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This thesis is focused on folic acid and its preventive effects on human health. Folic acid has many important functions in the human body and is especially important during pregnancy. It is essential for the proper development of the fetus and its adequate intake is the prevention of serious congenital malformations, such as neural tube defects, premature birth, abortion and other pathologies. The increased need for this vitamin is not easy to supplement with a regular diet. It is therefore recommended to take it in the form of food supplements (400-600 µg/day) during pregnancy and at least one month before the planned conception. For these reasons, it is necessary to focus on supporting the primary prevention of folic acid use, preferably in all fertile women. Folic acid also plays a crucial role in hematopoiesis and the main manifestation of its deficiency is megaloblastic anemia. Folic acid is further associated with the prevention of cardiovascular and cancer diseases and influencing the development of cognitive functions. The practical part of this thesis was focused on monitoring the saturation of pregnant women with this vitamin. The work contains a laboratory analysis of the content of the metabolite folic acid 5-methyltetrahydrofolate (5-MTHF) in the urine of pregnant women in order to...

Exposure to arsenic (As) is a major public health concern globally. Inorganic As (InAs) undergoes hepatic methylation to form monomethyl (MMAs)- and dimethyl (DMAs)-arsenical species, facilitating urinary As elimination. MMAsIII is considerably more toxic than either InAsIII or DMAsV, and a higher proportion of MMAs in urine has been associated with risk for a wide range of adverse health outcomes. One-carbon metabolism (OCM) is a biochemical pathway that provides methyl groups for the methylation of As, and is influenced by folate and other micronutrients, such as vitamin B12, choline, betaine and creatine. A growing body of evidence has demonstrated that OCM-related micronutrients play a critical role in As methylation. To analyze the impact of As exposure, it needs to be properly quantitated. Urinary As (uAs) is a biomarker of As exposure. Urinary creatinine (uCr) or specific gravity (SG) are used to correct uAs for urine dilution. However, uCr is correlated with As methylation, whereas SG has limitations in individuals with kidney damage. Therefore, it is important to determine which urine dilution proxy is appropriate in As-related research. In Chapter 2 we conducted a review that summarized observational epidemiological studies, interventions, and relevant experimental evidence examining the role that OCM-related micronutrients have on As methylation, toxicity of As, and risk for associated adverse health-related outcomes. People with higher relative percentage of MMAs (%MMAs) in urine (inefficient As methylation), have been shown to have a higher risk of cardiovascular disease and several cancers but appear to have a lower risk of diabetes and obesity in populations from the US, Mexico, and Taiwan. It is unknown if this opposite pattern with obesity is present in Bangladesh, a country with lower adiposity and higher As exposure in drinking water. Efficiency of As methylation differs substantially between species, between individuals, and across populations. In Chapter 3, we aimed to evaluate which urine dilution correction methods for uAs most accurately predicted blood As (bAs). We used data from the Folic Acid and Creatine Trial (FACT; N = 541) and Folate and Oxidative Stress (FOX; N = 343) study in Bangladesh. Three linear regression models were assessed using uAs (1) adjusted for uCr or SG as separate covariates, (2) standardized for uCr or SG, i.e., uAs/uCr, and (3) adjusted for residual corrected uCr or SG following adjustment for age, sex and BMI. Median uAs/bAs for FACT and FOX were 114/8.4 and 140/12.3 µg/L. In FACT, two-fold increases in uAs adjusted for uCr or SG were related to 34% and 22% increases in bAs, respectively, with similar patterns in FOX. In Chapter 4, we investigated the effects of folic acid (FA) and/or creatine supplementation on the concentrations of As species and primary (PMI: MMAs/InAs) and secondary (SMI: DMAs/MMAs) methylation indices in blood in Bangladeshi adults having a wide range of folate status. In a randomized, double-blinded, placebo-controlled trial, 622 participants were assigned to FA (400 or 800 μg/day), 3 g creatine/day, 3 g creatine + 400 μg FA/day, or placebo for 12 weeks. For the following 12 weeks, half of the FA participants were randomly switched to receive placebo. All participants received As-removal water filters at baseline. Blood As species were measured at baseline, and weeks 1, 12, and 24. In all groups, blood As species concentrations decreased due to filter use. After 1 week, the mean within-person increase in SMI for the creatine + 400FA group was greater than that of the placebo group (p = 0.05). The mean percent decrease (95% CI) in blood concentrations of MMAs (bMMAs) between baseline and week 12 was greater for all treatment groups compared to the placebo group [400FA: -10.3 (-11.9, -8.8); 800FA: -9.5 (-11.1, -8.0); creatine: -5.9 (-8.6, -3.0); creatine + 400FA: -8.4 (-10.0, -6.9); placebo: -2.0 (-4.0, 0.0)], and the percent increase in blood DMAs (bDMAs) concentrations for the FA treated groups all significantly exceeded that of placebo [400 FA: 12.8 (10.5, 15.2); 800 FA: 11.3 (8.90, 13.8); creatine + 400 FA: 7.40 (5.20, 9.70); placebo: -0.10 (-2.80, 2.60)]. The mean decrease in PMI and increase in SMI in all FA groups significantly exceeded placebo (p < 0.05). Data from week 24 showed evidence of a reversal of treatment effects on As species from week 12 in those who switched from 800FA to placebo, with significant decreases in SMI [-9.0% (-3.5, -14.8)] and bDMAs [-5.9% (-1.8, -10.2)] in those who switched from 800FA to placebo, whereas for those who remained on 800FA, PMI and bMMAs concentrations continued to decline [-7.2% (-0.5, -14.3) and -3.1% (-0.1, -6.2), respectively] for those who remained on 800FA supplementation. This trial was registered at https://clinicaltrials.gov as NCT01050556. In Chapter 5, we characterized the association between body mass index (BMI) and As methylation in Bangladeshi adults and adolescents participating in the FACT; FOX; and Metals, Arsenic, and Nutrition in Adolescents Study (MANAS). Arsenic species (InAs, MMAs, DMAs) were measured in urine and blood. Height and weight were measured to calculate BMI. The associations between concurrent BMI with urine and blood As species were analyzed using linear regression models, adjusting for nutrients involved in OCM such as choline. In FACT, we also evaluated the prospective association between weight change and As species. Mean BMIs were 19.2/20.4, 19.8/21.0, and 17.7/18.7 kg/m2 in males/females in FACT, FOX, and MANAS, respectively. BMI was associated with As species in female but not in male participants. In females, after adjustment for total urine As, age, and plasma folate, the adjusted mean differences (95% confidence) in urinary %MMAs and %DMAs for a 5 kg/m2 difference in BMI were -1.21 (-1.96, -0.45) and 2.47 (1.13, 3.81), respectively in FACT, -0.66 (-1.56, 0.25) and 1.43 (-0.23, 3.09) in FOX, and -0.59 (-1.19, 0.02) and 1.58 (-0.15, 3.30) in MANAS. The associations were attenuated after adjustment for choline. Similar associations were observed with blood As species. In FACT, a 1-kg of weight increase over 2 to 10 (mean 5.4) years in males/ females was prospectively associated with mean %DMAs that was 0.16%/0.19% higher. BMI was negatively associated with %MMAs and positively associated with %DMAs in females but not males in Bangladesh; associations were attenuated after plasma choline adjustment. In conclusion, we found that FA supplementation lowers bMMAs and increases bDMAs in a sample of primarily folate-replete adults, while creatine supplementation lowers bMMAs. Evidence of the reversal of treatment effects on As species following FA cessation suggests short-term benefits of supplementation and underscores the importance of long-term interventions such as FA fortification. Additionally, there is fairly robust evidence supporting the impact of folate on As methylation, and some evidence from case-control studies indicating that folate nutritional status influences risk for As-induced skin lesions and bladder cancer. However, the potential for folate to be protective for other As-related health outcomes, adverse health risks of high folate/FA levels (particularly in areas where folate supplements are common), and beneficial effects of other OCM-related micronutrients on As methylation and risk for health outcomes are not as well studied and warrant additional research. We also found that the role of body fat on estrogen levels that may influence OCM, e.g. by increasing choline synthesis. Research is needed to determine whether the associations between BMI and As species are causal and their influence on As-related health outcomes. Finally, we found that in assessing urine dilution correction approaches, models with uCr consistently had lower AIC values than SG across methods. The uAs associations with bAs were stronger after adjustment for uCr vs. SG. Decisions regarding urine dilution methods should consider whether the study outcomes are influenced by factors such as methylation or medical conditions.

This work focused on engineering bi-functionalized nanoparticles (NPs) based on carbon dots (CDs) to improve early cancer detection and treatment. Therefore, using folic acid (FA) as a targeting agent, the CDs were prepared to deliver high concentrations (HC) of doxorubicin (DOX) and gemcitabine (GEM) covalently and non-covalently to cancer cells. The prepared FA-CDs-DOX/GEM-HC NPs were characterized using ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. Assessment of the drug loading capacity (DLC) and drug loading efficiency (DLE) indicated that the non-covalent NPs have low DLC but high DLE compared to the relatively low DLE and high DLC of covalent NPs. In vitro drug release studies showed that the DOX/GEM release rate was faster at pH 5.0 in the non-covalent FA-CDs-DOX/GEM-HC NPs than covalent. Also, the non-covalent FA-CDs-DOX-HC NPs showed greater percentage cumulative drug release and lower cell viability in the MDA-MB-231 breast cancer cell line compared to covalent.

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Methylene tetrahydrofolate reductase has been purified 1000-fold from porcine kidney and 400-fold from mouse L1210-induced tumor tissue by classical methods. The enzyme preparations have been demonstrated to be essentially free of contaminating methionine synthetase and serine transhydroxymethylase activity. Studies of the kinetic properties of the kidney and tumor enzymes, with respect to the reverse reaction using N5-methyl tetrahydrofolate as the variable substrate, have indicated Km values of 2.0 and 2.4 x 10-4 M, respectively. Inhibition of this key branch point enzyme in folate metabolism by a number of antimetabolites indicates that several of these antifolate compounds exhibit enzyme inhibition superior to that of methotrexate, a druq extensively utilized in the maintenance of remission in cancer chemotherapy.

RNA epigenetics or Epitranscriptomics has emerged as a new field for understanding the post-transcriptional regulation of gene expression by RNA modifications. Among numerous types of RNA modifications, RNA cytosine-5 methylation (5-mrC) is recognized as an important epitranscriptomic mark that modulates mRNA transportation, stability and translation. In chapter 1, we summarize the currently available approaches to detect 5-mrC modification at global, transcriptome-wide and locus-specific levels, and compare the corresponding advantages and disadvantages of the techniques. We further focus on the bioinformatics data analysis of RNA bisulfite sequencing datasets by comparing existing packages with respect to key parameters for alignment and methylation calling and filtering of potentially false positive 5-mrC sites. To investigate the dynamic regulation of 5-mrC modification, as described in chapter 2, we adopt a widely used neuronal activity model, and perform RNA sequencing (RNA-seq) and RNA bisulfite sequencing (RNA BS-seq) to profile gene expression as well as transcriptome-wide 5-mrC modification. We have identified distinct gene expression profiles and differentially methylated 5-mrC sites (DMS) in neurons upon activation, and the genes with DMS sites are enriched with mitochondrial and synaptic functions. Moreover, it reveals a negative correlation between RNA methylation and mRNA expression in mouse cortical neurons during neuronal activity. Thus, these findings identify the dynamic regulation of 5-mrC modification during neuronal activity and reveal a potential link between RNA methylation and mRNA expression. In chapter 3, we investigate the effect of folate, a methyl-donor, on RNA cytosine-5 methylation (5-mrC) modification in adult mouse neural stem cells (NSCs). Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. RNA bisulfite sequencing of both total and polysome poly(A) RNA samples shows distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It also shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation. / Doctor of Philosophy / RNA epigenetics, a collection of RNA modifications, has recently emerged as an exciting, new field for understanding post-transcriptional regulation of gene expression. RNA cytosine-5 methylation (5-mrC) is one of the most well-known RNA modifications that modulates mRNA export, stability and translation. In the first chapter, we summarize the currently available methods for the measurement of 5-mrC modification. We highlight one of the techniques, RNA bisulfite sequencing (RNA BS-seq) and focus on the bioinformatics data analysis of RNA BS-seq datasets. We have compared several existing tools in regard of the key parameters in data analysis. In the second chapter, we adopt a widely used neuronal activity model to study the dynamic regulation of RNA cytosine-5 methylation (5-mrC). We perform RNA-seq and RNA BS-seq in neurons in response to stimulation. We have identified numerous differentially expressed genes and differentially methylated 5-mrC sites in activated neurons and find that these DMS-related genes are associated with mitochondrial and synaptic functions. Furthermore, we identify a negative correlation between RNA methylation and mRNA expression, indicating a potential role of 5-mrC modification in the regulation of mRNA expression. In the third chapter, we investigate the influence of a nutrient supplement, folic acid, on 5-mrC modification in adult mouse neural stem cells. Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. We perform RNA bisulfite sequencing of both total poly(A) RNA samples and polysome poly(A) RNA samples. We identify distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation.

Cardiovascular disease secondary to atherosclerosis is the most common cause of human morbidity and mortality. An early and fundamental event in the development of atherosclerosis is abnormal vascular endothelial and smooth muscle function. This can be measured by flow mediated dilatation and glyceryl trinitrate mediated dilatation in children at risk of atherosclerosis. Folic acid improves endothelial function (flow mediated dilatation) in adults with coronary artery disease. No studies have previously investigated the effects of folic acid on vascular function in at risk children with diabetes or obesity. In a cross sectional study an evaluation of vascular endothelial and smooth muscle function and their determinants was performed in 159 children with type 1 diabetes, 58 children with obesity, and 53 healthy children. Children with type 1 diabetes and children with mild to moderate obesity had comparable and severe vascular dysfunction but different determinants. Vascular function in healthy and obese children related to both body mass index and weight (adjusted for age and sex), and blood glucose. Children with obesity had lower folate levels and higher homocysteine levels than children with type 1 diabetes, an abnormal lipid profile and raised inflammatory markers. A randomised double blind placebo controlled cross over trial of 8 weeks of folic acid supplementation was performed in 38 children with type 1 diabetes. In these children, folic acid improved endothelial function with a sustained increase in folate levels but independent of homocysteine levels. Folic acid did not improve smooth muscle function. A randomised double blind placebo controlled parallel trial of 8 weeks folic acid supplementation was performed including 53 obese children. Folic acid did not improve vascular function in obese children in spite of sustained increase in folate levels, and a decrease in homocysteine levels. It was concluded that children with type 1 diabetes and obesity have comparable and severe endothelial and smooth muscle function. Determinants of vascular function in children, including weight and glucose, represent a continuum effect. Folic acid supplementation improved endothelial function in children with type 1 diabetes but not in children with obesity, whose metabolic changes causing endothelial dysfunction differ from children with diabetes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317003 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

[Truncated abstract] An elevated total plasma homocysteine concentration (tHcy) is associated with an increased risk of myocardial infarction and ischemic stroke. Folic acid, vitamin B6 and B12 supplements significantly reduce tHcy even in people who are not overtly vitamin deficient. If homocysteine is a causal risk factor for atherothrombotic events, treatment with B-vitamins might prove a simple and cost-effective means to reduce cardiovascular risk. However, it remains unclear whether elevated tHcy causes atherosclerosis or is simply a risk marker. To prove that homocysteine is a modifiable risk factor for cardiovascular disease it is necessary to show that lowering tHcy reduces vascular risk. The aim of this study was to determine whether long-term homocysteine-lowering with B-vitamins would improve vascular structure and function in people with a history of stroke. This study was a cross-sectional sub-study of the Vitamins TO Prevent Stroke trial (VITATOPS), a multi-centre, randomised, double-blind, placebo-controlled clinical trial designed to test the efficacy and safety of B-vitamins (folic acid 2mg, vitamin B6 25mg and vitamin B12 0.5mg) in the prevention of vascular events in patients with a recent history of stroke or transient ischemic attack. 173 VITATOPS participants were recruited for the current study. Age, sex, stroke type, medications, cardiovascular risk factors and smoking history were recorded and blood pressure, height, weight, waist and hip girth were measured in all subjects at least two years after randomisation. ... After a mean treatment period of 3.9 ± 0.9 years, the subjects randomised to vitamin treatment had significantly lower tHcy than the subjects randomised to placebo (7.9mol/L, 95%CI 7.5, 8.4 versus 11.8mol/L, 95%CI 10.9, 12.8; p<0.001). There were no significant differences between groups in CIMT (0.84 ± 0.17mm vitamins versus 0.83 ± 0.18mm placebo; p=0.74) or FMD (median of 4.0%, IQR 0.9, 7.2, vitamins versus 3.0%, IQR 0.6, 6.6 placebo; p=0.48). Pooled estimates from the meta-analyses showed that B-vitamin treatment reduces CIMT by 0.10mm (95%CI –0.20, -0.01mm) and increases FMD by 1.4%, (95%CI 0.7, 2.2), although these estimates may have been influenced by positive publication bias. The improvement in FMD was significant in studies of less than eight weeks duration but not in studies with longer treatment periods. The association between tHcy and CIMT and FMD was eliminated by adjustment for renal function and long-term B-vitamin treatment did not alter the strong linear relationship between tHcy and cystatin C. Lowering tHcy did not alter arterial wall inflammation assessed by 18FDG-PET, although small subject numbers meant we were unable to exclude a minor treatment effect. Long-term homocysteine-lowering with B-vitamin treatment did not improve CIMT or FMD or reduce arterial wall inflammation in people with a history of stroke. The relationship between tHcy and these markers of vascular risk was eliminated by adjustment for renal function. Our data are consistent with the hypothesis that elevated tHcy is a risk marker for cardiovascular disease rather than a modifiable causal risk factor.

Cardiovascular disease secondary to atherosclerosis is the most common cause of human morbidity and mortality. An early and fundamental event in the development of atherosclerosis is abnormal vascular endothelial and smooth muscle function. This can be measured by flow mediated dilatation and glyceryl trinitrate mediated dilatation in children at risk of atherosclerosis. Folic acid improves endothelial function (flow mediated dilatation) in adults with coronary artery disease. No studies have previously investigated the effects of folic acid on vascular function in at risk children with diabetes or obesity. In a cross sectional study an evaluation of vascular endothelial and smooth muscle function and their determinants was performed in 159 children with type 1 diabetes, 58 children with obesity, and 53 healthy children. Children with type 1 diabetes and children with mild to moderate obesity had comparable and severe vascular dysfunction but different determinants. Vascular function in healthy and obese children related to both body mass index and weight (adjusted for age and sex), and blood glucose. Children with obesity had lower folate levels and higher homocysteine levels than children with type 1 diabetes, an abnormal lipid profile and raised inflammatory markers. A randomised double blind placebo controlled cross over trial of 8 weeks of folic acid supplementation was performed in 38 children with type 1 diabetes. In these children, folic acid improved endothelial function with a sustained increase in folate levels but independent of homocysteine levels. Folic acid did not improve smooth muscle function. A randomised double blind placebo controlled parallel trial of 8 weeks folic acid supplementation was performed including 53 obese children. Folic acid did not improve vascular function in obese children in spite of sustained increase in folate levels, and a decrease in homocysteine levels. It was concluded that children with type 1 diabetes and obesity have comparable and severe endothelial and smooth muscle function. Determinants of vascular function in children, including weight and glucose, represent a continuum effect. Folic acid supplementation improved endothelial function in children with type 1 diabetes but not in children with obesity, whose metabolic changes causing endothelial dysfunction differ from children with diabetes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317003 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008