Determination of 3-mercaptopyruvate in rabbit plasma by high performance liquid chromatography tandem mass spectrometry

doi:10.1016/j.jchromb.2014.01.006

Journal of Chromatography B

Volumes 949–950, 15 February 2014, Pages 94-98

https://doi.org/10.1016/j.jchromb.2014.01.006 Get rights and content

Highlights

•
A method for the analysis of a cyanide antidote, 3-MP, was developed.
•
The method produced a linear range of 0.5–100 μM 3-MP in rabbit plasma.
•
The method was successful at detecting 3-MP from treated rabbits.

Abstract

Accidental or intentional cyanide poisoning is a serious health risk. The current suite of FDA approved antidotes, including hydroxocobalamin, sodium nitrite, and sodium thiosulfate is effective, but each antidote has specific major limitations, such as large effective dosage or delayed onset of action. Therefore, next generation cyanide antidotes are being investigated to mitigate these limitations. One such antidote, 3-mercaptopyruvate (3-MP), detoxifies cyanide by acting as a sulfur donor to convert cyanide into thiocyanate, a relatively nontoxic cyanide metabolite. An analytical method capable of detecting 3-MP in biological fluids is essential for the development of 3-MP as a potential antidote. Therefore, a high performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS) method was established to analyze 3-MP from rabbit plasma. Sample preparation consisted of spiking the plasma with an internal standard (¹³C₃-3-MP), precipitation of plasma proteins, and reaction with monobromobimane to inhibit the characteristic dimerization of 3-MP. The method produced a limit of detection of 0.1 μM, a linear dynamic range of 0.5–100 μM, along with excellent linearity (R² ≥ 0.999), accuracy (±9% of the nominal concentration) and precision (<7% relative standard deviation). The optimized HPLC-MS-MS method was capable of detecting 3-MP in rabbits that were administered sulfanegen, a prodrug of 3-MP, following cyanide exposure. Considering the excellent performance of this method, it will be utilized for further investigations of this promising cyanide antidote.

Introduction

Humans are exposed to cyanide (LD₅₀, human = 1.1 mg/kg) [1], [2] in a variety of ways, such as ingestion of some edible plants (spinach or cassava), industrial operations, smoke inhalation from fires and/or cigarettes, and terrorist activities [3], [4]. Once cyanide is absorbed, it inhibits the enzyme cytochrome c oxidase in the electron transport system, thereby disrupting aerobic metabolism. There are currently three U.S. Food and Drug Administration (FDA) approved cyanide treatments: hydroxocobalamin, sodium nitrite, and sodium thiosulfate [2], [5], [6], [7].

Hydroxocobalamin (vitamin B_12a) is a large molecular-weight cyanide antidote that detoxifies cyanide by sequestration. It forms a very strong bond with cyanide because of the high affinity of cyanide for the central cobalt atom (K_A ≈ 10¹² M⁻¹) [8]. Cyanide binds to cobalt to produce cyanocobalamin (vitamin B₁₂) [9], [10], [11], which resides in the plasma and is excreted in urine. The potential adverse effects of hydroxocobalamin are generally mild and include elevated blood pressure, decreased heart rate, rashes, and red coloring of the skin, tears, urine and sweat [12], [13]. The recommended dose of hydroxocobalamin is 5 g (administered over 15 min). Because of the high dose needed for optimum therapeutic effect, hydroxocobalamin must be administered intravenously [2], [14], limiting the applicability of hydroxocobalamin in mass casualty situations.

Similar to hydroxocobalamin, the mechanism of action of sodium nitrite is to sequester cyanide from cytochrome c oxidase. However, the sequestration of cyanide is indirect. Sodium nitrite causes the conversion of hemoglobin to methemoglobin, which has a high affinity towards cyanide [14], [15]. Recently, another mechanism of action of sodium nitrite was proposed as the prominent method of detoxification in which nitrite is converted to nitric oxide, which subsequently displaces cyanide bound to the active site of cytochrome c oxidase [16], [17]. Although sodium nitrite works well to detoxify cyanide, it is toxic at large concentrations [5], [18] and has a small therapeutic window. Sodium nitrite is especially toxic when smoke inhalation has occurred, due to the conversion of hemoglobin to methemoglobin, which reduces the oxygen carrying capacity of the blood [18]. Due to its limited therapeutic efficacy, sodium nitrite is typically administered in tandem with sodium thiosulfate.

Sodium thiosulfate detoxifies cyanide by donating a sulfur to convert cyanide to the much less toxic thiocyanate [18], [19], [20]. Therefore, sodium thiosulfate belongs to a class of cyanide therapeutics known as sulfur donors, which utilize sulfurtransferase enzymes as catalysts. Sodium thiosulfate utilizes rhodanese, which is mainly found in the liver and kidneys [14], [19], leaving the heart and central nervous system less protected and the main locations of cyanide toxicity [14]. It also has a slow onset of action, attributed to slow entry into cells and the mitochondria [5]. This necessitates its use in combination with faster acting therapeutics, typically sodium nitrite.

Considering that current cyanide antidotes each have major limitations, alternative cyanide antidotes are being investigated [5], [8], [21]. One such alternate antidote is 3-mercaptopyruvate (3-MP). Similar to sodium thiosulfate, 3-MP acts as a sulfur donor to produce thiocyanate but is instead catalyzed by 3-mercaptopyruvate sulfurtransferase (3-MST) [19], [21], [22]. Sulfanegen, a prodrug of 3-MP (i.e., sulfanegen converts to 3-MP upon administration), has been found to be highly effective in reversing cyanide toxicity [14], [20], [21], [23], [24]. Although a method for the detection of 3-MP by HPLC from mouse tissue has been proposed [25], a multistep, lengthy (>60 min), and high temperature (95 °C) modification of 3-MP is necessary. Therefore, the objective of this study was to develop a simple and sensitive analytical method for the analysis of 3-MP from rabbit plasma to facilitate further development of 3-MP as a cyanide antidote.

Section snippets

Reagents and standards

All solvents were LC–MS grade unless otherwise noted. Ammonium formate and 3-mercaptopyruvate (3-MP; HSCH₂COCOOH) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetone (HPLC grade, 99.5%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Isotopically-labeled 3-MP (HS¹³CH₂¹³CO¹³COOH) was synthesized and provided by the Center for Drug Design, University of Minnesota (Minneapolis, MN, USA) [21]. Millex tetrafluoropolyethylene syringe filters (0.22 μm, 4 mm, Billerica, MA, USA) were

HPLC-MS-MS analysis of 3-MP from rabbit plasma

Under biological conditions, 3-MP is in rapid equilibrium with its dimer [30]. This equilibrium is difficult to control and results in poor chromatographic behavior. Because MBB reacts with the thiol group of 3-MP [26], [27], which is essential for dimerization, a single 3-MPB complex is created (Fig. 1), which produced excellent chromatographic behavior. Fig. 2 shows representative chromatograms of spiked and nonspiked 3-MPB in plasma, with 3-MPB eluting at approximately 2.75 min. The method

Conclusion

An HPLC-MS-MS method for the detection of 3-MP was developed which features simple sample preparation, excellent accuracy and precision, an excellent detection limit, and has a linear range of over 2 orders of magnitude. While Ogasawara et al. [25] reported an HPLC-fluorescence method for the analysis of 3-MP in mouse tissue, the method presented here featured simple and low-temperature sample preparation (i.e., 3-MP is highly unstable at high temperatures), rapid analysis, a reduced lower

Acknowledgements

We gratefully acknowledge the support from a U.S. Dept. of Education, Graduate Assistance in Areas of National Need (GAANN) award to the Department of Chemistry & Biochemistry (P200A100103). We thank the National Science Foundation Major Research Instrumentation Program (Grant Number CHE-0922816) for funding the AB SCIEX QTRAP 5500 LC-MS-MS. We also would like to acknowledge the support by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National

References (31)

A.H. Hall et al.
J. Emerg. Med.
(1987)
H.A. Schwertner et al.
J. Chromatogr. B
(2012)
C. Brunel et al.
Forensic Sci. Int.
(2012)
M. Brenner et al.
Toxicol. Appl. Pharm.
(2010)
G.E. Isom et al.
A. Spallarossa et al.
J. Mol. Biol.
(2004)
Y. Ogasawara et al.
J. Chromatogr. B
(2013)
N.S. Kosower et al.
R.C. Fahey et al.
A.J.L. Cooper et al.
J. Biol. Chem.
(1982)

C.D. Barnes et al.

Drug Dosages in Laboratory Animals–A Handbook

(1973)

T.F. Cummings

Occ. Med.

(2004)

R.K. Bhandari et al.

Anal. Bioanal. Chem.

(2012)

B.A. Logue et al.

Crit. Rev. Anal. Chem.

(2010)

A.H. Hall et al.

Crit. Rev. Toxicol.

(2009)

Cited by (14)

3-Mercaptopyruvate sulfurtransferase: a review of past and present perspectives
2023, Sulfurtransferases: Essential Enzymes for Life
3-Mercaptopyruvate sulfurtransferase (3-MST) was first discovered in mammalian tissues in 1953 during a study on cysteine metabolism. Detailed studies since 1970 have elucidated the structure, catalysis mechanism, and subcellular localization of this enzyme and revealed its wide distribution in plants, animals, and bacteria. In mammals, 3-MST is most active in the liver and kidney and is highly localized in the heart, spleen, brain, and red blood cells. In the cells, 3-MST is mainly located in the mitochondria and a part of activity is in the cytosol. Mammalian 3-MST is a monomeric enzyme with a molecular weight of 31–34 kDa and an easily oxidizable cysteine residue in its active site. Splicing variants of 3-MST with varying molecular weights exist; however, their functional differences remain unclear. Despite differences in opinions among researchers regarding the physiological role of 3-MST, the 3-MST/3-MP pathway is regarded as an important part of cysteine metabolism. In particular, the role of 3-MST as a key enzyme in sulfane sulfur (bound sulfur) metabolism has recently attracted attention, and further research is underway to elucidate its physiological functions.
Discovery of an Inhibitor for Bacterial 3-Mercaptopyruvate Sulfurtransferase that Synergistically Controls Bacterial Survival
2020, Cell Chemical Biology
H₂S-producing enzymes in bacteria have been shown to be closely engaged in the process of microbial survival and antibiotic resistance. However, no inhibitors have been discovered for these enzymes, e.g., 3-mercaptopyruvate sulfurtransferase (MST). In the present study, we identified several classes of inhibitors for Escherichia coli MST (eMST) through high-throughput screening of ∼26,000 compounds. The thiazolidinedione-type inhibitors were found to selectively bind to Arg178 and Ser239 residues of eMST but hardly affected human MST. Moreover, the pioglitazone of this class concentration dependently accumulates the 3-mercaptopyruvate substrate and suppresses the H₂S and reactive sulfane sulfur products in bacteria. Importantly, pioglitazone could potentiate the level of reactive oxygen species in cellulo and consequently enhance the antimicrobial effects of gentamicin and macrophages in culture. This study has identified the bioactive inhibitor of eMST, paving the way for the pharmacological targeting of eMST to synergistically control the survival of E. coli.
Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations
2020, Journal of Biological Chemistry
3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H₂S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser–His–Asp triad, proposed to serve a general acid–base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 μmol·kg⁻¹ in brain to 1.4 μmol·kg⁻¹ in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the K_M for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 μm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.
Determination of free cyano-cobinamide in swine and rabbit plasma by liquid chromatography tandem mass spectrometry
2019, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
In recent years, Cobinamide (Cbi) has shown promise as a therapeutic for cyanide poisoning. There are several forms of Cbi based on the identity of the ligands bound to the cobalt in Cbi and these different forms of Cbi have divergent behavior (e.g., the aquo and hydroxo forms of Cbi readily bind to proteins, limiting their distribution significantly, whereas [Cbi(CN)₂] does not). While current analysis techniques only measure total Cbi, methods to elucidate the behavior of ‘available’ Cbi versus cyanide-complexed Cbi would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanide-complexed Cbi in plasma via liquid chromatography tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. Cyanide-complexed Cbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 μM, and the linear dynamic range was between 1 and 200 μM. The accuracy was 100 ± 17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography was eliminated using a polymeric-based stationary phase (PLRP-S) and a controlled washing protocol. The method allowed evaluation of the cyanide-bound and ‘available’ Cbi from treated animals and, when paired with a method for total Cbi analysis, allows for estimation of Cbi utilization when treating cyanide poisoning.
Analysis of potential cyanide antidote, dimethyl trisulfide, in whole blood by dynamic headspace gas chromatography–mass spectroscopy
2019, Journal of Chromatography A
Cyanide is a rapidly acting and highly toxic chemical. It inhibits cytochrome c oxidase in the mitochondrial electron transport chain, resulting in cellular hypoxia, cytotoxic anoxia and potentially death. In order to overcome challenges associated with current cyanide antidotes, dimethyl trisulfide (DMTS), which converts cyanide to less toxic thiocyanate in vivo, has gained much attention recently as a promising next-generation cyanide antidote. While there are three analysis methods available for DMTS, they each have significant disadvantages. Hence, in this study, a dynamic headspace (DHS) gas chromatography–mass spectroscopy method was developed for the analysis of DMTS from rabbit whole blood. The method is extremely simple, involving only acidification of a blood sample, addition of an internal standard (DMTS-d₆) and DHS-GC–MS analysis. The method produced a limit of detection of 0.04 μM for DMTS with dynamic range from 0.2 to 50 μM. Inter- and intraassay accuracy (100 ± 15% and 100 ± 9%, respectively), and precision (<10% and <9% relative standard deviation, respectively) were good. The validated method performed well during pharmacokinetic analysis of DMTS from the blood of rats treated with DMTS, producing excellent pharmacokinetic parameters for the treatment of cyanide exposure. The method produced significant advantages over current methods for analysis of DMTS and should be considered as a “gold standard” method for further development of DMTS as a potential next-generation cyanide countermeasure.
Methionine restriction leads to hyperhomocysteinemia and alters hepatic H<inf>2</inf>S production capacity in Fischer-344 rats
2018, Mechanisms of Ageing and Development
Citation Excerpt :
This enzyme produces H2S from 3-mercaptopyruvate (3-MP). Several studies reported that the endogenous level of 3-MP is very low and it is often below the quantitation level or not found at all (Ogasawara et al., 2013; Stutelberg et al., 2016, 2014). Therefore, we measured the activity of MPST in the liver to test for possible compensatory changes in response to MR. The activity of MPST was elevated in MR animals compared to controls at both 4 and 8 weeks (Fig. 6).
Dietary methionine restriction (MR) increases lifespan in several animal models. Despite low dietary intake of sulphur amino acids, rodents on MR develop hyperhomocysteinemia. On the contrary, MR has been reported to increase H₂S production in mice. Enzymes involved in homocysteine metabolism also take part in H₂S production and hence, in this study, the impact of MR on hyperhomocysteinemia and H₂S production capacity were investigated using Fischer-344 rats assigned either a control or a MR diet for 8 weeks. The MR animals showed elevated plasma homocysteine accompanied with a reduction in liver cysteine content and methylation potential. It was further found that MR decreased cystathionine-β-synthase (CBS) activity in the liver, however, MR increased hepatic cystathionine-γ-lyase (CGL) activity which is the second enzyme in the transsulfuration pathway and also participates in regulating H₂S production. The relative contribution of CGL in H₂S production increased concomitantly with the increased CGL activity. Additionally, hepatic mercaptopyruvate-sulphur-transferase (MPST) activity also increased in response to MR. Taken together, our results suggest that reduced CBS activity and S-Adenosylmethionine availability contributes to hyperhomocysteinimia in MR animals. Elevated CGL and MPST activities may provide a compensatory mechanism for maintaining hepatic H₂S production capacity in response to the decreased CBS activity.

View all citing articles on Scopus

View full text