Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.

Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (Jo2) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo2 and hydrogen peroxide release (Jh2o2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in Jh2o2 across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2] but a linear decline in Jh2o2 of 5-15% per 50-µM decrease in chamber [O2] in all samples. Jo2 was generally stable in Mt and Hm across [O2]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh2o2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2 sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O2] at which Jo2 and Jh2o2 are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.

Independent evaluation: insights from public accounting.

BACKGROUND: Maintaining the independence of contract government program evaluation presents significant contracting challenges. The ideal outcome for an agency is often both the impression of an independent evaluation and a glowing report. In this, independent evaluation is like financial statement audits: firm management wants both a public accounting firm to attest to the fairness of its financial accounts and to be allowed to account for transactions as it sees fit. In both cases, the evaluation or audit is being conducted on behalf of outsiders--the public or shareholders--but is overseen by a party with significant interests at stake in the outcome-the agency being evaluated or executive management of the firm. METHOD: We review the contracting strategies developed to maintain independence in auditing. We examine evidence on the effectiveness of professionalism, reputation, liability and owner oversight in constraining behavior in auditing. We then establish parallels with contracting for evaluations and apply these insights to changes that might maintain and improve evaluator independence. CONCLUSIONS AND RECOMMENDATIONS: By analogy with the Sarbanes Oxley Act of 2002 reforms in auditing, we recommend exploring using a reformulated Technical Working Group to encourage more prompt release of more evaluation results and to help insulate evaluators from inappropriate pressure to change their results or analysis approach.