Canada fortifies certain products with folic acid and has periconceptional supplementation guidelines – policies designed to improve folate status and reduce the incidence of poor birth outcomes. Though optimal folate concentrations have been linked to health benefits, concerns have been raised regarding potential associations with adverse health outcomes. Direct biochemical assessment of the folate status of Canadians based on a nationally representative sample has not been done in more than 40 years. The overall purpose of this research was to investigate the folate status of the Canadian population. All analyses used the nationally representative 2007–2009 Canadian Health Measures Survey (CHMS). Red blood cell (RBC) folate was measured by Immulite 2000 immunoassay. Key findings indicate that folate deficiency (<305 nmol/L) was virtually non-existent in the Canadian population (6–79 years old). Still, one-fifth of women of childbearing age (WCBA; 15–45 years old) had sub-optimal concentrations for the prevention of neural tube defects (<906 nmol/L). Folic acid supplement intake was a primary determinant of WCBA achieving a RBC folate concentration ≥906 nmol/L. A distinct shift towards elevated RBC folate concentrations emerged. Three hypothetical cut-offs (1450 nmol/L, 1800 nmol/L and 2150 nmol/L) were examined to create dialogue since a universal definition of high RBC folate concentration does not exist. Females, participants aged 60¬–79 years, and those who were overweight or obese had the greatest prevalence of having high RBC folate at each cut-off. We conducted the first national-level comparison of RBC folate concentrations between the United States and Canada. Two different folate assay methods – microbiologic assay (NHANES) and Immulite 2000 immunoassay (CHMS) – necessitated the application of a conversion equation. Median Canadian RBC folate concentrations (adjusted to microbiologic assay) were lower than those of Americans but unadjusted Canadian median RBC folate values were higher. Canadian WCBA were less likely than American WCBA to have RBC folate ≥906 nmol/L, though Canadian WCBA with unadjusted RBC folate values were more likely to achieve this cut-off. These results indicate a need for strategies targeting WCBA to improve compliance with folic acid supplement recommendations. The strength and necessity of supplements for the general population should be re-assessed. Further, harmonization of folate measurement procedures in future surveillance efforts would support comparisons and inform policy directions.

A prospective observational study was conducted to investigate the effects of nutrition and related factors on the outcome of pregnancy in nulliparous African American women 16-35 years of age. Blood samples from a subset of these subjects were taken during the first (1st), second (2nd) and third (3rd) trimesters of pregnancy and at delivery. Cord blood samples were also collected at delivery. Levels of selected biochemical variables including serum ferritin, vitamin B12 and folate as well as whole blood folate, and selected hematologic indices were determined and correlated with pregnancy outcome variables. During the second trimester of pregnancy, values for hematocrit and hemoglobin were less than 30% and 11 g/dL, respectively, in 16% and 30% of the participants, respectively. Serum and whole blood (WB) folate increased sequentially during pregnancy. Cord concentrations of serum folate were significantly higher than maternal concentrations at delivery (P<0.05). Serum ferritin declined significantly from 36±5.6 ng/ml in the first trimester to 17±1.5 ng/ml during the 3rd trimester (P<0.05), and returned to the 2nd trimester level (26±2.0 ng/ml) at delivery. Second trimester WB folate was positively related to birth weight (R2=0.21), while gestational age was inversely correlated with 3rd trimester vitamin B12 (R2=0.34). These data suggest that vitamin B12 and folate play an important role in the outcome of pregnancy in this population.

Dihydrofolate reductase is an enzyme that catalyzes the reaction of dihydrofolate to tetrahydrofolate with nicotinamide adenine dinucleotide phosphate. Haloferax volcanii is a “salt-loving” microorganism found in the Dead Sea. Such microorganisms have adapted to their extreme environments and now require extremely high salt concentrations to survive. The study of dihydrofolate reductase from Haloferax volcanii with the substrate folate bound, which is the focus of the work presented in this dissertation, offers further understanding of this adaptation. The effect that high salt concentration has on this enzyme is not fully understood; however, this dissertation includes the investigation of the structure of a halophilic enzyme with substrate bound, providing new information in this uncertain field of research. In particular, the results shown here (along with future studies) can be applied towards the question of how an extremely salty environment affects enzyme function, stability, solubility, and flexibility.Triple resonance nuclear magnetic resonance spectra of the 17.9 kDa enzyme with bound substrate have been interpreted. 1H, 13C, and 15N backbone and side chain chemical shifts were specifically assigned to the amino acids that make up the enzyme. These assignments revealed overall similarities to the chemical shifts of the apo enzyme, with some exceptions. These exceptions are of particular interest as they are due to the binding of folate. Secondary structural analysis of the nuclear magnetic resonance data based on the chemical shift index showed that the binary complex has similar secondary structural features to the folate-bound dihydrofolate reductase from mesophilic Escherichia coli.The structure of dihydrofolate reductase from Haloferax volcanii with folate bound has been investigated using Crystallography and nuclear magnetic resonance system structure calculations, suggesting an overall similarity to the X-ray crystallography and nuclear magnetic resonance structures of apo dihydrofolate reductase from Haloferax volcanii. The results of the structure calculation and secondary structural analysis of the chemical shift assignments suggest secondary structural features including a beta-sheet in the core of the enzyme composed of 8 beta-strands, surrounded by 4 alpha-helices, and 4 major loops. The structure of the binary complex was compared to its mesophilic counterpart, the folate-bound dihydrofolate reductase from Escherichia coli, which was investigated by X-ray crystallography. The results of the work described in this dissertation do not agree with loop structure of the mesophilic complex.

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A series of studies were conducted to evaluate effects of lactation, exercise, and anti-folate drugs on folate status in the horse, and the bioavailability of supplement and feed folate in the horse. In the first study, mares and foals had adequate plasma folate, RBC folate, and plasma homocysteine concentrations during 6 mo of lactation and growth. Therefore, mares and foals maintained on quality grass/legume pastures and offered a pasture supplement did not require additional folate supplementation to maintain folate status during lactation and growth. In the second study, 25 mg of oral folic acid (FA) supplemented 5 times/wk to 11 mature horses engaged in routine submaximal exercise did not improve folate status, submaximal athletic performance, or combat the increase in oxidative stress during the 12 wk supplementation period compared to 11 horses not given supplemental folate. The common practice of supplementing horses with oral FA in vitamin supplements appears to be of little benefit to horses engaged in routine submaximal exercise. In the third study, daily oral administration of pyrimethamine (PYR) and sulfadiazine (SDZ) for 9 wk followed by 6 wk of coadministration of either Peptidoglycan or FA was associated with a decline in folate status resulting in moderate hyperhomocysteinemia, but not clinical signs of anemia. Peptidoglycan as a source of formylated folate and FA were not effective in improving folate status in horses coadministered PYR and SDZ, two anti-folate drugs commonly administered in equine veterinary practice. The last study assessed the bioavailability of oral and i.v. 5-methyltetrahydrofolate (5-mTHF), 5-formyltetrahydrofolate (5-fTHF), or FA, and the bioavailability of folate from concentrates fed to horses. The minimum efficiency of absorption for supplemental FA was 11 %. The low bioavailability of FA indicates a need for further research on the potential benefits of alternative sources of folate, including 5-fTHF, on increasing folate status in the horse. / Ph. D.

Lactating women have an increased requirement for folate which contributes to their risk for suboptimal folate status. Although milk folate secretion appears to be maintained independent of folate intake and maternal folate status, studies with animal species have demonstrated a relationship between iron deficiency and impaired milk folate secretion. Objectives of this study were to monitor the folate status of lactating women and to examine the relationship among folate intake, dietary iron, folate status, iron status and milk folate. Seven-day dietary records, milk samples, and blood samples were collected monthly for four months from five lactating women. Dietary iron and folate was analyzed. Milk folate, serum ferritin, serum folate, and red blood cell (rbc) folate concentrations were measured. Mean folate and iron intakes were 495 ± l05μg/d and 24 ± 4 mg/d, respectively. All women had normal rbc folate and serum ferritin values during the study. Milk folate increased (p=.06) from 35± 10 μg/L in month one to 69 ± 30 μg/L in month three. Dietary and rbc folate were not significantly correlated with milk folate. There was a significant positive correlation between milk folate and serum folate (r = .48, p= .04) and between milk folate and iron intake (r=.63, p=.003). Results indicate that the folate intake in this population of lactating women was sufficient to maintain adequate folate stores. Results also suggest a relationship between iron intake and milk folate. Research is needed to determine dietary requirements during lactation and to investigate the relationship between dietary iron and milk folate. / Master of Science

Conventional methods of cancer therapy have been severely limited by inefficient delivery of therapeutic doses without incidence of harsh and toxic side effects in normal tissues. Consequently, countless new methods for early detection and drug delivery have been investigated in the area of nanoparticles and hydrogels. Although many of these methods are promising, the complex nature of cancer increases the difficultly for the development of the perfect system. Cellulose nanocrystals (CNCs) have been studied widely for a variety of applications. Despite their advantages, investigations of their abilities in the biomedical field have not been explored. The goal of this project is to delve into the potential uses of CNCs in detection, targeted drug delivery, and potentiation of irreversible electroporation (IRE)-induced cell death in folate receptor (FR)-positive cancers. To accomplish this task we have prepared stable and reproducible CNCs from wood pulp via sulfuric acid hydrolysis. Furthermore, we have functionalized the surface of these nanoparticles and conjugated them with the targeting ligand folic acid (FA) and the fluorescent imaging agent fluorescein-5\'-isothiocyanate (FITC) to create FITC-CNC-FA; CNCs have also been conjugated with doxorubicin (DOX), a potent chemotherapeutic (DOX-ALAL-CNC-FA). We have determined FITC-CNC-FA's and DOX-ALAL-CNC-FA's ability to specifically target FR-positive cancer cells in vitro; meanwhile non-targeted CNCs (FITC-CNC) were shown unable to bind to these cell types. In addition, we have investigated FITC-CNC-FA's pharmacokinetic activity in vivo. To properly model the CNC conjugate's activity in vivo, a physiologically based pharmacokinetic (PBPK) model has been constructed. We have also examined CNCs' ability to potentiate a new technique for tumor ablation, IRE. Pre-incubation with FA-conjugated CNCs (CNC-FA) have shown an increase in cytotoxicity in FR-positive cancer cells induced by IRE. In addition, CNC-FA did not potentiate IRE-induced cytotoxicity in a FR-negative cancer cell type. For a more comprehensive understanding of CNC-FA's ability to potentiate IRE induced cytotoxicity, we optimized a 3D in vitro hydrogel system. Preliminary data suggest this method of experimentation will be more realistic to in vivo studies to be completed in the future. Together, these studies showcase CNCs as efficient and effective nano-carriers in tumor detection and treatment. / Ph. D.

Le terme folate (vitamine B9) désigne une famille de molécules ayant une structure de base composée de 3 parties : un noyau pterine, un acide para-aminobenzoïque (pABA) et une chaine de glutamates. Le rôle de ces cofacteurs est de transporter des groupements monocarbonés. Ils interviennent dans de nombreuses réactions comme la synthèse des bases nucléiques, la synthèse de méthionine et la synthèse et le turnover de la S-adenosylmethionine,. Le folate est synthétisé chez les plantes et un grand nombre de micro-organismes dont les parasites du phylum des apicomplexes, tels que Plasmodium falciparum, et Toxoplasma gondii. Les enzymes impliquées dans cette voie de biosynthèse étant absentes chez l'homme, elles représentent des cibles herbicides, antibiotiques et antiparasitaires potentielles. Les inhibiteurs de la voie de biosynthèse du folate (tels que les sulfamides, analogues du pABA et inhibiteurs de la dihydroptéroate synthase, ou les inhibiteurs de la dihydrofolate réductase), sont souvent utilisés comme antibiotiques et antiparasitaires. Un problème majeur dans le traitement de ces maladies infectieuses est la résistance développée contre ces molécules, ce qui nécessite la recherche permanente de nouveaux médicaments. Le pABA est synthétisé en plusieurs étapes qui sont autant de cibles intéressantes pour développer de nouveaux inhibiteurs. Tout d'abord l'aminodeoxychorismate (ADC) synthase transforme le chorismate en ADC, puis dans une seconde étape, l'ADC est transformé en pABA par une ADC lyase. Chez les plantes supérieures et les parasites apicomplexes l'ADC synthase est une enzyme bifonctionnelle composée de deux grands domaines, un domaine glutamine amidotranferase (GAT) qui permet de produire le NH3 nécessaire à l'amination du chorismate, et un domaine ADC synthase (ADCS). Nous avons pu déterminer les paramètres cinétiques de la GAT-ADCS d'Arabidopsis. Nous avons constaté que ces deux domaines fonctionnent indépendamment, c'est-à-dire soit en présence de glutamine seule pour le domaine GAT (pas de chorismate), soit en présence de chorismate et de NH3 pour le domaine ADCS (pas de glutamine). Toutefois, le fonctionnement en tandem des deux domaines (tous les substrats sont présents) améliore les propriétés cinétiques (kcat) de chacun d'eux. Nos résultats montrent aussi que le NH3 produit par le domaine GAT et nécessaire à la synthèse de l'ADC n'est pas relargué dans le milieu extérieur mais canalisé (channeling) vers le domaine ADCS. Finalement, nous avons observé que l'ADC, produit final de la réaction, retro-inhibe le domaine ADCS en absence d'ADC lyase. Pris dans son ensemble, nos résultats indiquent que l'amination du chorismate est l'étape la plus limitante de la synthèse du pABA,.. Des expériences de criblage à haut débit nous ont permis d'identifier une molécule, la rubreserine, qui inhibe in vitro le domaine GAT de l'ADC synthase d'Arabidopsis avec un Ki autour de 8 µM. Nous avons observé que cette molécule inhibe la croissance de plantules d'Arabidopsis thaliana et la prolifération des parasites Toxoplasma gondii et Plasmodium falciparum avec des IC50 respectif de 65 µM, 20 µM et 1.2 µM. Chez Arabidopsis, la concentration en folate des cellules traitées est abaissée d'environ 40% par rapport au contrôle, une diminution qui n'a plus lieu en présence de pABA. L'ajout de pABA et de 5-formyltetrahydrofolate dans les milieux de culture d'Arabidopsis ou de Toxoplasma supprime en grande partie l'inhibition de croissance liée à la rubreserine, ce qui montre bien la connexion entre rubreserine et voie de biosynthèse du folate. Avec Toxoplama gondii, la rubreserine apparait plus efficace que les sulfamides pour bloquer l'invasion et la prolifération de ces parasites dans les fibroblastes humains. Ces résultats valident la GAT-ADCS comme cible anti-folate et montrent que la rubreserine a des propriétés anti-parasitaires intéressantes. / The term folate (vitamin B9) is a family of molecules with a basic structure composed of 3 parts: a core pterin, a para amino benzoic acid (PABA) moiety and a chain of glutamate. The role of these cofactors is to carry one-carbon groups. They are involved in many reactions such as the synthesis of nucleic acids, the synthesis of methionine and the synthesis and turnover of S-adenosylmethionine. Folate is synthesized in plants and many micro-organisms including parasites of the Apicomplexa phylum such as Plasmodium falciparum and Toxoplasma gondii. Several enzymes involved in the pathway are absent in humans, and so they are potential targets for herbicide, antibiotic and antiparasitic drugs. Inhibitors of folate biosynthesis (such as dihydropteroate synthase inhibitors, or inhibitors of dihydrofolate reductase), are often used as antibiotics and pesticides. A major problem in treating these infectious diseases is the resistance developed against these molecules, which requires a constant search for new drugs. pABA is synthesized in several steps which are all attractive targets for developing new inhibitors. First the aminodeoxychorismate (ADC) synthase converts chorismate into ADC, and then, in a second step, the ADC is converted to pABA by an ADC lyase. In higher plants and apicomplexan parasites the ADC synthase is a bifunctional enzyme composed of two main domains: a glutamine amidotranferase (GAT) domain that produces NH3, and an ADC synthase domain (ADCS) that catalyzes the amination of chorismate. We determined the kinetic parameters of the Arabidopsis GAT-ADCS. We found that these two domains function independently, that is to say either in the presence of glutamine alone for the GAT domain (no chorismate) or in the presence of chorismate and NH3 for the ADCS domain (no glutamine). However, the tandem operation of the two domains (all substrates are present) improves the kinetic properties (kcat) of each one. Our results also show that the NH3 produced by the GAT domain and required for the synthesis of ADC is not released into the surroundings but rather channeled to the active site of ADCS. Finally, we observed that ADC, the final product of the reaction, retro-inhibits the ADCS domain in the absence of ADC lyase. Taken together, our results indicate that the amination reaction of chorismate is the most limiting step of the synthesis of pABA. Using high-throughput screening approaches we have identified a molecule, rubreserine, which inhibits in vitro the GAT domain of Arabidopsis GAT-ADCS with a Ki value around 8 µM. We observed that this molecule inhibits the growth of Arabidopsis thaliana seedlings and the proliferation of Toxoplasma gondii and Plasmodium falciparum parasites with respective IC50 of 65 µM, 20 µM and 1 µM. In Arabidopsis, the concentration of folate in rubreserine-treated cells is lowered by about 40% compared to controls, a decrease that is suppressed in the presence of pABA. The addition of pABA and 5-Formyltetrahydrofolate in the culture media of Arabidopsis and Toxoplasma partly reverses the growth inhibition due to rubreserine, which shows the connection between the drug and folate biosynthesis. Rubreserine appears more effective than sulfa-drugs to block the invasion and proliferation of Toxoplasma gondii in human fibroblasts. These results validate the GAT-ADCS as an anti-folate target and show that rubreserine has interesting anti-parasitic properties.

Neural tube defects (NTD) result from the failure of the neural tube to close properly very early in gestation. A child born with an NTD may experience an early death or life-long disability. In the 1990s, the critical role of folic acid in the prevention of NTDs was confirmed and as a strategy to increase blood folate concentrations of women of childbearing age, folic acid fortification programs were mandated in Canada and the US. However, this change impacted the entire population not just women of childbearing age and not everyone may benefit from the increased folate intake. The objective of this research was to investigate the impact of higher intakes of folates on the mortality rates of children with acute lymphoblastic leukemia (ALL) and the risk of colorectal cancer (CRC) in adult populations. To address the impact in children with ALL, a comparison of the mortality rates between the pre- and post-fortification time periods in Ontario was performed using data from the Pediatric Oncology Group of Ontario. A second comparison between the mortality rates in these children in non-folic acid fortifying countries and the US was also completed. These analyses suggest that folic acid fortification is not negatively impacting mortality. With respect to CRC, one systematic review and two meta-analyses were conducted investigating folate intake and the risk of CRC or adenoma recurrence. The first analysis, in observational studies, compared high versus low folate intake and the risk of CRC. The second examined folate intake within the various polymorphisms of the methylene tetrahydrofolate reductase enzyme. The final study examined the impact of supplementation of 1 milligram or more per day of folic acid and the risk of colorectal adenoma recurrence in those adults with a history of colorectal adenomas. The findings from the completed observational studies suggest that there is an associated risk reduction in colorectal cancer from the intake of higher levels of folates. The investigations into the impact of the folic acid fortification program suggest that the program is not associated with having a negative impact on mortality of children with ALL or on the risk of colorectal cancer.