Biosynthesis of amine oxides using calcium-alginate immobilized hog liver microsomes : an evaluation of process and reactor design

An evaluation of the biocatalytic ability and the potential utilization of Calcium-alginate immobilized hepatic microsomes (beads) for the biosynthesis of amine oxides has been performed, To accomplish this task, the following three areas Were investigated: immobilization, catalytic biooxidation ability, and reactor configurations. Immobilization was found to result in higher yields over free microsomes at the expense of higher cofactor requirements. Refrigerated drying of immobilized micro-somes appears to be a better storage technique than wet storage in buffer. Smaller beads resulted in higher rates than larger beads, but size did not affect the overall yield. Chlorpromazine (CPZ), diphenylamine (DPA) and 4-hydroxy-2,2,6,6-tetramethylpiperidine (TMP) were used as substrates to demonstrate the catalytic ability of microsomal enzymes to biooxidize tertiary and hindered secondary amines. The biooxidation of all three compounds to their oxides, by hog liver microsomes, required the presence of NADPH and oxygen. CPZ was converted to its N-oxide while, DPA and TMP were converted to nitroxide free radicals. Optimal reaction requirements for each substrate were established, NADPH is a reaction limiting factor for the oxidation of all three compounds. Biooxidation of the substrates increased, up to two-fold, by addition of n-tylamine, suggesting that mixed function amine oxidase (MFAO) is responsible for the N-oxidation of the amines. The progress of the previously described reactions was followed by means of oxygen uptake and HPLC. The identification of CPZ N-oxide was done using NMR, HPLC and TLC. ESR spectroscopy was used to verify the formation of nitroxide free radicals. A recirculation flow reactor is recommended for large scale production with immobilized whole microsomes. This configuration helps to maintain the physical integrity of the biocatalyst over longer reaction periods. The recirculation flow reactor also gives higher yields than a batch reactor

The Immune Response of a Burn Injury Compared to an Excisional Injury in a Murine Model

Cutaneous injury triggers a significant immune response. Cells of the innate and adaptive systems are recruited to coordinate repair, prevent infection and limit damage. It remains unclear if different aetiologies of injury influences the characteristics of the immune response as well as the long-term impact post injury. A murine model was used to characterise the immune response. A 8% TBSA full thickness dorsal burn or excision injury was used. At days 1,3,7,14,28 and 84 post injury mice were euthanased; whole blood was collected for haematology and cytokine profiling, and inguinal lymph nodes were harvested for immune cell populations including dendritic and T cells. Both the innate and adaptive immune responses differed between the two aetiologies. The burn injury generated a rapid and greater acute cytokine response displaying both Th1 and Th2 cytokine profiles resulting in changes to the innate cells, with a drive towards monocyte to macrophage differentiation following burn injury. The dendritic cell profile differed between the two injury groups, with reduced dendritic cell maturation that persisted to day 84 post-injury. The cytokine and dendritic cell profiles appeared to impact the adaptive response, with reduced T cell activation after burn injury which was sustained to the later time points. The data suggests there are significant differences in the immune response, which is dependent on injury aetiology. The burn injury had sustained an immunological change that was not apparent following excisional injury. This may explain in part the long-term implications of burn injury, such as increased incidence of infection and malignancy, and provides a basis for further investigation to guide clinical intervention after burn injury