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Applications  |  Nutrition: Sulforaphane in Broccoli Seedlings


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 analyzed 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 a 12C-CO2 atmosphere.

We conclude that sulforaphane exclusively originates from seed reserves and that de novo biosynthesis 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

Hellerstein MK, RA Neese. 1999.
Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.
American Journal of Physiology, Endocrinology and Metabolism 276: 1146-1170.

Jennings ME II, DE Matthews. 2005.
Determination of complex isotopomer patterns in isotopically labeled compounds by Mass Spectrometry.
Analytical Chemistry 77: 6435–6444.

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.
European Food Research Technology 226: 1057–1064.

 

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