This cross-over study compared SFN and ERN bioavailability from a whole food source and a dietary supplement. We demonstrate that the whole food had both higher bioavailability and altered kinetics compared to a myrosinase-inactivated supplement. The whole food, which contains myrosinase, produced peak plasma concentrations that were 7 and 12-fold higher for SFN and ERN, respectively, compared to peak plasma concentrations after consumption of the broccoli supplement, which did not contain myrosinase. Similarly in the urine total 24 h excretion was 5 and 8-fold higher for SFN and ERN, respectively, when subjects consumed the whole food versus the supplement. In this study we also show that subjects heterozygous for the (A313
G) transition in GSTP1
had similar metabolism and excretion of ITCs as subjects homozygous for fully functional GSTP1
alleles. It has been reported that GSTM1
polymorphism had a significant effect on metabolism and excretion of SFN after broccoli consumption [23
] Due to the contribution of several GST isoforms to the metabolism of ITCs, further investigation into the role of other GSTs is an important area of research. These data are the first to show detailed UHPLC-MS/MS analysis of both SFN and ERN in human subjects after consuming broccoli sprouts and a broccoli supplement.
The necessity for myrosinase for ITC absorption has been considered in many different studies involving glucosinolate and ITC metabolism [24
] and in this report we provide further evidence that myrosinase activity is necessary for maximal bioavailability of ITCs. Lack of myrosinase can affect two main aspects of metabolism; bioavailability and kinetics. For bioavailability, several studies in humans have examined the difference between cooked and raw broccoli [14
] and another even compared excretion when broccoli sprouts were chewed versus swallowed intact [18
]. In all cases it has been concluded that inactivation of myrosinase led to lower plasma and urine concentrations of the ITCs. Recently a paper was published testing the differences in absorption and excretion between a broccoli powder and broccoli sprouts [17
]. In this study the authors reported that only 19% of the SFN was recovered after consumption of the broccoli powder compared to 74% recovery after consumption of the broccoli sprouts. From these data we can conclude that the presence of myrosinase is important for maximal bioavailability of ITCs.
Not only were the ITCs more bioavailable from the whole food source but the peak plasma and urine concentrations occurred sooner when subjects consumed the whole food. Cramer et. al. concluded that there was a delay in the appearance of SFN in subjects who consumed the broccoli powder compared to those who consumed the broccoli sprouts [17
]. In congruence with that report, herein we observed that the peaks in plasma concentrations and urinary excretion were delayed when subjects consumed the broccoli supplement.
The lower bioavailability and delayed appearance of ITC metabolites in the plasma and urine likely reflects the reliance on microflora in the colon for glucosinolate hydrolysis. When subjects are given fresh sprouts the ITCs are formed and released when consumed and are likely absorbed through the gut wall as well as in the jejunum [25
]. In contrast, unhydrolyzed glucosinolates need to be metabolized by gut microflora before the ITCs can be absorbed. In fact, a recent report demonstrated that cecal microbiota can hydrolyze glucosinolates and that SFN can be absorbed through the cecal enterocytes in rats [26
]. These factors relating to glucosinolate metabolism and ITC absorption play important roles in the bioavailability and kinetics of ITCs in humans.
In this report we provide further evidence that SFN and ERN interconvert in humans. Only one other study in humans has reported interconversion between SFN and ERN [27
]. We show for the first time that conversion is variable between subjects, but consistent within subjects across time and glucosinolate source. In our study the ratio of glucoerucin to glucoraphanin in the broccoli sprouts and broccoli supplement was 0.47 and 0.32, respectively. This is significant because the ratio of ERN/SFN metabolites in the plasma of most subjects was ≥ 0.4, indicating that some SFN had been converted to ERN. The variability between subjects in the plasma ratio ranged from ~0.2 to ~1.2. The variability in the urine ratio between individual subjects was even wider ranging from ~0.1 to ~2.3. The conversion of SFN to ERN appears to occur after absorption because in the plasma the ratio started ~0.4 and did not reach ~0.8 until 12 to 24 h post consumption. In contrast, regardless of time, the average ratio in the urine was ~0.8. This implies that when the ITCs are absorbed they are closer to the starting ratio of glucoerucin to glucoraphanin but as they are metabolized and excreted in the urine some SFN is converted into ERN. It is not clear what drives the conversion of the sulfoxide SFN to the thioether ERN but is an important area for future research. Whether this conversion from SFN to ERN is important for the health promoting effects of glucosinolate containing foods still remains to be determined although several reports provide a glimpse into the possibility of differing activities between these two ITCs. In regards to phase II enzyme induction similar induction of phase II enzymes has been reported for ERN and SFN in rat lung [8
], duodenum and urinary bladder [9
], with some tissue specificity noted. ERN has been reported to more potently induce phase III transporters multidrug resistance pump 1 and 2 [10
]. Another report indicated that ERN was less potent in inhibiting proliferation and modulating p53 and p21 protein expression in human lung cancer A549 cells [11
]. In contrast, ERN was substantially more effective at inducing G2/M cell cycle arrest, cell death, phase II enzymes and MRP2 in Caco-2 colon cancer cells [12
]. These reports indicate that the potency of ERN and SFN may be dependent on the biological endpoint and/or cell line, although further investigation is required.
In conclusion, our data provide further evidence that bioavailability of SFN and ERN is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. Furthermore, we provide strong evidence that the interconversion between SFN and ERN is consistent within each subject but variable between subjects. There is increasing evidence that isothiocyanates such as sulforaphane play an important role in human health and the prevention of diseases such as cancer, ischemia reperfusion damage and others [5
]. The current study further characterizes the bioavailability and kinetics of ITCs from a whole food source versus a dietary supplement, and have implications regarding consumer choices of how to best incorporate the chemopreventive effects of sulforaphane into their diets.