Position-specific isotope analysis of the methyl group carbon in methylcobalamin for the investigation of biomethylation processes

Abstract

In the environment, the methylation of metal(loid)s is a widespread phenomenon, which enhances both biomobility as well as mostly the toxicity of the precursory metal(loid)s. Different reaction mechanisms have been proposed for arsenic, but not really proven yet. Here, carbon isotope analysis can foster our understanding of these processes, as the extent of the isotopic fractionation allows to differentiate between different types of reaction, such as concerted (SN2) or stepwise nucleophilic substitution (SN1) as well as to determine the origin of the methyl group. However, for the determination of the kinetic isotope effect the initial isotopic value of the transferred methyl group has to be determined. To that end, we used hydroiodic acid for abstraction of the methyl group from methylcobalamin (CH3Cob) or S-adenosyl methionine (SAM) and subsequent analysis of the formed methyl iodide by gas chromatography (GC) isotope ratio mass spectrometry (IRMS). In addition, three further independent methods have been investigated to determine the position-specific δ (13)C value of CH3Cob involving photolytic cleavage with different additives or thermolytic cleavage of the methyl-cobalt bonding and subsequent measurement of the formed methane by GC-IRMS. The thermolytic cleavage gave comparable results as the abstraction using HI. In contrast, photolysis led to an isotopic fractionation of about 7 to 9 ‰. Furthermore, we extended a recently developed method for the determination of carbon isotope ratios of organometal(loid)s in complex matrices using hydride generation for volatilization and matrix separation before heart-cut GC and IRMS to the analysis of the low boiling partly methylated arsenicals, which are formed in the course of arsenic methylation. Finally, we demonstrated the applicability of this methodology by investigation of carbon fractionation due to the methyl transfer from CH3Cob to arsenic induced by glutathione.

Position-specific isotope modeling of organic micropollutants transformation through different reaction pathways

“…Another advantage, offered by the capability of the model to simulate position-specific isotope fractionation, is the possibility to directly compare the model outcomes with the results of position-specific isotope analysis, a technique that is likely to see an increasing number of applications due to the fast advances of analytical techniques (e.g. Breider and Hunkeler, 2011;Mckelvie et al, 2010;Wuerfel et al, 2013). Finally, besides the assistance in the quantitative interpretation of existing experimental observations, the model can be used to evaluate scenarios not yet explored experimentally as well as to design new experiments.…”

A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR

“…In this respect, it is also known that the processes affecting the carbon isotopic composition of isoprenoid lipids would be better elucidated by an intramolecular 13 C composition study [13]. Position-specific isotope analysis (PSIA) can be achieved by several approaches such as (bio)chemical degradations or pyrolysis but with limited applications (only small molecules and/or partial intramolecular information and/or tedious protocols) [14][15][16]. Recently, analyses of molecular isotopic structure have been proposed by tandem mass spectrometry, using an Orbitrap mass analyzer [17][18][19][20].…”

Methyl sulfates as methoxy isotopic reference materials for δ13C and δ2H measurements

“…For example, the information gained from the isotope signatures of plant methoxy groups can be applied for climate reconstructions of various climate archives such as tree rings, sediments and peat cores on time scales from the Anthropocene until the Eocene . Furthermore, they are useful in food science for studies on the authenticity of vanillin, in atmospheric sciences to investigate the origin and fate of C 1 volatile organic compounds such as methanol, chloromethane and bromomethane in the atmosphere and in biochemical investigations to better understand bio‐methylation processes …”

“…[18][19][20][21][22][23] Furthermore, they are useful in food science for studies on the authenticity of vanillin, 17,[24][25][26] in atmospheric sciences to investigate the origin and fate of C 1 volatile organic compounds such as methanol, chloromethane and bromomethane in the atmosphere 3,6,8,27,28 and in biochemical investigations to better understand bio-methylation processes. 29,30 Stable carbon and hydrogen isotope ratios of methoxy groups from various plant origins have been determined by site-specific natural isotope fractionation nuclear magnetic resonance (SNIF-NMR) and isotope ratio mass spectrometry (IRMS). The principle of measurement by IRMS is the generation of gaseous iodomethane (CH 3 I) via the reaction of methoxy groups with hydriodic acid (HI).…”