Handbook of Vitamin C Research

Human Specific Vitamin C Metabolism… 71DAsA is almost 10% that of AsA on a molar basis, based on experiments using the inherentlyscorbutic ODS rat (devoid of GLO) [46]. However, human experiments (Table 2 and 3) [4, 8,70] have clearly shown that the nutritional activity of DAsA is nearly equal to that of AsA.This discrepancy may be explained by the difference in DAsA metabolism between humanswith high reducing power and low dehydroascorbatase [3] and ODS rats with 24- to 50-foldhigher dehydroascorbatase activity when compared with humans [3]. The specific activity ofhuman liver dehydroascobatase is only 0.4 to 1 mol/min/mg protein, while those in rats andcows are 24 and 37 mol/min/mg protein, respectively [3]. Both human [51] and rat [52]dehydroascorbatases have been sequenced and identified as senescent marker protein 30(SMP30) and gluconolactonase [53] with 299 amino acid residues. X-ray crystallography ofbacterial (Xanthomonas campestris) gluconolactonase identified four residues coordinatingwith a central calcium ion (E48, N134, N191 and D242), which are also conserved in thecorresponding residues of human and rat dehydroascorbatase (E17, N103, N155 and D204,respectively) [54]. However, four ionizing residues (E83, K(E)95, E231 and D254) conservedamong bovine, rabbit, rat [52] and mouse dehydroascorbatase were converted to K83, N95,V232 and N253, respectively, in human dehydroascorbatase [51]. These residue changes mayexplain the large species difference in dehydroascorbatase activity in the human liver [3]. Toconfirm its activity, human dehydroascorbatase was produced in E. coli BL21 via the pColdII-humanSMP30 vector (A. Ishigami, personal communication). The amino acid sequence ofhuman dehydroascorbatase was 91%, which is homologous to that of the bovine enzyme; theactivity was found to depend on divalent cations.The non-enzymatic delactonization of DAsA follows pseudo-first order reaction kinetics,-(d[DAsA]/dt) = k[DAsA], with k values (s -1 × 10 -3 M) at pH 6.8, 7.0, 7.2, 7.4 and 7.6 of0.77, 1.15, 1.76, 2.41 and 3.18, respectively, in the presence of 6.7 mM Mg 2+ [46]. The rapidnon-enzymatic hydrolysis of DAsA is prevented by rapid reduction by enzymes withdehydroascorbic acid reductase activity [1, 69], including glutaredoxin [2], thioredoxin [47],omega class glutathione transferase [48] and serum albumin [49], and by non-enzymaticreduction with SH compounds [1].SMP30 (dehydroascorbatase) is the most abundant anti-xenobiotic [80] and anti-agingprotein [55] in the liver, accounting for about 1% of total liver protein, as demonstrated bypurification to ultracentrifugal homogeneity [53]. SMP30 has lactonase activity towardvarious aldonolactones and requires Zn 2+ or Mn 2+ as a cofactor. The lactonase reaction is thepenultimate step in AsA biosynthesis, and the essential role of SMP30 in the AsA syntheticprocess was verified in a nutritional study using SMP30 knockout (KO) mice. SMP30 hasparaoxanase activity to hydrolyze organophosphorus compounds and to counteract oxidativestress [56]. Administration of organophosphorus compounds stimulate VC metabolism andretard growth, but excess intake of VC under toxic conditions is effective in counteractingtoxic effects and restores dehydroascorbatase levels [80]. Prevention of oxidative stressdepends on antioxidants such as VC and reactions by xenobiotic enzymes.Dehydroascobatase is also known as regucalcin, and its gene expression appears to be asensitive marker to evaluate the renal impairment caused by chemicals, while its downregulationseems to be related to damage [57].The metabolic network to counteract xenobiotics is complex. Stable isotope-labeled VC(30 mg l-[1- 13 C] AsA) was given alone or with Fe (100 mg as ferrous fumarate) to produce