Diketogulonic Acid Research Articles

Metabolism of L-Ascorbic Acid-l-14C in Guinea Pigs with Alimentary Cholesterol Atheromatosis

A diet containing 0.3% cholesterol fed to guinea pigs for 18 weeks caused an accumulation of cholesterol in blood serum, liver and adrenals, atheromatous changes in coronary arteries and a marked increase in the weight of liver and spleen, in comparison with controls fed the same diet without cholesterol. Ascorbic acid intake was the same in these two groups (10 mg per animal per day, given through a stomach tube). Ascorbic and dehydroascorbic acid concentration in liver and adrenals was not changed in the cholesterol-fed animals, but the total quantity of ascorbic acid in their liver and spleen was substantially higher. Urinary excretion of ascorbic, dehydroascorbic and diketogulonic acids was decreased in this group while oxidation of intraperitoneally administered ascorbic acid-1-14C to 14CO2 remained unaltered. Urinary excretion of 14C was lower in the cholesterol-fed group. Specific activity of ascorbic acid in the tissues of cholesterol-fed guinea pigs was found to be significantly decreased at 96 hours after administration of labeled ascorbic acid compared to that at 48 hours. Guinea pigs with experimental atheromatosis had a significantly larger body pool and a moderately, though significantly, prolonged biological half-life of ascorbic acid. In guinea pigs with dietary cholesterol atheromatosis, an increased ascorbic acid retention exists.

On the Fractional Determination of Ascorbic, Dehyroascorbic, and Diketogulonic Acids.

On the Fractional Determination of Ascorbic, Dehyroascorbic, and Diketogulonic Acids.

Reversal of copper inhibition in chloroplast reactions by manganese.

In the Mehler reaction, a Hill reaction utilizing molecular oxygen as the electron acceptor, rates of net oxygen uptake are stimulated by added manganous ions. Both whole cell photosynthesis and the Mehler reaction are inhibited by copper. Copper inhibition of the Mehler reaction can be reversed by manganese salts. Glutathione. which alone has no effect on Mehler reaction rates, enhances the effect of manganese in reversing copper inhibition. The effects of added Cu(2+), Cu(2+) and Mn(2+), or Cu(2+), Mn(2+), and glutathione exhibit no induction phenomena when measured manometrically. Furthermore, the order of addition of these factors is unimportant: final rates are dependent only on the composition of reaction mixtures. Compared to the Mehler reaction, conventional Hill reactions are less sensitive to copper poisoning, while certain chloroplast mediated photoxidations (e.g. the photoxidation of diketogulonic acid) are far more sensitive. In all of the chloroplast mediated photoreactions tested, manganese is effective in reducing the sensitivity to copper poisoning.

Determination of vitamin C in serum, urine, and other biological materials.

Abstract The specific determination of vitamin C consists of the following: procedure 1 oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHAA) with 2,6-dichloroindophenol; reaction of DHAA with boric acid; reduction of interfering substances such as reductones (R) with homocysteine at pH 7.1; hydrazone formation from the coupling of oxidized AA, DHAA, DKGA (diketogulonic acid), and R with 2,4-dinitrophenylhydrazine (DNPH) in the presence of thiourea; color development with nitric and phosphoric acid; procedure 2 blank determination of hydrazones from DKGA and R by a modification of procedure 1 where the oxidation step is omitted and DHAA is reduced to AA before the addition of boric acid. To determine AA only, another blank (procedure 1′) is obtained by omitting the oxidation step in order to obtain hydrazone formation from substances other than AA. With whole blood, only total vitamin C could be determined because of the oxidation of AA to DHAA in the extraction procedure. With this procedure, complete recoveries of AA and DHAA added to serum and urine were obtained. Reproducibility of assays on different days was found to be satisfactory.

Kinetics of chloroplast-mediated photoxidation of diketogluonate.

Abstract— Illuminated chloroplasts can mediate a photoxidation of diketogulonic acid (DKGA) with rates of oxygen uptake equivalent to rates of Hill reactions with ferricyanide or quinone. The photoxidation of DKGA is sensitive to dichlorophenyl dimethylurea (DCMU) and exhibits the drop in quantum yield at long wavelengths characteristic of photosystem II. Still, the reaction is only partially inactivated by heating chloroplasts at 50° for 10 min (which destroys oxygen evolution). The photoxidation is inhibited by copper and detergents; and is stimulated by added flavin (or methyl viologen) and manganous ions.A model system containing Mn3+ (as manganipyrophosphate) and DKGA, mimics the chloroplast system. Pre‐illuminated chloroplast suspensions can be substituted for Mn3+ in the model dark reaction.It seems that a light‐dependent oxidation of Mn2+ to Mn3+ by photosystem II is the essential contribution of the chloroplasts. Electrons from Mn2+ move through the electron transport system to ferricyanide or to photosystem I where, via flavin (or methyl viologen), oxygen is reduced to H2O2.

The mechanism of action of ceruloplasmin in inhibiting ascorbic acid-induced depolymerization of hyaluronic acid

Autoxidation of ascorbic acid in the presence of hyaluronic acid resulted in depolymerization of the polysaccharide. Degradation of diketogulonic acid, an intermediate produced in the course of autoxidation of ascorbic acid, appeared to be the reaction with which depolymerization of hyaluronic acid was associated. Enzymatic oxidation of ascorbic acid by ceruloplasmin failed to result in depolymerization of hyaluronic acid. This effect of ceruloplasmin appeared to be the result of its capacity to alter the pathway of degradation of diketogulonic acid. Its failure to alter the course of depolymerization of hyaluronic acid by ionizing radiation indicated that its action was not due to destruction of free radicals. Apoceruloplasmin, serum albumin and haptoglobin had no influence on ascorbic acid-induced depolymerization of hyaluronic acid.

Studies on the ascorbic acid-induced depolymerization of hyaluronic acid

Ascorbic acid and intermediates produced in the course of its autoxidation were studied for their capacity to depolymerize hyaluronic acid. Depolymerization was expressed in terms of increase in specific fluidity as determined viscometrically. Calcium diketogulonate was demonstrated to be more active in eliciting depolymerization of the polysaccharide than either dehydroascorbic acid or ascorbic acid. Dehydroascorbic acid was about half as active as ascorbic acid. In the presence of added H 2O 2, the rate of depolymerization by all three compounds was markedly increased, although H 2O 2 alone failed to depolymerize hyaluronic acid. The results indicate that when ascorbic acid undergoes autoxidation in the presence of hyaluronic acid, degradation of diketogulonic acid is one of the major reactions to which depolymerization of the polysaccharide is coupled. H 2O 2 appears to increase the rate of ascorbic acid induced depolymerization of hyaluronic acid by a catalytic or synergistic action diketogulonic acid or its products of degradation.

URINARY OXALIC ACID EXCRETION BY MAN FOLLOWING INGESTION OF LARGE AMOUNTS OF ASCORBIC ACID

Recently, large amounts of ascorbic acid are used for the treatments of various diseases, so that the investigation on the urinary excretion of oxalic acid, which is one of the metabolites of ascorbic acid, was carried out in man following the administration of large amounts of the vitamin and the following findings were obtained.1. No significant increase in urinary oxalic acid was observed following the ingestion of large amounts (1-2g) of ascorbic acid for a long period (90-180 days).2. After ingestion of large amounts of ascorbic acid, it was eliminated in urine mainly in the reduced form of the vitamin and little was determined as diketogulonic acid, the precursor of oxalic acid.

On the metabolites of ascorbic acid, especially oxalic acid, eliminated in urine, following the administration of large amounts of ascorbic acid.

Large amounts of ascorbic acid are recently used in curing pigment anomalies. Investigation was therefore carried out in animals and man on the fate of the vitamin administered in large amounts, especially on the excretion of oxalic acid and the following findings were obtained.1. After administration of the large amounts of ascorbic acid, it was eliminated in urine mostly in the form of dehydroascorbic acid in the majority of cases (about 80%).2. When ascorbic acid was administered orally or intramuscularly in a dose of 3g per day, there was no marked increase in the urinary oxalic acid. Oral administration of 9g of the vitamin resulted in an marked increase of the urinary oxalic acid (from 20 to 30mg).3. With a rise in the dose of ascorbic acid, urinary diketogulonic acid was increassed. It seems to indicate that the large dose of ascorbic acid promotes the degradation of dehydroascorbic acid into digekogulonic acid, but further oxidation to oxalic acid does not occur markedly.4. The amounts of oxalic acid-C14 detected in the urine of animals and man following the administration of AsA-C14 accounted for 1.0 to 1.5% and 0.16% of the C14 given in animals and man respectively.5. Investigation on the effect of an intravenous an injection of a daily dose of 0.5mg of ascorbic acid carried out for 17 consecutive days on the formation of calculi with a lead ball inserted in the urinary bladder as a nucleus showed that the calculi formed around the nuclei in experimental animals was 7 times as heavy as those formed in the control animals. The amount of oxalic acid contained in the calculi was 0.73mg as contrasted to 0.33mg in the calculi of the control animals.

HISTOCHEMISTRY 78. ASCORBIC ACID IN NORMAL MAST CELLS AND MACROPHAGES AND IN NEOPLASTIC MAST CELLS.

SummaryAscorbic acid and the sum of ascorbic, dehydroascorbic and diketogulonic acids in the mast cell fraction (about 92% mast cells) from peritoneal washings of normal rats, and in mouse ascites tumor mast cells (about 98% mast cells), were determined per 106 cells and per μg protein-nitrogen. Ascorbic acid (551 mμg/106 cells) comprised about 87% of the sum of the acids in the normal cells and about 98% (271 mμg/106 cells) of the sum in the tumor cells. The concentrations of ascorbic acid in the normal and tumor cells were 13 and 10 mμg/μxg protein-nitrogen, respectively. The ascorbic acid per 106 cells in the rat macrophage cell fraction (96% macrophages) was 38% of that in the mast cell fraction, while the ascorbic acid per μg protein-nitrogen in the former was 1 1/3 times that in the latter.

Enzyme‐Catalyzed Breakdown of Dehydroascorbic Acid in Plant Tissue

Homogenates of plant material were examined for enzymatic delactonization of dehydroascorbic acid and oxidation of diketogulonic acid. Bitter gourd fruit, cauliflower, and the leaves of the tapioca plant contain an enzyme that brings about conversion of dehydroascorbic acid to diketogulonic acid. Tapioca leaves are able to effect, in addition, a significant enzyme‐catalyzed degradation of diketogulonic acid. The 1eaves of the tapioca plant contain some material that stabilizes ascorbic acid against oxidation to dehydroascorbic acid during homogenization in a Waring blender.

Influence of Dietary Protein Levels on Ascorbic Acid Status of the Rat.

There appears to be no report regarding the influence of dietary protein on biosynthesis of ascorbic acid in the rat. Prompted by a chance observation that rats receiving a high protein diet excreted relatively large amounts of ascorbic acid in urine, we investigated the influence of dietary protein on the Vit. C status of the rat. In the first instance, the effect of variations in dietary protein levels was examined. Materials and methods. Adult male albino rats (Wistar strain) with body weights in the range 180-200 g were employed in all experiments. Animals were distributed according to the randomized block design for the various test treatments. Diets fed had the following composition: Protein (Polson's lactic casein)—according to choice; fat (refined peanut oil)—10%; vitaminized starch—1%; salt mixture—2%; and corn starch—to 100. The vitaminized starch contained thiamine hydrochloride 40 mg, riboflavin 80 mg. niacin 100 mg. pyridoxine hydrochloride 40 mg, calcium pantothenate 100 mg and folic acid 5 mg per 100 g. The rats were given necessary amounts of diluted shark liver oil twice a week to provide the requirements of Yit. A and D. Cages with wide mesh wire bottoms which were well covered with resistant paint were used to house the animals individually. Urine was collected in brown glass bottles containing 10% trichloracetic acid (TCA) over 3-day periods. Collecting funnels and bottoms of cages were rinsed with 10% TCA solution at least thrice a day during the collection period. Urine collections were made up to known volume and aliquots used for estimation of total Vit. C (ascorbic acid. dehydroascorbic acid and diketogulonic acid) according to the dinitrophenylhydrazine (DNPH) method of Roe and Keuther(1). The same procedure was employed for estimation of ascorbic acid in tissues and blood.

Enzymatic transformation of dehydroascorbic acid to diketogulonic acid.

THE conversion of dehydroascorbic acid to diketogulonic acid has been studied in chemical systems1 ; but, as Harris2 has pointed out, definite evidence is not available for any enzymatically catalysed transformation.

Metabolism of C 14-labeled ascorbic, dehydroascorbic and diketogulonic acids in guinea pigs

Experiments were carried out in guinea pigs with l-ascorbic acid-1-C 14, l-ascorbic acid-6-C 14, dehydroascorbic acid-1-C 14, dehydroascorbic acid-6-C 14, diketogulonic acid-1-C 14, and diketogulonic acid-6-C 14. The results obtained by measuring the incorporation of C 14 in expired CO 2, urinary oxalate, and liver glycogen are in accord with a pathway of metabolism of the vitamin via dehydroascorbic acid and diketogulonic acid. The labeling pattern observed in liver glycogen after administration of l-ascorbic acid-6-C 14, dehydroascorbic acid-6-C 14, and diketogulonic acid-6-C 14 was different from that obtained with l-gulonolactone-6-C 13. These results, along with those showing no detectable conversion of l-ascorbic acid-1-C 14 to urinary l-gulonic acid, indicate that l-gulonic acid is not a major intermediate in the metabolism of the vitamin in guinea pigs. Possible mechanisms for the conversion of l-ascorbic acid to glycogen through l-xylulose and trioses are considered.

Título en español.

1. Los valores obtenidos para los ácidos ascórbicos (AA), dehidroascórbico (ADH) y dicetogulónico (ADC) durante las diferentes etapas del desarrollo de la fruta indicaron que el AA era bastante estable en la fruta de la acerola y que el pequeño contenido de ADH y ADC, especialmente del segundo, son productos de descomposición más que de metabolismo. 2. En la fruta fresca, en todas las etapas de crecimiento estudiadas, el contenido de AA nunca fué menor del 90 por ciento de la suma de los tres compuestos estudiados. ADH o ADC nunca llegaron a tener valores individuales sobre 6 por ciento, sin embargo, el contenido de ADH fué siempre algo mayor que el de ADC. 3. En los botones de flor o en las frutas rudimentarias congeladas se observó un aumento muy significativo en ADH y ADC y una disminución correspondiente en AA. Sin embargo, en etapas más avanzadas de crecimiento, AA siempre se mantuvo en la vecindad del 90 por ciento. En el caso del fruto congelado el ADC se encuentra en cantidad algo mayor que el ADH. 4. Durante la formación del fruto rudimentario (etapa 1 a 2) se observó un rápido aumento en AA, debido probablemente a condiciones óptimas en el sistema encimático responsable de la síntesis de ácido ascórbico en el fruto. 5. En el fruto maduro se observó una disminución significativa de ácido ascórbico.

The Effects of X-Irradiation on the Vitamin Excretion of the Rhesus Monkey (Macaca Mulatta)

Ten monkeys were exposed to three levels (325, 400, 650 r) of x radiation. Anorexin, leucopenia, loss of hair, and at the high levels of irradiation, hemorrhage and diarrhea, followed. Observations on pair-fed non- irradiated controls demonstrated that the decreased urinary excretion of riboflavin, thiamine, and N/sup 1/-methylnicotinamide which followed irradiation was a result of food restriction, not a specific effect of the x-ray injury. No meaningful changes could be found in blood ascorbic acid at the levels of irradiation and intake. Diketogulonic acid excretion was increased in urine after irradiation. A temporary, moderately increased excretion of pteroylglutamic acid and of vitamin B/sub 6/ followed x-irradiation. No prolonged loss of ascorbic acid, riboflavin, thiamine, niacin (N/sup 1/- methylnicotinamide), pyridoxine, or pteroylglutamate occurs which might indicate that x radiation is a conditioning agent in producing a deficiency of these factors. (auth)

Observations on the administration of dehydroascorbic acid in diabetogenic doses to albino rats.

1. Observations were made on the ascorbic acid (ASA) and “total ascorbic acid” (TASA = ASA + (dehydroascorbic acid) DHA + (diketogulonic acid) DKA) concentrations in the liver, kidney, pancreas and adrenals before and after the intravenous injection of DHA in diabetogenic doses or of ASA to male and female albino rats.2. Immediately following the dose of DHA, the concentration of TASA rose about 50‐fold in the kidneys. The concentration rose in the liver (10‐ to 16‐fold) and pancreas (7‐ to 10‐fold). The DHA which entered the tissues was rapidly reduced. The extent of reduction in the liver was more than 80 per cent, whereas in the kidneys and pancreas only about 50 per cent reduction took place. Twenty‐four hours later most of the excess TASA had disappeared. Following a second dose, the concentrations in the tissues presumably rose temporarily, but at the end of 24 hours further accumulation had not taken place. Following a third dose, rapid reduction to ASA was again observed. The concentrations in the tissues were almost normal 8 days later.3. Compared with DHA, the administration of ASA under similar conditions led to a greater rise in the concentration in the pancreas; the kidneys concentrated to the same extent as before whereas the liver concentrated much less.4. The intravenous injection of diabetogenic doses of DHA or of similar amounts of ASA after preliminary desensitization caused an immediate depletion of adrenal ascorbic acid. Some depletion was observed following the intravenous injection of only 0·2 g./kg. body weight of dehydroascorbic acid (the desensitizing dose). The fall was more marked after dehydroascorbic acid and in the male rat. Twenty‐four hours later a restoration in concentration of adrenal ascorbic acid occurred together with a significant rise in adrenal weight.

The determination of vitamin C in normal human plasma and erythrocytes

1. 1. Various methods based on (a) reduction of indophenol, (b) formation of a derivative with 2: 4-dinitrophenylhydrazine, and designed to measure ascorbic acid, dehydroascorbic acid and diketogulonic acid have been applied to the analysis of normal human plasma and erythrocytes. 2. 2. It is concluded that measurement of indophenol reduction at pH ca. 4 in protein-free filtrates of plasma, or of carbon monoxide saturated erythrocytes, treated with p-chloromercuribenzoic acid provides the most accurate means of determining ascorbic acid in these materials. 3. 3. Comparison of values for total vitamin C and ascorbic acid of human plasma indicates that the amount of dehydroascorbic acid and/or diketogulonic acid in normal plasma is smaller than can be reliably determined by this procedure. The similar, and contrary, findings of others have been discussed in relation to the methods used. 4. 4. In erythrocytes the results obtained were consistent with the presence of a small amount of dehydroascorbic acid and/or diketogulonic acid. 5. 5. Measurement of indophenol reduction of plasma or erythrocyte filtrates treated with hydrogen sulphide gives results which are believed to include some non-ascorbic acid material and thus to overestimate any dehydroascorbic acid present. 6. 6. The concentration of total vitamin C in plasma is higher than that in erythrocytes, freed as far as possible from leucocytes, when the concentration in plasma is high. When the concentration in plasma is low the reverse relationship holds. In contrast, the concentration of ascorbic acid in plasma tends to be higher than that in erythrocytes at all levels.

Ascorbic acid—photochemical oxidation in solutions containing oxalic acid. I.—catalysis by traces of iron salts

AbstractAscorbic acid is lost when a solution at pH 1.7 containing 0.25% oxalic acid, 0.02% ascorbic acid and a trace of iron salt is exposed to light from a mercury vapour lamp. It is demonstrated that this is a photochemical reaction dependent upon a number of factors: irradiation, access of air, presence of oxalate ions and a trace of iron salt; and the absence of any one of these factors inhibits the reaction. The ascorbic acid is apparently oxidized to dehydroascorbic acid and diketogulonic acid.

Catabolism of ascorbic acid by animal tissues.

SummaryThe catabolism of ascorbic acid in animal tissues has been studied using rat and guinea pig liver homogenates in vitro. It has been shown that ascorbic acid is oxidized to its dehydro form by a heat-stable, non-enzymatic factor in the insoluble portion of liver. Dehydroascorbic acid is then further degraded beyond the diketogulonic acid stage by an enzyme system in the soluble portion of the liver.