One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation and dose schedule of the intervention agent. This crossover study was designed to evaluate the bioavailabilty and tolerability of two preparations of beverages prepared from 3-day-old broccoli sprouts in order to administer the chemopreventive agent sulforaphane: [i] enterically generated from its cognate glucosinolate (glucoraphanin) by thioglucosidases found in the gut microflora (GRR), and [ii] pre-released when the GRR beverage was treated with myrosinase from the daikon plant Raphanus sativus to catalyze hydrolysis of glucoraphanin to sulforaphane (SFR). Substantial differences were observed between the two formulations in terms of bioavailability and palatability, both of which influence the utility of broccoli sprouts beverages as useful agents for chemoprevention studies in humans.
Doses of sulforaphane (150 μmol) and glucoraphanin (800 μmol) within each beverage were selected on the basis of literature reports of earlier studies and were intended to provide bioequivalence in SFT
elimination together with reasonable tolerability. First, we had previously used a single dose of 200 μmol sulforaphane in a broccoli sprout preparation in women undergoing reduction mammoplasty to determine uptake of the isothiocyanate into breast tissue (30
). Second, very small in-patient studies in healthy volunteers had defined uptake and elimination kinetics with a single dose of approximately 150 μmol sulforaphane (23
) or three 25-μmol doses administered daily for 7 days (31
). In no instances were any adverse effects reported. Additionally, from these studies it was estimated that ~70% of the administered sulforaphane was excreted as derivatives of glutathione conjugates in urine over the subsequent 24 hour interval. Third, our 2003 intervention trial in Qidong utilized a glucosinolate-rich formulation of broccoli sprouts containing 400 μmol glucoraphanin. Estimates of total sulforaphane excretion in these individuals indicated 18% of the administered glucosinolate was excreted as isothiocyanate derivatives over the subsequent 24 hours, with a range of 2–50% amongst the 100 study participants (20
). No adverse events or issues with compliance were noted in this study as well. Shapiro et al (31
) also noted an 18% recovery of SFT
following dosing with 100 μmol GRR. Based upon these outcomes, a dose of 800 μmol glucoraphanin in the GRR beverage was chosen in anticipation of providing equivalent elimination totals (~100 μmol) of sulforaphane and metabolites between the two formulations. This prediction was not met.
The bioavailability of sulforaphane administered as SFR was far superior to GRR. Despite the attempt to match overall excretion yields between the two formulations, participants receiving the SFR had a 3-fold greater excretion of SFT
than did those receiving GRR. As seen from and Supplemental Table 1
, in both formulations excretion of SFT
was essentially complete within 24 hours of dosing. Estimates of the AUCs derived from the excretion patterns depicted in suggest that 70% of the administered sulforaphane in the SFR beverage was eliminated in 24 hours; a finding very much in accord with earlier results (15
). However, in the current study, only 5% of the administered glucoraphanin in GRR beverage was recovered as sulforaphane metabolites (SFT
). Thus, despite doubling the dose of glucoraphanin compared to the 2003 intervention, somewhat less sulforaphane was hydrolyzed, absorbed, metabolized and eliminated in the current study. The reasons underlying the diminished bioavailability of GRR in the current study are not clear. A technical explanation could arise from the use of different analytical methods to measure SFT
in the two studies: a colorimetric cyclocondensation assay (23
) in 2003 and isotope dilution mass spectrometry (26
) in 2009. However, a follow-up comparison of the two methods indicated they provided comparable measures of SFT
in urine samples from the 2003 study (26
). A biological basis for the differences could lie in different rates of hydrolysis of glucoraphanin to sulforaphane in the gastrointestinal tract between the two study populations. Intestinal microflora contribute substantially to this hydrolysis (15
). At this time it is not clear which species of microflora harbor the thioglucosidases that catalyze this conversion, nor is it known to what extent this catalytic capacity varies under the influence of health status, age or dietary changes in humans. It is possible that other enzymes in the microflora or the host tissues divert sulforaphane to alternative, unmeasured products such as nitriles. That such factors are important can be inferred from the observation that the interindividual variability for excretion of SFT
was significantly greater for participants receiving GRR compared to SFR beverage, irrespective of whether GRR was administered on the first or second wave (). Not all of the administered glucoraphanin was hydrolyzed in the gastrointestinal tract, as low amounts (typically 2–4 μmol) of glucoraphanin were excreted in urine daily in individuals receiving GRR. This is the first evidence of absorption and urinary elimination of intact glucoraphanin in humans, although Cwik et al. (33
) have recently reported the excretion of unmetabolized glucoraphanin following its oral administration to dogs and rats.
Nonetheless, there was substantial interindividual variability in the AUCs measured for the participants also drinking the SFR beverage. It has been hypothesized that polymorphisms in GSTs, which participate in the conjugation of sulforaphane to ultimately yield the major excretion product, the mercapturic acid, could contribute to this variability (see ) (25
). Null genotypes of GSTs could be related to decreased metabolism and urinary excretion of isothiocyanates such as sulforaphane, thereby increasing the body burden of sulforaphane following dosing and leading to a more pronounced or protracted pharmacodynamic action. Such a notion was triggered by results from a case-control study in a Chinese population demonstrating that reduction in risk of lung cancer was associated with increased consumption of cruciferous vegetables, primarily among individuals null for GSTM1
). By contrast, in a small broccoli feeding study (n=16) Gasper et al. (25
) reported that individuals null for GSTM1
excreted sulforaphane at a faster rate and to a greater extent than did GSTM1-positive individuals. In a larger study (n = 100), Steck et al. (34
) also observed that a greater proportion of individuals with the null GSTM1
genotype, but not the GSTT1
genotype, had higher urinary excretion rates of isothiocyanates following feeding of frozen/microwaved broccoli (myrosinase-inactivated) than did those with GSTM1
alleles present. They also observed substantial interindividual variability in the amount of SFT
excreted in the 24 h following broccoli ingestion. Seow et al. (37
) found an association between genotypes and urinary isothiocyate levels only among the consumers of the highest tertiles of cruciferous vegetable intake. In the current study we observed no effect of GST genotype, for either GSTM1
, on the AUCs (either for the initial 24 h or for the cumulative excretion with 7 days of dosing) for urinary elimination of total sulforaphane metabolites, regardless of whether they received GRR or SFR beverage. Similarly, in the 2003 Qidong intervention with a glucosinolate-rich beverage, no interaction between GST genotype and sulforaphane excretion was observed. Levels of sulforaphane intake in the two Qidong clinical trials, as adjudged by urinary SFT
excretion levels, were substantially higher than those reported in the case-control or broccoli feeding studies. Collectively, the feeding studies with various formulations of broccoli indicate that GST genotypes are unlikely to have profound effects on sulforaphane disposition in humans. Whereas differential rates of hydrolysis of glucoraphanin may account for some of the variability in excretion rates with GRR, additional factors remain to be described that influence SFR elimination.
Although the wash-out phase of our cross-over study demonstrated clearly that virtually all sulforaphane metabolites were eliminated within 24 hours, irrespective of whether GRR or SFR was administered, there were substantive differences in the rates of elimination between the two formulations. With administration of SFR, 95% of the SFT
was eliminated in the first 12 hours, compared to only 60% of the SFT
when participants were dosed with GRR. Vermeulen et al. (38
) have reported that in a small, randomized, free-living cross-over feeding trial of raw and cooked broccoli, higher amounts of SFT
were found in the blood and urine when broccoli was eaten raw. In this instance the predominant component is sulforaphane whereas it is glucoraphanin in the cooked broccoli in which the myrosinase has been denatured. Peak plasma concentrations were achieved at 1.6 and 6 h, respectively, for raw and cooked broccoli. Clearly, excretion of SFT
is delayed when broccoli or broccoli sprout preparations contain glucoraphanin instead of sulforaphane. Elimination half-lives of the sulforaphane mercapturic acid were comparable (~2.5 h), indicating again that the rate-limiting step for whole body elimination of glucoraphanin is likely the initial hydrolysis step. Interestingly, in this raw/cooked broccoli cross-over feeding study, the investigators also noted a better bioavailability when subjects were fed raw broccoli (SFT
= 37% of the administered dose) compared to cooked broccoli (3.4%), a finding very much in accord with our results comparing SFR and GRR.
The different kinetics for uptake and elimination of SFT
following administration of GRR versus SFR present a dilemma for the optimization of a broccoli-based food or beverage intervention. Sulforaphane exerts its cancer chemopreventive effects in experimental models through regulation of diverse molecular mechanisms, including activation of Keap1-Nrf2 signaling, inhibition of NFκB, inhibition of histone deacetylases, induction of apoptosis, and cell cycle arrest (10
). Agents affecting multiple processes in carcinogenesis are likely to be more useful chemopreventive agents than those affecting a single target. However, the mechanisms of pathway activation and inhibition require different pharmacokinetic optimizations; pulsed, high peak concentrations for pathway activation and sustained concentrations for pathway or enzyme inhibition. SFR produces the former, whilst GRR favors the latter. Moreover, not all molecular actions of sulforaphane are triggered at the same concentrations. For example, activation of Keap1-Nrf2 signaling occurs at substantially lower concentrations than does induction of apoptosis (2
). Thus, in the absence of clear understanding of the primary modes of action of sulforaphane and a consequent lack of understanding of how to optimize dose and duration within the target cells, we propose a blended approach may be most suitable for future interventions with broccoli sprout preparations, especially for large-scale, population-based interventions. Advantages of SFR include its better bioavailability and more rapid uptake, which are tempered by its more astringent taste. The taste in beverage formulation could be partially masked by admixing with mango juice, but more suitable masking materials are still required. SFR is intrinsically less stable than GRR, leading to additional logistical concerns about formulation, storage and distribution in settings of large, long interventions. By contrast, GFR, which is very stable and more tolerable to study participants, manifests variable but overall poor bioavailability. It does, however, provide a means to produce a more stable steady-state level of sulforaphane within the body. Future studies comparing the pharmacodynamic effects of these two formulations, individually, and perhaps combined, will be required to fully optimize food-based formulations containing sulforaphane, in addition to further evaluations of the impacts of dose and schedule on mechanistic or disease-based biomarkers.