In this work, different analytical speciation schemes have been used to study the reduction of Cr(VI) by a chromate-resistant strain of filamentous fungi Ed8 (Aspergillus sp), indigenous to contaminated industrial wastes. As demonstrated previously, this strain has the capability to reduce chromate present in the growth medium without its accumulation in the biomass, yet the reduced chromium end-products have not been characterized. Liquid growth medium, initially containing 50 mg L(-1) Cr(VI), was analyzed for Cr(III)/Cr(VI) and for total Cr at different time intervals (0-24 h) after inoculation with fungi. Three hyphenated procedures, based on the Cr(III)-EDTA formation and species separation by anion-exchange or ion-pairing reversed-phase chromatography with ICP-MS or DAD detection were used. The results obtained for Cr(VI) in each case were consistent, demonstrating efficient reduction of chromate during 24 h of Ed8 growth. However, pre-column complexation with EDTA did not ensure complete recovery of the reduced forms of chromium in the above procedures. An alternative speciation scheme, based on extraction of Cr(VI)-benzyltributylammonium bromide (BTAB) ion pairs into chloroform and subsequent determination of residual chromium by ICP-MS has provided evidence on the effective conversion of chromate into reduced chromium species in the growth medium. The results indicate the feasibility of using Ed8 strain for chromate bioremediation purposes. Analytically it can be concluded that speciation of chromium in biological systems should not be limited to its two most common oxidation states, because the actual reduced chromium species are not converted quantitatively to Cr(III)-EDTA.
Ceruloplasmin (Cp), a copper metalloprotein in human serum has been a valuable diagnostic marker in Wilson's disease where Cp levels tend to be low while high levels in serum were associated with myocardial infarction, neoplastic and inflammatory conditions. There is no standardized reference method for Cp and current immunologic and bichromatic assays have a number of drawbacks. The method described here uses immunoaffinity chromatography to remove six of the most abundant proteins from a serum sample and high-pressure liquid chromatography (HPLC) with a size-exclusion column to separate Cp from other serum proteins and any free Cu prior to analysis of 63 Cu and 65 Cu by inductively-coupled plasma mass spectrometry (ICPMS). Identification of Cp is based on retention time match of the unknown protein in the serum sample with the Cp external standard and the presence of 63 Cu and 65 Cu at a ratio of 2.2 ± 0.1. The method accuracy, as established independently by two of the authors with a reference serum certified for Cp, is 98 to 101% and the coefficient of variation is 6.4% and 5.4%, respectively. The assay was used to analyze a total of 167 human sera for Cp from patients with myocardial infarction (MI), pulmonary embolism (PE), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), other forms of arthritis, and a set of healthy patients as normal controls (NC). Our data show that Cp concentrations tend to be higher in MI, RA, and SLE patients, higher in female as compared to male patients, and we did not observe a correlation between Cp concentration and patient's age for the set of 70 patients for which we had gender and age information.
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