Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin

Abstract

Aims: To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage.

Methods: F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment.

Results: Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the warfarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound.

Conclusions: The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage.

Figure 1

Pathways of photodegradation and glucuronidation of F.

Figure 2

H.p.l.c. chromatograms of F solution after 6 h irradiation (a) in the absence of HSA and (b) in the presence of HSA.

Figure 3

Comparison of fluorescence spectra of HSA obtained under various conditions. Conditions; (a) 0 h in the absence of F (b) 0 h in the presence of F (c) 8 h in the absence of F and (d) 8 h in the presence of F. Numbers on spectra indicate excitation wavelengths.

Figure 4

H.p.l.c. chromatograms of constituents released from HSA by alkali treatment. (a) HSA irradiated in the absence of F (b) HSA irradiated in the presence of F (c) HSA not irradiated in the presence of F.

Figure 5

Rosenthal plots of the reversible binding of F (a) and Nap (b) to HSA after irradiation in the presence and absence of F for 8 h. Closed squares and open squares indicate the values in the absence of F and the values in the presence of F, respectively. Lines represent the simulation data.

Figure 6

Photodegradation of F and Fgnd by sunlight in the presence of HSA.Conditions: photo-exposure time, 30 min; F and Fgnd concentrations, 500 μm; HSA concentration, 500 μm. Data represent mean±s.e.mean (n=3). An asterisk represents significant difference (P<0.01).

Figure 7

Effect of sunlight on the covalent binding of F and Fgnd to HSA.Conditions: photo-exposure time, 30 min; F and Fgnd concentration, 500 μm; HSA concentration, 500 μm. Data represent mean±s.e.mean (n=3). An asterisk represents significant difference (P<0.01).

Figure 8

Scheme showing the photoinduced covalent binding of F and Fgnd to HSA.