LC–MS-based absolute metabolite quantification:Application to metabolic flux measurement in trypanosomes

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism

Hematologic Changes in Visceral Leishmaniasis/Kala Azar

Visceral Leishmaniasis (VL) or Kala Azar is a chronic infectious disease caused by parasites of the Leishmania donovani complex that can cause various hematologic manifestations. It is characterized by fever, enlargement of liver and spleen, weight loss, pancytopenia and hypergammaglobinemia. It is endemic in the Indian subcontinent, mainly seen in the states of Bihar and West Bengal. Patients with VL can present to the haematologist for various haematological problems prior to receiving the diagnosis of VL. Anaemia is the most common haematological manifestation of VL. VL may also be associated with leucopenia, thrombocytopenia, pancytopenia, hemophagocytosis and disseminated intravascular coagulation. Hematological improvement is noted within a week and complete hematological response occurs in 4–6 weeks of treatment. Relapses are rare and increased risk of being diagnosed with hematolymphoid malignancies on long term follow up is not noted