Bound Sulfane Sulfur Research Articles

Treatment with aripiprazole and N-acetylcysteine affects anaerobic cysteine metabolism in the hippocampus and reverses schizophrenia-like behavior in the neurodevelopmental rat model of schizophrenia.

Preclinical and clinical studies have shown that the antipsychotic drug aripiprazole and the antioxidant N-acetylcysteine have unique biological properties. The aim of the study was to investigate, in a rat model of schizophrenia, the effects of chronic administration of these drugs on schizophrenia-like behaviors and anaerobic cysteine metabolism in the hippocampus (HIP). The schizophrenia-type changes were induced in Sprague-Dawley rats by repeated administration of the glutathione synthesis inhibitor l-butionine-(S,R)-sulfoximine in combination with the dopamine reuptake inhibitor GBR 12909 in the early postnatal period. Adult model rats were chronically treated with aripiprazole (0.3 mg·kg-1 , i.p.) or N-acetylcysteine (30 mg·kg-1 , orally), and their effects on schizophrenia-like behaviors were assessed using the social interaction test and novel object recognition test. In the HIP, the level of anaerobic cysteine metabolites, H2 S, and bound sulfane sulfur were determined by a fluorescence method, while the expression of H2 S-synthetizing enzymes: cystathionine β-synthase (CBS) and mercaptopyruvate sulfurtransferase (MST) by western blot. Long-term treatment with aripiprazole or N-acetylcysteine reversed social and cognitive deficits and reduced the exploratory behaviors. In the HIP of 16-day-old model pups, H2 S levels and MST protein expression were significantly decreased. In adult model rats, H2 S levels remained unchanged, bound sulfane sulfur significantly increased, and the expression of CBS and MST slightly decreased. The studied drugs significantly reduced the level of bound sulfane sulfur and the expression of tested enzymes. The reduction in bound sulfane sulfur level coincided with the attenuation of exploratory behavior, suggesting that modulation of anaerobic cysteine metabolism in the HIP may have therapeutic potential in schizophrenia.

PH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus.

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

Tissue-dependent variation of hydrogen sulfide homeostasis in anoxic freshwater turtles.

Hydrogen sulfide (H2S) controls numerous physiological responses. To understand its proposed role in metabolic suppression, we measured free H2S and bound sulfane sulfur (BSS) in tissues of the freshwater turtle Trachemys scriptaelegans, a species undergoing strong metabolic suppression when cold and anoxic. In warm normoxic turtles, free H2S was higher in red blood cells (RBCs) and kidney (∼9-10 µmoll-1) than in brain, liver and lung (∼1-2 µmoll-1). These values overall aligned with the tissue H2S-generating enzymatic activity. BSS levels were similar in all tissues (∼0.5 µmoll-1) but ∼100-fold higher in RBCs, which have a high thiol content, suggesting that RBCs function as a circulating H2S reservoir. Cold acclimation caused significant changes in free and bound H2S in liver, brain and RBCs, but anoxia had no further effect, except in the brain. These results show tissue-dependent sulfide signaling with a potential role in brain metabolic suppression during anoxia in turtles.

Abstract 122: Increased Plasma Sulfide in Vascular Surgery Patients Correlates with Reduced Post-Operative Mortality

Objective: Hydrogen sulfide (H 2 S) is an endogenously produced gaseous signaling molecule with the potential to modulate vascular functions. Free plasma sulfide can be measured by various techniques, but no consistent relationship with cardiovascular disease has yet emerged. For example, sulfide levels are decreased in CHF patients, but elevated in PAD. We therefore sought to compare plasma sulfide levels from PAD patients to matched controls, and explore links between mortality rates and sulfide levels using two assays. Approach & Results: Patients undergoing carotid endarterectomy (n=49), open lower extremity revascularization (n=44) or leg amputation (n=22) were enrolled (mean age 68.9±9.6, 67% male). Blood was collected from 20 matched control patients, without PAD or CAD (mean age 67.9± 1.3, male 65%). Plasma sulfide was measured using two methods, first detection using lead acetate, and second using mass spectrometry. Controls had increased plasma sulfide levels measured by both methods (lead acetate, Fig. A; mass spec, Fig. B) compared to PAD patients (p<0.001, p=0.013). Also, PAD patients were divided into high (n=57) and low (n=58) sulfide (lead acetate) groups by median split. Low sulfide PAD patients had increased probability of post-op mortality (p=0.0337, Fig. C). To determine the source of plasma H 2 S detected by lead acetate, we tested the effects of detergent and proteolytic denaturation of plasma as well as of reducing agents on H 2 S release. We found denaturation increased plasma sulfide release, and that dithiothreitol was most effective at liberating H 2 S, suggesting bound sulfane sulfur as source of H 2 S detected using the lead acetate assay. Conclusions: Plasma free and bound sulfide were reduced in PAD patients compared to controls, and correlated with mortality. These findings provide evidence linking circulating sulfide to clinically meaningful events, and support directed H 2 S investigations toward diagnostic and therapeutic purposes.

Total sulfane sulfur bioavailability reflects ethnic and gender disparities in cardiovascular disease

Hydrogen sulfide (H2S) has emerged as an important physiological and pathophysiological signaling molecule in the cardiovascular system influencing vascular tone, cytoprotective responses, redox reactions, vascular adaptation, and mitochondrial respiration. However, bioavailable levels of H2S in its various biochemical metabolite forms during clinical cardiovascular disease remain poorly understood. We performed a case-controlled study to quantify and compare the bioavailability of various biochemical forms of H2S in patients with and without cardiovascular disease (CVD). In our study, we used the reverse-phase high performance liquid chromatography monobromobimane assay to analytically measure bioavailable pools of H2S. Single nucleotide polymorphisms (SNPs) were also identified using DNA Pyrosequencing. We found that plasma acid labile sulfide levels were significantly reduced in Caucasian females with CVD compared with those without the disease. Conversely, plasma bound sulfane sulfur levels were significantly reduced in Caucasian males with CVD compared with those without the disease. Surprisingly, gender differences of H2S bioavailability were not observed in African Americans, although H2S bioavailability was significantly lower overall in this ethnic group compared to Caucasians. We also performed SNP analysis of H2S synthesizing enzymes and found a significant increase in cystathionine gamma-lyase (CTH) 1364 G-T allele frequency in patients with CVD compared to controls. Lastly, plasma H2S bioavailability was found to be predictive for cardiovascular disease in Caucasian subjects as determined by receiver operator characteristic analysis. These findings reveal that plasma H2S bioavailability could be considered a biomarker for CVD in an ethnic and gender manner. Cystathionine gamma-lyase 1346 G-T SNP might also contribute to the risk of cardiovascular disease development.

(Invited) In Vivo Electrochemical Monitoring of Free Endogenous Hydrogen Sulfide

Increasing researches have demonstrated that hydrogen sulfide is an important neuromodulator in the CNS and the third gasotransmitter joined with nitric oxide and carbon monoxide. It not only protects neurons and gila, and induces hippocampal long-term potential, but also is related to CNS diseases, such as Alzheimer’s disease, Parkinson’s disease, ischemic stroke and traumatic brain injury. Although there are some methods to determine hydrogen sulfide, the transformation of different forms of sulfide (S2-, HS-, H2S, bound sulfane sulfur et al.) and the chemical instability (easily oxidized) of hydrogen sulfide in physiological conditions render a great challenge to in vivo track accurately of the free hydrogen sulfide in the brain under the physiological condition. Motivated by this need, we are dedicating to develop in vivo electrochemical method for selective monitoring free hydrogen sulfide in the brain. Based on the specific H2S-induced chemical reaction (i.e. precipitate metals salts), we synthesized an electrochemiluminescent (ECL) probe and developed a reaction-based turn-on ECL sensor to selectively detect extracellular hydrogen sulfide in the microdialysate of rat brain taking advantage of the volatile property of hydrogen sulfide. More recently, we find one electrochemical mechanism that ammineruthenium(III) (Ru(NH3)6 3+) can catalyze the electrochemical oxidation of free sulfide including both forms of HS- and H2S in a neutral solution. Based on our mechanism, we developed one simple and effective method to in vivo track the dynamic of free hydrogen sulfide in the free moving animal, which can’t reach by other developed probes and methods. These methods open a new opportunity and offers a simple and effective platform for investigations of hydrogen sulfide in physiological and pathological processes.

Reduction of Ammineruthenium(III) by Sulfide Enables In Vivo Electrochemical Monitoring of Free Endogenous Hydrogen Sulfide.

The discovery of endogenous sulfide in mammalian brain opens up a door to understanding of the physiological function of hydrogen sulfide (H2S). The transformation of different forms of sulfide (i.e., S2-, HS-, H2S, bound sulfane sulfur, et al.) in various physiological conditions hurdles the direct detection of hydrogen sulfide in vivo. Here, we find that ammineruthenium(III) (Ru(NH3)63+) can catalyze the electrochemical oxidation of free sulfide including HS- and H2S in a neutral solution (pH 7.4). This property is used to constitute an electrochemical mechanism for selective detection of hydrogen sulfide. By coupling in vivo microdialysis with selective electrochemical detection, we successfully developed an integrated microchip-based online electrochemical system (OECS) for continuous monitoring of free endogenous hydrogen sulfide in the central nervous system (CNS). The microchip-based OECS is well responsive toward hydrogen sulfide with high stability, sensitivity and selectivity. Compared with the existing methods, the OECS does not require offline treatment of brain tissue or adjustment of the detection solutions into acidic or strong basic atmosphere. These priorities essentially enable the system to accurately and reliably track dynamics of hydrogen sulfide in the CNS.

Hydrogen sulfide metabolism regulates endothelial solute barrier function

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in the cardiovascular system. In addition to free H2S, H2S can be oxidized to polysulfide which can be biologically active. Since the impact of H2S on endothelial solute barrier function is not known, we sought to determine whether H2S and its various metabolites affect endothelial permeability. In vitro permeability was evaluated using albumin flux and transendothelial electrical resistance. Different H2S donors were used to examine the effects of exogenous H2S. To evaluate the role of endogenous H2S, mouse aortic endothelial cells (MAECs) were isolated from wild type mice and mice lacking cystathionine γ-lyase (CSE), a predominant source of H2S in endothelial cells. In vivo permeability was evaluated using the Miles assay. We observed that polysulfide donors induced rapid albumin flux across endothelium. Comparatively, free sulfide donors increased permeability only with higher concentrations and at later time points. Increased solute permeability was associated with disruption of endothelial junction proteins claudin 5 and VE-cadherin, along with enhanced actin stress fiber formation. Importantly, sulfide donors that increase permeability elicited a preferential increase in polysulfide levels within endothelium. Similarly, CSE deficient MAECs showed enhanced solute barrier function along with reduced endogenous bound sulfane sulfur. CSE siRNA knockdown also enhanced endothelial junction structures with increased claudin 5 protein expression. In vivo, CSE genetic deficiency significantly blunted VEGF induced hyperpermeability revealing an important role of the enzyme for barrier function. In summary, endothelial solute permeability is critically regulated via exogenous and endogenous sulfide bioavailability with a prominent role of polysulfides.

117 - Hydrogen Sulfide Increases Endothelial Permeability by Bound Sulfane Sulfur

117 - Hydrogen Sulfide Increases Endothelial Permeability by Bound Sulfane Sulfur

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.

S1-1 H2S and beyond

Hydrogen sulfide (H 2 S) is a toxic gas that emits an unpleasant smell of rotten eggs. About 20 years ago, the discovery of endogenous sulfide in mammalian brain urged us to study the role of H 2 S in neuronal transmission. We found a neuromodulatory role with cystathionine beta–synthase (CBS) as H 2 S-producing enzyme in the brain. In the following year we demonstrated that H 2 S relaxes vascular smooth muscle in synergy with nitric oxide (NO) with cystathionine gamma–lyase (CSE) as another enzyme. Our finding of a cytoprotective effect of H 2 S on neurons from oxidative stress led to the identification of protecting various tissues and organs from ischemia–reperfusion injury. In addition to the H 2 S-producing pathway with 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT), we recently found a novel pathway with 3MST and d -amino acid oxidase (DAO) that produces H 2 S from d -cysteine. This pathway is more efficient than l -cysteine pathways in the kidney, and promises the therapeuric application of d -cysteine to the renal ischemic diseases. H 2 S is oxidized to polysufides that exist in the brain and activate transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes more efficiently than parental H 2 S. H 2 S exerts its effect by reducing a target such as a cysteine disulfide bond, whereas polysulfides by adding bound sulfane sulfur to protein thiols (sulfhydration or sulfuration). The physiological roles of H 2 S and polysulfides will be discussed.

OP04 Microbiota regulation of host hydrogen sulfide bioavailability and metabolism

Hydrogen sulfide (H2S), generated through various endogenous enzymatic and nonenzymatic pathways, is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in the gastrointestinal tract also produce significant amounts of H2S that regulates microflora growth and virulence responses. However, the impact of the microbiota on host global H2S bioavailability and metabolism remains unknown. To address this question, we examined H2S bioavailability in its various forms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levels in tissues from germ-free versus conventionally housed mice. Free H2S levels were significantly reduced in plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by 50–80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantly reduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevated compared to conventional mice. These data reveal that the microbiota profoundly regulates systemic bioavailability and metabolism of H2S.

Microbial regulation of host hydrogen sulfide bioavailability and metabolism

Hydrogen sulfide (H2S), generated through various endogenous enzymatic and nonenzymatic pathways, is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in the gastrointestinal tract also produce significant amounts of H2S that regulates microflora growth and virulence responses. However, the impact of the microbiota on host global H2S bioavailability and metabolism remains unknown. To address this question, we examined H2S bioavailability in its various forms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levels in tissues from germ-free versus conventionally housed mice. Free H2S levels were significantly reduced in plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by 50–80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantly reduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevated compared to conventional mice. These data reveal that the microbiota profoundly regulates systemic bioavailability and metabolism of H2S.

Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Hydrogen sulfide (H2S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H2S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H2S. The complexity of analytical H2S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H2S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H2S. Acid-labile H2S is released by incubating the sample in an acidic solution (pH 2.6) of 100mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H2S. Volatilized H2S is then trapped in 100mM Tris–HCl (pH 9.5, 0.1mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100mM phosphate buffer (pH 2.6, 0.1mM DTPA), and H2S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H2S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H2S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.

Metabolic Turnover of Hydrogen Sulfide

OPINION article Front. Physiol., 18 April 2012Sec. Membrane Physiology and Membrane Biophysics https://doi.org/10.3389/fphys.2012.00101

Hydrogen sulfide: its production, release and functions

Hydrogen sulfide (H(2)S), which is a well-known toxic gas, has been recognized as a signal molecule as well as a cytoprotectant. It is produced by three enzymes, cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase along with cysteine aminotransferase. In addition to an immediate release of H(2)S from producing enzymes, it can be stored as bound sulfane sulfur, which may release H(2)S in response to physiological stimuli. As a signal molecule, it modulates neuronal transmission, relaxes smooth muscle, regulates release of insulin and is involved in inflammation. Because of its reputation as a toxic gas, the function as a cytoprotectant has been overlooked: the nervous system and cardiovascular system are protected from oxidative stress. In this review, enzymatic production, release mechanism and functions of H(2)S are focused on.

3-Mercaptopyruvate Sulfurtransferase Produces Hydrogen Sulfide and Bound Sulfane Sulfur in the Brain

Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant. Currently, pyridoxal-5'-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) is thought to be the major H(2)S-producing enzyme in the brain. We recently found that brain homogenates of CBS-knockout mice, even in the absence of PLP, produce H(2)S at levels similar to those of wild-type mice, suggesting the presence of another H(2)S-producing enzyme. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) produces H(2)S from cysteine. In addition, 3MST is localized to neurons, and the levels of bound sulfane sulfur, the precursor of H(2)S, are greatly increased in the cells expressing 3MST and CAT but not increased in cells expressing functionally defective mutant enzymes. These data present a new perspective on H(2)S production and storage in the brain.

3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide that is stored as bound sulfane sulfur in the brain

3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide that is stored as bound sulfane sulfur in the brain

Selective effects of diallyl disulfide, a sulfane sulfur precursor, in the liver and Ehrlich ascites tumor cells

The present in vivo studies demonstrated that diallyl disulfide (DADS), occurring in garlic, elevated hepatic sulfane sulfur level and activities of γ-cystathionase and 3-mercaptopyruvate sulfotransferase in healthy mice but did not affect the hepatic glutathione level. DADS efficiently corrected the concentrations of glutathione and sulfane sulfur, and ameliorated γ-cystathionase activity that had been lowered in the livers of Ehrlich ascites tumor-bearing mice. In Ehrlich ascites tumor cells, diallyl disulfide did not alter bound sulfane sulfur level, sulfotransferases activity or glutathione level. These data indicate that this compound is capable of acting efficiently and selectively only in the liver and can be used for hepatoprotection during chemotherapy.