Are Raw Brassica Vegetables Healthier Than Cooked Ones? A Randomized, Controlled Crossover Intervention Trial on the Health-Promoting Potential of Ethiopian Kale.

The present human intervention trial investigated the health-promoting potential of B. carinata, with a focus on effects of thermal processing on bioactivity. Twenty-two healthy subjects consumed a B. carinata preparation from raw (allyl isothiocyanate-containing) or cooked (no allyl isothiocyanate) leaves for five days in a randomized crossover design. Peripheral blood mononuclear cells were exposed to aflatoxin B1 (AFB1), with or without metabolic activation using human S9 mix, and subsequently analyzed for DNA damage using the comet assay. Plasma was analyzed for total antioxidant capacity and prostaglandin E2 (PGE2) levels. Cooked B. carinata significantly reduced DNA damage induced by AFB1 as compared to baseline levels (+S9 mix: 35%, -S9 mix: 33%, p ≤ 0.01, respectively). Raw B. carinata only reduced DNA damage by S9-activated AFB1 by 21% (p = 0.08). PGE2 plasma levels were significantly reduced in subjects after consuming raw B. carinata. No changes in plasma antioxidant capacity were detectable. A balanced diet, including raw and cooked Brassica vegetables, might be suited to fully exploit the health-promoting potential. These results also advocate the promotion of B. carinata cultivation in Eastern Africa as a measure to combat effects of unavoidable aflatoxin exposure.

Diverse Excretion Pathways of Benzyl Glucosinolate in Humans after Consumption of Nasturtium (Tropaeolum majus L.)-A Pilot Study.

SCOPE: Different metabolic and excretion pathways of the benzyl glucosinolate breakdown products benzyl isothiocyanate and benzyl cyanide are investigated to obtain information about their multiple fate after ingestion. Detailed focus is on the so far underestimated transformation/excretion pathways-protein conjugation and exhalation. METHODS AND RESULTS: Metabolites, protein conjugates, and non-conjugated isothiocyanates are determined in plasma, urine, and breath of seven volunteers after consuming freeze-dried nasturtium or bread enriched with nasturtium. Samples are collected up to 48 h at selected time points. The metabolites of the mercapturic acid pathway are detectable in plasma up to 24 h after consumption. Additionally, mercapturic acid is the main metabolite in urine, but non-conjugated benzyl isothiocyanate is detectable as well. Protein conjugates show high amounts in plasma even 48 h after consumption. In breath, benzyl isothiocyanate and benzyl cyanide are detectable up to 48 h after consumption. CONCLUSION: Isothiocyanates are not only metabolized via the mercapturic acid pathway, but also form protein conjugates in blood and are exhaled. To balance intake and excretion, it is necessary to investigate all potential metabolites and excretion routes. This has important implications for the understanding of physiological and pharmacological effects of isothiocyanate-containing products.

In Vitro Determination of Protein Conjugates in Human Cells by LC-ESI-MS/MS after Benzyl Isothiocyanate Exposure.

Glucosinolates and their breakdown products, especially isothiocyanates (ITCs), are hypothesized to exert a broad range of bioactivities. However, physiological mechanisms are not yet completely understood. In this study, formation of protein conjugates after incubation with benzyl isothiocyanate (BITC) was investigated in vitro. A survey of protein conjugates was done by determining BITC cysteine and lysine amino acid conjugates after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated. Stability studies showed that cysteine conjugates are not stable under alkaline conditions, and lysine conjugates did not show any correlation to pH values, although stability increased at low temperatures. Lysine conjugates were the preferred form of protein conjugates, and longer BITC exposure times led to higher amounts. Knowledge about the reaction sites of ITCs in eukaryotic cells may help to understand the mode of action of ITCs leading to health promoting as well as toxicological effects in humans.

Leaching and degradation kinetics of glucosinolates during boiling of Brassica oleracea vegetables and the formation of their breakdown products.

Domestic processing methods, such as boiling, significantly affect the glucosinolate content and the formation of breakdown products in Brassica vegetables. Here, we comprehensively describe the effect of aqueous heat treatment on the degradation and leaching kinetics of glucosinolates on the formation of their enzymatic and non-enzymatic hydrolysis and breakdown products. The results were correlated with the inactivation kinetics of myrosinase and epithiospecifier protein activity in the Brassica oleracea vegetables kohlrabi, white cabbage, and red cabbage. Short-term heating increased isothiocyanate formation due to inactivation of the epithiospecifier protein. Myrosinase was inactivated shortly after that. Boiling led to leaching of glucosinolates and their hydrolysis products into the boiling water. Heating to 99 °C resulted in thermally-induced glucosinolate breakdown and nitrile formation, both in vegetables and boiling water. Finally, kinetic modeling not only revealed differences in myrosinase inactivation among the vegetables, but also glucosinolate leaching and degradation kinetics differed between individual glucosinolates and vegetables.

Determination of isothiocyanate-protein conjugates in milk and curd after adding garden cress (Lepidium sativum L.).

Isothiocyanates (ITC) play an important role in health promotion and cancer prevention due to their anti-bacterial, anti-inflammatory, and anti-cancerogenic properties. However, ITC are highly reactive so that a reaction with further food components is very likely. For example, a reaction of ITC with nucleophilic amino acid side chains of proteins such as cysteine and lysine can occur, reducing the bioavailability of indispensable amino acids and protein functions may be altered. Therefore, it is of great interest to investigate the fate of ITC in the food matrix. Accordingly, the aim of the present study was to investigate the interaction of milk proteins and the ITC benzyl isothiocyanate (BITC) and allyl isothiocyanate (AITC) forming dithiocarbamates and thioureas in milk and curd. After incubating milk and curd with pure ITC or ITC-containing garden cress (Lepidium sativum L.), proteins were isolated, digested, and analyzed via LC-ESI-MS/MS as amino acid derivatives ("conjugates"). Protein conjugates of AITC and BITC were detected in all samples investigated. Further, the acidic pH value in curd favored the formation of dithiocarbamates over the formation of thioureas. Slightly acidic or neutral conditions like in fresh milk favored the formation of thioureas. The investigations also indicated that AITC shows a higher reactivity and dithiocarbamates are formed preferably, whereas incubation with BITC lead to less protein conjugates and the ratio of thioureas and dithiocarbamates was more balanced. In addition, amino acid modifications were often analyzed with indirect methods like measuring the decline of the amino acid residues. In this study, the modified amino acids were analyzed directly leading to more reliable results concerning the amount of modification.

Bioavailability and metabolism of benzyl glucosinolate in humans consuming Indian cress (Tropaeolum majus L.).

SCOPE: Benzyl isothiocyanate (BITC), which occurs in Brassicales, has demonstrated chemopreventive potency and cancer treatment properties in cell and animal studies. However, fate of BITC in human body is not comprehensively studied. Therefore, the present human intervention study investigates the metabolism of the glucosinolate (GSL) glucotropaeolin and its corresponding BITC metabolites. Analyzing BITC metabolites in plasma and urine should reveal insights about resorption, metabolism, and excretion. METHODS AND RESULTS: Fifteen healthy men were randomly recruited for a cross-over study and consumed 10 g freeze-dried Indian cress as a liquid preparation containing 1000 µmol glucotropaeolin. Blood and urine samples were taken at several time points and investigated by LC-ESI-MS/MS after sample preparation using SPE. Plasma contained high levels of BITC-glutathione (BITC-GSH), BITC-cysteinylglycine (BITC-CysGly), and BITC-N-acetyl-L-cysteine (BITC-NAC) 1-5 h after ingestion, with BITC-CysGly appearing as the main metabolite. Compared to human plasma, the main urinary metabolites were BITC-NAC and BITC-Cys, determined 4-6 h after ingestion. CONCLUSION: This study confirms that consumption of Indian cress increases the concentration of BITC metabolites in human plasma and urine. The outcome of this human intervention study supports clinical research dealing with GSL-containing innovative food products or pharmaceutical preparations.

Determination of benzyl isothiocyanate metabolites in human plasma and urine by LC-ESI-MS/MS after ingestion of nasturtium (Tropaeolum majus L.).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine the concentration of benzyl isothiocyanate (BITC) metabolites in human plasma and urine. In this study, the following BITC metabolites have been considered: BITC-glutathione, BITC-cysteinylglycine, BITC-cysteine, and BITC-N-acetyl-L-cysteine. The assay development included: (1) synthesis of BITC conjugates acting as reference substances; (2) sample preparation based on protein precipitation and solid-phase extraction; (3) development of a quantitative LC-MS/MS method working in the multiple-reaction monitoring mode; (4) validation of the assay; (5) investigation of the stability and the reactivity of BITC conjugates in vitro; (6) application of the method to samples from a human intervention study. The lower limits of quantification were in the range of 21-183 nM depending on analyte and matrix, whereas the average recovery rates from spiked plasma and urine were approximately 85 and 75 %, respectively. BITC conjugates were found to be not stable in alkaline buffered solutions. After consumption of nasturtium, containing 1,000 µM glucotropaeolin, the primary source of BITC, quantifiable levels of BITC-NAC, BITC-Cys, and BITC-CysGly were found in human urine samples. Maximum levels in urine were determined 4 h after the ingestion of nasturtium. With regard to the human plasma samples, all metabolites were determined including individual distributions. The work presented provides a validated LC-MS/MS method for the determination of BITC metabolites and its successful application for the analysis of samples collected in a human intervention study.