CAN CHOLINE SPARE METHIOININE FROM CATABOLISM IN LACTATING MICE AND DAIRY COWS?

Several studies have demonstrated that supplementation of rumen-protected choline (RPC) improves milk production in the lactating dairy cow; however, there are an equal number of studies failing to observe production responses. To date, there are only three studies that provide quantitative information in ruminants on the metabolic fates of methyl groups derived from choline and Methionine (Met). This has limited the ability to predict when, and under what conditions, RPC supplementation will be beneficial. The objectives of this thesis were to determine the interaction of choline and Met methyl group metabolism and the extent of methyl group transfer during lactation, and define what role, if any, is there for RPC in remethylation of homocysteine and in the sparing of Met in lactating animals. A preliminary study with lactating mice consuming a low protein basal diet (10%) was conducted. From 11 to 15 d postpartum, stable isotopes of [methyl,2H3] choline and [methyl,2H3] Met replaced the unlabeled choline and Met in the basal diet to measure the metabolic fates of choline and Met including Met remethylation and sources of Met methyl in the mammary gland. Isotopic analysis revealed that the liver is a major site of Met remethylation from dietary choline with a minimum choline methyl group contribution to Met remethylation of 35%. Mammary tissue was not a major site of Met remethylation from dietary choline (< 10% of Met methyl) as measure by Met methyl in mammary tissue and milk casein. However, there was a significant unlabeled source of methyl groups contributing at a minimum of 45% Met remethylation in the mammary tissue, presumably by de novo synthesis. This suggested that in addition to the liver, the mammary gland is an active site of methyl group transactions. In a subsequent study, lactating dairy cows were fed a total mixed ration formulated to meet the nutrient requirements with exception of metabolizable Met that was restricted to 1.49 % of metabolizable protein. Treatments included a Control (basal diet) and RPC supplemented diet where the basal diet was top dressed with 15g/d RPC, diets were fed in a single reversal design with 2 week experimental periods. Stable isotopes of Met, [1-13C] Met, [13CH3] Met, and [methyl-2CH3] choline were continuously infused on d 14 of each period to determine the metabolic fate and methyl transactions of Met methyl as measured in blood and milk casein. Treatment had no effect on milk production or composition. However, plasma free Met from choline transmethylation was shown to act as a significant contributor to casein synthesis. Fractional Met remethylation rates in the control and RPC treatments were 26 and 23%, respectively. Methionine Met methyl loss within the mammary tissue appears to be minimal. Based on casein Met enrichment, upwards of 40% of Met present in casein had undergone transmethylation with choline serving as the ultimate methyl donor. Furthermore, plasma versus casein Met methyl enrichment data suggested that a significant amount of de novo methyl group synthesis occurs in the dairy cow's mammary gland with choline serving as a major methyl donor

The lipidome of Crithidia fasiculata and its plasticity

We would like to thank the Engineering and Physical Sciences Research Council, University of St. Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support [Ph.D. studentship to MC; Grant code: EP/L016419/1].Crithidia fasiculata belongs to the trypanosomatidae order of protozoan parasites, bearing close relation to other kinetoplastid parasites such as Trypanosoma brucei and Leishmania spp. As an early diverging lineage of eukaryotes, the study of kinetoplastid parasites has provided unique insights into alternative mechanisms to traditional eukaryotic metabolic pathways. Crithidia are a monogenetic parasite for mosquito species and have two distinct lifecycle stages both taking place in the mosquito gut. These consist of a motile choanomastigote form and an immotile amastigote form morphologically similar to amastigotes in Leishmania. Owing to their close relation to Leishmania, Crithidia are a growing research tool, with continuing interest in its use as a model organism for kinetoplastid research with the added benefit that they are non-pathogenic to humans and can be grown with no special equipment or requirements for biological containment. Although comparatively little research has taken place on Crithidia, similarities to other kinetoplast species has been shown in terms of energy metabolism and genetics. Crithidia also show similarities to kinetoplastids in their production of the monosaccharide D-arabinopyranose similar to Leishmania, which is incorporated into a lipoarabinogalactan a major cell surface GPI-anchored molecule. Additionally, Crithidia have been used as a eukaryotic expression system to express proteins from other kinetoplastids and potentially other eukaryotes including human proteins allowing various co- and post-translational protein modifications to the recombinant proteins. Despite the obvious usefulness and potential of this organism very little is known about its lipid metabolism. Here we describe a detailed lipidomic analyses and demonstrate the possible placidity of Crithidia’s lipid metabolis. This could have important implications for biotechnology approaches and how other kinetoplastids interact with, and scavenge nutrients from their hosts.Publisher PDFPeer reviewe

Studies on some potentially cytotoxic compounds in the nitrogen- and sulphur-mustard series

Imperial Users onl

The role of folate status in formate metabolism and its relationship to antioxidant capacity during alcohol intoxication

Alcohol abuse during pregnancy has been associated with Fetal Alcohol Spectrum Disorder (FASD). Research to date has focused on the role played by ethanol in the development of this disorder. In addition to ethanol, alcoholic drinks also contain methanol. Hence, consumption of alcohol can also lead to methanol accumulation. Methanol is metabolized to formaldehyde, which is then rapidly metabolized to formate, a toxic metabolite. Folate, a B-vitamin and antoxidant, is a cofactor in the metabolism of formate. This study assessed the relationship between formate and folate, formate kinetics in folate deficiency and, changes in antioxidant capacity during formate insult in folate deficiency. The findings of this study would lead to a better understanding of the role of formate in the development of the etiology of FASD and form the basis of future research. The relationship between formate and folate was investigated in intoxicated human female subjects, sober drug rehabilitating females and, pregnant women. A negative (inverse) relationship was observed between plasma formate and folate in pregnant sober women (correlation coefficient = -0.4989). Such a relationship, however, was not observed in whole blood in alcohol intoxicated (correlation coefficient = 0.0899) and detox women (correlation coefficient = 0.2382). Because of the health promoting ingredients in grain and fruit based alcoholic drinks, antioxidant B-vitamins were higher during intoxication while homocysteine levels were lower.Formate kinetics during folate deficiency and changes in the body antioxidant capacity was investigated in folate deficient young swine. Folate deficiency altered formate kinetics leading to decreased systemic clearance (by approximately 2.3 fold), increased half-life (by 2.5 fold) and, consequently increased exposure (by 2.7 fold). Folate deficiency alone compromised antioxidant capacity. However, the combination of folate deficiency and formate insult further compromised antioxidant capacity.In conclusion, methanol accumulates after alcohol intoxication, which can lead to formate build up in the body. During folate deficiency formate kinetics is altered leading to reduced formate clearance and increased exposure. Exposure to formate coupled to folate deficiency compromises antioxidant capacity, which can have deleterious effects on the fetus

Molecules and Evolution

Biochemical basis of evolution - evolution and DNA, genetic code, microbiology and heredity, mutations, and evolution and hemoglobin

Assessing the role of soil chemoautotrophs in carbon cycling: An investigation into isotopically labelled soil microorganisms

Recently observed increases in atmospheric CO2 have created great interest in carbon capture technologies and natural sinks of this major component of the carbon cycle. Humic substances are a large, operationally defined fraction of soil organic matter. It was thought that humic substances consist of cross-linked macromolecular structures forming a distinct class of compounds. However, it was recently concluded by members of my research group that the vast majority of humic material in soils, are a complex mixture of microbial/plant biopolymers and degradation products, and not a distinct chemical category. The postulation that microbial inputs to soil carbon are greatly underestimated was put forward by my research group in 2007. Therefore, I have attempted to demonstrate the inputs made by soil chemoautotrophic bacteria. A method was developed where soil samples were measured for chemoautotrophic activity by subjecting them to a suite of scientific techniques. A growth chamber was used to propagate extant soil chemoautotrophic bacteria from different soils and subjected to an array of chemical and biological analyses. The growth chamber was used to measure CO2 concentrations and introduce stable isotopic 13CO2. Estimations of CO2 sequestration were made using direct measurements for Irish soils and one Eurasian soil. Isotope labelled DNA was isolated using cesium chloride gradient ultracentrifugation. The dominant chemoautotrophic bacteria uncovered were Thiobacillus denitrificans and Thiobacillus thioparus. Labelled biomass was isolated and described using GCMS-IRMS and NMR, where an array of PLFAs, protein/peptide, carbohydrates and aliphatics were observed. Finally, an attempt to mimic common agricultural practice was performed to measure soil chemoautotrophic activity. This demonstrated the capability of this approach to benefit carbon flux estimations and hopefully in the future help to elucidate carbon flow into soils for the greater environment