Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1⁺) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.     

Inhibition ofCitrobacter freundii by lactic acid, ascorbic acid, citric acid,Thymus vulgaris extract and NaCl at 31 °C and 5 °C

Citrobacter freundii has been implicated in food spoilage and food poisoning outbreaks. This study examines the effects of some compounds (e.g. citric acid, ascorbic acid, lactic acid, sodium chloride, andThymus vulgaris extract) on growth of two strains of Citrobacter freundii at 31 °C and 5 °C. At 31 °C, lactic acid (0.2%) or ascorbic acid (0.2%) alone completely inhibited growth of the tested strains, as there was 100% reduction in growth of the strains after 24 h incubation in nutrient broth containing these compounds.Thymus vulgaris extract (0.3%) reduced the growth rate (p<0.05), the percentages of inhibition after 24 h incubation were about 60% for both strains. NaCl (5%) greatly reduced growth, the percentages of inhibition were about 84% for both strains. Combination ofT. vulgaris extract (0.3%) and NaCl (4%) together completely inhibited growth ofC. freundii species tested. Ascorbic acid (0.1%) or citric acid (0.03%) did not affect growth of the strains (p>0.05), but a lag occurred before increase in number could be observed. In chicken and fish homogenates, combination of NaCl (4%) and ascorbic acid (0.1%) reduced the growth (p < 0.05) (growth inhibition was 40%). At 5 °C, lactic acid (0.1%) alone greatly reduced the growth (p<0.05). The activity of NaCl, or ascorbic acid alone against the tested strains was greatly increased (p<0.05). ForC. freundii 4, the percentage of growth inhibition after 6 days incubation in broth containing 3% NaCl or 0.1% ascorbic acid were 88% and 72%, respectively. ForC. freundii 38, the percentage of growth inhibition after 6 days incubation in broth containing these compounds were 60% and 54%, respectively.     

An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines.

The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1–3) since its introduction in 1960 by Satakeet al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15–30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS.     

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.     

An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites

2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.     

Determination of ascorbic acid and its related compounds in foods and beverages by hydrophilic interaction liquid chromatography

A new hydrophilic interaction liquid chromatography method for the simultaneous determination of ascorbic acid (AA), erythorbic acid (EA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG) was developed using a diol column with an isocratic solution of acetonitrile-66.7 mM ammonium acetate solution (85:15, v/v) at a detection wavelength of 260 nm. The calibration curves were found to be linear in the range of 1-50 microg/ml for AA and EA and in the range of 2.5-100 microg/ml for AA-2G and AA-2betaG. Detection limits of AA, EA, AA-2G and AA-2betaG were 0.3, 0.3, 0.03 and 0.03 microg/ml, respectively. This method was satisfactorily applied to the determination of AA, EA, AA-2G and AA-2betaG in a fruit, a food and beverages. The results show that the procedure is simple and sensitive and that it can be employed for the simultaneous determination of AA and its related compounds in foods and beverages.     

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.     

The ascorbic acid paradox

Ascorbic acid (AA) is a common culture medium and dietary supplement. While AA is most commonly known for its antioxidant properties, it is also known to function as a pro-oxidant under select conditions. However, the complexity and often unknown composition of biological culture systems makes prediction of AA behaviour in supplemented cultures challenging. The frequent observation of outcomes inconsistent with antioxidant behaviour suggests that AA may be playing a pro-oxidant role more often than appreciated. In this work we explored the intracellular and extracellular impact of AA supplementation on KG1a myeloid leukaemia cells over a 24-h culture period following the addition of the AA supplement. At 24 h we found that supplementation of AA up to 250 μM resulted in intracellular antioxidant behaviour. However, when these same cultures were evaluated at 2 or 4 h we observed pro-oxidant activity at the higher AA concentrations indicating that the outcome was very much time and dose dependent. In contrast, pro-oxidant activity was never observed in the extracellular medium. Paradoxically, and to our knowledge not previously reported, we observed that intracellular pro-oxidant activity and extracellular antioxidant activity could occur simultaneously. These results indicate that the precise activity of AA supplementation varies as a function of dose, time and cellular location. Further, these results demonstrate how in the absence of careful culture characterization the true impact of AA on cultures could be underappreciated.     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.     

Testicular and plasma ascorbic acid levels in mice following dietary intake: a high-performance liquid chromatographic analysis

A modified buffer system is reported for the analysis of vitamin C in mouse plasma and testes, on a reversed-phase high-performance liquid chromatographic column with ultraviolet detection. The buffer, consisting of 0.1 M NaH₂PO₄ and 0.2 mM Na₂EDTA adjusted to pH 3.1 with orthophosphoric acid, resolved the ascorbic acid (AA) peak allowing clear quantitation of the chemical. The method is also applicable to the assay of dehydroascorbic acid after its reduction to ascorbic acid, and overcomes problems of AA stability encountered in previously reported procedures. Using the present technique, variations in the vitamin levels of plasma and testes are studied from 3 to 29 days after the commencement of feeding a vitamin C-rich diet (1%, w/w) in mice. The plasma AA levels were elevated above the controls by a factor of 2.5 by day 8. Contrary to this, testicular AA levels increased marginally (1.2-fold) by day 12 and were maintained at levels close to the control values thereafter. It appears that the feedback inhibition mechanism which is effective in plasma is not operational in the testes. The findings are of clinical significance in that testicular AA levels do not change significantly as a result of dietary intake of vitamin C, whereas plasma AA levels do show an increase.     

Chemometric-assisted kinetic-spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)₃]³⁺) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer’s law is followed in the concentration ranges of 4.3–74.1, 4.3–78.3, and 2.0–33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Ion exchange separation of ascorbic acid and isolation of the 2,4-dinitrophenylosazone

A method has been developed for the separation of small amounts of ascorbic acid from the neutral and basic constituents of rat urine by adsorption on Amberlite IR-4B anion-exchange resin and subsequent elution with hydrochloric acid. The procedure separates the ascorbic acid from the nonacidic substances present, with an over-all recovery of 85–90% of the ascorbic acid originally present. A detailed study of optimum conditions for the formation of the ascorbic acid 2,4-dinitrophenylosazone was made, resulting in a method which permits osazone formation to be complete in 3 hr. The ascorbic acid 2,4-dinitrophenylosazone was shown to be chemically and radioactively pure by accepted standards, including data for the near ultraviolet, visible, and infrared absorption spectra.     

2,3-dehydro-4-epi-N-acetylneuraminic acid; a neuraminidase inhibitor

Treatment of N-acetylneuraminic acid methyl ester with sulfuric acid and acetic anhydride at 50° followed by deacetylation gave 2,3-dehydro-2-deoxy-N-acetylneuraminic acid methyl ester and methyl 5-acetamido-2,6-anhydro-2,3,5-trideoxy-d-glycero-d-talo-non-2-enonate (2,3-dehydro-4-epi-NeuAc methyl ester) in equal yields (～40% each). The structure of the latter was ascertained primarily from analysis of its mass spectrum and ¹H- and ¹³C-nuclear magnetic resonance spectra. The relative proportions of these two glycals in the foregoing reaction was dependent on temperature, as at 0°, the yield of 2,3-dehydro-4-epi-NeuAc was markedly diminished. A minor by-product of this acetylation reaction was 2-methyl-(methyl 7,8,9- tri-O-acetyl-2,6-anhydro-2,3,5-trideoxy-d-glycero-d-talo-non-2-enonate)-[4,5-d]-2-oxazoline. Based upon this finding and additional interconversion experiments, a mechanism involving the intermediacy of the latter oxazoline to account for the epimerization is proposed. These glycals and their methyl esters are competitive inhibitors of Arthrobacter sialophilus, neuraminidase, suggesting that the 4-hydroxyl group must be equatorially oriented for maximal enzyme inhibition.     

A comparison of leukocyte ascorbate levels measured by the 2,4-dinitrophenylhydrazine method with high-performance liquid chromatography using electrochemical detection

In leukocytes a dynamic relationship between the reduced form of ascorbic acid (AA) and its oxidized product dehydro-AA has been described. It is therefore important to know which form of the vitamin predominates when choosing a methodology. The purpose of this study was to find out if the majority of ascorbate in human leukocytes isolated by centrifugation through Percoll is in the reduced AA form by measuring reduced AA by HPLC and comparing the values to those obtained by using the 2,4-dinitrophenylhydrazine (DNPH) method which measures total ascorbate, and quantifying the reduced and oxidized forms of the vitamin in leukocytes using a modification of the DNPH method. There was no significant difference (P greater than .05) between the HPLC and DNPH values for 12 individuals and 87% of the AA was found to be in the reduced form. These results support the assumption that the majority of AA found in a mixed leukocyte population isolated through Percoll is in the reduced form and that both methods can be used for AA measurements.