Formation of phosphoglycosides in Caenorhabditis elegans: a novel biotransformation pathway.

BACKGROUND: Caenorhabditis elegans (C. elegans) has become a widely used model to explore the effect of food constituents on health as well as on life-span extension. The results imply that besides essential nutrients several flavonoids are able to impact the aging process. What is less investigated is the bioavailability and biotransformation of these compounds in C. elegans. In the present study, we focused on the soy isoflavone genistein and its metabolism in the nematode as a basis for assessing whether this model system mimics the mammalian condition. PRINCIPAL FINDINGS: C. elegans was exposed to 100 µM genistein for 48 hours. The worm homogenate was extracted and analyzed by liquid chromatography (LC). 11 metabolites of genistein were detected and characterized using LC electrospray ionization mass spectrometry. All genistein metabolites formed by C. elegans were found to be sugar conjugates, primarily genistein-O-glucosides. The dominant metabolite was identified as genistein-7-O-phosphoglucoside. Further interesting metabolites include two genistein-di-O-glycosides, a genistein-O-disaccharide as well as a genistein-O-phosphodisaccharide. CONCLUSIONS/SIGNIFICANCE: Our study provides evidence for a novel biotransformation pathway in C. elegans leading to conjugative metabolites which are not known for mammals. The metabolism of genistein in mammals and in C. elegans differs widely which may greatly impact the bioactivity. These differences need to be appropriately taken into consideration when C. elegans is used as a model to assess possible health or aging effects

Electrospray mass spectrometric detection (QTrap system) of genistein-<i>O</i>-monohexosides formed by <i>C. elegans</i>.

<p>(A) Total ion chromatogram (TIC) of the enhanced product ion (EPI) scan of the <i>m/z</i> 431 precursor showing four genistein-<i>O</i>-monohexoside metabolites. The two major ones are named M10 and M11; two additional minor metabolites are marked as M8 and M9. (B) MS/MS spectrum of the standard compound genistein-7-<i>O</i>-ß-D-glucoside. (C) MS/MS spectrum of metabolite M10, identified as genistein-7-<i>O</i>-ß-D-glucoside using the corresponding standard compound. (D) Proposed structures of the fragment ions observed in the MS/MS spectra shown under (B) and (C).</p

Electrospray mass spectra (QTrap system) of the deprotonated genistein metabolite M4 assigned as a genistein-phosphodisaccharide.

<p>(A) MS spectrum. (B) MS/MS spectrum of <i>m/z</i> 673. (C) Proposed structures of the product ions observed in the MS/MS spectra. Hex, Hexose.</p

Representative LC-UV chromatogram of the organic <i>C. elegans</i> extract after exposure of nematodes to genistein.

<p>Incubations were performed with genistein (black line) and without genistein (controls, grey line) for 48 hours. The chromatogram was monitored at 260 nm and is displayed on two different scales: (A) Full scale chromatogram showing the dominance of M5 and M10, which represent around 80% of the genistein metabolites based on the peak areas. (B) Zoomed chromatogram showing the detailed genistein metabolite spectrum. M2 (marked by a dashed line) was too small to be visualized using UV detection but was detected by mass spectrometry.</p

Proposed pathway for the conjugative metabolism of <i>C. elegans</i> leading to the main metabolites.

<p>UGT, UDP-glycosyltransferase.</p

Predicted formula, determined and theoretical mass/charge ratios as well as the mass error of fragment ions observed in the product ion mass spectrum of <i>m/z</i> 511.

<p>Predicted formula, determined and theoretical mass/charge ratios as well as the mass error of fragment ions observed in the product ion mass spectrum of <i>m/z</i> 511.</p

Electrospray mass spectra (QTrap system) of the deprotonated main genistein metabolite M5, identified as a genistein-monophosphoglucoside.

<p>(A) MS spectrum. (B) MS/MS spectrum of <i>m/z</i> 511. (C) MS/MS/MS spectrum of <i>m/z</i> 511→<i>m/z</i> 241.</p

Electrospray mass spectrometric detection (QTrap system) of genistein-di-<i>O</i>-hexosides and genistein-<i>O</i>-disaccharides formed by <i>C. elegans</i>.

<p>(A) MS/MS spectrum of metabolite M7, assigned to a genistein-disaccharide with a proposed chemical structure. (B) MS/MS spectrum of metabolite M1, identified as genistein-4′,7-di-<i>O</i>-ß-D-glucoside using a standard compound. (C) MS/MS spectrum of metabolite M3, characterized as a genistein-di-<i>O</i>-hexoside. (D) Proposed structures of the product ions observed in the MS/MS spectra shown under (A), (B) and (C). Hex, Hexose.</p