Carlos Simões Nunes

In vitro and in vivo degradation of myo-inositol hexakisphosphate by a phytase from Citrobacter braakii

Phytases (EC 3.1.3) are widely used in animal feed to increase the availability of phosphorus and decrease the anti nutritive effect of myo-inositol hexakisphosphate (InsP6). The aim of this work was to investigate the stereospecific degradation of InsP6 in vitro and in vivo by a phytase from Citrobacter braakii (C. braakii), and to study gastric survival of the phytase as well as the site of action in the gastrointestinal tract. The in vitro results showed that the C. braakii phytase belongs to the group of 6-phytases (EC 3.1.3.26). However, in approximately one out of 10 instances the phytase initiated hydrolysis at the D-3 (L-1) position, demonstrating that phytase specificity is not unambiguous. Following the main degradation pathway, InsP6 was degraded by stepwise removal of the phosphate groups on positions 6/1/5. The stereospecificity was found to be similar under in vitro and in vivo conditions. The phytase was found to be stable in the gastric environment and to be active in the stomach and possibly also in the proximal small intestine. While InsP4 was accumulated under in vitro conditions this was not the case in vivo, where both InsP5 and InsP4 were seen to be hydrolysed in the small intestine, possibly as a combined action of the C. braakii phytase and endogenous phosphatases present in the mucosa. The ability of the C. braakii phytase to focus its activity on degrading InsP6 to InsP4 is believed to be a favourable complement to the endogenous phosphatases.

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay.

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 microg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8+/-3.5 ng/g), followed by kidney (14.2+/-1.5 ng/g), liver and muscle (5.7+/-0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5+/-13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.