Liquid chromatographic measurement of -ascorbic acid and -ascorbic acid in biological samples

- and -Ascorbic acids have been separated using liquid chromatography (LC) on a polymer-coated silica-based NH₂ column and the -isomer has been quantified in human serum, rat serum, rat lung, rat lung perfusate, infant formula (SRM 1846) and mixed food sample (SRM 2383). The -isomer was observed only in trace amounts in the mixed food sample. The results demonstrate that ascorbic acid was stable on the column and completely recovered from supplemented samples of human serum and that this method of analysis is accurate, precise and has broad application exhibiting no dependence on the nature of the matrices evaluated herein.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

Preparation of dehydro-l-(+)-ascorbic acid dimer by oxidation of ascorbic acid with arsenic acid/iodine and formation of complexes between arsenious acid and ascorbic acid

Ascorbic acid in the presence of a catalytic amount of iodine reduces arsenic acid in methanol giving the arsenious acid bound to the 2-methyl hemi-ketal of dehydroascorbic acid, 5, in 1:1 and in a more stable 2:1 5/As(III) molar ratio. Removal of the As(III) and treating the 2-methyl hemi-ketal of dehydroascorbic acid with refluxing acetonitrile affords the pure, crystalline dehydroascorbic acid dimer in good yields. Ascorbic acid also binds to As(III) of H(3)AsO(3) in a 1:1 and 2:1 ascorbic acid/As(III) molar ratio. The 1:1 complex is not stable and by expulsion of H(3)AsO(3) is transformed to the more stable 2:1 complex. The data do not permit distinguishing the 2:1 complexes between [AsL(2)(H(2)O)](-)H(+) or AsL(LH)(H(2)O) where L is the bis deprotonated and LH is the mono deprotonated 2-methyl hemi-ketal of dehydroascorbic acid or ascorbic acid. The 2:1 ascorbic acid/As(III) complex is oxidized by dioxygen, in a solvent-dependent manner, to dehydroascorbic acid implying dioxygen activation by the bound As(III). With thiophenol the same complex gives quantitatively triphenyl trithioarsenite, As(SPh)(3).     

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.     

The half-life and uptake of 14c-1-ascorbic acid in selected organs of rainbow trout (salmo gairdneri)

• 1.1. The uptake and apparent half-life of ¹⁴C-1-ascorbic acid was determined in selected organs of rainbow trout over an 84 day period.
• 2.2. Brain ascorbic acid concentrations in the trout were consistently higher than those in the other tissues measured.
• 3.3. Uptake of ¹⁴C radioactivity by whole brain tissues of the trout was initially lower than in the other tissues; however, ¹⁴C radioactivity continued to increase in this organ until 42 days post-injection. This would indicate that whole brain tissues of the trout take a long time to reach an equilibrium state and suggests the existence of a blood-brain barrier to ascorbic acid in the central nervous system of the trout.
• 4.4. The half-life of ascorbic acid in selected organs of the rainbow trout appears to be very much longer than that of other animals.
• 5.5. Specific activity measurements suggest that the rainbow trout has a slow rate of synthesis of ascorbic acid.