Glucosinolates Are Mainly Absorbed Intact in Germfree and Human Microbiota-Associated Mice.

Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-l-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small.

Bioavailability and biotransformation of sulforaphane and erucin metabolites in different biological matrices determined by LC-MS-MS.

The food-related isothiocyanate sulforaphane (SFN), a hydrolysis product of the secondary plant metabolite glucoraphanin, has been revealed to have cancer-preventive activity in experimental animals. However, these studies have often provided inconsistent results with regard to bioavailability, bioaccessibility, and outcome. This might be because the endogenous biotransformation of SFN metabolites to the structurally related erucin (ERN) metabolites has often not been taken into account. In this work, a fully validated liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous determination of SFN and ERN metabolites in a variety of biological matrices. To reveal the importance of the biotransformation pathway, matrices including plasma, urine, liver, and kidney samples from mice and cell lysates derived from colon-cancer cell lines were included in this study. The LC-MS-MS method provides limits of detection from 1 nmol L(-1) to 25 nmol L(-1) and a mean recovery of 99 %. The intra and interday imprecision values are in the range 1-10 % and 2-13 %, respectively. Using LC-MS-MS, SFN and ERN metabolites were quantified in different matrices. The assay was successfully used to determine the biotransformation in all biological samples mentioned above. For a comprehensive analysis and evaluation of the potential health effects of SFN, it is necessary to consider all metabolites, including those formed by biotransformation of SFN to ERN and vice versa. Therefore, a sensitive and robust LC-MS-MS method was validated for the simultaneous quantification of mercapturic-acid-pathway metabolites of SFN and ERN.

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples.

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-L-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.