A simple, highly sensitive and improved method for the measurement of bleomycin-detectable iron: the ‘catalytic iron index’ and its value in the assessment of iron status in haemochromatosis

Abstract

In the presence of ferrous ions (Fe(2+)), the anti-tumour agent bleomycin will induce DNA degradation. Degradation of DNA into substances detectable by the thiobarbituric acid test has been used previously for the detection of iron in a form that is capable of catalysing the formation of the potentially harmful hydroxyl free radical. In the present paper, we describe the application of the ethidium-binding assay of DNA damage to the measurement of bleomycin-detectable iron, comparing its performance with the conventional method in the assessment of iron standard solutions and plasma samples from haemochromatosis patients. The ethidium-binding assay proved to be more responsive than the thiobarbituric acid test in the detection of DNA damage induced by very low concentrations of iron, but became saturated at higher iron concentrations. Agreement between the two versions of the assay in the identification of plasma samples containing bleomycin-detectable iron was good, but agreement on the actual concentrations of such iron in the positive samples was poor. This discrepancy is believed to be due to interference with the thiobarbituric acid assay by plasma. Consequently, it was not possible to obtain reliable estimates of free iron concentrations in plasma when using the conventional version of the bleomycin assay. We have devised a parameter of iron status called the catalytic iron index. For healthy, non-haemochromatotic individuals, the mean value of this parameter was found to be 0.81 (range 0.78-0.84; n=20). Elevated values were observed in some plasma samples from haemochromatosis patients, but these showed no correlation with serum ferritin levels. In contrast, correlations were seen with both serum iron and transferrin saturation levels, but only when these were above the normal range.

Bacterial Siderophores Hijack Neutrophil Functions

“…To ascertain whether Ent could quench the reactivity of LIP, we modified the bleomycin-based LIP detection assay that was originally developed to quantify LIP in biological samples (19). In our modified assay system, we instead prepared a series of samples with known concentrations of labile iron and tested whether Ent could quench their detection.…”

“…The assay was performed as outlined by Burkitt et al (19) with modifications described previously (20). In principle, ascorbic acid converts Fe +3 into Fe +2 , which in combination with bleomycin, causes DNA breaks.…”

Chemical, Biological and Medical Controversies Surrounding the Fenton Reaction

“…The assay has been shown to be unresponsive to iron bound to storage, transport and other proteins, being negative for the presence of free iron in plasma or serum from healthy individuals [98]. In contrast, the assay has confirmed the presence of free iron in individuals with iron overload (genetic haemochromatosis) [30,99] and in certain pathological situations, such as the rheumatoid joint [91,98] and during cancer chemotherapy [100,101]. Using NMR spectroscopy, Grootveld and colleagues have demonstrated that the free iron in plasma and serum from haemochromatosis patients is associated with citrate.…”

“…Various methods have been developed to detect 'free' iron (assumed to be complexed to low molecular weight molecules, such as citrate and the adenine nucleotides), but, invariably, these involve the use of powerful iron chelating agents that, it can be argued, may mobilise iron from sites where it would not otherwise be capable of catalysing • OH generation [91][92][93][94][95][96]. Perhaps the most successful of these assays has been the so-called bleomycin assay, which relies on the ability of the anti-tumour agent bleomycin to degrade DNA in the presence of iron [30,91,97]. The assay has been shown to be unresponsive to iron bound to storage, transport and other proteins, being negative for the presence of free iron in plasma or serum from healthy individuals [98].…”

Physicochemical properties of ferumoxytol, a new intravenous iron preparation

“…The procedure followed for the BDI assay was based on previous published methods [4,7,8]. Briefly, for each serum sample or standard, the following reagents were combined in polypropylene tubes in the following order: 100 lL 1 M trishydroxymethylaminomethane buffer, 250 lL 1 mg mL )1 deoxyribonucleic acid solution, 25 lL 1AE5 units mL )1 bleomycin solution, 25 lL serum blank, serum containing sample or standard, 50 lL 50 mM magnesium chloride solution and 50 lL 8 mM ascorbic acid solution.…”