Figure 1.
The structures of quercetin aglycone and plasma metabolites.
Quercetin is part of the flavonol subclass of flavonoids. Metabolites of quercetin include methylated (4′-O-methyl quercetin: tamarixetin), sulphated (quercetin-3′-sulphate) and glucuronidated (quercetin-3-glucuronide) counterparts.
Figure 2.
Platelets internalise quercetin and tamarixetin.
Platelets suspended to a density of 2×108 cells.mL−1 were incubated with quercetin (100 µM: (B.i-iii) and (C.i-iii)), tamarixetin (40 µM: (D.i-iii) and (E.i-iii)) or solvent control (DMSO (0.2% (v/v)): (A.i-iii)) for 30 min. Fluorescence was detected at 480 nm–500 nm after excitation at 430 nm with an argon laser. Images of a single middle layer from z-stacks are shown (DMSO control: (A.i-iii), quercetin: (B.i-iii) tamarixetin: (C.i-iii)) and higher magnifications of areas of interest are also shown (quercetin: (D.i-iii), tamarixetin: (E.i-iii)). Images represent results from at least 3 individual experiments.
Figure 3.
Quercetin and tamarixetin associate with platelets.
Quercetin (50 µM) and tamarixetin (50 µM) were incubated with platelets (8×108 cells.mL−1) for 5, 40, 60 or 120 min prior to lysis with 50% (v/v) methanol and 0.1% (v/v) HCl. Compounds within extracts obtained from platelet lysates spiked with myricetin (external control compound of similar structure) were separated over 60 min using HPLC analysis with photodiode array detection. Chromatograms show quercetin ((A)-RT: 46.1 min) and tamarixetin ((B)-RT: 51.6 min) associated with platelets through comparison with standards (quercetin: D; tamarixetin: 3E), untreated platelets (plasma control: C) and the external control, myricetin ((A, B)-RT: 39 min; F: standard). Insets show UV absorbance spectrum (λ: 360 nm) of detected compounds. Data represent 3 individual experiments.
Figure 4.
Quercetin is metabolised by platelets.
Platelets (8×108 cells.mL−1) pretreated with quercetin (50 µM) and tamarixetin (50 µM) for 5, 40, 60 or 120 min were lysed with 50% methanol and 0.1% formic acid. Extracts from lysed platelets were separated by HPLC utilising UV detection (λ: 210 nm) over a period of 20 min, before identification of the protonated masses of compounds using mass spectrometry. Mass spectrums show the ([M+H+]+) of quercetin (molecular mass: 303 Da), the platelet metabolite (molecular mass: 317 Da) and the external control spike, myricetin (molecular mass: 319 Da) at 5 min (A: with plasma control inset) and 60 min (B). EIC and mass spectrums of standards show tamarixetin (EIC: C.i-RT: 7.5 min; mass spectrum: C.ii-molecular mass: 317 Da), quercetin (EIC: (D.i)-RT: 7 min; mass spectrum: (D.ii)-molecular mass: 303 Da) with inset MS spectrum showing the absence of tamarixetin (ii) and myricetin (EIC: (E.i)-RT: 6.5 min; mass spectrum: (E.ii)-molecular mass: 319 Da). Data represent 3 individual experiments using platelets isolated from 3 different blood donors.