Analysis and characterization of sulfane sulfur.

In the late 1970s, sulfane sulfur was defined as sulfur atoms covalently bound only to sulfur atoms. However, this definition was not generally accepted, as it was slightly vague and difficult to comprehend. Thus, in the early 1990s, it was defined as "bound sulfur," which easily converts to hydrogen sulfide upon reduction with a thiol-reducing agent. H2S-related bound sulfur species include persulfides (R-SSH), polysulfides (H2Sn, n ≥ 2 or R-S(S)nS-R, n ≥ 1), and protein-bound elemental sulfur (S0). Many of the biological effects currently associated with H2S may be attributed to persulfides and polysulfides. In the 20th century, quantitative determination of "sulfane sulfur" was conventionally performed using a reaction called cyanolysis. Several methods have been developed over the past 30 years. Current methods used for the detection of H2S and polysulfides include colorimetric assays for methylene blue formation, sulfide ion-selective or polarographic electrodes, gas chromatography with flame photometric or sulfur chemiluminescence detection, high-performance liquid chromatography analysis with fluorescent derivatization of sulfides, liquid chromatography with tandem mass spectrometry, the biotin switch technique, and the use of sulfide or polysulfide-sensitive fluorescent probes. In this review, we discuss the methods reported to date for measuring sulfane sulfur and the results obtained using these methods.

Sodium thiosulfate: A donor or carrier signaling molecule for hydrogen sulfide?

Sodium thiosulfate has been used for decades in the treatment of calciphylaxis and cyanide detoxification, and has recently shown initial therapeutic promise in critical diseases such as neuronal ischemia, diabetes mellitus, heart failure and acute lung injury. However, the precise mechanism of sodium thiosulfate remains incompletely defined and sometimes contradictory. Although sodium thiosulfate has been widely accepted as a donor of hydrogen sulfide (H2S), emerging findings suggest that it is the executive signaling molecule for H2S and that its effects may not be dependent on H2S. This article presents an overview of the current understanding of sodium thiosulfate, including its synthesis, biological characteristics, and clinical applications of sodium thiosulfate, as well as the underlying mechanisms in vivo. We also discussed the interplay of sodium thiosulfate and H2S. Our review highlights sodium thiosulfate as a key player in sulfide signaling with the broad clinical potential for the future.

Solubilization and stabilization of lipoic acid trisulfide by creation of various ß-cyclodextrin clathrates.

Lipoic acid trisulfide, a sulfane sulfur-containing trisulfide of α-lipoic acid, holds promise in pharmaceuticals, yet knowledge gaps persist regarding its synthesis, properties, and stability. Here, we synthesized the lipoic acid trisulfide with a purity exceeding 99% from α-lipoic acid on a gram scale and obtained novel ß-cyclodextrin clathrates (84%-95% yield). Differential scanning calorimetry confirmed the inclusion of lipoic acid trisulfide in ß-cyclodextrins. The resulting ß-cyclodextrin clathrates exhibited significant improvements in water solubility and thermal stability. This pioneering study demonstrated a novel approach to the practical preparation of trisulfide and its ß-cyclodextrin clathrates as active ingredients, paving the way for clinical development.

α-Lipoic acid increases the viability of nephrocytes and elevates sulfane sulfur level in plasma of patients with chronic kidney disease.

BACKGROUND: Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. METHODS: The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. RESULTS: The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.

Orthogonal Persulfide Generation through Precision Tools Provides Insights into Mitochondrial Sulfane Sulfur.

The sulfane sulfur pool, comprised of persulfide (RS-SH) and polysulfide (RS-SnH) derived from hydrogen sulfide (H2S), has emerged as a major player in redox biochemistry. Mitochondria, besides energy generation, serve as significant cellular redox hubs, mediate stress response and cellular health. However, the effects of endogenous mitochondrial sulfane sulfur (MSS) remain largely uncharacterized as compared with their cytosolic counterparts, cytosolic sulfane sulfur (CSS). To investigate this, we designed a novel artificial substrate for mitochondrial 3-mercaptopyruvate sulfurtransferase (3-MST), a key enzyme involved in MSS biosynthesis. Using cells expressing a mitochondrion-localized persulfide biosensor, we demonstrate this tool's ability to selectively enhance MSS. While H2S was previously known to suppress human immunodeficiency virus (HIV-1), we found that MSS profoundly affected the HIV-1 life cycle, mediating viral reactivation from latency. Additionally, we provide evidence for the role of the host's mitochondrial redox state, membrane potential, apoptosis, and respiration rates in managing HIV-1 latency and reactivation. Together, dynamic fluctuations in the MSS pool have a significant and possibly conflicting effect on HIV-1 viral latency. The precision tools developed herein allow for orthogonal generation of persulfide within both mitochondria and the cytosol and will be useful in interrogating disease biology.

Transcription factor OxyR regulates sulfane sulfur removal and L-cysteine biosynthesis in Corynebacterium glutamicum.

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. IMPORTANCE C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effects of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids.

Yohimbine Alleviates Oxidative Stress and Suppresses Aerobic Cysteine Metabolism Elevated in the Rat Liver of High-Fat Diet-Fed Rats.

Yohimbine is a small indole alkaloid derived from the bark of the yohimbe tree with documented biological activity, including anti-inflammatory, erectile dysfunction relieving, and fat-burning properties. Hydrogen sulfide (H2S) and sulfane sulfur-containing compounds are regarded as important molecules in redox regulation and are involved in many physiological processes. Recently, their role in the pathophysiology of obesity and obesity-induced liver injury was also reported. The aim of the present study was to verify whether the mechanism of biological activity of yohimbine is related to reactive sulfur species formed during cysteine catabolism. We tested the effect of yohimbine at doses of 2 and 5 mg/kg/day administered for 30 days on aerobic and anaerobic catabolism of cysteine and oxidative processes in the liver of high-fat diet (HFD)-induced obese rats. Our study revealed that HFD resulted in a decrease in cysteine and sulfane sulfur levels in the liver, while sulfates were elevated. In the liver of obese rats, rhodanese expression was diminished while lipid peroxidation increased. Yohimbine did not influence sulfane sulfur and thiol levels in the liver of obese rats, however, this alkaloid at a dose of 5 mg decreased sulfates to the control level and induced expression of rhodanese. Moreover, it diminished hepatic lipid peroxidation. It can be concluded that HFD attenuates anaerobic and enhances aerobic cysteine catabolism and induces lipid peroxidation in the rat liver. Yohimbine at a dose of 5 mg/kg can alleviate oxidative stress and reduce elevated concentrations of sulfate probably by the induction of TST expression.

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R-S-S-Alk) rapidly convert into the corresponding thioethers (R-S-Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N-t-butyl-iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R-S-S-Alk) to tautomerize into the thiosulfoxide (R-(S=S)-Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

Sulfane Sulfur is an intrinsic signal activating MexR-regulated antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1, an opportunistic human pathogen, deploys several strategies to resist antibiotics. It uses multidrug efflux pumps, including the MexAB-OprM pump, for antibiotic resistance, and it also produces hydrogen sulfide (H2 S) that provides some defense against antibiotics. MexR functions as a transcriptional repressor of the mexAB-oprM operon. MexR responds to oxidative stresses caused by antibiotic exposure, and it also displays a growth phase-dependent derepression of the mexAB-oprM operon. However, the intrinsic inducer has not been identified. Here, we report that P. aeruginosa PAO1 produced sulfane sulfur, including glutathione persulfide and inorganic polysulfide, produced from either H2 S oxidation or from L-cysteine metabolism. Sulfane sulfur directly reacted with MexR, forming di- and trisulfide cross-links between two Cys residues, to derepress the mexAB-oprM operon. Levels of cellular sulfane sulfur and mexAB-oprM expression varied during growth, and both reached the maximum during the stationary phase of growth. Thus, self-produced H2 S and sulfane sulfur may facilitate antibiotic resistance via inducing the expression of antibiotic resistance genes.

3-Mercaptopyruvate sulfurtransferase: an enzyme at the crossroads of sulfane sulfur trafficking.

3-Mercaptopyruvate sulfurtransferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate to generate an enzyme-bound hydropersulfide. Subsequently, MPST transfers the persulfide's outer sulfur atom to proteins or small molecule acceptors. MPST activity is known to be involved in hydrogen sulfide generation, tRNA thiolation, protein urmylation and cyanide detoxification. Tissue-specific changes in MPST expression correlate with ageing and the development of metabolic disease. Deletion and overexpression experiments suggest that MPST contributes to oxidative stress resistance, mitochondrial respiratory function and the regulation of fatty acid metabolism. However, the role and regulation of MPST in the larger physiological context remain to be understood.

Recent advances in probe design to detect reactive sulfur species and in the chemical reactions employed for fluorescence switching.

Reactive sulfur species, including hydrogen sulfide, hydropersulfide, and polysulfide, have many roles in biological systems. For example, hydrogen sulfide is involved in the relaxation of vascular smooth muscles and mediation of neurotransmission, while sulfane sulfur, which exists in cysteine persulfide/polysulfide, and glutathione persulfide/polysulfide, is involved in physiological antioxidation and cytoprotection mechanisms. Fluorescence imaging is well suited for real-time monitoring of reactive sulfur species in living cells, and many fluorescent probes for reactive sulfur species have been reported. In such probes, the choice of detection chemistry is extremely important, not only to achieve effective fluorescence switching and high selectivity, but also because the reactions may be applicable to develop other chemical tools, such as reactive sulfur species donors/scavengers. Here, we present an overview of both widely used and recently developed fluorescent probes for reactive sulfur species, focusing especially on the chemical reactions employed in them for fluorescence switching. We also briefly introduce some applications of fluorescent probes for hydrogen sulfide and sulfane sulfur.

The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate.

Heterotrophic bacteria actively participate in the biogeochemical cycle of sulfur on Earth. The heterotrophic bacterium Cupriavidus pinatubonensis JMP134 contains several enzymes involved in sulfur oxidation, but how these enzymes work together to oxidize sulfide in the bacterium has not been studied. Using gene-deletion and whole-cell assays, we determined that the bacterium uses sulfide:quinone oxidoreductase to oxidize sulfide to polysulfide, which is further oxidized to sulfite by persulfide dioxygenase. Sulfite spontaneously reacts with polysulfide to produce thiosulfate. The sulfur-oxidizing (Sox) system oxidizes thiosulfate to sulfate. Flavocytochrome c sulfide dehydrogenase enhances thiosulfate oxidation by the Sox system but couples with the Sox system for sulfide oxidation to sulfate in the absence of sulfide:quinone oxidoreductase. Thus, C. pinatubonensis JMP134 contains a main pathway and a contingent pathway for sulfide oxidation.IMPORTANCE We establish a new pathway of sulfide oxidation with thiosulfate as a key intermediate in Cupriavidus pinatubonensis JMP134. The bacterium mainly oxidizes sulfide by using sulfide:quinone oxidoreductase, persulfide dioxygenase, and the Sox system with thiosulfate as a key intermediate. Although the purified and reconstituted Sox system oxidizes sulfide, its rate of sulfide oxidation in C. pinatubonensis JMP134 is too low to be physiologically relevant. The findings reveal how these sulfur-oxidizing enzymes participate in sulfide oxidation in a single bacterium.

Effect of S-Allyl -L-Cysteine on MCF-7 Cell Line 3-Mercaptopyruvate Sulfurtransferase/Sulfane Sulfur System, Viability and Apoptosis.

The S-Allyl-L-cysteine ​​(SAC) component of aged garlic extract (AGE) is proven to have anticancer, antihepatotoxic, neuroprotective and neurotrophic properties. -Cystathionase (CTH), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) are involved in H2S/sulfane sulfur endogenous formation from L-cysteine. The aim of the study was to determine the effect of SAC on MCF-7 cells survival and apoptosis, which is a widely known approach to reduce the number of cancer cells. An additional goal of this paper was to investigate the effect of SAC on the activity and expression of enzymes involved in H2S production. The experiments were carried out in the human breast adenocarcinoma cell line MCF-7. Changes in the cell viability were determined by MTT assay. Cell survival was determined by flow cytometry (FC). Changes in enzymes expression were analyzed using Western blot. After 24 h and 48 h incubation with 2245 µM SAC, induction of late apoptosis was observed. A decrease in cell viability was observed with increasing SAC concentration and incubation time. SAC had no significant cytotoxic effect on the MCF-7 cells upon all analyzed concentrations. CTH, MPST and CBS expression were confirmed in non-treated MCF-7 cells. Significant decrease in MPST activity at 2245 µM SAC after 24 h and 48 h incubation vs. 1000 µM SAC was associated with decrease in sulfane sulfur levels. The presented results show promising SAC effects regarding the deterioration of the MCF-7 cells' condition in reducing their viability through the downregulation of MPST expression and sulfate sulfur level reduction.

Biosynthesis of Sulfur-Containing Small Biomolecules in Plants.

Sulfur is an essential element required for plant growth. It can be found as a thiol group of proteins or non-protein molecules, and as various sulfur-containing small biomolecules, including iron-sulfur (Fe/S) clusters, molybdenum cofactor (Moco), and sulfur-modified nucleotides. Thiol-mediated redox regulation has been well investigated, whereas biosynthesis pathways of the sulfur-containing small biomolecules have not yet been clearly described. In order to understand overall sulfur transfer processes in plant cells, it is important to elucidate the relationships among various sulfur delivery pathways as well as to investigate their interactions. In this review, we summarize the information from recent studies on the biosynthesis pathways of several sulfur-containing small biomolecules and the proteins participating in these processes. In addition, we show characteristic features of gene expression in Arabidopsis at the early stage of sulfate depletion from the medium, and we provide insights into sulfur transfer processes in plant cells.

A smartphone based device for the detection of sulfane sulfurs in biological systems.

Sulfane sulfur species are newly recognized signaling molecules that play physiological roles in many biological events. The development of new technologies for the detection of sulfane sulfurs is important. Point-of-care (POC) devices are in-field rapid and low-cost detectors that are more convenient to use than bulky and expensive standard instruments. In this report, a new fluorescent probe (SSP5) was designed to detect sulfane sulfurs using a POC sulfane sulfur smartphone spectrum apparatus (S4A). This probe proved to be sensitive and selective for sulfane sulfur species over other biologically relevant sulfur species such as cysteine and H2S. The low-cost and compact S4A has achieved comparable performance to standard laboratory equipment in both a standard buffer system and a synthetic urine system. The proposed system (SSP5 + S4A) has the potential for high accuracy and rapid detection of sulfane sulfur species in remote and low resource settings.

Thiotaurine: From Chemical and Biological Properties to Role in H2S Signaling.

In the last decade thiotaurine, 2-aminoethane thiosulfonate, has been investigated as an inflammatory modulating agent as a result of its ability to release hydrogen sulfide (H2S) known to play regulatory roles in inflammation. Thiotaurine can be included in the "taurine family" due to structural similarity to taurine and hypotaurine, and is characterized by the presence of a sulfane sulfur moiety. Thiotaurine can be produced by different pathways, such as the spontaneous transsulfuration between thiocysteine - a persulfide analogue of cysteine - and hypotaurine as well as in vivo from cystine. Moreover, the enzymatic oxidation of cysteamine to hypotaurine and thiotaurine in the presence of inorganic sulfur can occur in animal tissues and last but not least thiotaurine can be generated by the transfer of sulfur from mercaptopyruvate to hypotaurine catalyzed by a sulfurtransferase. Thiotaurine is an effective antioxidant agent as demonstrated by its ability to counteract the damage caused by pro-oxidants in the rat. Recently, we observed the influence of thiotaurine on human neutrophils functional responses. In particular, thiotaurine has been found to prevent human neutrophil spontaneous apoptosis suggesting an alternative or additional role to its antioxidant activity. It is likely that the sulfane sulfur of thiotaurine may modulate neutrophil activation via persulfidation of target proteins. In conclusion, thiotaurine can represent a biologically relevant sulfur donor acting as a biological intermediate in the transport, storage and release of sulfide.

Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium.

(1) The beneficial effects of hydrogen sulfide (H2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H2S biosynthesis, previously used as a marker for H2S production, is dramatically increased in circulation in uremia, while H2S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H2S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine-ß-synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca2+ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid.

A possible mechanism of inhibition of U87MG and SH-SY5Y cancer cell proliferation by diallyl trisulfide and other aspects of its activity.

The study was conducted to elucidate the mechanism of antiproliferative and antioxidative action of diallyl trisulfide (DATS), a garlic-derived organosulfur compound. Changes in the L-cysteine desulfuration, and the levels of cystathionine and non-protein thiols in DATS-treated human glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cells were investigated. The inhibition of proliferation of the investigated cells by DATS was correlated with an increase in the inactivated form of Bcl-2. In U87MG cells, an increased level of sulfane sulfur and an increased activity of 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese, the enzymes involved in sulfane sulfur generation and transfer, suggest that DATS can function as a donor of sulfane sulfur atom, transferred by sulfurtransferases, to sulfhydryl groups of cysteine residues of Bcl-2 and in this way lower the level of active form of Bcl-2 by S-sulfuration. Diallyl trisulfide antioxidative effects result from an increased level of cystathionine, a precursor of cysteine, and an increased glutathione level. MPST and rhodanese, the level of which is increased in the presence of DATS, can serve as antioxidant proteins.

Lipoic Acid as a Possible Pharmacological Source of Hydrogen Sulfide/Sulfane Sulfur.

The aim of the present study was to verify whether lipoic acid (LA) itself is a source of H2S and sulfane sulfur. It was investigated in vitro non-enzymatically and enzymatically (in the presence of rat tissue homogenate). The results indicate that both H2S and sulfane sulfur are formed from LA non-enzymatically in the presence of environmental light. These results suggest that H2S is the first product of non-enzymatic light-dependent decomposition of LA that is, probably, next oxidized to sulfane sulfur-containing compound(s). The study performed in the presence of rat liver and kidney homogenate revealed an increase of H2S level in samples containing LA and its reduced form dihydrolipoic acid (DHLA). It was accompanied by a decrease in sulfane sulfur level. It seems that, in these conditions, DHLA acts as a reducing agent that releases H2S from an endogenous pool of sulfane sulfur compounds present in tissues. Simultaneously, it means that exogenous LA cannot be a direct donor of H2S/sulfane sulfur in animal tissues. The present study is an initial approach to the question whether LA itself is a donor of H2S/sulfane sulfur.

Possible Involvement of Hydrosulfide in B12-Dependent Methyl Group Transfer.

Evidence from several fields of investigation lead to the hypothesis that the sulfur atom is involved in vitamin B12-dependent methyl group transfer. To compile the evidence, it is necessary to briefly review the following fields: methylation, the new field of sulfane sulfur/hydrogen sulfide (S°/H2S), hydrosulfide derivatives of cobalamins, autoxidation of hydrosulfide radical, radical S-adenosylmethionine methyl transfer (RSMT), and methionine synthase (MS). Then, new reaction mechanisms for B12-dependent methyl group transfer are proposed; the mechanisms are facile and overcome difficulties that existed in previously-accepted mechanisms. Finally, the theory is applied to the effect of S°/H2S in nerve tissue involving the "hypomethylation theory" that was proposed 50 years ago to explain the neuropathology resulting from deficiency of vitamin B12 or folic acid. The conclusions are consistent with emerging evidence that sulfane sulfur/hydrogen sulfide may be beneficial in treating Alzheimer's disease.