ascorbic-acid and gulonolactone

The reviewed studies addressed the possibility of using gene transfer for correction of L-ascorbic acid biosynthesis and carbohydrate utilization in rainbow trout. Analyses of enzymatic activities in the L-AAB pathway indicated that reasons for the lack of L-AA production can be common in fish and scurvy-prone animals. Rat gulonolactone oxidase cDNA was transferred into trout. Regardless of the fact that rGLO transcription occurred in embryos, neither GLO protein, nor enzyme activity were detected. There was no production of L-AA in transgenic fish raised on vitamin C-free diets or injected with L-gulonolactone. These results indicated that the conditions required for translation or stability of rGLO were not present in trout tissues. To augment carbohydrates utilization, human glucose transporter 1 and rat hexokinase II cDNAs were tested. In the transfected embryos. HK activity, rates of hexose uptake and glucose oxidation were increased. The effect of hGLUT1 on glucose metabolism was greater than that of rHKII. Trout carrying hGLUT1 and rHKII with viral or piscine promoters were created. Though interpretation of the metabolic effects of the transgenes was complicated with mosaicism, a tendency to improved carbohydrate utilization was revealed in some of the transgenic individuals.

Topics: Animals; Animals, Genetically Modified; Ascorbic Acid; Dietary Carbohydrates; DNA, Complementary; Genetic Engineering; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Hexokinase; Humans; L-Gulonolactone Oxidase; Monosaccharide Transport Proteins; Muscle Proteins; Oncorhynchus mykiss; Phosphorylation; Pilot Projects; Rats; Recombinant Fusion Proteins; Species Specificity; Sugar Acids; Sugar Alcohol Dehydrogenases; Transgenes

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

Topics: Animals; Ascorbic Acid; Bombyx; Hexoses; Larva; Pupa; Sugar Acids

Topics: Animals; Ascorbic Acid; Biological Transport; Cell Membrane; Dehydroascorbic Acid; Erythrocytes; Gene Knockout Techniques; Glucose; Hemolysis; Mice; Mice, Knockout; Oxidation-Reduction; Oxidoreductases; Phenotype; Sugar Acids

The L-ascorbate (AsA) content and the expression of six L-galactose pathway-related genes were analyzed in peach flesh during fruit development. Fluctuation of AsA during peach fruit development was divided into four phases based on the overall total AsA (T-AsA) content per fruit: AsA I, 0-36 days after full bloom (DAFB); AsA II, 37-65 DAFB; AsA III, 66-92 DAFB and AsA IV, 93-112 DAFB. Phase AsA III was a lag phase for AsA accumulation, but did not coincide with the lag phase for fruit development. The T-AsA concentration was highest at the early stage until 21 DAFB [2-3 micromol per gram of fresh weight (g(-1) FW)], and decreased to 1/4 and 1/15 of this value at 50 and 92 DAFB, respectively. T-AsA then remained at 0.15-0.20 micromol g(-1) FW until harvest at 112 DAFB. More than 90% of the T-AsA was in the reduced form until 21 DAFB. The proportion of reduced form of AsA then decreased concomitantly with the decrease in AsA concentration. To determine the main pathway of AsA biosynthesis and the AsA biosynthetic capacity of peach flesh, several precursors were incubated with immature whole fruit (59 DAFB). The AsA concentration increased markedly with L-galactono-1,4-lactone or L-galactose (Gal), but d-galacturonate and L-gulono-1,4-lactone failed to increase AsA, indicating dominance of the Gal pathway and potent AsA biosynthetic capabilities in immature peach flesh. The expression of genes involved in the last six steps of the Gal pathway was measured during fruit development. The genes studied included GDP-d-mannose pyrophosphorylase (GMPH), GDP- d-mannose-3',5'-epimerase (GME), GDP- L-galactose guanylyltransferase (GGGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose-1-dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH). GMPH, GME and GGGT had similar expression patterns that peaked at 43 DAFB. GPP, GDH and GLDH also had similar expression patterns that peaked twice at 21 and 91 DAFB, although the expression of GDH was quite low. High level of T-AsA concentration was roughly correlated with the level of gene expression in the early period of fruit development (AsA I), whereas no such relationships were apparent in the other periods (e.g. AsA III and IV). On the basis of these findings, we discuss the regulation of AsA biosynthesis in peach fruit.

Topics: Ascorbic Acid; DNA, Complementary; DNA, Plant; Fruit; Galactose; Gene Expression Regulation, Plant; Genes, Plant; Hexuronic Acids; Lactones; Prunus; Sugar Acids

Inability to synthesize vitamin C, because of a deficiency in gulonolactone oxidase (GULO) expression, is a genetic deficiency shared by a small number of animals including humans. Although the most overt symptom of vitamin C deficiency, scurvy, can be readily corrected by modest consumption of vitamin C, there is increasing interest in the effect of high-level administration in treating human disease. Using a previously derived Gulo-expressing vector, which produces murine GULO under the control of the murine cytomegalovirus (mCMV) promoter, we constructed and validated a recombinant helper-dependent adenovirus (HDAd-mCMV-Gulo) that can be used to correct this genetic defect. A human liver cell line (Hep G2) infected with the HDAd-mCMV-Gulo vector expressed GULO in a time- and gene dose-dependent manner. These cells also produced ascorbic acid when exogenous gulonolactone was supplemented in the medium. Likewise, Gulo(-/-) mice treated with HDAd-mCMV-Gulo at 2 x 10(11) VP expressed GULO in the liver and produced ascorbic acid. Serum ascorbic acid concentrations in Gulo(-/-) mice injected with GULO-expressing HDAd were elevated to levels comparable to those of wild-type mice (62 +/- 15 microM) after 4 days of infection and were maintained at significantly higher levels compared with those in untreated Gulo(-/-) mice for at least 23 days. A similar elevation was observed in urine and tissue ascorbic acid concentrations in vector-treated animals. In conclusion, we demonstrate here that gene therapeutic HDAd-mCMV-Gulo vectors can mediate the expression of GULO and endogenous production of ascorbic acid in human cells and in Gulo(-/-) transgenic mice. Taken together, these data show that a gene therapy approach can be successfully employed in the treatment and further study of vitamin C deficiency in scurvy-prone mammals.

Topics: Adenoviridae; Animals; Ascorbic Acid; Base Sequence; Cell Line; DNA Primers; Genetic Vectors; Helper Viruses; Humans; L-Gulonolactone Oxidase; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Sugar Acids

Gulonolactone treatment of mice resulted in the elevation of hepatic ascorbate and hydrogen peroxide levels accompanied by transient liver swelling and reversible dilatation of endoplasmic reticulum cisternae. Although a decrease in glutathione (reduced form)/total glutathione ratio was observed in microsomes, the redox state of luminal foldases remained unchanged and the signs of endoplasmic reticulum stress were absent. Increased permeability of the microsomal membrane to various compounds of low molecular weight was substantiated. It is assumed that Gulonolactone-dependent luminal hydrogen peroxide formation in the endoplasmic reticulum provokes a temporary increase in non-selective membrane permeability, which results in the dilation of the organelle and in enhanced transmembrane fluxes of small molecules.

Topics: Animals; Ascorbic Acid; Endoplasmic Reticulum; Glutathione; Hydrogen Peroxide; Intracellular Membranes; Liver; Male; Mice; Mice, Inbred Strains; Microscopy, Electron; Microsomes, Liver; Oxidation-Reduction; Permeability; Sugar Acids

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.

Topics: Amino Acid Sequence; Animals; Ascorbic Acid; Biopterins; Blotting, Western; Calcimycin; Cattle; Cells, Cultured; Endothelial Cells; Gene Expression; Intracellular Fluid; Ionophores; L-Gulonolactone Oxidase; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Synthase Type III; Plasmids; Sugar Acids; Transfection

A convenient method to obtain unknown chiral C2- and C3-functionalized aldono-1,4-lactone derivatives starting from l-ascorbic acid, which would be valuable in the synthesis of derivatives of various pharmacologically active agents for structure-activity studies, is described. The practicality of this approach is demonstrated by the synthesis of a series of 5,6-O-isopropylidene-2-allyl-3-keto-l-galactono-gamma-lactone and 5,6-O-isopropylidene-3-allyl-2-keto-l-galactono-gamma-lactone derivatives using the thermal Claisen rearrangement of the corresponding 3-O- and 2-O-allyl derivatives of 5,6-O-isopropylidene-l-ascorbic acid, respectively, followed by stereospecific reduction to the corresponding alcohols. The synthetic steps are shown to be efficient, and enantiospecific, and they proceed with high yields.

Topics: Ascorbic Acid; Magnetic Resonance Spectroscopy; Sugar Acids

Modifications of the analytical method to determine L-gulono-gamma-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-gamma-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-gamma-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-gamma-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-gamma-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-gamma-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-gamma-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.

Topics: Animals; Ascorbic Acid; Cold Temperature; Dehydroascorbic Acid; L-Gulonolactone Oxidase; Liver; Reproducibility of Results; Spectrophotometry; Sugar Acids; Sugar Alcohol Dehydrogenases; Swine; Time Factors

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore-forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione-loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.

Topics: Alamethicin; Animals; Ascorbic Acid; Enzyme Activation; Glutathione; Glutathione Disulfide; L-Gulonolactone Oxidase; Light; Male; Microsomes, Liver; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Sugar Acids; Sugar Alcohol Dehydrogenases; Uncoupling Agents

Ascorbate synthesis causes glutathione consumption in the liver. Addition of gulonolactone resulted in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed. In hepatic microsomal membranes, ascorbate synthesis stimulated by gulonolactone caused an almost equimolar consumption of GSH. This effect could be counteracted by the addition of catalase or mercaptosuccinate, indicating the role of hydrogen peroxide formed during ascorbate synthesis in the depletion of GSH. The observed phenomenon may be one of the reasons why the evolutionary loss of ascorbate synthesis could be advantageous.

Topics: Amitrole; Animals; Ascorbic Acid; Catalase; Cells, Cultured; Glutathione; Kinetics; Liver; Male; Mice; Mice, Inbred Strains; Microsomes, Liver; Sugar Acids

Six experiments were conducted to study the effect of diet on growth and plasma ascorbic acid in chickens. D-Glucuronolactone failed to improve growth with either a crude yeast-fish meal diet or a purified diet based on casein and gelatin. With the purified diet, D-glucuronic acid and L-gulonolactone also failed to improve growth and did not influence plasma ascorbic acid levels. Dietary ascorbic acid improved growth of chicks with a purified diet in most cases, but not with a corn-soybean diet. Meat meal and fish meal caused slight increases in plasma ascorbic acid, whereas soybean meal, safflower meal, and cottonseed meal caused greater increases when used in a purified diet. Gulonolactone oxidase activity in the kidney was not different between chicks fed the purified or the corn-soybean diets, but was reduced by 0.1% dietary ascorbic acid. The mechanism for the increase in plasma ascorbic acid with the addition of soybean meal and other plant protein sources to the diet is not known.

Topics: Animals; Ascorbic Acid; Body Weight; Chickens; Food, Fortified; Glucuronates; Glucuronic Acid; Male; Sugar Acids

The relationship between glutathione deficiency, glycogen metabolism and ascorbate synthesis was investigated in isolated murine hepatocytes. Glutathione deficiency caused by various agents increased ascorbate synthesis with a stimulation of glycogen breakdown. Increased ascorbate synthesis from UDP-glucose or gulonolactone could not be further affected by glutathione depletion. Fructose prevented the stimulated glycogenolysis and ascorbate synthesis caused by glutathione consumption. Reduction of oxidised glutathione by dithiothreitol decreased the elevated glycogenolysis and ascorbate synthesis in diamide or menadione treated hepatocytes. Our results suggest that a change in GSH/GSSG ratio seems to be a sufficient precondition of altering glycogenolysis and a consequent ascorbate synthesis.

Topics: Acetaminophen; Animals; Ascorbic Acid; Bucladesine; Buthionine Sulfoximine; Cyclic AMP; Diamide; Fructose; Glutathione; Glycogen; Liver; Male; Methionine Sulfoximine; Mice; Sugar Acids; Uridine Diphosphate Glucose; Vitamin K

1. The synthesis of ascorbic acid in rat-liver extracts is impaired during starvation, and more from glucuronolactone and glucuronate than from gulonate and gulonolactone. 2. The formation of xylulose from gulonate and from gulonolactone is greatly enhanced during starvation, whereas it is decreased from glucuronolactone and from glucuronate. 3. The activity of the enzymes of the glucuronic acid pathway during starvation has been determined in rat-liver preparations. Gulonolactone oxidase is decreased, NAD-linked gulonate dehydrogenase is enhanced, and uronolactonase, aldonolactonase and NADP-linked hexonate dehydrogenase are unchanged. 4. The impairment of ascorbic acid synthesis from gulonate observed during starvation can be accounted for by the depressed activity of gulonolactone oxidase. 5. The cause of the enhanced formation of xylulose has been located in the sedimentable fraction of liver homogenate. 6. The hypothesis is formulated of an increased utilization of the glucuronic acid pathway during starvation.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Carbohydrate Dehydrogenases; Carbohydrate Metabolism; Esterases; Glucuronates; Glucuronic Acid; Lactones; Liver; Liver Extracts; Metabolism; NADP; Oxidoreductases; Pentoses; Rats; Research; Starvation; Sugar Acids; Xylulose

Topics: Ascorbic Acid; Humans; Liver; Oxidoreductases; Sugar Acids

Topics: Ascorbic Acid; Lipid Metabolism; Lipid Peroxidation; Lipids; Oxidoreductases; Sugar Acids

Topics: Animals; Ascorbic Acid; Carbohydrate Metabolism; Glucuronates; Lactones; Sugar Acids

Topics: Ascorbic Acid; Carbohydrate Metabolism; Hexoses; Sugar Acids

Topics: Ascorbic Acid; Carbohydrate Metabolism; Hexoses; Lactones; Lipid Metabolism; Lipids; Sugar Acids

Topics: Ascorbic Acid; Humans; Malondialdehyde; Oxidoreductases; Sugar Acids; Vitamin E Deficiency; Vitamins

Topics: Animals; Ascorbic Acid; Carbohydrate Metabolism; Glucuronates; Hexoses; Rats; Sugar Acids