Hydrogen peroxide (H₂O₂) is a reactive oxygen species (ROS) generated by mitochondria, which in small amounts participates in redox signaling and in large amounts contributes to oxidative stress. It is produced as a byproduct of oxidative phosphorylation resulting from the partial reduction of oxygen to form superoxide, which is then partitioned by superoxide dismutase to form H₂O₂ and O₂. Measuring fluctuations in H₂O₂ concentration is relevant to understanding both normal biological signaling and pathological diseases. Accurate measurements of mitochondrial H₂O₂ have been difficult to obtain in vivo, though, due to limitations in many of the currently available tools. For instance, isolated mitochondria have been used to indirectly extrapolate H₂O₂ flux results in vitro to estimate that of in vivo conditions. Fluorescent probes used to detect oxidative stress work best in cells or in simple organisms such as zebrafish with optically accessible systems in vivo. Moreover, most of these probes are not specific for any individual ROS and may require conditional activation to produce a signal. Oxidative damage markers, including DNA damage markers (e.g., 8-hydroxy-2’-deoxyguanosine) and lipid peroxidation products (e.g., 8-isoprostane), offer an indirect means of assessment by detecting the consequences of changes in H₂O₂.

A highly sensitive, ratiometric mass spectrometry probe has been developed to overcome the difficulties of directly measuring mitochondrial H₂O₂in vivo. MitoB (MitoBoronic acid) contains a lipophilic triphenylphosphonium cation moiety that drives its accumulation into the negatively charged mitochondrial matrix. It rapidly passes through biological membranes and once taken up by mitochondria, its arylboronic moiety selectively reacts with H₂O₂ to produce a biologically stable phenol product, MitoP (MitoPhenol). The ratio of the conversion of MitoB to MitoP over time thus indicates the mitochondrial matrix H₂O₂ concentration. The rate of this reaction within the mitochondrial matrix is slow (~3.8 M^-1s^-1 at pH 8.0, 25°C), and MitoB will not disrupt cytoplasmic H₂O₂ levels that reside at a lower pH (~7.2). With these given parameters, the reaction remains contained within the mitochondria. To determine the MitoP/MitoB ratio, the newly produced MitoP and any remaining MitoB are extracted from a given sample, and their proportion is quantified by LC-MS/MS relative to deuterated internal standards, MitoB-d₁₅ and MitoP-d_15, which are added to correct for variations that arise during extraction and detection. Higher ratio values will indicate a higher content of H₂O₂ in the mitochondria. The workflow of this method and example data results are depicted below.

The MitoB method has been used in mouse, Drosophila, and fish to report mitochondrial H₂O₂ levels in whole organisms.^1-6 In these instances, MitoB is injected directly into the animal, then after a period of time to allow the MitoB to respond to endogenous H₂O₂, the desired tissue is removed, homogenized, and spiked with deuterated internal standards before chemical extraction and quantification by LC-MS/MS. The MitoB method is also well-suited to cell culture systems, wherein the MitoB and MitoP pools will equilibrate between the mitochondrial matrix and the extracellular environment, making it possible to sample the culture medium to infer mitochondrial H₂O₂ concentration.² This avoids the need to perform sample extraction prior to LC-MS/MS analysis. Note that in both whole animals and cell culture, MitoB will also respond to peroxynitrite (ONOO^-), which can form in the presence of superoxide and nitric oxide. The contribution of ONOO^- can be controlled for by inhibiting nitric oxide synthases using L-NAME, a nitric oxide synthase inhibitor, or by suppressing the expression of nitric oxide synthases. This is an important factor to determine, since the MitoB method assumes that the only substantial contribution to the conversion of MitoB to MitoP is the direct reaction with H₂O₂ inside the mitochondria. Changes in the mitochondrial membrane potential (ΔΨ_m) are an additional factor that may influence the MitoP/MitoB ratio, since uptake of MitoB into mitochondria is ΔΨ_m-dependent. In living tissues, a ΔΨ_m within a normal range of 120-160 mV should not affect MitoB uptake.

No matter the sample type, a major advantage of the MitoB methodology is that there are pause points along the sampling and extraction protocol in which the samples can be held at -80°C for at least two months. This is especially important for laboratories that do not have ready access to mass spectrometry facilities or the experience to perform LC-MS/MS analysis. For these researchers, Cayman offers MitoP/MitoB ratio analysis as a contract service. Frozen samples of any biological type can be shipped to our mass spectrometry laboratory where our specialists will efficiently quantify mitochondrial H₂O₂, using state-of-the art triple quad and Orbitrap instruments. We offer the MitoB probe plus all necessary internal and calibration standards in our catalog (see table below), enabling you to conduct your experiment as needed before handing off your samples to us for analysis.

Our mitochondrial experts are always open to discuss the results and help with the interpretation and generation of new hypotheses stemming from your analysis. Email our services team at contractresearch@caymanchem.com to find out more.

For those laboratories with the ability to perform mass spec analysis on their own, Cayman offers a Hydrogen Peroxide Ratiometric MaxSpec^® Kit, which includes MitoB, MitoP, and their respective deuterated standards plus a detailed protocol for use. Each compound in the kit is formulated as a ready-to-use, quantitative solution to streamline sample and standard preparation for mass spec analysis. These formulations are guaranteed to meet rigorous quality standards, ensuring consistency and reproducibility from run to run.

See complete details of the Hydrogen Peroxide Ratiometric MaxSpec^® Kit

For researchers that prefer to collect data using a fluorescent plate reader or flow cytometer, Cayman offers a Mitochondrial ROS Detection Assay Kit that includes a fluorescent detection reagent that measures the production of mitochondrial ROS under specific conditions. For additional resources on measuring ROS, download our Guide to Oxidative Stress Assays (PDF) and read our article on Probes to Detect Oxidative Stress. For additional resources to assess mitochondrial function and toxicity, download our Mitochondrial Function Assays brochure (PDF).

References

1. Cochemé, H.M., Quin, C., McQuaker, S.J., et al. Measurement of H₂O₂ within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix. Cell Metab. 13(3), 340-350 (2011).

2. Cochemé, H.M., Logan, A., Prime, T.A., et al. Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H₂O₂ in living Drosophila. Nat. Protoc. 7(5), 946-958 (2012).

3. Logan, A., Shabalina, I.G., Prime, T.A., et al. In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice. Aging Cell13(4), 765-768 (2014).

4. Chouchani, E.T., Pell, V.R., Gaude, E., et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515(7527), 431-435 (2014).

5. Latorre-Pellicer, A., Moreno-Loshuertos, R., Lechuga-Vieco, A.V., et al. Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing. Nature 535(7613), 561-565 (2016).

6. Salin, K., Auer, S.K., Villasevil, E.M., et al. Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress. Sci. Rep.7(41228), (2017).