Determination of Glucosinolates and Specific Metabolites in Brussels Sprouts and Human Tissues

Abstract

Numerous studies have investigated the relationship between glucosinolates and their metabolites, such as isothiocyanates in Brassica vegetables, and their impact on human health. Even though several studies have analysed glucosinolates in certain types of Brassica vegetables, such as broccoli, there is a lack of studies which have monitored glucosinolates in other varieties, such as Brussels sprouts. Consequently, there is little reliable data on the distribution of these compounds among commonly consumed vegetables and other sources, particularly cooked vegetables. Additionally, some animal studies have observed that glucosinolates (mainly glucoraphanin) can be absorbed intact after the thermal inactivation of myrosinase. However, there is a lack of research on the absorption of many different glucosinolates and metabolites in human tissue following consumption. Accordingly, there is limited knowledge about the extent to which these compounds are absorbed into the body and their duration of stay. Therefore, the aim of the study was to improve and implement methods to identify and quantify glucosinolates in raw and cooked Brussels sprouts and relevant metabolites in human tissue after Brussels sprouts consumption. An analytical method using tandem liquid chromatography and mass spectrometry was developed for the identification and quantitation of intact glucosinolates in freeze-dried Brussels sprouts and validated using labelled stable internal standards. It was then applied to investigate the impact of 3 cooking methods: boiling, steaming and roasting, on the retention of intact glucosinolates in Brussels sprouts. Finally, intact sinigrin and its metabolites in human tissue were measured after consuming steamed Brussels sprouts. The method was successfully able to separate and quantify 11 glucosinolates, comprising 98 % of total glucosinolates, in freeze-dried Brussels sprouts. The limits of quantification ranged from 0.21 to 1.9 µM. The relative standard deviation (RSD) values of intraday and interday precision ranged from 2.6 to 13.9% and from 2.6 to 18.9%, respectively. Steaming was found to be the optimal method to increase high amounts (˃ 100%) of glucosinolates compared to boiling (65%) and roasting (54%). The human study showed that sinigrin and its metabolites (time for highest measured value in brackets) were found and quantified in blood plasma: allyl isothiocyanate-cysteineglycine (6 hours); allyl isothiocyanate-cysteine (6 hours); and allyl isothiocyanate-N-acetylcysteine 6 hours); in urine: sinigrin (4 hours); allyl isothiocyanate cysteine (8 hours) and allyl isothiocyanate-N-acetylcysteine (8 hours); and in faeces: allyl isothiocyanate-cysteine (24 hours); and allyl isothiocyanate- N-acetylcysteine (24 hours). In conclusion, the project provided a validated analytical method for glucosinolates in Brussels sprouts, evidence for the advantage of steaming on glucosinolate retention and for the absorption and excretion of sinigrin and its metabolites.