Figure 4.

The reaction of 5-HMF and VC in vitro and, thereby, a reduction in the VC content in PC12 cells. (A) The UV-visible absorption spectra of 5-HMF. A total of 20 μmol/L 5-HMF in 10 mmol/L phosphate buffer, pH 7.4, was titrated with increasing concentrations (0, 2.5, 5, 10, 15 and 20 μmol/L) of Fe²⁺, 2-OG or VC in vitro. After incubation for 2 min, the absorption spectra of 5-HMF was obtained from 200 to 500 nm by UV-visible spectroscopy analysis. The absorption peak of 5-HMF is 285 nm. (B) The correlation curves of concentration and absorbance difference compared with 285 nm (Δabs 285 nm). There is no correlation between 5-HMF and Fe²⁺ or 5-HMF and 2-OG; however, there is a positive correlation between 5-HMF and VC, which indicates the reaction of 5-HMF and VC. (C) Determination of the content of 5-HMF in the intracellular cytoplasm. The content of 5-HMF in PC12 cells was detected by HPLC after the cells were exposed to 5-HMF in the medium for 1 h. 5-HMF was detectable in the cytoplasm, and the content increased with increasing concentrations of ex-tracellular 5-HMF. (D) The reduction of VC content by 5-HMF. The VC content was detected by UV-visible spectroscopy analysis after the cells were exposed to 5-HMF (100 μg/mL) for 1 h. The data are representative of four different experiments and are expressed as the means ± SE. **p < 0.01, compared with the control.