Effect of acute administration of ethylmalonic acid (EMA) on Mn-SOD and CuZn-SOD activities in cerebral cortex and skeletal muscle of rats.

<p>Values are mean ± standard error of mean for five independent experiments (animals) per group. Data were expressed as nmol. min^-1. mg of protein^-1. No differences between groups were detected (Student’s <i>t</i> Test).</p><p>Effect of acute administration of ethylmalonic acid (EMA) on Mn-SOD and CuZn-SOD activities in cerebral cortex and skeletal muscle of rats.</p

Effect of acute ethylmalonic acid (EMA) administration (6 μmol/g) on DCFH oxidation <i>(A)</i> and superoxide generation <i>(B)</i> in cerebral cortex and skeletal muscle from 30-day-old rats.

<p>The experiments were performed in duplicate and the data represent mean ± standard error of the mean and are expressed as DCFH oxidation: nmol. mg protein^-1; superoxide: nM. min^-1. mg of protein^-1 (n = 5–8 per group). *<i>p</i> < 0.05 compared to control group (Student <i>t</i> test for independent samples).</p

Effect of acute administration of ethylmalonic acid (EMA) on respiratory chain complex I-III activity in cerebral cortex and skeletal muscle of rats.

<p>Values are mean ± standard error of mean for five independent experiments (animals) per group. Data were expressed as nmol. min^-1. mg of protein^-1. No differences between groups were detected (Student’s <i>t</i> Test).</p><p>Effect of acute administration of ethylmalonic acid (EMA) on respiratory chain complex I-III activity in cerebral cortex and skeletal muscle of rats.</p

Effect of acute ethylmalonic acid (EMA) administration (6 μmol/g) on thiobarbituric acid-reactive species (TBA-RS) levels in cerebral cortex and skeletal muscle from 30-day-old rats.

<p>The experiments were performed in duplicate and the data represent mean ± standard error of the mean and are expressed in nmol. mg protein^-1 (n = 5 per group). ***<i>p</i> < 0.001 compared to control group (Student <i>t</i> test for independent samples).</p

Effect of acute ethylmalonic acid (EMA) administration (6 μmol/g) on carbonyl <i>(A)</i> and sulfhydryl <i>(B)</i> content in cerebral cortex and skeletal muscle from 30-day-old rats.

<p>The experiments were performed in duplicate and the data represent mean ± standard error of the mean and are expressed in nmol. mg protein^-1 (n = 5 per group). ***<i>p</i> < 0.001 compared to control group (Student <i>t</i> test for independent samples).</p

Global ischemia/reperfusion study.

<p>Post-reperfusion survival times are shown in (A) for animals treated with ammonium tetrathiomolybdate (ATTM) versus vehicle (control). *<i>p</i> < 0.05, log-rank test, <i>n</i> = 16 per group. Sequential changes in core temperature, heart rate, cardiac output, and blood pressure are shown (B–E). *<i>p</i> < 0.05 using a two-way repeated measures ANOVA plus Bonferroni test. Note that this test was only performed up to 2 h post-reperfusion due to early mortality. Measurements of (F) blood levels of reduced glutathione (GSH; antioxidant reserve capacity), (G) the ratio of GSH to oxidized glutathione (GSSG) (lower values indicate greater oxidative stress), (H) protein carbonyls (oxidative damage), and (I) interleukin-6 (IL-6; systemic inflammation) were performed at 2 h post-reperfusion, before the onset of significant mortality. *<i>p</i> < 0.05, unpaired <i>t</i>-test.</p

In vitro release of gaseous H₂S from ATTM and NaHS under different environmental conditions.

<p>Comparison of ammonium tetrathiomolybdate (ATTM) and NaHS with changes in (A) concentration and (B) time. In (A), the molarity of each compound was adjusted for equal total sulfur content; drugs were incubated for 1 h at physiological pH (7.4) and temperature (37°C). In (B), fixed concentrations were used: ATTM 100 mM (total sulfur) and NaHS 0.3 mM. The effects of pH, temperature, and the presence of thiols on H₂S gas released from ATTM are shown in (C–E). Here, fixed concentrations (100 mM total sulfur) and incubation time (1 h) were employed. Peak H₂S concentrations are displayed in parts per million (ppm). The thiols used were reduced glutathione (GSH; 5 mM) and L-cysteine (Cys; 5 mM). The dotted lines reflect typical H₂S gas levels (3–4 ppm) obtained from ATTM (100 mM total sulfur) following 1 h incubation at normal physiological pH and temperature. <i>n</i> = 3–6 per group.</p

Inhibition of oxygen consumption by sulfide-containing drugs.

<p>(A) Ex vivo concentration response curves for ammonium tetrathiomolybdate (ATTM) and NaHS in relative normoxia. Experiments were performed at 150–250 μM O₂. In (B), tissues respired to hypoxia. Vehicle, ATTM (0.5 mM, corresponding to 2 mM total sulfur), or NaHS (0.5 mM) was added at 200 μM O₂. Note that oxygen consumption in vehicle-treated tissues also decreases at lower [O₂] (supply dependence) when tissues respire towards hypoxia. (C) shows a representative trace of tissues respiring to hypoxia, with the timing of the following events indicated: <i>a</i>, sensitivity test; <i>b</i>, addition of tissue to the chamber; <i>c</i>, oxygenation; <i>d</i>, baseline measurements; <i>e</i>, addition of ATTM or vehicle (control). (D) and (E) show the effects of ATTM in vivo following increasing, hourly IV bolus doses or a continuous infusion (10 mg/kg/h), respectively. (F) shows core temperature and (G) shows echocardiography-derived heart rate at the end (24 h) of continuous infusion. Panels A, B, D, and E show percentage inhibition compared to baseline values, before the addition of drugs. *<i>p <</i> 0.05 versus control using a two-way repeated measures ANOVA (plus Bonferroni’s test in D and E) or unpaired <i>t</i>-test (in F and G). <i>n</i> = 3–12 for ex vivo experiments, and <i>n</i> = 4 per group for in vivo studies.</p

Safety studies.

<p>Effects of ammonium tetrathiomolybdate (ATTM) via either IV bolus dosing (A) or continuous infusion (B) on the arterial partial pressure of oxygen (PaO₂) and percentage of oxygenated hemoglobin (oxy Hb). For the continuous infusion study, changes in acid/base balance, hemodynamics, and muscle tissue oxygen tension (tPO₂) at experiment end (5 h) are shown in (C–H); dotted lines denote the average baseline value. Where applicable, supplemental oxygen was commenced from 3 h. (I) shows the absorbance spectrum of oxy- and sulfhemoglobin, used for calculation of sulfhemoglobin levels in vivo (in J; infusion study). Note that there was no absorbance overlap between either hemoglobin form and ATTM (1 mM) at λ577/620. Formation of sulfhemoglobin ex vivo using either ATTM or NaHS to spike naïve rat blood is shown in (K). Here, the dotted line represents the maximum sulfhemoglobin level. *<i>p <</i> 0.05 versus baseline (i.e., before the addition of ATTM) in panels A, B, and J using a two-way ANOVA followed by Bonferroni’s testing; *<i>p <</i> 0.05 versus control (and ATTM versus ATTM + O₂) in panels C–H using a one-way ANOVA followed by Dunn’s multiple comparison test. <i>n</i> = 5–10 (in vivo), and <i>n</i> = 3 per group (ex vivo) in (K).</p

Pharmacokinetic/pharmacodynamic studies.

<p>Maximal changes in mean arterial blood pressure (A) and detection of (peak) exhaled H₂S gas (in parts per million [ppm]) (B) following increasing IV bolus doses of ammonium tetrathiomolybdate (ATTM) or NaHS. No exhaled H₂S was detectable following ATTM administration. Acid/base interactions following ATTM treatment are shown in (C); the top left <i>y</i>-axis denotes (arterial) partial pressure of carbon dioxide (PCO₂); bottom left and right <i>y</i>-axes are (arterial) base excess and pH, respectively. Alterations in (arterial) glucose and lactate following ATTM treatment are shown in (D). (E) shows the absorbance (ultra violet—visible) spectrum of ATTM with (inset) a row of microplate wells used to construct a standard curve. (F) and (G) respectively show changes in ATTM plasma levels (measured using the absorbance peak at 468 nm at 2 min after ATTM administration) against the quantity of drug administered and subsequent (25 min later) changes in arterial pH. <i>n</i> = 3–4/group.</p