No de novo sulforaphane biosynthesis in broccoli seedlings.

Introduction

The isothiocyanate sulforaphane, present in significant amounts in broccoli (Brassica oleracea L.) seedlings in the form of its precursor glucoraphanin, has been identified as an inducer of quinine reductase, a phase-II detoxification enzyme known for its anticarcinogenic properties. However, the concentration glucoraphanin/sulforaphane in broccoli seedlings usually decreases during the first weeks after germination, and no conclusive data are available in the literature that glucoraphanin/sulforaphane is either or not biosynthesized de novo during these first weeks of seedling development.

Stable Isotope Solution: Cross-Labelling broccoli with ¹²C and ¹³C

To solve this issue, we performed a cross labelling experiment with two different carbon isotopes in co-operation with the University Medical Centre in Groningen. Both ¹²C (99 atom % ¹²C) and ¹³C (98 atom % ¹³C) broccoli seeds were first produced and subsequently germinated and grown either in a ¹³CO₂ or a ¹²CO₂ environment. Afterwards, the labeling degree of sulforaphane in seeds and in seedlings was analysed on a HPLC-MS system (Gorissen et al., 2011).

Result

Figure 1 shows the mass distribution of non-labeled ¹²C-sulforaphane (C6H11NOS2) – originating from ¹²C-seeds grown in a 98 atom % ¹³C-CO₂ atmosphere – with the [M+H+] signal at 178.04 m/z. The dominant signal at the expected m/z value of 178.04 for 12C6-sulforaphane, and the minor signals at [M+H+]+1 to +6, show that the broccoli was not enriched with ¹³C (as compared to a ¹²C standard). The mass spectrum in Figure 2 shows the [M+H+] peaks of 13C6-sulforaphane from ¹³C-seedlings at 178.04 + 6 = 184.05 m/z and 13C512C-sulforaphane at 183.05 m/z in a ratio of 89:11. This ratio indicates an overall ¹³C-abundance of sulforaphane-C in the labeled seeds of 98.2 atom %, calculated using the binomial distribution (Hellerstein & Neese, 1999; Jennings & Matthews, 2005). Consistent with this result, virtually no ¹²C-sulforaphane (178.04 m/z) was detected in ¹³C-seedlings and no ¹³C-sulforaphane in ¹²C-seedlings.

Figure 1. High resolution mass spectrum (175.0-190.0 m/z, [12C6H12ONS2+H+] 178.04) of sulforaphane in broccoli seedlings grown from ¹²C-seeds in a ¹³C-CO₂ atmosphere.

Figure 2. High resolution mass spectrum (175.0-190.0 m/z, [13C512CH12ONS2+H+] 183.05 m/z and [13C6H12ONS2+H+] 184.05) of sulforaphane in broccoli seedlings grown from ¹³C-seeds in a¹²C-CO₂ atmosphere.

We conclude that sulforaphane exclusively originates from seed reserves and that de novobiosynthesis is not detectable in broccoli seedlings. Our findings confirm the conclusion of Nakagawa et al. (2006) that consumption of younger broccoli seedlings may be recommended above older seedlings with regard to health aspects and optimal dosage, since they contribute to a higher intake of protective glucosinolates (Rychlik & Adam, 2008).

References

Gorissen A, NU Kraut, R de Visser, M de Vries, H Roelofsen, RJ Vonk. 2011.
No de novo sulforaphane biosynthesis in broccoli seedlings.
Food Chemistry 127: 192-196. Ask a reprint.

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Jennings ME II, DE Matthews. 2005.
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Nakagawa K, T Umeda, O Higuchi, T Tsuzuki, T Suzuki, T Miyazawa. 2006.
Evaporative light-scattering analysis of sulforaphane in broccoli samples: quality of broccoli products regarding sulforaphane contents.
Journal of Agricultural and Food Chemistry 54: 2479-2483.

Rychlik M, ST Adam. 2008.
Glucosinolate and folate content in sprouted broccoli seeds.
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