An analytical method to investigate the metabolites and co-factors of the methionine-homocysteine cycle in women using combined oral contraceptives and non-users

Abstract

Biotransformation protects the body from endogenous and exogenous substances like combined oral contraceptives (COCs). During biotransformation, estrogen undergoes methylation with the assistance of catechol-O-methyltransferase (COMT) to limit the production of carcinogenic products. Effective COMT activity is crucial to limit the formation of these carcinogenic products. The importance of estrogen methylation is demonstrated by the fact that women with polymorphisms in the COMT-pathway, which negatively affect the methylation rate, are more prone to develop breast cancer. The conversion of catechol estrogens to methoxy-estrogens seems to be reduced in COC users. However, whether this is the result of inhibition of the COMT enzyme or due to the limited availability of co-factors is not known. Analytes that form part of the methionine-homocysteine cycle, such as betaine, S-adenosyl-methionine (SAM), and 5-methyl-tetrahydro folic acid (5-MTHFA,) act as methyl group donors during the methylation of catechol estrogens. The methionine-homocysteine cycle is a multi-step cycle including a significant number of metabolites and co-factors. To determine how the methionine-homocysteine cycle is influenced by COCs (and other xenobiotics), an analytical method is required to successfully quantify all the metabolites and co-factors of the methionine-homocysteine cycle. During the first part of this study, the focus was to implement and further optimise a liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method to quantify 18 metabolites and co-factors of the methionine-homocysteine cycle. This method included the use of a C18 column together with perfluoroheptanoic acid (PFHpA) as an ion-pairing agent. Cystine and homocystine were added to this method, and sample preparation was optimised. The LC-ESI-MS/MS method was successfully developed and partially validated by assessing parameters such as linearity, repeatability, precision, accuracy, and stability. The analytes of interest gave good linearity and repeatability, although some analytes showed less than ideal precision and accuracy, particularly those that were investigated at low concentrations. The final part of this study focused on implementing the LC-ESI-MS/MS method to determine how COC use affects the methylation metabolism in pre-menopausal women. Serum samples obtained for the estrogen biotransformation and oxidative stress status (eBOSS) study were used and included COC users and non-users. The method was successfully applied to these samples and indicated a significant difference in the levels of betaine, choline, dimethyl glycine (DMG), total cysteine, and ratios such as betaine:choline and DMG:betaine when the two groups were compared. These results suggest that COC use may affect the methionine-homocysteine cycle. Further investigation is needed to better understand the results obtained and the potential impact it may have.