Manipulation of Continuous and End-of-Day Red/Far-Red Light Ratios Affects Glucobrassicin and Gluconasturtiin Accumulation in Cabbage (Brassica oleracea) and Watercress (Nasturtium officinale), Respectively.

Cabbage (Brassica oleracea) and watercress (Nasturtium officinale) produce glucobrassicin (GBS) and gluconasturtiin (GNST), precursors of chemopreventive compounds. Their accumulation is affected by environmental signals. We studied the impact of the red to far-red light (R/FR) ratio on GBS concentration in red ″Ruby Ball″ and green ″Tiara″ cabbage. Foliar shading, via weed surrogates that competed with cabbage plants for specific durations, induced R/FR variation among treatments. ″Ruby Ball″ GBS concentrations were the highest when R/FR within the canopy was the lowest. ″Tiara″ was unaffected by competition. The same trend was observed in a controlled environment using R and FR LEDs without weeds present. ″Ruby Ball″ subjected to an R/FR = 0.3 treatment had 2.5- and 1.4-fold greater GBS concentration compared to R/FR = 1.1 and 5.0 treatments combined. Watercress given end-of-day (EOD) R and/or FR pulses after the main photoperiod had the lowest GNST concentrations after an EOD FR pulse but the highest concentrations after an R followed by FR pulse.

Using Near-infrared reflectance spectroscopy (NIRS) to predict glucobrassicin concentrations in cabbage and brussels sprout leaf tissue.

BACKGROUND: Glucobrassicin (GBS) and its hydrolysis product indole-3-carbinol are important nutritional constituents implicated in cancer chemoprevention. Dietary consumption of vegetables sources of GBS, such as cabbage and Brussels sprouts, is linked to tumor suppression, carcinogen excretion, and cancer-risk reduction. High-performance liquid-chromatography (HPLC) is the current standard GBS identification method, and quantification is based on UV-light absorption in comparison to known standards or via mass spectrometry. These analytical techniques require expensive equipment, trained laboratory personnel, hazardous chemicals, and they are labor intensive. A rapid, nondestructive, inexpensive quantification method is needed to accelerate the adoption of GBS-enhancing production systems. Such an analytical method would allow producers to quantify the quality of their products and give plant breeders a high-throughput phenotyping tool to increase the scale of their breeding programs for high GBS-accumulating varieties. Near-infrared reflectance spectroscopy (NIRS) paired with partial least squares regression (PLSR) could be a useful tool to develop such a method. RESULTS: Here we demonstrate that GBS concentrations of freeze-dried tissue from a wide variety of cabbage and Brussels sprouts can be predicted using partial least squares regression from NIRS data generated from wavelengths between 950 and 1650 nm. Cross-validation models had R2 = 0.75 with RPD = 2.3 for predicting µmol GBS·100 g-1 fresh weight and R2 = 0.80 with RPD = 2.4 for predicting µmol GBS·g-1 dry weight. Inspections of equation loadings suggest the molecular associations used in modeling may be due to first overtones from O-H stretching and/or N-H stretching of amines. CONCLUSIONS: A calibration model suitable for screening GBS concentration of freeze-dried leaf tissue using NIRS-generated data paired with PLSR can be created for cabbage and Brussels sprouts. Optimal NIRS wavelength ranges for calibration remain an open question.

Identification and analysis of a mercapturic acid conjugate of indole-3-methyl isothiocyanate in the urine of humans who consumed cruciferous vegetables.

Glucobrassicin, a quantitatively significant constituent of Brassica vegetables, gives rise to indole-3-carbinol (I3C) and its dimer di-indolylmethane (DIM) when the vegetables are chewed. I3C and DIM have been extensively studied with respect to their anti-carcinogenic properties. However, the presumed intermediate isothiocyanate in their formation, indole-3-methyl isothiocyanate (IMITC), has to our knowledge never been observed, despite the fact that isothiocyanates derived from cruciferous vegetables are known to have anti-carcinogenic properties. Therefore, we investigated the formation and presence in human urine of IMITC by analyzing for its N-acetylcysteine conjugate, IMITC-NAC, in order to gain a more complete understanding of the biochemical pathways leading to formation of I3C and DIM upon consumption of vegetables rich in glucobrassicin. Standard IMITC-NAC was synthesized and its structure confirmed by NMR and MS. IMITC-NAC was identified in extracts of Brussels sprouts chopped in the presence of N-acetylcysteine. An LC-ESI-MS/MS-SRM method for analysis of IMITC-NAC, with [13C,15N]IMITC-NAC as internal standard, was developed and validated. Then, ten subjects (7 females) consumed a salad of Brussels sprouts and cabbage (containing 100-500µmol glucobrassicin) once daily for 3days. Urine was collected at intervals up to 24h after vegetable consumption. Levels of IMITC-NAC in the urine of these 10 subjects ranged from 0.2 to 30.2pmol/mL urine. These results provide the first evidence for the presumed intermediacy of IMITC in the formation of I3C and DIM in humans who consumed Brussels sprouts and cabbage as a source of glucobrassicin.

Harnessing the Power of Cruciferous Vegetables: Developing a Biomarker for Brassica Vegetable Consumption Using Urinary 3,3'-Diindolylmethane.

Glucobrassicin in Brassica vegetables gives rise to indole-3-carbinol (I3C), a compound with potent anticancer effects in preclinical models. We previously showed that the urinary metabolite 3,3'-diindolylmethane (DIM) could discriminate between volunteers fed high and low doses of Brassica vegetables. However, the quantitative relationship between glucobrassicin exposure and urinary DIM level is unclear. We conducted a clinical trial to examine the hypotheses that a range of glucobrassicin exposure from Brassica vegetables is reflected in urinary DIM and that this effect plateaus. Forty-five subjects consumed vegetables, a mixture of brussels sprouts and/or cabbage, at one of seven discrete dose levels of glucobrassicin ranging from 25 to 500 µmol, once daily for 2 consecutive days. All urine was collected for 24 hours after each vegetable-eating session. Urinary DIM was measured using our published liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC/ESI-MS/MS-SRM) method. Urinary DIM excretion increased predictably with increasing glucobrassicin dose and plateaued between 200 and 300 µmol of glucobrassicin. The association between glucobrassicin dose and urinary DIM was strong and positive (R2 = 0.68). The majority of DIM was excreted in the first 12 hours after vegetable consumption. We conclude that urinary DIM is a reliable biomarker of glucobrassicin exposure and I3C uptake and that feeding glucobrassicin beyond 200 µmol did not consistently lead to more urinary DIM, suggesting a plateau in potential chemopreventive benefit. Cancer Prev Res; 9(10); 788-93. ©2016 AACR.

Urinary 3,3'-diindolylmethane: a biomarker of glucobrassicin exposure and indole-3-carbinol uptake in humans.

BACKGROUND: Brassica vegetable consumption may confer a protective effect against cancer, possibly attributable to their glucosinolates. Glucobrassicin is a predominant glucosinolate and is the precursor of indole-3-carbinol (I3C), a compound with anticancer effects. However, objective assessments of I3C uptake from Brassica vegetables have not been successful. METHODS: We conducted a randomized, crossover trial to test whether 3,3'-diindolylmethane (DIM, a metabolite of I3C) excreted in the urine after consumption of raw Brassica vegetables with divergent glucobrassicin concentrations is a marker of I3C uptake from such foods. Twenty-five subjects were fed 50 g of either raw "Jade Cross" Brussels sprouts (high glucobrassicin concentration) or "Blue Dynasty" cabbage (low glucobrassicin concentration) once daily for 3 days. All urine was collected for 24 hours after vegetable consumption each day. After a washout period, subjects crossed over to the alternate vegetable. Urinary DIM was measured using a novel liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) method with [(2)H2]DIM as internal standard. RESULTS: Urinary DIM was consistently and significantly higher after Brussels sprouts feeding than after cabbage feeding, as evidenced by an average difference of 8.73 pmol/mg creatinine (95% confidence interval, 5.36-12.10; P = 0.00002). CONCLUSION: We have successfully quantified urinary DIM after uptake of I3C from food, and demonstrated that differences in glucobrassicin exposure are reflected in urinary DIM levels. IMPACT: Our LC-ESI-MS/MS-SRM method and the results of our study indicate urinary DIM is a measure of I3C uptake from Brassica vegetables, a finding that can be utilized in prospective epidemiologic and chemoprevention studies.