ascorbic-acid and threo-pentos-2-ulose

Vitamin C (ascorbic acid; AA) is a well-known reducing agent and has been evaluated for its antitumor activity. However, the mechanism for its antitumor action remains unclear. Tracking the metabolism of AA may help to elucidate its antitumor mechanism. In this study, a near-infrared fluorescent probe (Arg-Cy) for monitoring the metabolic products of AA in living cells was developed based on the reaction of the guanidine group in Arg-Cy with the adjacent diketone involved in the metabolites of AA. Consequently, the probe can respond to L-xylosone, a metabolite of AA, with high selectivity and sensitivity and was successfully used to visualize the real-time changes of L-xylosone levels in living cells incubated under normoxic conditions. Considering that the tumor microenvironment suffers from hypoxia, the L-xylosone levels in the process of HepG2 cell death induced by pharmacological doses of AA were also monitored under hypoxic conditions. Surprisingly, no obvious fluorescence change appeared during this process. Furthermore, detection of the intracellular redox state using a reported H2O2 probe confirmed that AA can be metabolized to L-xylosone only under normoxic conditions due to the oxidative stress, but not under hypoxic conditions. Therefore, we hypothesize that the mechanism for cell death induced by AA under hypoxia is different from that under normoxia. Thus, the developed probe can provide a tool for monitoring the metabolism of AA and may help to clarify the mechanism for the antitumor activity of vitamin C in the tumor microenvironment.

Topics: Apoptosis; Ascorbic Acid; Cell Hypoxia; Dipeptides; Fluorescent Dyes; Hep G2 Cells; Humans; Hydrogen Peroxide; Ketoses; Microscopy, Fluorescence; Spectrometry, Mass, Electrospray Ionization

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.

Topics: Antibodies, Monoclonal; Ascorbic Acid; Cell Culture Techniques; Ketoses; Molecular Structure; Oxidation-Reduction; Recombinant Proteins

Topics: Ascorbic Acid; Hydrogen-Ion Concentration; Ketoses

Topics: Ascorbic Acid; Ketoses; Phenylhydrazines; Vitamins; Xylose