst responses and generate lipid oxidation products such as 4-hydroxynonenal (4-HNE). In this study we hypothesized that since 4-HNE can target glycolysis then this could modify the oxidative burst. To address this the oxidative burst was determined in freshly isolated healthy subject neutrophils using 13-phorbol myristate acetate (PMA) and the extracellular flux analyzer. Neutrophils pretreated with 4-HNE exhibited a significant decrease in the oxidative burst response and phagocytosis. Mass spectrometric analysis of alkyne-HNE treated neutrophils followed by click chemistry detected modification of a number of cytoskeletal, metabolic, redox and signaling proteins that are critical for the NADPH oxidase mediated oxidative burst. These modifications were confirmed using a candidate immunoblot approach for critical proteins of the active NADPH oxidase enzyme complex (Nox2 gp91phox subunit and Rac1 of the NADPH oxidase) and glyceraldehyde phosphate dehydrogenase, a critical enzyme in the metabolic regulation of oxidative burst. Taken together, these data suggest that 4-HNE-induces a pleiotropic mechanism to inhibit neutrophil function. These mechanisms may contribute to the immune dysregulation associated with chronic pathological conditions where 4-HNE is generated.