title: Investigation of the fate of dietary flavonols in humans and rats using HPLC-MS2 techniques
Creators: name: Mullen, William
Publisher: name: University of Glasgow
Year: 2009
Subjects: subject: Flavonols, metabolism, LCMS, onions, quercetin

description: There is a growing evidence of the potential health benefits of a diet rich in fruits and vegetables. National nutritional guidelines advise the consumption of at least five portions (400 g ) of these foods per day with the goal being a reduction in the levels of coronary heart disease (CHD) strokes and certain cancers. 
The beneficial properties of fruit and vegetables may be ascribed, in part to the presence of antioxidants and recent attention in this regard has focused on phenolic and polyphenolic compounds. These compounds are present in a wide variety of commonly consumed foods and beverages.
Onions are a rich source of the polyphenolic flavonol quercetin-4′-glucoside. For this compound to have some health effects it must be absorbed and reach target organs in a concentration and form where they can exert an effect. To-date interest has focussed on the levels of the intact quercetin aglycone circulating in plasma and excreted in urine. However, it is now known that quercetin does not circulate in the plasma as the parent compound or the aglycone. However, at the outset of this project, the exact form(s) and concentration of metabolites circulating in plasma were unknown.
The need to know what compounds are actually circulating, and at what concentration, is important if in vitro studies are to be made into the mechanisms by which quercetin could, potentially, exert a health benefit.
The reasons why these issues have not been addressed are due to a number of factors. The main methodology used in studies into absorption, distribution, metabolism and excretion or (A.D.M.E) as it is know in the drug industry, is by use of chromatography coupled to various detection systems. This can range from a simple isocratic single pump linked to a single wavelength absorbance detector, to a gradient pumping system with an autoinjector linked in series to a diode array absorbance detector and mass spectrometer. The latter instruments, although initially expensive are now becoming more affordable.
The original methodology used to determine the level of quercetin in plasma involved hydrolysis of the quercetin conjugates back to the aglycone. The information, which is lost by using this hydrolysis method, is vital if we are to gain a better understanding of the A.D.M.E process. There have been a large number of feeding studies carried out using onions or the flavonol contained in them. However, very little additional information was gained after the initial investigations. 
The objectives of the studies presented in this thesis were to develop methodology to identify and quantify the major metabolites of quercetin in man after ingestion of onions. This would initially require the use of radiolabelled [2-14C]quercetin-4´-glucoside fed to rats to facilitate the development of the method. Having successfully developed methods that would work both in rats and in man, it was of great interest to establish the fate of the complete dose of [2-14C]quercetin-4´-glucoside in rats.
In Chapter 2 radiolabelled quercetin-4´-glucoside was used as a tracer to follow the metabolism of the compound as it was acted on by the digestive system of the rat. After 1 h 93% of the ingested dose was recovered in the gastrointestinal tract (GIT). Analysis using HPLC with a photodiode array (PDA) detector in series with a radioactivity monitor connected to an electrospray ion trap mass spectrometer facilitated the separation, quantification and partial identification of 18 out of 19 metabolites. The 1 h sample was part of a larger study that investigated the fate of the radiolabelled compounds up to 5 h after dosing. The latter samples formed part of another study not reported on in this thesis.
Having developed the methodology, using the radiolabelled compound, it was then applied to a feed of onions to healthy human volunteers to determine if metabolite detection, identification and quantification could be carried out without the use of the radioactive tracer. In Chapter 3 plasma samples collected 1 h after a feed of onions and urine collection from 0-4 h post feed were used to test if the method could be transferred to a non labelled assay. A total of 22 metabolites plus the parent compound were identified. The metabolic profile of the plasma and urine showed marked differences, again pointing to major post absorption metabolism. The successful transfer of the method from the initial radiolabelled study to the onion feed allowed pharmacokinetic data to be obtained from all plasma samples taken over a 24 h period, along with the 0-24 h urine samples. In Chapter 4 it was seen that the metabolites are both rapidly absorbed and excreted, with plasma levels returning almost back to baseline by 6 h. The total excretion in urine accounted for 4.5% of the ingested dose. These results were controversial, as the pioneer of this field had published that the elimination half life of quercetin was of the order of 18 h. The differences between the two methods employed are discussed in Chapters 3 and 4.
The fact that only 4.5% could be accounted for in this study, which was in agreement with other studies, leaves the question of what happens to the other 95.5%. It is possible that the potential health benefit attributed to this compound may have nothing to do with the parent compound but could be coming from something in the other 95.5%.
Studies using patients who have undergone an ileostomy have been used to provide further information into what happens to the majority of the dose. By collecting the ileal fluid after a flavonol feed the amount of intact compound can be measured in ileal fluid (Hollman et al., 1995b; Walle et al., 2000). This work and some results from a similar trial study are discussed in Chapter 4, with regard to the process of metabolite absorption and formation. 
 The only way to follow the parent compound throughout its passage through the body is by use of a labelled compound. In Chapter 5 a second feed of [2-14C]quercetin-4´-glucoside, which focuses on the overall fate of the compound, has samples collected for up to 72 h. The fate of the dose was monitored both in terms of the level of radioactivity excreted and found in the tissues and also the identity of the radioactive compounds detected in these samples. 
In Chapter 5 the results from this study and what impact they could have on quercetin’s potential ability to be the compound responsible for the health benefits are discussed.