<p> Gulonolactone oxidase enzyme is important in the final stage of ascorbic acid biosynthesis. Gulonolactone oxidase is encoded by the Gulo gene. Most animals, such as mice, have the Gulo gene, through which they produce ascorbic acid from glucose, while humans, guinea pigs and primate animals carry a non functional Gulo gene. Ascorbic acid plays an important role in many biological processes. However, it is primarily essential as an antioxidant. Ascorbic acid protects genomic DNA from free radicals resulting from oxidative stress that might otherwise cause a variety of diseases such as cancer or heart disease. This thesis focuses on investigating the role of ascorbic acid in the elimination of oxidative stress-induced mutagenesis.</p> <p> To investigate how vitamin C decreases level of the DNA mutation frequency and protects DNA from free radicals, knockout Gulo and Big Blue mice were used as models to determine the ability of vitamin C to minimize oxidative stress. The Big Blue mice carry the cll gene which is a reporter gene through which DNA mutation rate can be detected in any part of body. Therefore, we generated double transgenic mice which are Gulo deficient or a Big Blue background. Homozygote Gulo cll positive (Gulo-/- cll+) were obtained by crossing heterozygote Gulo cll Positive and homozygote Gulo mice. Five Gulo-/-cll mice were placed under vitamin C deficient diet and another five were supplemented with vitamin C. DNA mutation frequency was analyzed in the two groups. There were no significant differences in mutation frequencies between homozygote Gulo-/- cll mice on vitamin C deficient diet and homozygote Gulo-/- cll+ mice fed vitamin C rich diet. One treatment mouse showed increased frequency in mutations but a second did not. Further tests can be done on other treated knockout mice to identify the mutation types generated by oxidative stress in the absence of vitamin C.</p> / Thesis / Master of Science (MSc)

M.S.

Series or experiments employing laboratory methods of homogenization were run at temperatures or 100°F, 110°F, 120°F, 130°F, 140°F, 150°F, 160°F, and 170°F and with pressures of 1000, 1750, and 2500 pounds at each temperature. In all series, at all temperatures and at all pressures, the results of homogenization indicated a reduction of the titratable ascorbic acid content of the milk processed. The amount of destruction of ascorbic acid was variable, ranging from 7.4 per cent to 14.3 per cent with an average reduction or 11.34 per cent. Supplement experiments, employing commercial experiment and commercial methods corroborated the results obtained in the laboratory. / Master of Science

1. A saturation study was conducted on three normal college girls, weighing 50, 43, and 54 kg. Subject I was found to require more than 2.2 mg. per kg. of ascorbic acid or a total of 110 mg. To maintain tissue saturation and subjects II and III were found to require 1.6 mg. per kg. or a total of 67 mg. And 86 mg. respectively. 2. The data for subjects II and III suggest that they may be a relationship between body size and the ascorbic acid requirement, since on the per kg. basis, they both needed the same (1.6 mg. per kg.). However, the fact that subject I required a much greater amount to maintain tissue saturation, more than 2. mg. per kg. Indicates that individual difference may overshadow body size in its effect on requirement of vitamin C. 3. The effect of various factors on the ascorbic acid excretion were observed: a. The correlations between the percentage of ascorbic acid intake excreted and the urinary pH were -.2122, -.315, and -/8588 for subjects I, II, and III respectively, only that of subject III being significant. b. Insignificant correlations of .293, -.2015, and -.0507 were found between the urinary volume and the percentage of ascorbic acid excreted. c. Correlations for subjects I and II between ascorbic acid excretion and cigarettes smoked were .0243 and .3198. Neither was considered significant. d. Insignificant correlations, -.149 and .00286, were found between ascorbic acid excretion and the coffee intake. e. A slightly significant correlation of -.3848 was found between the percentage (Average for the three subjects) of the ascorbic acid intake excreted and the maximum daily temperature. 4. A comparison of the results of this saturation study with two other saturation studies (2, 24) points out that 1.6-1.7 mg. per kg. May be an adequate requirement in most of the cases studied. However, all three studies give data on only thirteen subjects. 5. Suggestions are offered which it is hoped will so simplify the procedure of the saturation test that with efficient planning a much larger number of subjects may be studied: a. Collection and analysis of urine only on test dose days. b. Ad libitum intake of foods containing negligible amounts of vitamin C. c. Determination of the urinary pH at the time of collection. / Master of Science

Vitamin C (ascorbic acid) is one of the most essential organic compounds required by the human body for normal metabolic function. Unfortunately, this valuable nutrient is not produced in the human body but most plants and animal can produce this molecule. Although ascorbic acid was not isolated until the early part of the twentieth century, it was known that eating limes and other citrus fruits could ward off the affects of scurvy as early as the 1500's. Ascorbate serves many critical functions in plants as well as the human body. In both, it works as a cofactor in the production of hydroxyproline-rich compounds and helps protect molecules such as proteins, lipids and fatty acids from oxidation. Although the biochemical pathway in animals has been known since the 1950's (Jackel et al., 1950), the exact process by which ascorbic acid is made in plants has eluded scientists. It was shown in 1963 that the inversion of the hexose carbon chain, which occurs in the animal pathway, is not a possible mode of synthesis in plants (Loewus, 1963). As an alternative, a non-inversion pathway was proposed, which achieves ascorbic acid using D-mannose and L-galactose as intermediates, referred to as the Smirnoff-Wheeler pathway (Wheeler et al., 1998). It was shown that transforming lettuce (cv. Grand Rapids and Black Seeded Simpson) and tobacco (cv. Xanthi) with the terminal enzyme in the animal biosynthetic pathway (GLO; L-gulono-gamma-lactone oxidase) increases the ascorbic acid content between 4 and 7 fold. It was also shown through feeding studies that wild type tobacco plants had elevated ascorbate levels when fed the animal precursor (Jain and Nessler, 2000). These data suggest that at least part of the animal pathway could be present in plants, along with the Smirnoff-Wheeler (1998) pathway. To further investigate this discovery, wild type and ascorbic acid-deficient Arabidopsis thaliana were transformed with the glo. Homozygous lines of these transformants were generated and the ascorbic acid levels were compared to the untransformed wild type and mutant plants. Although the wild type plants containing glo did not show a significant increase in ascorbic acid production, all five of the vtc mutant lines had an increased ascorbic acid content relative to wild type level. These data suggest that an alternative pathway is present in plants that does not require many of the steps in the published Smirnoff-Wheeler (1998) pathway to produce ascorbic acid. / Master of Science

Background: The adequacy of serum ascorbic acid (vitamin C) concentrations in young Canadian adults is unknown. Individuals have varied serum ascorbic acid response to dietary vitamin C, possibly due to genetic variation. Objective: To investigate the prevalence of serum ascorbic acid deficiency in young Canadians and to determine whether common genotypes modify the association between dietary vitamin C and serum ascorbic acid. Methods: Subjects were 1277 men and women aged 20-29 years from the Toronto Nutrigenomics and Health study. Vitamin C intakes were estimated by a 196-item FFQ. Fasting blood was collected to measure serum ascorbic acid by HPLC and to genotype for common polymorphisms in genes that code for glutathione S-transferase (GST) (GSTM1, GSTT1 and GSTP1), haptoglobin (Hp), and vitamin C transporters (SLC23A1 and SLC23A2). Results: 53% of subjects had adequate, 33% had suboptimal and 14% had deficient serum ascorbic acid. Subjects with deficiency had higher mean C-reactive protein, waist circumference, BMI and blood pressure than subjects with adequate serum ascorbic acid. The odds ratio (95% confidence interval) for serum ascorbic acid deficiency was 3.43 (2.14, 5.50) for subjects who did not meet the vitamin C recommendation compared to those who did. The corresponding odds ratios were 2.17 (1.10, 4.28) and 12.28 (4.26, 33.42) for individuals with the GSTT1 functional and null genotypes respectively (interaction p=0.01), and 2.29 (0.96, 5.45) and 4.03 (2.01, 8.09) for the GSTM1 functional and null genotypes (interaction p=0.04). These odds ratios were 4.77 (2.36, 9.65) for the Hp2-2 genotype, but 1.69 (0.80, 3.63) for carriers of the Hp1 allele (interaction p=0.02). Serum ascorbic acid concentrations (mean +/- SE) differed among SLC23A1 rs4257763 genotypes (GG: 24.4 +/- 1.3, GA: 26.8 +/- 1.1, AA: 29.7 +/- 1.4, p=0.002). Conclusions: Serum ascorbic acid deficiency is prevalent and associated with markers of chronic disease. Individuals with GST null or Hp2-2 genotypes had an increased risk of deficiency if they did not meet the recommendation for vitamin C, suggesting that GSTs and haptoglobin may spare ascorbic acid when dietary vitamin C is insufficient, thus protecting against serum ascorbic acid deficiency.

Background: The adequacy of serum ascorbic acid (vitamin C) concentrations in young Canadian adults is unknown. Individuals have varied serum ascorbic acid response to dietary vitamin C, possibly due to genetic variation. Objective: To investigate the prevalence of serum ascorbic acid deficiency in young Canadians and to determine whether common genotypes modify the association between dietary vitamin C and serum ascorbic acid. Methods: Subjects were 1277 men and women aged 20-29 years from the Toronto Nutrigenomics and Health study. Vitamin C intakes were estimated by a 196-item FFQ. Fasting blood was collected to measure serum ascorbic acid by HPLC and to genotype for common polymorphisms in genes that code for glutathione S-transferase (GST) (GSTM1, GSTT1 and GSTP1), haptoglobin (Hp), and vitamin C transporters (SLC23A1 and SLC23A2). Results: 53% of subjects had adequate, 33% had suboptimal and 14% had deficient serum ascorbic acid. Subjects with deficiency had higher mean C-reactive protein, waist circumference, BMI and blood pressure than subjects with adequate serum ascorbic acid. The odds ratio (95% confidence interval) for serum ascorbic acid deficiency was 3.43 (2.14, 5.50) for subjects who did not meet the vitamin C recommendation compared to those who did. The corresponding odds ratios were 2.17 (1.10, 4.28) and 12.28 (4.26, 33.42) for individuals with the GSTT1 functional and null genotypes respectively (interaction p=0.01), and 2.29 (0.96, 5.45) and 4.03 (2.01, 8.09) for the GSTM1 functional and null genotypes (interaction p=0.04). These odds ratios were 4.77 (2.36, 9.65) for the Hp2-2 genotype, but 1.69 (0.80, 3.63) for carriers of the Hp1 allele (interaction p=0.02). Serum ascorbic acid concentrations (mean +/- SE) differed among SLC23A1 rs4257763 genotypes (GG: 24.4 +/- 1.3, GA: 26.8 +/- 1.1, AA: 29.7 +/- 1.4, p=0.002). Conclusions: Serum ascorbic acid deficiency is prevalent and associated with markers of chronic disease. Individuals with GST null or Hp2-2 genotypes had an increased risk of deficiency if they did not meet the recommendation for vitamin C, suggesting that GSTs and haptoglobin may spare ascorbic acid when dietary vitamin C is insufficient, thus protecting against serum ascorbic acid deficiency.

The objective of this dissertation was to study the disposition and absorption of riboflavin and ascorbic acid in healthy male subjects as a function of age. Deficiencies of these vitamins have been found in the elderly and malabsorption is suspected in this population. Absorption of riboflavin was examined by administration of an oral dose of 200 mg of riboflavin-5'-phosphate (FMN) and disposition was studied following a 30 min constant rate IV infusion of FMN equivalent to 25 mg of riboflavin. Ascorbic acid absorption was examined by administration of a 5 g oral dose and disposition was studied following a 1 g IV dose. Test doses of these vitamins were administered to subjects ranging in age from 33 to 85 years. High performance liquid chromatographic assays were developed for the determination of riboflavin and ascorbic acid in plasma and urine. The method for riboflavin separated FMN and flavin adenine dinucleotide under isocratic conditions. Quantification of the three flavins was achieved by fluorescence detection. The assay system for ascrobic acid used a postcolumn reaction for the fluorometric detection of a dehydroascorbic acid derivative. This method used isoascorbic acid as the internal standard. Finally, similar specificity between a "methoxyaniline" colorimetric method and the chromatographic method was observed for the quantification of ascorbic acid in plasma and urine. Albumin plasma concentration and riboflavin protein binding were decreased with age (p < 0.05). The in vitro stability of ascorbic acid in whole blood and plasma decreased with age (p < 0.05, n = 17). The results of this study indicate no age-related alterations in parameters associated with the gastrointestinal absorption of these vitamins. Increased relative renal excretion of riboflavin with age may explain the deficiency of this vitamin. On the other hand, ascorbic acid deficiency with age may be explained by decreased stability of the vitamin in blood.

In controlled release applications a drug is molecularly dispersed in a polymer phase. In the presence of a thermodynamically compatible solvent, swelling occurs and the polymer releases its content to the surrounding medium. The rate of the drug release can be controlled by interfering with the swelling rate of the beads or by influencing diffusion through the viscosity of the polymer. Beads that contain chitosan were prepared through the ionotropic gelation method where tripolyphosphate (TPP) was used as the crosslinking agent. Beads that consisted of 3% w/v isoniazid (lNH) and 5% w/v chitosan were prepared in a 5% w/v TPP solution (pH 8.7) as the primary beads. To improve the drug loading of chitosan isoniazid beads (ClB) the TPP concentration, pH of the TPP solution and the INH concentrations were altered for maximum drug loading. To increase the porosity of the beads of chitosan beads Explotab® (EXPL), Ac-Di-Sol® (ADS) and Vitamin C (VC) were added individually to chitosan solutions at concentrations of 0.1, 0.25 and 0.5% w/v before adding the mixture to the TPP solution. Morphology, swelling and drug loading studies were used to evaluate the different formulations. After these excipients were added individually they were also added in combinations of two excipients respectively and characterised. From the results of the drug loading studies the beads that contained only chitosan and isoniazid showed a percentage drug loading of (43.92%) which is the best of all the beads that were analyzed. The multi excipient combination of Ac-Di-Sol® and Explotab® showed the best swelling capability at both pH levels. Dissolution studies were conducted on all the formu lations over a period of 6 hours (360 minutes) at pH 5.6 and pH 7.4. From the dissolution results it were clear that no chitosan dissolved at both pH values. The dissolution of single pharmaceutical excipient (SPE) and multi pharmaceutical excipient (MPE) formulations can be arranged in the following order: VC/ADS < VC < ADS/EXPL < ADS < VC/EXPL < CIB < EXPL. Explotab® is a potential excipient for enhanced drug release over a wide pH range. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

The most apparent symptom of boron deficiency in higher plants is a cessation of growth. Deficiency causes a reduction in ascorbate concentration and the absorption of nutrient ions. Addition of ascorbate temporarily relieves deficiency symptoms. In boron sufficient plants the addition of ascorbate to media causes an increased uptake of nutrients. In an attempt to discover if ascorbate addition to deficient plants causes increased ion uptake, radish plants were grown hydroponically in four different strengths of boron solution. A colorimetric assay for phosphorus was performed both before and after supplementation. Results, however, were inconclusive.