Reduction of dehydroascorbic acid by homocysteine

To determine the reductive process of extracellular dehydroascorbic acid (DHA), molecules (homocysteine, homocysteine thiolactone, methionine, cysteine, and homoserine) were tested to identify those with the potential to reduce DHA to ascorbic acid (AA). Homocysteine (Hcy) was the most potent of the molecules tested. The efficacy of Hcy was compared with that of other molecules able to reduce DHA (reduced glutathione (GSH) and cysteine (Cy)). Although all three molecules were able to reduce DHA, GSH and Cy were not to reduce DHA to AA at concentrations lower than 100 micromol/l, and only less than 5% DHA was reduced to AA at concentrations of 200-300 micromol/l. In contrast, Hcy reduced DHA to AA stoichiometrically at concentrations as low as 10 micromol/l. In Jurkat and U937 cells, the increasing concentrations of extracellular Hcy suppressed intracellular dehydroascorbic acid uptake, indicating that extracellular reduction of DHA by Hcy leads to decreasing extracellular DHA available for its intracellular uptake. Simultaneous oxidation and reduction of Hcy and DHA were accelerated extracellularly in the presence of quercetin, an inhibitor of DHA uptake, suggesting that extracellular ascorbic acid concentration increased via blocking DHA uptake by quercetin and reducing extracellular DHA by Hcy. The effect of homocysteine on DHA reduction and uptake was confirmed with human umbilical vein endothelial cells. The oxidation of Hcy also prevented the decrease in DNA synthesis in human umbilical vein endothelial cells, which would occur following exposure to Hcy.     

Estimation of dehydroascorbic acid in blood of diabetic patients.

A modified method has been described for the estimation of ascorbic acid (AA) and dehydroascorbic acid (DHA) in blood and plasma. DHA is practically absent in the blood of normal human beings. On the other hand, diabetic patients have a persistently high blood DHA level. The DHA from diabetic blood has been isolated as the 2,4-dinitrophenylhydrazone derivative and identified by thin-layer chromatography and spectrophotometry.     

The physiological role of dehydroascorbic acid

The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub-nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln(135) in transmembrane (TM) 3, Leu(227) in TM5, and Leu(284) in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28-fold lower affinity to hY2[L227A], suggesting the Leu(227) interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three-dimensional models of the widely divergent NPY receptor subtypes.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Glucose-independent transport of dehydroascorbic acid in human erythrocytes

It has been previously reported that glucose and its structural analogs inhibit dehydroascorbic acid (DHA) transport across the membranes of nonpolar cells, which led to the suggestion that the hexose transporter mediates dehydroascorbic acid transport. The present study examines the role of the erythrocyte hexose transport system in dehydroascorbic acid uptake. We have confirmed that dehydroascorbic acid may be a ligand of the hexose transport system under certain experimental conditions. However, there is an additional pathway of dehydroascorbic acid transport that is uninfluenced by external glucose. This pathway is one of facilitated diffusion, demonstrating saturation kinetics of transport, cis-inhibition, and trans-stimulation. The Km for the system is 412 microM. It is suggested that this previously undescribed sugar-independent transporter is the physiologically important route of DHA uptake in erythrocytes.