ABSTRACT
This paper describes a comparison of two analytical techniques, one employing bathophenanthrolinedisulfonate (BPT), a most commonly-used reagent for Fe (II) determination, as chromogen and an electrothermal atomic absorption spectroscopy (ETAAS) for the quantification of non-transferrin bound iron (NTBI) in sera from thalassemic patients. Nitrilotriacetic acid (NTA) was employed as the ligand for binding iron from low molecular weight iron complexes present in the serum but without removing iron from the transferrin protein. After ultrafiltration the Fe (III)-NTA complex was then quantified by both methods. Kinetic study of the rate of the Fe (II)-BPT complex formation for various excess amounts of NTA ligand was also carried out. The kinetic data show that a minimum time duration (> 60 minutes) is necessary for complete complex formation when large excess of NTA is used. Calibration curves given by colorimetric and ETAAS methods were linear over the range of 0.15-20 microM iron (III). The colorimetric and ETAAS methods exhibited detection limit (3sigma) of 0.13 and 0.14 microM, respectively. The NTBI concentrations from 55 thalassemic serum samples measured employing BPT as chromogen were statistically compared with the results determined by ETAAS. No significant disagreement at 95% confidence level was observed. It is, therefore, possible to select any one of these two techniques for determination of NTBI in serum samples of thalassemic patients. However, the colorimetric procedure requires a longer analysis time because of a slow rate of exchange of NTA ligand with BPT, leading to the slow rate of formation of the colored complex.
Subject(s)
Chromogenic Compounds/chemistry , Colorimetry/methods , Humans , Iron/blood , Phenanthrolines/chemistry , Spectrophotometry, Atomic/methodsABSTRACT
In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.