While assessment of folate requirements has been based only on dietary intakes, folate produced by the colonic microflora can exceed amounts consumed in food. Bacterially synthesized folate is absorbed across the rat and piglet colon. In vitro studies suggest, but direct evidence is lacking that folate is absorbed across the intact human colon. If indeed folate is absorbed, the amount synthesized may be susceptible to manipulation by fibre and prebiotics intake. We therefore performed two studies to investigate folate absorption across the colon. To confirm absorption across the intact human colon, in our first study, 684 nmol (320 µg) 13C5-glutamyl-[6S]-5-formyltetrahydrofolate was infused into the cecum of six adults and blood samples were collected. Tandem mass spectrometry confirmed folate absorption across the colon by appearance in plasma of 13C5-[6S]-5-methyltetrahydrofolate, at a rate of 0.6±0.2 nmol/h versus 7±1.2 nmol/h after intravenous injection of 172 nmol 13C5-5-formyltetrahydrofolate. Since bifidobacteria are potent folate producers, in our second study we evaluated the influence of bifidogenic oligosaccharides on colonic folate production and host folate status, using a piglet animal model. Piglets (n=12) were randomly assigned a milk-based formula with 5g/L inulin + 5g/L galactooligosaccharides, or 5g/L maltodextrin (control). After 28 days, the weights of colon contents (178 %) and colon tissue (37.9 %) of piglets fed oligosaccharides were greater than controls (P=0.0003, P=0.0044, respectively). The bacterial load and folate contents in the colons of piglets fed oligosaccharides were greater than controls (P=0.0022, P=0.0218, respectively). Body weights, blood folate status and liver and kidney folate concentrations did not differ. In conclusion, folate is absorbed across the human colon. Supplementation of the piglet diet with 5g/L inulin and 5g/L galactooligosaccharides increased the amounts of microbial folate, and the weights of colon tissue and contents, but folate concentrations in colon contents, blood and organs were not affected.

While assessment of folate requirements has been based only on dietary intakes, folate produced by the colonic microflora can exceed amounts consumed in food. Bacterially synthesized folate is absorbed across the rat and piglet colon. In vitro studies suggest, but direct evidence is lacking that folate is absorbed across the intact human colon. If indeed folate is absorbed, the amount synthesized may be susceptible to manipulation by fibre and prebiotics intake. We therefore performed two studies to investigate folate absorption across the colon. To confirm absorption across the intact human colon, in our first study, 684 nmol (320 µg) 13C5-glutamyl-[6S]-5-formyltetrahydrofolate was infused into the cecum of six adults and blood samples were collected. Tandem mass spectrometry confirmed folate absorption across the colon by appearance in plasma of 13C5-[6S]-5-methyltetrahydrofolate, at a rate of 0.6±0.2 nmol/h versus 7±1.2 nmol/h after intravenous injection of 172 nmol 13C5-5-formyltetrahydrofolate. Since bifidobacteria are potent folate producers, in our second study we evaluated the influence of bifidogenic oligosaccharides on colonic folate production and host folate status, using a piglet animal model. Piglets (n=12) were randomly assigned a milk-based formula with 5g/L inulin + 5g/L galactooligosaccharides, or 5g/L maltodextrin (control). After 28 days, the weights of colon contents (178 %) and colon tissue (37.9 %) of piglets fed oligosaccharides were greater than controls (P=0.0003, P=0.0044, respectively). The bacterial load and folate contents in the colons of piglets fed oligosaccharides were greater than controls (P=0.0022, P=0.0218, respectively). Body weights, blood folate status and liver and kidney folate concentrations did not differ. In conclusion, folate is absorbed across the human colon. Supplementation of the piglet diet with 5g/L inulin and 5g/L galactooligosaccharides increased the amounts of microbial folate, and the weights of colon tissue and contents, but folate concentrations in colon contents, blood and organs were not affected.

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Colorectal cancer (CRC) is the second most common cancer, in both sexes, in developed countries and the incidence rates are rising in many populations. Less than 10% of cases are thought to be due to recognised familial syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Epidemiological studies suggest that dietary factors play an important role in the aetiology of CRC; disease incidence is inversely associated with diets high in folate and has been positively associated with red meat consumption. Folate is important in DNA methylation and synthesis. The increased risk associated with red meat is proposed to be due to the formation of heterocyclic amines during cooking rather than meat consumption per se. The current study, a population-based case-control study (269 case and 408 control subjects) was carried out in the Grampian region and investigated polymorphisms in genes involved in folate and xenobiotic metabolism. Inter-individual differences in the activation and detoxification of xenobiotics and the metabolism of folate might alter risk of CRC. Mouthwash samples were collected from all participants and genomic DNA was extracted for genotypic analysis of MTHFR, CYP1A1, NAT2, GSTM1 and GSTT1. Homozygous possession of the MTHFR A1298C substitution was associated with a reduced risk of colon cancer. The reduced risk of rectal cancer associated with possession of the MTHFR C677T substitution approached statistical significance. A proposed mechanism is presented to explain the inverse association between CRC and MTHFR allelic variants. The CYP1A1 C2453A substitution was inversely associated with CRC risk. No significant alteration of risk was associated with deletions of GSTM1 or GSTT1 genes or acetylation status imputed from NAT2 genotype. Genomic DNA extracted from mouthwash samples, collected by post from an elderly population, was effective as template for PCR/RFLP methods of genotyping.

Poverty stricken areas of the world are affected by Neglected Tropical Diseases, with an estimated 1 billion sufferers. As well as inadequate living conditions and healthcare, there has been very little pharmaceutical incentive to tackle these diseases. As a result, the diseases are still spreading. Drugs available on the market suffer from poor efficacy, high toxicity, increasing resistance and inappropriate dosing for rural treatment. The nature of many NTDs prevents the use of vaccinations. Therefore, more efficacious and safe treatments are sought after. The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited in a number of drugs and drug targets. The same cannot be said for the kinetoplastids. Drug discovery programmes have focused on targeting enzymes of the folate metabolism with very little clinical success. Despite showing significant inhibition of the parasitic enzymes, potency is seen to decrease in cellular and animal models. Understanding how the folate pathway operates in these organisms could provide insight into where and how anti-folate compounds bind. This information could then be used to facilitate better drug treatments for the kinetoplastids. This thesis describes a number of approaches undertaken to better understand folate metabolism in kinetoplastids. Clinical and literature anti-folate compounds were immobilized onto resins, followed by chemical proteomics, utilizing novel techniques (iTRAQ), to allow for target identification. Using competition studies, specific and non-specific targets were identified in parasitic lysate (T. brucei and L. major) for each anti-folate compound. This method was further exploited by creating a folate resin (Folate beads). The resin had the potential to pull down 9 proteins from the “folate-ome”. In future studies, the resin can be used to enrich for the folate proteins in kinetoplastids and related organisms. Alongside the studies of the folate proteins, it was also desired to study proteins involved in the essential pterin pathway. This pathway has not been extensively studied in kintoplastids, with the exception of PTR1 (by-pass protein for DHFR). The failure to synthesise pterin derivatives for bead coupling led to a fragment screening campaign being carried out on QDPR in leishmania major. Working through a triage workflow, two moderately potent fragments were identified, showing inhibition against LmQDPR. Through structure-free optimization strategies, greater than 100 optimized fragments were synthesised in a bid to understand SAR. Although this work remains incomplete, LmQDPR has been successfully crystalized with 23 hit fragments, which are awaiting further biophysical analysis to understand binding.

The purpose of this study was to determine the absorption of [13C]5-formyltetrahydrofolic acid across the human colon with an intact microflora. Bioavailability was determined after administration of a pH-dependent acrylic co-polymer coated barium sulphate caplet containing 855 nmol and measuring the plasma appearance of [13C5]5-methyltetrahydrofolic acid compared to an intravenous injection of the same compound (214 nmol). Blood samples serially collected after both test doses were analyzed by microbiological assay and tandem mass spectrometry to determine total folates and ratio of labeled to unlabeled folates, respectively. Caplet disintegration in the colon was quantitatively assessed by fluoroscopic imaging for six of nine subjects. The plasma [13C5]5-methyltetrahydrofolic acid appearance rate was 4 + 0.9 nmol/h (intravenous) and 0.35 + 0.02 nmol/h (caplet). Mean apparent absorption across the colon was 35%. These findings suggest that physiological doses of natural folate are absorbed across the human colon in the presence of an undisturbed microbiota.

The purpose of this study was to determine the absorption of [13C]5-formyltetrahydrofolic acid across the human colon with an intact microflora. Bioavailability was determined after administration of a pH-dependent acrylic co-polymer coated barium sulphate caplet containing 855 nmol and measuring the plasma appearance of [13C5]5-methyltetrahydrofolic acid compared to an intravenous injection of the same compound (214 nmol). Blood samples serially collected after both test doses were analyzed by microbiological assay and tandem mass spectrometry to determine total folates and ratio of labeled to unlabeled folates, respectively. Caplet disintegration in the colon was quantitatively assessed by fluoroscopic imaging for six of nine subjects. The plasma [13C5]5-methyltetrahydrofolic acid appearance rate was 4 + 0.9 nmol/h (intravenous) and 0.35 + 0.02 nmol/h (caplet). Mean apparent absorption across the colon was 35%. These findings suggest that physiological doses of natural folate are absorbed across the human colon in the presence of an undisturbed microbiota.