Azathioprine is a thiopurine prodrug clinically used for immunosuppression in the treatment of inflammatory diseases and in pharmacological regimens of organ transplantations. Its pharmacological action is based on the release of 6-mercaptopurine, but the biochemical processes underlying this biotransformation have remained obscure. In this investigation, human glutathione transferases (GSTs) from seven distinct classes were assayed with azathioprine. GSTs A1-1, A2-2, and M1-1, all abundantly expressed in human liver, displayed the highest activity among the 14 GSTs tested. The uncatalyzed reaction of azathioprine with glutathione was estimated to be less than 1% of the GST-catalyzed biotransformation. GST M1-1 is polymorphic with a frequently occurring null allele, and GSTs A1-1 and A2-2 show variable expression levels in human subjects, implying significant differences in the rate of 6-mercaptopurine release from azathioprine. Individuals expressing high GST activity are apparently predisposed for adverse reactions to azathioprine treatment, both by promoting excessively high concentrations of free 6-mercaptopurine and its toxic metabolites and by depleting cellular glutathione. These novel aspects of GST-dependent azathioprine biotransformation have not been considered previously.

Concentrations up to 2 and 12 nM of the hydroxamate siderophores ferrichrome and ferricrocin, respectively, were identified in soil solutions of podzolic forest soils at four sites in both northern and southern Sweden. No ferrichrysin was detected. As with the dissolved organic carbon and low molecular mass organic acids, the highest concentrations of the siderophores were found in the upper layers i.e. the mor layer, the eluvial and upper illuvial horizons. At the southern sites, the concentrations of ferrichrome and ferricrocin were both of similar magnitude and did not differ between the two sites. In contrast, soil solutions at the two northern sites contained more ferricrocin than ferrichrome; the ferricrocin concentrations were also higher at the northern sites than at the southern sites. Analyses were performed by high performance liquid chromatography with a porous graphitic carbon column on which ferrichrome, ferricrocin and ferrichrysin were separated. Detection by electrospray ionization mass spectrometry (ESI-MS) combined with on-line sample pre-concentration, by means of column-switching, enabled detection limits of 0.1-0.2 nM for ferrichrome, ferrichrysin and ferricrocin. The structural identities of the siderophores were further verified by MS/MS fragmentation. Fragmentation of ferrichrome, ferricrocin and ferrichrysin occurred mainly via peptide cleavage. The most intense fragments were typified by the loss of one of the three iron(III) chelating hydroxamate residues, i.e N(5)-acyl-N(5)-hydroxy ornithine.

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Column-switching liquid chromatography followed by low-resolution ICPMS was evaluated as a tool for speciation analysis of metal-containing biomolecules. The strategy was applied on siderophores, strong iron chelators of low molecular weight (M(w) < 1500). Prior to the LC-ICPMS analysis, reductive displacement of iron by gallium was performed using ascorbate as the reducing agent to increase the sensitivity. Different experimental conditions during the exchange reaction were tested using ferrichrysin and ferrichrome for evaluation. A reaction time of 30 min and a pH of 3.9 gave an exchange yield of 27 and 83% for ferrichrysin and ferrichrome, respectively. A gradient elution profile was also developed to separate gallium-chelated siderophores on a PGC column. Detection limits for standard solutions of ferrichrysin and ferrichrome in the low-nanomolar range were obtained by monitoring the gallium-69 isotope. The combined use of LC-ICPMS and LC-ESI-MS/MS was also evaluated as a tool to identify unknown metal complexes, here siderophores, in field soil solution samples.