Using exomarkers to assess mitochondrial reactive species in vivo

Background: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. Scope of review: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. Major conclusions and general significance: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Abbreviations: EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio

Collagen-induced Arthritis Is Reduced in 5-Lipoxygenase-activating Protein-deficient Mice

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycaemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs following the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal and disruption to mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear due to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycaemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycaemia in both cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

Asymmetries between the production of D+ and D- mesons from 500 GeV/c pi- nucleon interactions as a function of xF and pt**2

We present asymmetries between the production of D+ and D- mesons in Fermilab experiment E791 as a function of xF and pt**2. The data used here consist of 74,000 fully-reconstructed charmed mesons produced by a 500 GeV/c pi- beam on C and Pt foils. The measurements are compared to results of models which predict differences between the production of heavy-quark mesons that have a light quark in common with the beam (leading particles) and those that do not (non-leading particles). While the default models do not agree with our data, we can reach agreement with one of them, PYTHIA, by making a limited number of changes to parameters used

Mass Splitting and Production of Σc0\Sigma_c^0 and Σc++\Sigma_c^{++} Measured in 500GeV500 {GeV} π−−\pi^- -N Interactions

From a sample of $2722 \pm 78$ $\Lambda_c^+$ decaying to the $pK^-\pi^+$ final state, we have observed, in the hadroproduction experiment E791 at Fermilab, $143 \pm 20$ $\Sigma_c^0$ and $122 \pm 18$ $\Sigma_c^{++}$ through their decays to $\Lambda_c^+ \pi^{\pm}$. The mass difference $M(\Sigma_c^0) - M(\Lambda_c^+$) is measured to be $(167.38\pm 0.29\pm 0.15) {MeV}$; for $M(\Sigma_c^{++}) - M(\Lambda_c^+)$, we find $(167.76\pm 0.29\pm0.15) {MeV}$. The rate of $\Lambda_c^+$ production from decays of the $\Sigma_c$ triplet is (22\pm 2\pm 3) {%} of the total $\Lambda_c^+$ production assuming equal rate of production from all three, as measured for $\Sigma_c^0$ and $\Sigma_c^{++}$. We do not observe a statistically significant $\Sigma_c$ baryon-antibaryon production asymmetry. The $x_F$ and $p_t^2$ spectra of $\Lambda_c^+$ from $\Sigma_c$ decays are observed to be similar to those for all $\Lambda_c^+$'s produced.Comment: 15 pages, uuencoded postscript 3 figures uuencoded, tar-compressed fil

Flexibility but no coordination of visits in provisioning riflemen

Parental care strategies occupy a continuum from fixed investments that are consistent across contexts to flexible behaviour that largely depends on external social and environmental cues. Determining the flexibility of care behaviour is important, as it influences the outcome of investment games between multiple individuals caring for the same brood. We investigated the repeatability of provisioning behaviour and the potential for turn taking among breeders and helpers in a cooperatively breeding bird, the rifleman, Acanthisitta chloris. First, we examined whether nest visit rate is an accurate measure of investment by assessing whether carers consistently bring the same size of food, and whether food size is related to nest visit rate. Our results support the use of visit rate as a valid indicator of parental investment. Next, we calculated the repeatability of visit rate and food size to determine whether these behaviours are fixed individual traits or flexible responses to particular contexts. We found that riflemen were flexible in visit rate, supporting responsive models of care over ‘sealed bids’. Finally, we used runs tests to assess whether individual riflemen alternated visits with other carers, indicative of turn taking. We found little evidence of any such coordination of parental provisioning. We conclude that individual flexibility in parental care appears to arise through factors such as breeding status and brood demand, rather than as a real-time response to social partners

Relations between lipoprotein(a) concentrations, LPA genetic variants, and the risk of mortality in patients with established coronary heart disease: a molecular and genetic association study

Background: Lipoprotein(a) concentrations in plasma are associated with cardiovascular risk in the general population. Whether lipoprotein(a) concentrations or LPA genetic variants predict long-term mortality in patients with established coronary heart disease remains less clear. Methods: We obtained data from 3313 patients with established coronary heart disease in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. We tested associations of tertiles of lipoprotein(a) concentration in plasma and two LPA single-nucleotide polymorphisms ([SNPs] rs10455872 and rs3798220) with all-cause mortality and cardiovascular mortality by Cox regression analysis and with severity of disease by generalised linear modelling, with and without adjustment for age, sex, diabetes diagnosis, systolic blood pressure, BMI, smoking status, estimated glomerular filtration rate, LDL-cholesterol concentration, and use of lipid-lowering therapy. Results for plasma lipoprotein(a) concentrations were validated in five independent studies involving 10 195 patients with established coronary heart disease. Results for genetic associations were replicated through large-scale collaborative analysis in the GENIUS-CHD consortium, comprising 106 353 patients with established coronary heart disease and 19 332 deaths in 22 studies or cohorts. Findings: The median follow-up was 9·9 years. Increased severity of coronary heart disease was associated with lipoprotein(a) concentrations in plasma in the highest tertile (adjusted hazard radio [HR] 1·44, 95% CI 1·14–1·83) and the presence of either LPA SNP (1·88, 1·40–2·53). No associations were found in LURIC with all-cause mortality (highest tertile of lipoprotein(a) concentration in plasma 0·95, 0·81–1·11 and either LPA SNP 1·10, 0·92–1·31) or cardiovascular mortality (0·99, 0·81–1·2 and 1·13, 0·90–1·40, respectively) or in the validation studies. Interpretation: In patients with prevalent coronary heart disease, lipoprotein(a) concentrations and genetic variants showed no associations with mortality. We conclude that these variables are not useful risk factors to measure to predict progression to death after coronary heart disease is established. Funding: Seventh Framework Programme for Research and Technical Development (AtheroRemo and RiskyCAD), INTERREG IV Oberrhein Programme, Deutsche Nierenstiftung, Else-Kroener Fresenius Foundation, Deutsche Stiftung für Herzforschung, Deutsche Forschungsgemeinschaft, Saarland University, German Federal Ministry of Education and Research, Willy Robert Pitzer Foundation, and Waldburg-Zeil Clinics Isny

Effects of Acute Febrile Diseases on the Periodontium of Rhesus Monkeys with Reference to Poliomyelitis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67269/2/10.1177_00220345510300050301.pd