Investigating the non-enzymatic methylation of arsenite by methylcobalamin B₁₂ using high-performance liquid chromatography on-line with inductively coupled plasma-mass spectrometry

Abstract

In this study, we have demonstrated the use of HPLC-ICP-MS as a powerful tool for studying the reaction of arsenite with methylcobalamin in the presence of glutathione. Even though this reaction has previously been investigated using radioactive ⁷³As, HPLC-ICP-MS provides considerable advantages relating to sample throughput, sensitivity, and separation efficiency of the arsenic-containing reaction products. As a result of these advantages, kinetic studies were easily conducted, potentially providing valuable insight into the reaction itself. In addition, the HPLC-ICP-MS approach allowed for the tentative identification of methylarsonous acid, an arsenic species that had escaped detection in previous studies of the same reaction. Overall, this study confirmed that methylcobalamin acts as a methylating agent for arsenite in the presence of glutathione in an abiotic environment. A series of methylated arsenic species, identified as methylarsonic acid, dimethylarsinic acid and methylarsonous acid were detected.

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Introduction

Arsenic is ubiquitous in the environment, and as a result humans are exposed to it continuously.^1,2 In particular, chronic exposure to elevated concentrations of inorganic arsenic in drinking water is believed to cause adverse health effects and contribute to cancers of the lung, bladder and skin.^3–7 It is for this reason that the maximum permissible concentration of arsenic in drinking water has been set at 10 µg l⁻¹ by the EPA and WHO.^8,9

To date, the majority of epidemiological studies on arsenic have assumed that inorganic arsenic is by far the most toxic form of the element, and it is believed to cause the most adverse health effects. Until recently, the process of arsenic methylation, which occurs in certain organisms including humans, was considered to be a means of detoxification as it gives rise to the less toxic methylarsonic acid (MMAs^V) and dimethylarsinic acid (DMAs^V) species which are readily excreted in urine.^10–12 However, this view is now being challenged as a result of several recent studies reporting on the presence of additional methylated arsenic species, i.e. methylarsonous acid (MMAs^III) and dimethylarsinous acid (DMAs^III), in urine samples from people exposed to relatively high levels of arsenic in their drinking water.^13–16 In some cases, MMAs^III and DMAs^III have been found to exhibit greater cytotoxicity and genotoxicity than the inorganic forms of the metalloid.^17–20

In view of these latest findings, it is of particular interest to revisit the ideas concerning arsenic methylation and biomethylation with the intention of improving our understanding of factors affecting the formation of various methylated forms of arsenic. Numerous studies have demonstrated that S-adenosylmethionine acts as a source of carbocations (CH₃⁺) responsible for the oxidative methylation of inorganic arsenite (iAs^III)^1,21via nucleophilic substitution. This view is widely shared amongst chemists investigating the behaviour of arsenic in environmental and biological systems. In contrast, only a few studies have reported on the possibility of vitamin B₁₂ or any of its derivatives acting as methylating agents for arsenic. For example, it has been suggested that methylcobalamin or methylvitamin B₁₂ (CH₃B₁₂), a derivative of vitamin B₁₂ in human metabolism, functions as a methyl donor in the biosynthesis of dimethylarsine from arsenate or arsenite in cell extracts of methanobacillus.²² Also, Buchet and Lauwerys suggested that CH₃B₁₂ methylates arsenite at a low rate in the presence or absence of rat liver extracts.²³ Furthermore, Styblo et al. added 100 µg CH₃B₁₂/ml in their arsenite methyltransferase studies, although experimental evidence for its requirement was not presented.^24,25 Broader acceptance of CH₃B₁₂ acting as a methylating agent for arsenic has been hampered by lack of a theory suitable to explain the mechanism by which it acts. With respect to this, Zakharyan and Aposhian conducted a more fundamental study in which they demonstrated that CH₃B₁₂ methylates arsenic in the absence of any enzymes, providing glutathione is present.²⁶

The purpose of the present study was to initially confirm the findings of the Zakharyan and Aposhian study,²⁶ and to subsequently try to achieve higher methylation efficiencies. It was also our intention to improve the analytical methodology that was used in the original study, i.e. avoid using radioactive ⁷³As and open column chromatography and instead use modern state-of-the-art analytical techniques such as high-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). These analytical techniques have the potential to make arsenic methylation studies simpler and more efficient to conduct. In fact, using HPLC–ICP-MS allows for the efficient separation of various arsenic species and their subsequent very sensitive and selective detection.

Experimental

Reagents

Ultrapure 18 MΩ deionized water, DIW (Millipore, MA, USA) was used in the preparation of all reagents and standards. A stock standard of arsenite (iAs^III, sodium m-arsenite, Sigma, St. Louis, MO, USA) used in the methylation experiments was prepared at a concentration of 360 µg mL⁻¹ As. A 0.1 M solution of Tris buffer (Tris(hydroxymethyl)aminomethane hydrochloride, Sigma) was prepared and the pH was adjusted to 7.8. Methylcobalamin (CH₃B₁₂) and glutathione (GSH, γ-Glu-Cys-Gly; reduced form) were also obtained from Sigma and reagent solutions were prepared at concentrations of 1 mM and 0.18 mM, respectively. GSH and CH₃B₁₂ solutions were stored at 4 °C in amber-colored HDPE bottles and were prepared weekly because it was observed that freshly prepared reagents were required to achieve optimum as well as reproducible methylation efficiencies. A stock solution of selenite (iSe^IV, sodium selenite, Alfa Products, Danvers, MA, USA) at a concentration of 1000 µg mL⁻¹ was also prepared. Hydrogen peroxide used as an oxidizing agent was obtained from J. T. Baker (Ultrex II 30%, Phillipsburg, NJ, USA)

Ammonium phosphate, ammonium nitrate and ammonium hydroxide obtained from J. T Baker (Phillipsburg, NJ, USA) were used to prepare and adjust the pH of the chromatographic mobile phase used for Separation 1 (see Procedures section). Ammonium carbonate (J. T. Baker) was used to prepare the chromatographic mobile phase used for Separation 2 (see Procedures section).

Standards for arsenic speciation analysis

Stock standards of arsenite (iAs^III) and arsenate (iAs^V) (1000 µg As mL⁻¹ as As₂O₃ in 2% HCl and H₃AsO₄·½ H₂O in 2% HNO₃, respectively) were obtained from Spex Industries (Metuchen, NJ, USA). Disodium methylarsonate (MMAs^V, 99%) and dimethylarsinic acid (DMAs^V, 98%) were obtained from ChemService (West Chester, PA, USA). Methyloxoarsine originally obtained from Dr. William R. Cullen at the University of British Columbia, was a gift from the US EPA, National Exposure Research Laboratory in Cincinnati, Ohio. Stock standard solutions (∼1000 µg As mL⁻¹) of MMAs^III, MMAs^V and DMAs^V were prepared in DIW and stored at 4 °C in amber-colored HDPE bottles.

Instrumentation

A model PQ3 ICP-MS (VG Elemental, Franklin, MA, USA) was used for element-specific chromatographic detection of arsenic-containing species. A concentric nebulizer and cooled conical spray chamber were used to introduce the chromatographic effluent into the plasma. The operating parameters were as follows: forward power 1350 W; coolant argon flow 12 L min⁻¹; auxiliary argon flow 1.0 L min⁻¹; nebulizer gas flow 0.8 L min⁻¹. Chromatographic data were collected and integrated using PlasmaLab software. Masses 75 and 77 were monitored with a 250 ms dwell time and 10 min data acquisition time. Integrated peak areas were imported into a spreadsheet program for further data reduction.

The chromatographic system used consisted of a GP50 gradient pump (Dionex, Sunnyvale, CA, USA) and AS3500 autosampler (Thermo Separations Products, San Jose, CA, USA). The sample injection volume was 25 µL. A non-metallic automated switching valve (Waters, Milford, MA USA) with a 25 µL sample loop was used to introduce a flow injection peak (20 ng As mL⁻¹) post-column at the beginning of each chromatographic run. The flow injection peak was used to correct for instrument drift throughout the day. A length (∼60 cm ) of 0.25 mm id PEEK^® tubing was used to interface the chromatographic system to the ICP-MS nebulizer.

Procedures

Tris buffer solutions (pH 7.8) containing GSH and iSe^IV were left to stand for 30 min prior to adding iAs^III and CH₃B₁₂. The resulting reaction mixtures were incubated at 37 °C for various time intervals. Microlitre aliquots of the reaction mixtures were taken, diluted five-fold with DIW and immediately analyzed using HPLC-ICP-MS. Two different anion exchange HPLC separations were used throughout this study. The main HPLC column used was a PRP-X100 column (Hamilton, Reno, NV, USA, 250 × 4.1 mm id). The eluent consisted of 10 mM NH₄H₂PO₄/10 mM NH₄NO₃ (pH 6.3) at a flow rate of 1.0 mL min⁻¹ (Separation 1). A second anion-exchange HPLC column was also used to confirm identification of the arsenic products formed. This was an IC-Pak Anion HR column (Waters, Milford, MA, USA, 75 × 4.6 mm id), eluted with 10 mM (NH₄)₂CO₃ (pH 10) at a flow rate of 1 mL min⁻¹ (Separation 2).

The arsenic methylation reaction was monitored using HPLC–ICP-MS as described above. Methylation efficiency was defined according to the following equation using integrated chromatographic peak areas:

100%(DMAs^V + MMAs^V + MMAs^III)/(iAs^III + iAs^V+ DMAs^V + MMAs^V + MMAs^III)

By using peak areas rather than As concentrations, uncertainty associated with the purity of the MMAs^III standard could be avoided.

Results and discussion

Identification of reaction products by using HPLC–ICP-MS

During the reaction of CH₃B₁₂, GSH, iSe^IV and iAs^III, samples of the reaction mixture were taken at various time intervals, diluted five-fold and analyzed immediately by using HPLC–ICP-MS with Separation 1 (Fig. 1). For the chromatograms shown in Fig. 1B, the starting reaction mixture consisted of 3.4 µg mL⁻¹ iAs^III, in 0.07 M Tris buffer with 0.17 mM CH₃B₁₂, 20 µg mL⁻¹ iSe^IV, and 22 µM GSH. After approximately 4 hours incubation at 37 °C, the resulting chromatogram revealed the presence of four arsenic species in addition to the starting material, iAs^III. Upon comparing the retention times of various arsenic standards (Fig. 1a) with those of the reaction products, it was possible to identify the presence of MMAs^V, DMAs^V and iAs^V. Identification of these arsenic species through the use of HPLC–ICP-MS is currently considered routine as numerous publications have repeatedly described a variety of HPLC methods and applications suitable for this purpose.^27–32 However, only recently have HPLC methods been developed that are suitable for the identification of methylated arsenic species in which arsenic exists in the three oxidation state, i.e. MMAs^III and DMAs^III.^13–15,33 Several studies have recently used this approach to determine MMAs^III in human urine^13–15 and DMAs^III and MMAs^III in wool from sheep exposed to arsenosugars.³⁴ One of the reasons for the delayed development of this methodology has been the lack of pure and well-characterized standards for both of these species. Overcoming this problem to some extent has made it possible to use HPLC retention times for the identification of these species in “real” samples, especially urine samples. In the present study, MMAs^III was identified to be one of the reaction products. MMAs^III eluted between DMAs^V and MMAs^V when using Separation 1 (Fig. 1a).

Fig. 1 HPLC–ICP-MS chromatograms showing arsenic species detected at m/z 75 using Separation 1. (a) Standard chromatogram, each peak approximately 20 ng As mL⁻¹; (b) sample chromatograms taken after 2, 4, and 24 hours incubation of 3.4 µg mL⁻¹ iAs^III with 0.17 mM CH₃B₁₂, 22 µM GSH, 20 µg mL⁻¹ iSe^IV in 0.07 M TRIS buffer (note: iAs^III peak is off scale), also shown is the chromatogram obtained for 24 h incubation after the addition of H₂O₂.

In order to further support the validity of these identifications, a second HPLC method (Separation 2) was used to analyze the reaction mixture. Both of the separations make use of resin-based anion exchange columns with quaternary ammonium fuctional groups. The PRP-X100 column (Separation 1) utilizes a poly(styrene-divinyl)benzene polymeric support while the IC-Pak A HR column (Separation 2) is based on a polymethacrylate resin. These stationary phase differences resulted in significant selectivity differences for the arsenic species studied, and so agreement in the speciation findings of the two methods increased the validity of our identifications. With Separation 1 the order of elution was: iAs^III, DMAs^V, MMAs^III, MMAs^V and iAs^V (Fig. 1a), while with Separation 2 the order of elution was: MMAs^III, DMAs^V, iAs^III, MMAs^V and iAs^V (Fig. 2a).

Fig. 2 HPLC–ICP-MS chromatograms showing arsenic species detected at m/z 75 using Separation 2. (a) Standard chromatogram, original concentrations were approximately 20 ng As mL⁻¹ each peak (partial oxidation of MMAs^III and iAs^III species in the standard is noted); (b) sample chromatograms taken after 24 h incubation of 3.4 µg mL⁻¹ iAs^III with 0.17 mM CH₃B₁₂, 22 µM GSH, 20 µg mL⁻¹ iSe^IV in 0.07 M TRIS buffer, also shown is the chromatogram obtained for 24 h incubation after the addition of H₂O₂. (Note: iAs^V peak is off scale.)

Complimentary evidence supporting the presence of MMAs^III was obtained when adding 20 µL of 30% H₂O₂ to the 1∶5 diluted reaction mixture and subsequently analyzing it (Fig. 1B). Upon doing so it was observed that the peak assigned to be MMAs^III completely disappeared, while the peak area corresponding to MMAs^V increased. At the same time, the iAs^III peak decreased in size and the iAs^V peak area increased as expected. Similar results were obtained using Separation 2. In this case, both the MMAs^III and iAs^III species were completely oxidized as more time had passed between the addition of H₂O₂ and the analysis. Unfortunately, an analytical method capable of a more detailed identification of MMAs^III is currently unavailable. Attempts in our laboratory to identify this species using electrospray mass spectrometry have not been successful to date; however, efforts in this area are continuing.

Zakharyan and Aposhian²⁶ did not report the presence of MMAs^III in their report describing the non-enzymatic methylation of arsenic, even though reaction conditions were almost identical with those of the present study. The reason for this is probably because H₂O₂ was used to quench the reaction prior to analysis. This would have prevented detection of MMAs^III as it would have oxidized it to MMAs^V.

In the present study, it is possible that an arsenic-containing complex was observed during the early stages of the reaction. It was noted that sample aliquots taken early in the reaction exhibited excessive tailing of the peak at the retention time of iAs^III. Fig. 3 shows the chromatograms obtained after 2 h incubation of a mixture containing 7.2 µg mL⁻¹ iAs^III, in 0.06 M Tris buffer with 0.18 mM CH₃B₁₂, and 18 µM GSH. The tailing was no longer observed upon further reaction or if H₂O₂ was added to the sample. After addition of H₂O₂ considerable amounts of the methylated species appeared almost immediately, mainly MMAs^V (Fig. 3b). It is possible that a GSH-MMAs^III and/or GSH-iAs^III complex forms early in the reaction which later hydrolyses releasing MMAs^III. Styblo et al. reported on the use of H₂O₂ for the improved quantification of methylated arsenic species using thin layer chromatography.³⁵ It is possible that the improvement seen resulted from the release of MMAs^III bound to GSH and subsequent oxidation to MMAs^V. The detection and identification of arsenic–glutathione species has been reported.^36–38

Fig. 3 HPLC–ICP-MS chromatograms showing arsenic species detected at m/z 75 using Separation 1. (a) Chromatogram for sample taken after 2 h incubation of 7.2 µg mL⁻¹ iAs^III with 0.18mM CH₃B₁₂, and 18 µM GSH in 0.06 M TRIS buffer. (b) Chromatogram for same sample after addition of H₂O₂ (note: iAs^III is off scale).

Reaction kinetics

The nonenzymatic methylation of iAs^III using two different GSH concentrations, each in duplicate, was monitored over a 24 h period. The average kinetic profiles of the resulting methylated arsenic species are presented in Fig. 4. When a 22 µM GSH solution was used (Fig. 4a), it was observed that the overall average methylation efficiency was significantly greater than that observed when an 11 µM GSH solution was used (Fig. 4b), i.e., 17% vs. 5.5%. At both GSH levels examined, the efficiency of arsenic methylation reached a relatively constant level after 3–4 h. In both cases DMAs^V remained relatively constant for the remainder of the reaction. However, MMA^V levels continuously increased, while MMAs^III levels continuously decreased for the remainder of the reaction. Based on this data it seems that MMAs^III is being continuously oxidized to MMAs^V during the reaction.

Fig. 4 Kinetic profiles of individual methylated arsenic species and sum of all methylated species (methylation efficiency). Methylation efficiency was defined according to the following equation using integrated chromatographic peak areas: 100%(DMAs^V + MMAs^V + MMAs^III)/(iAs^III + iAs^V+ DMAs^V + MMAs^V + MMAs^III). Experimental conditions: a) mixture containing 3.4 µg mL⁻¹ iAs^III, 0.17mM CH₃B₁₂, 22 µM GSH, 20 µg mL⁻¹ iSe^IV in 0.07 M TRIS buffer incubated at 37 °C for various time intervals, (b) 3.6 µg mL⁻¹ iAs^III with 0.18 mM CH₃B₁₂, 11 µM GSH, 20 µg mL⁻¹ iSe^IV in 0.07 M TRIS buffer incubated at 37 °C for various time intervals.

Comparison with previous studies

It was observed that more reproducible results and higher methylation efficiencies were obtained by mixing the TRIS buffer initially with iSe^IV and GSH and allowing the mixture to stand for 30–60 minutes prior to the addition of iAs^III and CH₃B₁₂. Using peak areas and Separation 1, the methylation efficiency obtained for 11 µM GSH increased from 3% (n = 2) to 5.6% (n = 5, RSD 6%) when allowing 30–60 min mixing time (reaction conditions as in Fig. 4b). The maximum arsenic methylation efficiency recorded after 24 hours reaction time for duplicate preparations was 15% and 18% (reaction conditions as in Fig. 4a). If measured after the addition of H₂O₂, the calculated methylation efficiency was 18% and 20%.

Methylation efficiency could also be calculated based on the concentration of DMAs^V and MMA^V determined after addition of H₂O₂, thereby eliminating the need to use a MMA^III standard in the calculation. This would also eliminate any potential problems with the calculation of methylation efficiency using peak areas that could arise from slight differences in HPLC-ICP-MS response for each of the arsenic species. In this case, methylation efficiency is defined as the following:

100%([DMAs^V] + [MMAs^V])/[iAs^III]_starting

Using this method to calculate methylation efficiency, both separations gave comparable results for methylation efficiency to those reported using peak areas above. The average maximum 24 h methylation efficiencies for Separation 1 and Separation 2 were 22% and 21%, respectively (reaction conditions as in Fig. 4a).

These numbers compare reasonably well to the 12% methylation reported by Zakharian and Aposhian.²⁶ However, the concentrations of the reagents used in the two studies were different. In agreement with the Zakharyan and Aposhian²⁶ study, we observed that in the absence of GSH, arsenic methylation did not occur.

As suggested previously,²⁶ it seems that the most likely explanation for the non-enzymatic methylation of arsenic by CH₃B₁₂ is that under the highly reducing conditions of this reaction, methylation occurs via nucleophilic attack of an arsenite-GSH complex on the Co–C bond. In support of this is previous work by Hogenkamp et al. demonstrating the heterolytic cleavage of the C–Co bond of methylcobalamin by the nucleophilic thiolate anion under relatively mild conditions.^39,40

Conclusions

In this study we have confirmed that CH₃B₁₂ acts as a methylating agent for iAs^III in the presence of GSH. This study has been carried out without the use of radioactive chemicals. Instead what are now considered to be routine analytical techniques were used, i.e. HPLC–ICP-MS. This analytical approach is rapid, extremely sensitive, and thus allows for detailed kinetic studies to be carried out without any requirements for radioactive arsenic.

However, even though the HPLC–ICP-MS approach has been shown to be extremely powerful for the speciation of arsenic, it is desirable to further confirm the presence of MMAs^III using other independent analytical techniques. In addition, it is our intention to study further the methylation of arsenic by CH₃B₁₂, in particular, the mechanism by which it occurs. This will be carried out by employing HPLC–ICP-MS methods that also allow for the speciation of selenium and Co in the various forms of vitamin B₁₂. It is also our intention to monitor the relative amounts of the reduced and oxidized GSH throughout the reaction.

Acknowledgements

This work was supported in part under IAG DW75939361 between the US FDA and the US EPA. S.A.P. and M.M.-R. would like to thank the FDA for funding. M.M.-R. would also like to thank the Engineering and Physical Sciences Research Council (EPSRC) of the United Kingdom for funding (Grant numbers GR/N08974). Finally, we would like to thank Dr John Creed, USEPA for the gift of methyloxoarsine.

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Footnote

† Current address: Department of Chemistry, Environmental Chemical Processes Laboratory, University of Crete, PO Box 1470, 71409, Heraklion, Greece. E-mail: spergantis@chemistry.uoc.gr

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