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

Organ-specific ischemia/reperfusion and cellular anoxia/reoxygenation.

<p>Ammonium tetrathiomolybdate (ATTM) confers cardioprotection (A–C, <i>n</i> = 6), neuroprotection (D–F, <i>n</i> = 6), and cytoprotection (G and H, ATTM 5.5 mM, <i>n</i> = 3). In (C), Evans blue dye shows the myocardial area not at risk. In (C) and (F), viable tissue appears pink/red, with non-viable tissue depicted as white/beige. In (G) a representative flow cytometry plot denotes positive (+ve) and negative (−ve) labeling for propidium iodide (PI) and Annexin V, expressed as median fluorescence intensity (MFI). Here, ATTM and controls are depicted in dark red and black, respectively. Live cells (negative for both labels) are shown in the bottom left quadrant and as a percentage of the total in the figure (G, left panel). The dotted line (G, left panel) shows viability in untreated cells (no ischemia/reperfusion or drugs). A representative fluorescence histogram of MitoSOX (mitochondrial superoxide production) is shown in (H), right panel. AAR, area at risk; BNP, B-type natriuretic peptide; IS, infarct size; S100β, S100 calcium binding protein β. *<i>p</i> < 0.05 using an unpaired <i>t</i>-test.</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

Plasma bilirubin and (un)conjugated bile acid levels in patients on the day of diagnosis of severe sepsis.

<p>(A) The plot depicts median log₂ fold changes of bilirubin (Bili), and unconjugated as well as glycine- and taurine-conjugated bile acid quantities in plasma of severely septic patients (<i>n</i> = 48) fulfilling American College of Chest Physicians/Society of Critical Care Medicine consensus criteria compared to non-septic controls (<i>n</i> = 20) (*<i>p</i><0.05 compared to controls). (B) Receiver operating characteristics of bilirubin, CDCA, TDCA, or the combined performance of CDCA+TDCA in predicting 28-d mortality. DCA, deoxycholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GLCAS, glycolithocholic acid sulphate; GUDCA, glycoursodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholic acid; TLCAS, taurolithocholic acid sulphate; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.</p

Clinical characteristics of septic patients subjected to targeted metabolomic analysis of bile acids.

<p>APACHE II, Acute Physiology and Chronic Health Evaluation II; SAPS II, Simplified Acute Physiology Score; SOFA, Sequential Organ Failure Assessment.</p

Polymicrobial sepsis severely impairs the activity of enzymes responsible for phase I and II biotransformation.

<p>(A) Activities of CYP1A, CYP2A, CYP2B, CYP2C, and CYP2E were assessed by ethoxycoumarin O-deethylation, while (B) CYP3A activity was quantified using the ethylmorphine N-demethylation model reaction for phase I biotransformation. Glutathione-S-transferase activity (C) and bilirubin glucuronidation (D), representing typical phase II conjugation reactions, were assessed by the model reaction 1-chloro-2,4-dinitrobenzene conjugation, resulting in the formation of dinitrobenzene-glutathione conjugate (GS-DNB), or the Burchell method, respectively (<i>n</i> = 5 for sham, <i>n</i> = 8 for sepsis, *<i>p</i> = 0.004 compared to sham).</p

Polymicrobial sepsis causes deranged bile acid conjugation and transport.

<p>At 15 h after sepsis induction, plasma, liver tissue, and bile were subjected to targeted metabolomics. Expression of BAAT, facilitating conjugation to taurine and glycine, was quantified by immunoblotting. (A) The plot depicts median log₂ fold changes of unconjugated as well as glycine- and taurine-conjugated bile acid in plasma, liver, and bile, comparing septic to sham-operated rats (<i>n</i> = 12 per group, *<i>p</i><0.05 or **<i>p</i><0.01 compared to sham). (B) Conjugation index as a surrogate for the observed conjugation defect reflected by the ratio of unconjugated bile acids CA and CDCA to the corresponding taurine (TCA and taurochenodeoxycholic acid) and glycine (GCA and glycochenodeoxycholic acid) conjugates in plasma, liver and bile (ratio given as log₂ fold change, <i>n</i> = 12 per group). (C and D) Representative immunoblots of BAAT 15 h after sepsis induction in cytosolic (c) as well as peroxisomal (p) fractions, with corresponding densitometric analysis (<i>n</i> = 5 for sham, <i>n</i> = 8 for sepsis; BAAT (c): *<i>p</i> = 0.002; BAAT (p): *<i>p</i> = 0.006 compared to sham). Densitomentric values are normalised to β-actin. DCA, deoxycholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GLCAS, glycolithocholic acid sulphate; GUDCA, glycoursodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholic acid; TLCAS, taurolithocholic acid sulphate; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.</p