Use of a new vitamin C-deficient diet in a depletion/repletion clinical trial.

To conduct an inpatient study on the recommended dietary allowance (RDA) for vitamin C, we developed a unique vitamin C-deficient diet using a nutrient database and selective menus. Fourteen different menus were developed offering > 300 items with 0-2.4 mg vitamin C per serving. During the 4-6 mo volunteers were hospitalized, daily dietary vitamin C was restricted to < or = 5.0 mg. The mean daily dietary vitamin C intake was < 3.9 mg for the seven study subjects. With concurrent supplementation, the diet provided > or = 85% of the RDA for 17 essential nutrients. Within 3 wk of admission the diet induced vitamin C deficiency as indicated by plasma concentrations, which decreased from 23 +/- 6.9 to 6.9 +/- 2.0 mumol/L. Daily intake of vitamin C and five other nutrients was determined by nutrient database analyses. Mean energy, protein, the iron were 105-185% of the RDA and total and saturated fat were 32% and 10% of energy, respectively. Weight and nutritionally relevant indexes remained normal. Dietary adherence, calculated by the number of days with < or = 5.0 mg vitamin C per total study days, was 88-98% per repletion dose. Computer analyses of menu selections permitted individual preferences to be met while restricting vitamin C intake to < or = 5.0 mg/d. There were no complications from the diet during the depletion and repletion phase. With this diet, ascorbic acid pharmacokinetics for escalating doses could be determined in healthy volunteers.

Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.

Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value.

Determination of optimal vitamin C requirements in humans.

Although the recommended dietary allowance provides an estimate for vitamin C ingestion in humans, optimal vitamin C requirements are unknown. We define optimal vitamin C requirements operationally based on the following: dose-function relations, availability in the food supply, steady state concentrations in plasma and tissues achieved at each dose of vitamin C, urinary excretion, bioavailability, toxicity, and epidemiologic observations. Optimal vitamin C requirements can be estimated when information is available for at least some of these criteria.

Role of Mg-ATP in norepinephrine biosynthesis in intact chromaffin granules.

Dopamine beta-monooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (delta psi) because norepinephrine biosynthesis was unchanged whether delta psi was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (delta pH). Nevertheless, norepinephrine biosynthesis was inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N,N-dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining delta psi and delta pH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine beta-monooxygenase.

Cellular functions of ascorbic acid: a means to determine vitamin C requirements.

Optimal ascorbic acid (vitamin C) requirements in humans are unknown. In situ kinetics is a biochemical approach to determine requirements for vitamin C and other vitamins. In situ kinetics requires that cellular functions of ascorbic acid are characterized. Vitamin-C-dependent cellular reactions are directly related to vitamin C concentrations inside and outside cells. By coupling intracellular and extracellular functions of ascorbic acid to vitamin concentration, in situ kinetics provides a novel approach to determining vitamin C requirements.

A parkinsonian syndrome induced in the goldfish by the neurotoxin MPTP.

Parkinson's disease has been modeled in humans, lower primates, and to a lesser extent in some other vertebrates by administration of the potent neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine). The MPTP model has thus drawn considerable attention as a system to search for anti-Parkinson's disease drugs, although the cost and scarcity of primates has limited extensive applications. We now report that a parkinsonian syndrome can be elicited in the common goldfish (Carassius auratus) by a single dose of MPTP. The syndrome is characterized by profound bradykinesia (slow movement), the full extent of which is reached 3 days after MPTP administration. The reduction in movement is paralleled by loss of dopamine and norepinephrine from the forebrain and midbrain and in other brain regions as well. The toxic oxidative product of MPTP, MPP+, is also accumulated predominantly in forebrain and midbrain, and pretreatment with the monoamine oxidase blocker tranylcypromine substantially reduces accumulation of the toxic metabolite. A barely perceptible coarseness in balance adjustment also occurs in treated animals. The MPTP-treated goldfish recover normal movement and normal brain monoamine levels within 10-13 days after administration of the drug. We interpret these and other data to indicate that MPTP can induce a Parkinson's disease-like syndrome in the goldfish that is similar in many aspects to the syndrome induced by MPTP in humans and other primates. This remarkable parallel may permit the goldfish to supplement expensive and scarce primates for the purpose of searching and screening neuroprotective drugs with specific relevance to Parkinson's disease.

Ascorbic acid and in situ kinetics: a new approach to vitamin requirements.

Ascorbic acid requirements are based on preventing the deficiency disease scurvy and on urinary excretion of vitamin C. We proposed the first quantitative approach to determining optimal requirements for ascorbic acid and other vitamins, called in situ kinetics. In situ kinetics biochemically is based on the application of Michaelis-Menten reaction kinetics to ascorbic acid-dependent reactions in situ. Clinically in situ kinetics is based on determining vitamin availability to tissues so that cell-specific reactions can occur. The biochemical concepts of in situ kinetics are verified for the first time through studying ascorbic acid regulation of norepinephrine biosynthesis. The principles of in situ kinetics can now be applied to humans and human cells and for determining optimal requirements for ascorbic acid and for other vitamins.

Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum.

We investigated whether circulating ascorbic acid in humans is protein bound or free and whether ascorbic acid exists in its reduced form alone as ascorbic acid or in its reduced and oxidized forms as ascorbic acid and dehydroascorbic acid, respectively. Ascorbic acid and dehydroascorbic acid were determined by using HPLC with coulometric electrochemical detection, and protein binding was determined by centrifugal ultrafiltration. Ascorbic acid was free in plasma and serum of normal, healthy volunteers, 10 men and 10 women. Ascorbic acid was detectable only in its reduced form. However, dehydroascorbic acid could be made to appear in samples processed under oxidizing conditions. Because circulating ascorbic acid is free and is detected only as reduced vitamin, ascorbic acid may be available without intermediates for peripheral utilization. Dehydroascorbic acid may not be present in plasma and serum of normal humans unless assay conditions permit ascorbic acid oxidation.

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.

Ascorbic acid regeneration in chromaffin granules. In situ kinetics.

We have investigated in intact chromaffin secretory vesicles the kinetics, specificity, and mechanism of intragranular ascorbic acid regeneration by extragranular ascorbic acid. The apparent Km of internal ascorbic acid regeneration for external ascorbic acid was 280 microM by Lineweaver-Burk analysis and 287 microM by Eadie-Hofstee analysis. Intragranular ascorbic acid regeneration was specifically mediated by extragranular ascorbic acid or its isomer isoascorbic acid; the reducing agents glutathione, thiourea, homocysteine, NADH, and NADPH did not support regeneration. The structural analog D-glucose did not inhibit regeneration by external ascorbic acid, suggesting specificity at the membrane site of electron transfer. The driving force for regeneration of intragranular ascorbic acid was independent of membrane potential, absolute intragranular and extragranular pH, and ATPase activity, but might be coupled to the pH difference across the chromaffin granule membrane. Since the apparent Km of regeneration was approximately 10-fold below the cytosolic concentration of ascorbic acid, the reaction may proceed at Vmax in situ.

Determination of dehydroascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection.

A method for the detection of dehydroascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection is described. Samples were first assayed for ascorbic acid, then reduced with 2,3-dimercapto-1-propanol to convert dehydroascorbic acid in the sample to ascorbic acid, and subsequently reassayed for total ascorbic acid. The dehydroascorbic acid content was the difference between the two measurements. The dehydroascorbic acid assay provides complete recovery of dehydroascorbic acid, without affecting the ascorbic acid content present prior to reduction. The assay is highly sensitive and reproducible with both standards and biological samples, and was used for routine detection of less than or equal to 1 pmol per sample injection of dehydroascorbic acid. Prior to reduction, dehydroascorbic acid standards frozen at -80 degrees C were stable for at least 1 month; after reduction, stability was limited to 3 days. Dehydroascorbic acid was added to human neutrophil samples; the samples were reduced and ascorbic acid was measured. Ascorbic acid in these samples was stable for greater than or equal to 12 h in a refrigerated autosampler (0-2 degrees C). With a run time for each sample of only 4 min, multiple samples can be prepared and placed in the autosampler for unattended assaying.

Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. Depletion and reaccumulation.

Ascorbic acid (vitamin C) was found in isolated human mononuclear leukocytes and their purified components in millimolar concentration. Intracellular ascorbic acid was depleted greater than 96% during cell culture and was rapidly reaccumulated after addition of physiologic concentrations of ascorbic acid to the extracellular medium. Purified cells maintained concentration gradients of ascorbic acid as large as 100-fold across the plasma membrane. The ability to vary intracellular ascorbic acid concentrations over such a wide range makes it possible for the first time in these cells to study ascorbic acid function in direct relationship to intracellular concentration.

Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis.

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.

Concerning hydrolysis of mycolate esters, of phthiocerol dimycocerosates and of related mycobacterial lipids: an anecdotal account.

We review the experimental difficulties that have been encountered in hydrolyzing mycolic acid esters, their beta-O-substituted analogues, permethylated cord factor, phthiocerol dimycocerosates and similar mycobacterial lipids. Hydrolysis of the beta-O-substituted methyl mycolates is invariably sluggish and is accompanied by considerable beta-elimination to generate mycolenoic acids. Historical evidence for this often undesirable side reaction is presented. Improvements in methodology are described in which hydrolysis is promoted and beta-elimination is minimized. The reaction systems developed were found applicable to hydrolysis of the quite inert phthiocerol dimycocerosates. Permethylation of the recovered phthiocerol provides an excellent derivative for mass spectrometric analysis to define its complete structure.