For pre-menopausal women, these results suggest that consumption of a daily dosage of 2 capsules of the treatment offers beneficial outcomes, in that the mean 2-OHE concentration and the mean 2-OHE:16α-OHE ratio significantly increased during a 28-day supplementation. In contrast, there were no significant differences in the placebo group. The estrogen metabolite 2-OHE2 undergoes conjugation to 2-methoxyestradiol (2-MeO-E2), which has been shown to inhibit cell proliferation.27
This 2-OHE2 conjugation product has also been identified as an inhibitor of angiogenesis and tumour growth; thus, an increase in 2-OHE concentration, and the subsequent increase in 2-MeO-E2, is considered a beneficial outcome independent of the 2-OHE:16α-OHE ratio.28–80
The increased ratio, having been clearly identified as a biomarker of a reduction in breast cancer risk in pre- menopausal women,2,5,31
is a very positive outcome, and an outcome that has previously been observed in pre-menopausal women taking either I3C or dietary lignans.10,12,14
For post-menopausal women, the results are more complex. There was a significant increase in the concentration of 2-OHE from day 0 to day 28 in the treatment group, but not in the placebo group. As with pre-menopausal women, this represents a significant beneficial effect, because previous research has indicated that an increase in the concentration of 2-OHE in the urine is correlated independently with a reduction in breast cancer risk.28–30
Furthermore, this metabolite is regarded as a weak estrogen, with minimal negative effects, and possibly anti-estrogenic effects, compared to the much more potent negative effects of 16α-OHE.3
Given the expectation that there is a limited pool of estrogen substrates for the various metabolic pathways, one would expect that an increase in the activity of the pathway producing 2-OHE would concomitantly result in a reduction in the activity of the 16α-OHE pathway, and this was the hypothesis confirmed in an IC3 supplementation trial.32
In another I3C supplementation trial, the authors noted that although a significant increase in the 2:16 ratio was observed, 3 of 20 subjects experienced no change in this ratio at any time-point.10
They noted that “ … some individuals may be resistant to change”. In our study, 4 of 21 pre-menopausal and 5 of 17 post-menopausal women experienced a decrease in this ratio. In a study on soy consumption in post-menopausal women, only women who were equol producers increased the 2:16 ratio.33
It may be that future nutrigenomic studies may elucidate genetic differences in the way some women metabolize estrogen, and/or in how they digest, absorb and metabolize dietary components that influence estrogen metabolism.
For the combined results in both pre- and postmenopausal women, the increase in 2-OHE in the treatment group was highly significant, and there was a trend towards significance in the 2:16a-OHE ratio. The unexpected trend towards an increase in the concentration of 16α-OHE in both the pre- and post-menopausal groups undoubtedly attenuated what might have been a significant increase in the 2-OHE:16α-OHE ratio when the results for both preand post-menopausal women were combined. The P
-value for the 2:16 ratio, in the combined groups, was 0.074, and this is most likely a combination of the significant pre-menopausal P
-value of 0.016, and the non-significant post-menopausal P
-value of 0.387. However, the small increase in average levels of 16α-OHE in both pre- and post-menopausal women needs to be investigated further, to assess whether this is a spurious finding or the result of metabolic alterations in estrogen metabolism. This estrogen metabolite is associated with an increase in breast cancer risk;1–7
thus, any supplement that appears to increase the level of this metabolite requires further investigation.
The analyses of 4-OHE and the so-called carcinogenic ratio provided somewhat interesting results. Although there were no significant differences in 4-OHE1 concentrations among any of the groups or sub-groups, the apparent reduction in 4-OHE levels in the treatment group, with only 12 subjects, in comparison with an increase in 4-OHE levels in the placebo group of 10 subjects, warrants further research, although a larger trial may show these results to be extraneous. The carcinogenic ratio, which combines E3, 4-OHE (1 and 2), 16α-OHE1 and 2-OHE (1 and 2) provided more interesting results, given the significant or close-to-significant results for the treatment groups and lack thereof in the placebo groups.
Although there may be dispute as to whether E3 belongs among the carcinogenic metabolites, Takahashi et al.9
reported on the carcinogenic effects of estriol and its metabolites in young adult mice. After initiation of endometrial carcinogenesis with N
-nitroguanidine, the researchers measured the incidences of endometrial hyperplasia/ proliferative lesions and adenocarcinomas following implantation with estrogen metabolite pellets. The metabolites that produced significant incidences of proliferative lesions were 2-hydroxyestriol (2-OH-E3), 2-methoxyestriol (2-MeO-E3), 2-methoxyestradiol (2-MeO-E2) and 16-epiestriol, and those that resulted in adenocarcinomas were estrone (E1), estradiol (E2), E3, 16α-OHE1, 16β-OHE1 and 17-epiestriol (a metabolite of 16α-OHE1). In effect, two estriol metabolites produced lesions, and two produced adenocarcinomas. Human studies are less definitive, but there is some evidence that estriol metabolites may be carcinogenic in some instances.34
In this trial, it was expected that serum enterolactone concentrations would rise as dietary HMR lignan intake rose. The fact that there were no significant increases in the average serum enterolactone concentrations in any of the groups or sub-groups is puzzling. Originally, the manufacturer of this specific lignan (Linnea, Inc., Switzerland) stated, in a brochure for public consumption, that a significant increase in serum enterolactone concentration may only be expected if dietary intake of HMR lignans is 25 to 50 mg/day.35
However, a later brochure indicated that 10 to 50 mg/day would be sufficient to increase serum enterolactone concentrations,36
and this was based on a study by Cosentino.37
The daily intake of HMR lignan in this trial was 20 mg, and this may or may not have been sufficient to see a significant increase in serum enterolactone concentrations. Further research on the most efficacious dosage of HMR lignans is warranted. Serum quantification of the HMR lignin metabolite 7-hydroxy-HMR lignin and the enterolactone precursor enterodiol would also be advisable, in order to verify adequate HMR lignan absorption and metabolism.
Although it was determined, by power analysis, that 44 subjects per group would be needed in order to obtain statistically robust results, the unexpected reduction in group numbers to 38 women consuming the treatment and 30 women consuming the placebo attenuated the number and power of statistical findings. These findings were further attenuated when the treatment and placebo groups were subdivided into pre- and post-menopausal women, and when non-detectable concentrations of other metabolites of interest, such as 4-OHE, were analysed statistically. Thus, the fact that several statistically-significant results were obtained with only 21 pre-menopausal women in the treatment group, and 17 post-menopausal women in the treatment group, for the major metabolites, and even fewer for other metabolites, indicates that these findings are important, and that future research with a greater number of participants, ie, sufficient to subdivide into pre- and post-menopausal groups while retaining statistical robustness, may well elicit more favourable results.