Vitamin C activity of dehydroascorbic acid in humans--association between changes in the blood vitamin C concentration or urinary excretion after oral loading.

We performed oral loading of AsA or DAsA (1 mmol) in subjects who had consumed a diet low in vitamin C (C) (C< or =5 mg/d) for 3 d before loading, and measured urinary and blood vitamin C. Since the crossover method was used, the same experiment was repeated after an interval of about 1 mo in each subject. The results of the experiment including a total of 17 subjects for 2005 and 2006, were as follows. (1) There were marked individual differences in urinary C excretion. (2) The C level in 24-h urine after C loading did not differ between the two orally administered C forms (AsA and DAsA). (3) C excretion between 0 and 3 h after C loading was significantly higher (p<0.05) for the DAsA group, while those between 3 and 6, 6 and 9, 9 and 12, and 12 and 24 h after C loading were significantly higher (p<0.05 or p<0.01) for the AsA group. (4) The blood C concentration and the increase in C 1 h after C loading were significantly higher (p<0.05 and p<0.01, respectively) in the DAsA than in the AsA group. (5) Evaluation of the association between C metabolism and the single nucleotide polymorphisms of glutathione S-transferase P (GSTP) 1-1 showed a lower urinary C excretion and a significantly lower C level in 24-h urine (p<0.05) after AsA loading, and a significantly lower urinary C excretion between 0 and 3 h after DAsA loading (p<0.05) for the GA heterozygotes than for the AA homozygotes. Considering the activity of C as DAsA in humans, based on urinary and blood C levels after a single loading of C, the utilization of DAsA is equivalent to that of AsA, although the metabolic turnover time is different. The involvement of polymorphisms in the xenobiotic metabolizing enzyme, GSTP1-1, in C metabolism, particularly urinary C excretion, was also clarified. This demonstrates the necessity of considering gene polymorphisms in determining individual C requirements. An abstract of this paper was reported by the Vitamin C Research Committee (Ochanomizu University) in 2007.

Vitamin C metabolomic mapping in experimental diabetes with 6-deoxy-6-fluoro-ascorbic acid and high resolution 19F-nuclear magnetic resonance spectroscopy.

Metabolomic mapping is an emerging discipline geared at providing information on a large number of metabolites as a complement to genomics and proteomics. Here we have probed ascorbic acid homeostasis and degradation in diabetes using 6-deoxy-6-fluoro ascorbic acid (F-ASA) and 750 MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy with proton decoupling In vitro, Cu(2+)-mediated degradation of F-ASA revealed the formation of 4 major stable degradation products at 24 hours. However, when normal or diabetics rats were injected with F-ASA intraperitoneally (IP) for 4 days, up to 20 fluorine-labeled compounds were observed in the urine. Their composition resembled, in part, metal catalyzed degradation of F-ASA and was not explained by spontaneous degradation in the urine. Diabetes led to a dramatic increase in urinary F-ASA loss and a relative decrease in most other urinary F-compounds. Diabetes tilted F-ASA homeostasis toward oxidation in liver (P <.01), kidney (P <.01), spleen (P <.01), and plasma (P <.01), but tended to decrease oxidation in brain, adrenal glands, and heart. Surprisingly, however, besides the major oxidation product fluoro-dehydroascorbic acid (F-DHA), no F-ASA advanced catabolites were detected in tissues at 5 micromol/L sensitivity. These findings not only confirm the key role of the kidney in diabetes-mediated loss of ascorbic acid, but demonstrate that only selected tissues are prone to increased oxidation in diabetes. While the structure of most degradation products needs to be established, the method illustrates the power of high resolution (19)F-NMR spectroscopy for the mapping of complex metabolomic pathways in disease states.

Dehydroascorbic acid in urine as a possible indicator of surgical stress.

BACKGROUND/AIM: L-Ascorbic acid (AA) is the predominant circulating form of vitamin C found in human blood. It has been hypothesized that surgical stress increases the vitamin C metabolite dehydroascorbic acid (DHAA). Vitamin C is mainly excreted through the kidneys. In this study, the ratio of AA to DHAA excreted in urine was determined in patients who had undergone total hip joint endoprosthesis surgery (n = 12), and the results were compared with data obtained from healthy controls (n = 12). METHODS: All subjects received 1,000 mg sodium ascorbate intravenously three times a day (every 8 h) for 8 days, starting 2 days prior to surgery. Total urine was collected daily while subsequent determinations of AA and DHAA were performed photometrically. RESULTS: Administration of vitamin C led to average daily excretions of the combined products AA + DHAA of 2,343 +/- 438 mg/day (mean value +/- confidence intervals). The initial average ratio DHAA/AA of all 24 probands was 0.064 (6% DHAA; 153 +/- 76 mg/day). One day after surgery, an increase in the DHAA/AA ratio to 0.165 (15% DHAA; 332 +/- 107 mg/day) was measured in the patients. The ratio decreased 2 days after surgery and returned to normal within 5 days. CONCLUSION: Our data indicate that surgery increases the oxidation of AA and urinary excretion of DHAA, as a result of the enhanced formation of free radicals.

[The excretion of ascorbic acid and its metabolites in uremia and hemodialysis]. / Vyvedenie askorbinovoi kisloty i ee metabolitov pri uremii i gemodialize.

Renal excretion of ascorbic, dehydroascorbic and diketogulonic acids in uremia and relevant loss in hemodialysis are measured in comparison with those in patients with uremic syndrome (prior to hemodialysis) and in healthy subjects (control). Renal elimination of ascorbic acid was higher while of dehydroascorbic acid lower vs control. Elimination of diketogulonic acid was similar to control. In a session of hemodialysis, the organism loses 132.0 +/- 13.6 mg of ascorbic, 132.0 +/- 10.0 mg of dehydroascorbic and 204.0 +/- 9.0 mg of diketogulonic acid. 48-hour urinary losses of the patients reached 8.4 +/- 1.4, 19.6 +/- 1.1, 75.6 +/- 1.5 mg, respectively. Compared to control, hemodialysis patients lose the above acids 24.3, 2.7 and 4.6 times more.

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate.

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250x4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 microM CDA (S/N=3). The linear range found applying the optimized conditions was 0.2 to 200 microM. The intra- and inter-day assay precision (R.S.D.) of CDA (10 microM) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.

Measurement of ascorbate and dehydroascorbate contents in biological fluids.

Instabilities of ascorbate and dehydroascorbate throughout sample processing are clearly a significant aspect of quantifying of them. Contents of ascorbate in biological fluids decrease with measurable oxidation occurring within minutes to hours. Similarly, dehydroascorbate disappears with chemical or enzymatic degradation within minutes. The half-life of dehydroascorbate in human heparinized plasma was approximately 2 min. These results indicated that the amount of dehydroascorbate present in sample solutions is a function of both the oxidation of ascorbate and the degradation of dehydroascorbate during the processing of biological fluids. To quantify ascorbate and dehydroascorbate concentrations in biological fluids including circulating blood plasma and urine, we established a high-performance liquid chromatographic method, which requires no pretreatment of sample solutions.

Roles of urinary sodium ion concentration and pH in promotion by ascorbic acid of urinary bladder carcinogenesis in rats.

Since the sodium salt of ascorbic acid (AA) promoted two-stage urinary bladder carcinogenesis in rats, whereas AA itself did not, the roles of the urinary sodium ion concentration and pH on urinary bladder carcinogenesis were investigated. Male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks and then treated with basal diet containing 5% AA plus 3% sodium bicarbonate (NaHCO3), 5% AA, 3% NaHCO3 or 5% sodium L-ascorbate (SA), 5% SA plus 1% ammonium chloride (NH4Cl) or 1% NH4Cl, or no added chemical for 32 weeks. NaHCO3 significantly increased the induction of neoplastic and preneoplastic lesions of the urinary bladder. Like SA, AA plus NaHCO3 induced high incidences of neoplastic and preneoplastic lesions of the urinary bladder, whereas AA alone did not. NH4Cl reduced the promoting activity of SA in urinary bladder carcinogenesis. These results suggest important roles for urinary sodium ion concentration and pH in modulating urinary bladder carcinogenesis. Moreover, AA was found to act as a copromoter under conditions of increased urinary pH and sodium ion concentration.

A specific method for determination of total ascorbic acids in urine by the alpha,alpha'- dipyridyl method.

Application ot the alpha,alpha'- dipyridyl method for determination of ascorbic acid in urine is described. The urine sample was acidified with trichloracetic acid and shaken with activated carbon to remove interfering substances. The acid filtrate was first neutralized (pH 7.0) by adding Na2HPO4. The dehydroascorbic acid was then reduced back to ascorbic acid by incubation with dithiothreitol. After removal of the excess dithiothreitol with N-ethylmaleimide, ascorbic acid was determined by measuring the reduction of ferric ion. The ferrous ion produced was coupled to alpha,alpha'-dipyridyl in the presence of H3PO4. Ferrous ion in urine samples, which theoretically interferes with the method, was removed by a combination of Na2HPO4 and H3PO4.