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Sulphoraphane originates from glucoraphanin in broccoli and is associated with anti-cancer effects. A preclinical study suggested that daily consumption of broccoli may increase the production of sulphoraphane and sulphoraphane metabolites available for absorption. The objective of this study was to determine whether daily broccoli consumption alters the absorption and metabolism of isothiocyanates derived from broccoli glucosinolates. We conducted a randomised cross-over human study (n 18) balanced for BMI and glutathione S-transferase μ 1 (GSTM1) genotype in which subjects consumed a control diet with no broccoli (NB) for 16 d or the same diet with 200 g of cooked broccoli and 20 g of raw daikon radish daily for 15 d (daily broccoli, DB) and 100 g of broccoli and 10 g of daikon radish on day 16. On day 17, all subjects consumed a meal of 200 g of broccoli and 20 g of daikon radish. Plasma and urine were collected for 24 h and analysed for sulphoraphane and metabolites of sulphoraphane and erucin by triple quadrupole tandem MS. For subjects with BMI >26 kg/m2 (median), plasma AUC and urinary excretion rates of total metabolites were higher on the NB diet than on the DB diet, whereas for subjects with BMI <26 kg/m2, plasma AUC and urinary excretion rates were higher on the DB diet than on the NB diet. Daily consumption of broccoli interacted with BMI but not GSTM1 genotype to affect plasma concentrations and urinary excretion of glucosinolate-derived compounds believed to confer protection against cancer. This trial was registered as NCT02346812.

To examine the relationship between dietary cruciferous vegetable intake and selected tumour biomarkers for histone acetylation (H3K9ac, H3K18ac, HDAC3 and HDAC6), proliferation (Ki-67) and cell-cycle regulation (p21) from breast tissue. The study used baseline data of women recruited to participate in a clinical trial of sulforaphane supplement. Dietary cruciferous vegetable intake was collected through a validated Arizona Cruciferous Vegetable Intake Questionnaire. Breast tissue was obtained from biopsy samples. Spearman correlations were calculated between intake of specific cruciferous vegetables and biomarkers. Tissue biomarkers were log2-transformed to obtain approximate normality. Linear regression analyses were conducted to examine associations between cruciferous vegetable intake and biomarkers adjusting for age and use of non-steroidal anti-inflammatory drugs. False discovery rate (FDR) was used to account for multiple comparisons. Clinical trial baseline. Fifty-four women who had abnormal mammogram findings and were scheduled for breast biopsy. Mean intake of total cruciferous vegetables from all food sources was 81·7 (sd 57·3) g/d. Mean urinary total sulforaphane metabolites was 0·08 (sd 0·07) µm/mm creatinine. Total cruciferous vegetable intake was inversely associated with Ki-67 protein expression in breast ductal carcinoma in situ (DCIS) tissue (β=-0·004; se=0·001; FDR q value=0·03), but not in benign or invasive ductal carcinoma (IDC) tissue. No association was found for other biomarkers measured (HDAC3, HDAC6, H3K9, H3K18 and p21) in all tissues examined (benign, DCIS and IDC). The present study sought to provide additional evidence for the potential role of sulforaphane in histone acetylation and cell proliferation. Here, we report that total cruciferous vegetable intake is associated with decreased cell proliferation in breast DCIS tissue.

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.

Sulforaphane (SF) is a chemopreventive isothiocyanate (ITC) derived from glucoraphanin (GRP) hydrolysis by myrosinase, a thioglucoside present in broccoli. The ability of broccoli powders sold as supplements to provide dietary SF is often of concern as many supplements contain GRP, but lack myrosinase. In a previous study, biomarkers of SF bioavailability from a powder rich in GRP, but lacking myrosinase, were enhanced by co-consumption of a myrosinase-containing air-dried broccoli sprout powder. Here, we studied the absorption of SF from the GRP-rich powder used in the previous study, but in combination with fresh broccoli sprouts, which are commercially available and more applicable to the human diet than air-dried sprout powder. A total of four participants each consumed four meals (separated by 1 week) consisting of dry cereal and yogurt with sprouts equivalent to 70 μmol SF, GRP powder equivalent to 120 μmol SF, both or neither. Metabolites of SF were analysed in blood and urine. The 24 h urinary SF-N-acetylcysteine recovery was 65, 60 and 24 % of the dose ingested from combination, broccoli sprout and GRP powder meals, respectively. In urine and plasma, ITC appearance was delayed following the GRP powder meal compared with the sprout and combination meals. Compared with the GRP powder or sprouts alone, combining broccoli sprouts with the GRP powder synergistically enhanced the early appearance of SF, offering insight into the combination of foods for improved health benefits of foods that reduce the risk for cancer.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).

Background Isothiocyanates (ITCs), hydrolysis products from glucosinolates, are a family of biologically active compounds originating from cruciferous vegetables. Many ITCs are assumed to have cancer preventive effects and to further evaluate these potential health effects, reliable biomarkers of ITC exposure are needed. Aim of the study In this study we investigated the ability of urinary ITC excretion to reflect a low or high daily intake of cruciferous vegetables. Methods The design was a controlled human crossover study (n = 6). Subjects consumed a self-restricted glucosinolate-free diet 48 h before the study-day where a basic diet supplemented with 80 or 350 g of mixed cruciferous vegetables was consumed. All urine was collected in intervals during the 48 h period after ingestion of the cruciferous vegetables. Total ITC in the cruciferous mixture and total ITC and their metabolites in urine was quantified as the cyclocondensation product of 1,2-bezenedithiol by high performance liquid chromatography. Results The total urinary excretion of ITCs correlated significantly with the two doses of ITC from diets with high or low cruciferous content (r s = 0.90, P < 0.01). The fraction of urinary ITC excreted was 69.02 ± 11.57% and 74.53 ± 8.39% of the amounts ingested for 80 and 350 g cruciferous vegetables, respectively. Conclusion The results in this study indicate that the urinary excretion of ITCs, measured by use of the cyclocondesation reaction, is a useful and precise tool that may be used as a biomarker of ITC exposure in population based studies.

Allyl-isothiocyanate (AITC) is an organosulfur phytochemical found in abundance in common cruciferous vegetables such as mustard, wasabi, and cabbage. Although AITC is metabolized primarily through the mercapturic acid pathway, its exact pharmacokinetics remains undefined and the biological function of AITC metabolites is still largely unknown. In this study, we evaluated the inhibitory effects of AITC metabolites on lipid accumulation in vitro and elucidated the pharmacokinetics and tissue distribution of AITC metabolites in rats. We found that AITC metabolites generally conjugate with glutathione (GSH) or N-acetylcysteine (NAC) and are distributed in most organs and tissues. Pharmacokinetic analysis showed a rapid uptake and complete metabolism of AITC following oral administration to rats. Although AITC has been reported to exhibit anti-tumor activity in bladder cancer, the potential bioactivity of its metabolites has not been explored. We found that GSH-AITC and NAC-AITC effectively inhibit adipogenic differentiation of 3T3-L1 preadipocytes and suppress expression of PPAR-γ, C/EBPα, and FAS, which are up-regulated during adipogenesis. GSH-AITC and NAC-AITC also suppressed oleic acid-induced lipid accumulation and lipogenesis in hepatocytes. Our findings suggest that AITC is almost completely metabolized in the liver and rapidly excreted in urine through the mercapturic acid pathway following administration in rats. AITC metabolites may exert anti-obesity effects through suppression of adipogenesis or lipogenesis.

Background/objectivesIsothiocyanate (ITC) is formed via the hydrolysis of glucosinolates by myrosinase, found in cruciferous vegetables. Although myrosinase is inactivated by the cooking process, no studies have incorporated the effect of cooking into the estimation of dietary ITC intake or evaluated the validity. We evaluated the validity of dietary ITC intake estimated from a food frequency questionnaire (FFQ), and urinary ITC levels using 24 h urine samples or a WFR (weighed food record), and evaluated the reproducibility of dietary ITC in two FFQs administered at an interval of 1-year.Subjects/methodsThe JPHC-NEXT Protocol Area included a total of 255 middle-aged participants across Japan. We calculated dietary ITC intake from WFR and two FFQs by assuming that cooked cruciferous vegetables contain zero ITC. Urinary ITC excretion was measured at two points during summer and winter. The validity and reproducibility of dietary ITC intake estimated by FFQ were assessed using Spearman’s correlation coefficients.ResultsAlthough we observed a moderate correlation between dietary ITC intake derived from a 12-day WFR and urinary ITC excretion, notwithstanding the cooking process, the correlation between dietary ITC intake estimated by FFQ and mean urinary ITC excretion was low. However, the correlation was improved when we compared urinary ITC excretion and a 3-day WFR or FFQ collected during winter. Our FFQ showed good reproducibility.ConclusionAlthough seasonality is a critical factor, dietary ITC intake estimated using an FFQ showed moderate validity and reproducibility and can be used in future epidemiological studies.

Glucosinolates, phytochemicals found in cruciferous vegetables, are metabolised to bioactive isothiocyanates (ITC) by certain bacteria in the human gut. Substantial individual variation in urinary ITC excretion has been observed in previous cruciferous vegetable-feeding studies. We hypothesised that individual differences in gut microbial community contribute to the observed variation in glucosinolate metabolism, i.e. gut microbiota composition between high- and low-ITC excreters differs. We recruited twenty-three healthy individuals and fed them a standardised meal containing 200 g of cooked broccoli. After the meal, 24 h urinary ITC excretion was measured. Study participants with the highest (n 5) and lowest (n 5) ITC excretion provided faecal samples for ex vivo bacterial cultivation with 50 μm-glucoraphanin, the major glucosinolate found in broccoli. When grown ex vivo, faecal bacteria from the selected high-ITC excreters were able to degrade more glucoraphanin than those from the low-ITC excreters (P = 0·05). However, bacterial fingerprints of faecal and ex vivo culture microbiota revealed no statistically significant differences between the high- and low-ITC excreters in terminal restriction fragment length polymorphism analysis of the bacterial 16S ribosomal RNA gene. In conclusion, glucosinolate degradation by faecal bacteria ex vivo may be associated with in vivo bacterial glucosinolate metabolism capacity, but no direct link to specific bacterial species could be established, possibly due to the complexity and functional redundancy of the gut microbiota.

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. Figure Chemical structure of the benzyl glucosinolate present in nasturtium (Indian cress; Tropaeolum majus L.) and the corresponding benzyl isothiocyanate (BITC). Further, the metabolism via the mercapturic acid pathway in human beings is shown in detail.