| Transsulphuration is a metabolic pathway in which homocysteine is metabolized to give rise to cysteine, a precursor for endogenous synthesis of glutathione and taurine in vivo. The reduced form of glutathione plays a critical role in maintaining reducing intracellular red-ox environment. Pyridoxal 5'-phosphate (PLP), the major biological form of vitamin B 6, serves as a cofactor for key enzymes in the transsulphuration pathway and one-carbon metabolism. Therefore, studies examining the nutritional regulation of transsulphuration via vitamin B6 deficiency are of importance to the health of humans and young animals. In the first study, changes in biochemical indices of sulphur amino acid metabolism were characterized in piglets subjected to dietary vitamin B6 depletion. Despite the use of a pair-feeding design, growth and feed efficiency in B6 deficient pigs were lower than control pigs starting from the fourth week of depletion. Pigs consuming a vitamin B6 deficient semi-purified diet showed reduced plasma PLP concentrations throughout the 6-week experiment. Decreases in hepatic cystathionine beta synthase (CBS), cystathionine gamma lyase (CGL), serine hydroxymethyltransferase (SHMT) activities and hepatic and plasma cysteine concentrations due to vitamin B6 deficiency were observed. By contrast, severe hyperhomocysteinemia was observed starting from the fourth week of depletion. At the end of the 6th week of depletion, plasma methionine and serine concentrations were increased while glycine concentration was decreased by vitamin B6 depletion. A depletion-repletion study was then conducted to further characterize biochemical indices of sulphur amino acid metabolism in response to graded levels of vitamin B6 repletion (0.75, 1.5, 2.25, 3 mg/kg pyridoxine·HCl) in pigs deficient of vitamin B6. Significant dose dependent increases in plasma PLP and cysteine, and decreases in homocysteine to vitamin B 6 repletion were demonstrated, and these were sensitive to the duration of repletion. To have a better understanding of the quantitative impact of vitamin B6 deficiency on transsulphuration, kinetic measures of cysteine metabolism were undertaken. In a pilot study, a stable isotope model using L-[2,3,3-d3]serine and L-[3-13C]cysteine to quantify the rate of transsulphuration (TS), was established. The model was preliminarily established with constant infusion doses for L-[3-13 C]cysteine and L-[2,3,3-d3]serine at 7.5 micromol/(kg·h) and 60 micromol/(kg·h), respectively. This study was used to establish infusion doses for the measurement of serine and cysteine kinetics. Results from the pilot study were then incorporated into a study designed to examine the effect of vitamin B6 deficiency on serine and cysteine kinetics. The results show that whole body serine and cysteine fluxes were reduced by vitamin B6 deficiency. Due to the extensive dilution of hepatic serine label, transfer to cysteine was not measurable in either group, and therefore the rate of transsulphuration could not be calculated. However, reductions in enzyme activity support impairment in TS that may have led to a reduction in cysteine rate of appearance. In summary, the current research has led to the development and characterization of a model of vitamin B 6 deficiency in the pig that can be used to establish quantitative estimates of the impact of vitamin B6 status, as well as other nutrients, on whole body sulphur amino acid metabolism. |
