Studies on the mutagenic activity of ascorbic acid in vitro and in vivo

In vitro data are presented to show that ascorbic acid does not have intrinsic mutagenicity towards strain TA100 of S. typhimurium if deionized water is used to prepare the incubation medium. The addition of Cu2+ ions to the bacterial medium that contains ascorbic acid, or the use of tap water and ascorbic acid alone, causes a mutagenic and cytotoxic response that is blocked by EDTA. Additional in vitro data demonstrate that hydrogen peroxide is mutagenic to S. typhimurium strain TA100 and it is suggested that ascorbic acid may be mutagenic and cytotoxic through the generation of hydrogen peroxide. In vivo studies using a sensitive intrahepatic host-mediated mutagenicity assay indicate that ascorbic acid is not genotoxic in guinea pigs even when the dietary intake of vitamin C is above the level required for tissue saturation (5000 mg/kg body weight/day).     

Effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice

The effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice has been studied. After the mice were on diets containing added ascorbic acid for two months, the activities of ascorbic acid synthesizing enzymes in the mouse liver homogenates were measured using L-gulono-gamma-lactone as a substrate. Exogenous ascorbic acid intake (0.5, 1 or 5% in the diet) was able to increase the concentration of ascorbic acid in the blood and to decrease the activities of ascorbic acid synthesizing enzymes in mouse liver. The results suggest that ascorbic acid synthesis was controlled by local regulatory mechanism or by the concentration of ascorbic acid in the hepatic portal blood. Ingestion of dietary erythorbic acid, a stereoisomer of ascorbic acid, had no effect on the activities of ascorbic acid synthesizing enzymes.     

Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1.2x10(6) and 6.5x10(4) respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.     

Hypercholesterolemia due to ascorbic acid.

Rats fed a purified diet containing ascorbic acid developed hypercholesterolemia. Because rats do not require exogenous ascorbic acid, they may be comparable to humans who supplement their diets with ascorbic acid in capsular form. The amount of ascorbic acid in this experiment was equivalent to 82 to 630 mg of capsular ascorbic acid ingested by an average man and was well below the amount ingested by those in search of respiratory benefit. The data are consonant with those on humans consuming controlled diets.     

Studies on a new metabolite and its oxidation in the presence of ascorbic acid

1. A number of tissues, in particular, brain, liver, and kidney, incubated aerobically in vitro as slices or ground suspensions produce a compound which combines with p-aminobenzoic acid in acid solution to form a yellow color. 2. A study of this reaction in rat brain has shown that this compound can be produced when washed boiled brain protein is incubated aerobically with ascorbic acid. The latter acts as a catalyst to break the linkage between the protein and the compound. Oxygen is taken up in the process. 3. A number of aromatic hydroxy compounds such as epinephrine and catechol inhibit the reaction. Cyanide has little or no effect. No reaction occurs anaerobically. 4. The occurrence of the reaction in some animals has been described.     

Kinetic studies on the reaction of compound II of myeloperoxidase with ascorbic acid. Role of ascorbic acid in myeloperoxidase function

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 +/- 0.9 x 10(-4) to 2.0 +/- 0.3 x 10(-3)M and a first-order rate constant for reduction of 12.6 +/- 0.6 to 18.8 +/- 1.3 s-1. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-1. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide----compound I followed by compound I + chloride----HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.     

Pulmonary edema and ascorbic acid loss.

Loss of ascorbic acid from lung and pulmonary edema were produced in mice by intravenous injection of either adrenaline or noradrenaline (5 mumol/kg). While adrenalectomy performed before noradrenaline administration reduced the degree of pulmonary edema, a prior dose of hexamethonium accentuated this effect. Given alone, hexamethonium caused both loss of ascorbic acid and pulmonary edema. The results show that although endogenous catecholamines can potentiate the pulmonary edema produced by either adrenaline or noradrenaline, they play no specific role in the ascorbic acid loss. The evidence suggests that lung ascorbic acid levels are decreased following the development of pulmonary edema, irrespective of how it was caused.