Turnover ascorbate

Section 13.3.8) can increase the rate of ascorbate turnover several-fold in species for which it is not a vitamin. 

A number of xenobiotics - such as polychlorinated biphenyls, DDT, and aminopyrine - increase the urinary excretion and tissue concentrations of ascorbate in rats, and increase the incorporation of label from [ C]glucose into ascorbate. The rate of ascorbate turnover can increase 5- to 10-fold under... 

Mobilization and Metabolism. The total ascorbic acid body pool in healthy adults has been estimated to be approximately 1.5 g, which increases to 2.3—2.8 g with intakes of 200 mg/d (151—158). Depletion of the body pool to 600 mg initiates physiological changes, and signs of clinical scurvy are reported when the body pool falls below 300 mg (149). Approximately 3—4% of the body pool turns over daily, representing 40—60 mg/d of metabolized, or consumed, vitamin C. Smokers have a higher metaboHc turnover rate of vitamin C (approximately 100 mg/d) and a lower body pool than nonsmokers, unless compensated through increased daily intakes of vitamin C (159). The metaboHsm of ascorbic acid varies among different species. 

Ascorbic acid (vitamin C) depletion is the most consistent evidence of compromised antioxidant status in diabetes with reports of reduced levels and altered metabolic turnover in several tissues in experimentally induced diabetes in animals (Rikans, 1981 Yew, 1983 McLennan et al., 1988) and in patients with diabetes (Som et al., 1981 Jennings et al., 1987 Sinclair et al., 1991). 

So-called blue multinuclear copper oxidase enzymes, such as laccase and ascorbate oxidase, catalyze the stepwise oxidation of organic substrates (most likely in successive one-electron steps) in tandem with the four-electron reduction of O2 to water, i.e. no oxygen atom(s) from O2 are incorporated into the substrate (Eq. 4) [15]. Catechol oxidase, containing a type 3 center, mediates a two-electron substrate oxidation (o-diphenols to o-chinones), and turnover of two substrate molecules is coupled to the reduction of O2 to water [34,35]. The non-blue copper oxidases, e.g. galactose oxidase and amine oxidases [27,56-59], perform similar oxidation catalysis at a mononuclear type 2 Cu site, but H2O2 is produced from O2 instead of H2O, in a two-electron reduction. 

However, the average number of turnovers an enzyme molecule is able to perform before deactivation varies substantially among preparations two turnovers for ascorbate peroxidase [50], 238 for horseradish peroxidase A2 [28], 265 for horseradish peroxidase C [51], and 81,000 for Zo peroxidase [28]. 

Systematic examination of the catalytic properties of dimeric complexes was initiated shortly after the identification of dinuclear iron sites in metalloenzymes. The first report of a reactive dimeric system came from Tabushi et al. in 1980, who examined the catalytic chemistry of [Fe3+(salen)]20, 1 (salen is N,N -(salicylaldehydo)-l,2-ethylenediamine) (12). They reported interesting stereoselectivity in the oxidation of unsaturated hydrocarbons with molecular oxygen in the presence of mercaptoethanol or ascorbic acid and pyridine as a solvent ([l]<<[alkane]<<[2-mercaptoethanol]). With adamantane as substrate, they observed the formation of a mixture of (1- and 2-) adamantols and adamantanone (Table I) (12). Both the relative reactivity between tertiary and secondary carbons (maximum value is 1.05) and final yield ( 12 turnovers per 12 hr) were dependent on the quantity of added 2-mercaptoethanol. Because autoxidation of adamantane gave a ratio of 3°/2° carbon oxidation of 0.18-0.42, the authors proposed two coexisting processes autooxidation and alkane activation. 

There have been two major studies of ascorbate requirements in deple-tion/repletion studies, one in Sheffield during the 1940s (Medical Research Council, 1948) and the other in Iowa during the 1960s (Baker et al., 1969,1971 Hodges etal., 1969,1971). In addition, Kallnerand coworkers (1979,1981) have determined the body pool of ascorbate and the fractional rate of turnover under various conditions. Levine and coworkers (1995, 1996, 1999) have measured plasma and leukocyte ascorbate in studies of subjects maintained on more than minimally adequate amounts of vitamin C for relatively prolonged periods of time to determine optimum, rather than minimum, requirements. 

A priori, the best means of determining vitamin C requirement would seem to be determination of the total body pool and its fractional rate of loss or catabolism. An appropriate intake would then be that to replace losses and maintain the body pool. Clinical signs of scurvy are seen when the total body pool of ascorbate is below 1.7 mmol (300 mg). The pool increases with intake, reaching a maximum of about 8.5 mmol (1,500 mg) in adults -114 /rmol (20 mg) per kg of body weight. The fractional turnover rate of ascorbate is 3% to 4% daily, suggesting a need for 45 to 60 mg per day for replacement. The basis for the 1989 U.S. Recommended Daily Allowance (RDA) of 60 mg (National Research Council, 1989) was the observed mean fractional turnover rate of 3.2% of a body pool of 20 mg per kg of body weight per day, with allowances for incomplete absorption of dietary ascorbate and individual variation. 

Because the mean fractional turnover rate of 3.2% per day was observed during a depletion study, and the rate of ascorbate catabolism varies with intake, it has been suggested that this implies a rate of 3.6% per day before depletion. On this basis, and allowing for incomplete absorption and individual variation, various national authorities arrive at a reference intake of 80 mg per day. 

The rate of ascorbate catabolism is not constant. If it were, more or less complete depletion of the body pool would be expected within 25 to 33 days yet, in the Sheffield study, in which the subjects were initially maintained on 70 mg of ascorbate per day, they received a diet essentially free from the vitamin no changes were apparent for 17 weeks (Medical Research Council, 1948). In the Iowa study, the subjects were not initially saturated with vitamin C the first skin lesions did not develop for 5 to 6 weeks after the depletion period (Baker etal., 1969,1971 Hodges etal., 1969,1971). Kallner and coworkers (1979) showed that the turnover time of body ascorbate varied between 56 days at low intake (about 15 mg per day) and 14 days (at intakes of 80 mg per day). It is thus apparent that the rate of ascorbate catabolism is affected markedly by the intake, and the requirement to maintain the body pool cannot be estimated as an absolute value. A habitual low intake, with a consequent low rate of catabolism, will maintain the same body pool as a habitual higher intake with a higher rate of catabolism. 

Kallner A, Hartmann D, and Hornig D (1979) Steady-state turnover and body pool of ascorbic acid in man. American Journal of Clinical Nutrition 32, 530-9. 

In the case of laccase and ascorbate oxidase, the observed ET rates for the reduction of the type-3 coppers (see Table VIII) are lower than the observed turnover number. This can be explained only by the possibility that the enzymes are in a resting form under the experimental conditions. A considerable reorganization energy seems to be necessary to get to the reduced state of the type-3 coppers (release of the bridging OH" and movement of the copper GU2 and GU3). From these data it cannot be decided what the rate-limiting step is in the catalytic cycle, either this intramolecular ET or the reaction of the dioxygen at the trinuclear copper site. 

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