Hypochlorous acid and 3,4-dihydroxyphenylalanine increase the formation of serum protein lipofuscin-like fluorophores in vitro.

Introduction: Serum protein lipofuscin-like fluorophores (LLFs) that include fluorescent advanced glycation end products (AGEs), are an index of protein modification and levels are abnormally high in hemodialysis patients. To investigate the possibility that hypochlorous acid (HOCl) and 3,4-dihydroxyphenylalanine (DOPA) may contribute to high LLFs concentrations, we have examined the effect of these factors on serum protein LLF formation in vitro.

Methods: Protein LLF concentration was measured at excitation 350 nm and emission 460 nm and was expressed in arbitrary units relative to quinine sulphate fluorescence. Oxidation of serum or other solutions with HOCl was carried out at room temperature for 30 minutes and serum was delipidated before measurement of protein LLFs.

Results: Serum protein LLF concentration increased non-linearly by a maximum 247% with increasing HOCl concentration in the range 6.5-32.9 mmol/L and this was mirrored by a decrease in protein tryptophan fluorescence. HOCl (32.9 mmol/L) increased LLFs in human gamma-globulin solutions (15-fold in 12 mg/mL and 5-fold in 60 mg/mL solutions) and did not alter LLFs appreciably in human serum albumin solution (60 mg/mL). Addition of DOPA (265 micromol/L) significantly (P<0.001) increased LLF formation in serum by nearly 2-fold during 3 days incubation under air.

Conclusions: These data suggest that HOCl and DOPA are capable of generating serum protein LLFs and that gamma-globulins appear to be an important substrate for protein LLF formation in human serum. These findings may be relevant to the abnormally high concentrations of serum protein LLFs and impaired immune response in hemodialysis patients.