Ferrozine assay

Non-haem iron in solution, plasma or associated with membranes, may be assessed utilizing the ferrozine assay (Ceriotti and Ceriotti, 1980). 

Ferrozine 3-(2-pyridyl)-5,6-bis(4-phenylsulphonic acid)-1,2,4-triaz-ine (Fig. 4.3) reacts with iron in the iron(II) ferrous state to form an intense pink complex which can be measured spectrophotometrically (lmax = 562 nm). The assay can be performed at pH 1.6 to eliminate interference from turbidity at pH values close to the isoelectric points of proteins which may be present in biological samples under investigation, e.g., serum, and in some instances to decrease interference in the assay from copper (see below). In determinations in which such problems are not considerations, the assay may be carried out at pH 7.4. (This might be more relevant for the assessment of membrane-bound iron under in vivo conditions.) 

1 % (w/v) ascorbic acid (prepared immediately prior to use) and 0.25% thiourea in 0.1 M HC1. Both these reagents are dissolved in water and HC1 subsequently added to 0.1 M concentration. 

Glycine buffer 0.2 M glycine in 5 mM HC1, pH 4.15. 20 mg/ml ferrozine in water (prepared freshly on the day). Standard iron stock solution for calibration consisting of 0.45 mM iron chloride, i.e., 2.23 mM iron(II) in glycine buffer (prepared immediately prior to use, since iron(II) salts oxidize very readily in air). 

2 ml ascorbate/thiourea solution are mixed in a glass tube (or directly 

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Soluble Cr(VI), Fe(II), and U(VI) were monitored spectrophotometrically, Cr at 540 nm using the s-diphenyl carbazide method (Bartlett James, 1979), Fe(II) at 562 nm using the ferrozine assay (Stookey, 1970), and U at 575 nm using 2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol (Johnson Florence, 1971). Total dissolved Cr, U, and Fe were determined by flame atomic absorption spectroscopy (AAS) or by inductively coupled plasma (ICP) optical emission spectroscopy. 

At the same time as the test solution two blank determinations are carried out, the first consisting of the assay system as above but in the absence of ferrozine with 0.1 ml water in lieu. This allows for the contribution to the absorbance from haem proteins or other absorbing components which may be present usually the absorbance of this blank is of the order of 0.005. The second consists of the assay system in the absence of the sample, replaced by 0.2 ml water, to allow for interaction of ferrozine with reactable iron in the reagents which evaded removal prior to assay. 

The major source of interference and inaccuracy is iron contamination. It is thus essential to ensure that all the reagents, especially the ascorbate and the hydrochloric acid, are iron-free. Copper ions may also interfere in this assay (Duffy and Gandin, 1977). This can be eliminated by incorporating thiourea which, at a low pH, forms a stable colourless complex with copper without affecting the reaction of ferrozine with iron, thereby reducing the colour formation of ferrozine with copper. The affinity of thiourea for copper is maximal in the low pH range used here. 

Metal chelating assay Percentage of inhibition of ferrozine-Fe + complex formation... 

With serum iron assays, iron is (1) released from transferrin by decreasing the pH of the serum, (2) reduced from Fe to Fe and (3) complexed with a chromogen such as bathophenan-throhne or ferrozine. Such iron-chromogen complexes have an extremely high absorbance at the appropriate wavelength, which is proportional to iron concentration. 

Rice EW, Fenner HE. Study of the ICSH proposed reference method for serum iron assay Obtaining optically clear filtrates and substitution of ferrozine. Clin Chim Acta 1974 53 391-3. 

Figure 15-8 Effect of the ratio of Fe(II) atoms per molecule on the rate of iron oxidation by some ferritins and their variants. Remaining Fe(II) was measured in a discontinuous assay by removing an aliquot and adding it to a solution of ferrozine. Protein solutions were in 0.1m Mes buffer pH 6.5. Fixed iron concentration of 48pM (NH4)2 Fe (804)2 at 1 pM protein. Y-axes indicate the fraction of remaining Fe(II). (A) EcFTNa -f 48 Fe atoms per molecule (B) EcFTNa - - 480 Fe atoms per molecule (C) EcFTNa - - 980 Fe atoms per molecule (D) HuHF + 40 Fe atoms per molecule (E) HuHF -(- 500 Fe atoms per molecule (F) HuHF -1- 2000 Fe atoms per molecule.

Figure 15-9 Oxidation of 48 Fe(II) atoms per molecule added in two amounts to EcFTNa. Comparison of the rates of oxidation after the first and second additions. (A-D) Remaining Fe(II) was assayed with ferrozine and conditions were as in Figure 15-8. The second additions of Fe(II) was made at the specified times after the first addition. (E and F) Stopped flow traces, second addition made 24 hours after the first. (A-D) Y-axes indicate the fraction of remaining Fe (II). (E and F) Y axes indicate absorbance. (A) EcFTNa (B) EcFTNa-E130A (C) EcFTNa-E49A (D) EcBFR (E) EcFTNa (F) EcFTNa-E130A.

Several chromogenic iron chelators have been developed [17] that can be used in the assay. Ferrozine [3-(2-pyridyl)5,6-bis(4-phenylsulfonic acid) 1,2,4 triazine] Is also popular because its ferrous complex has a molar absorptivity of 28000 at 562 nm, which is about 35% more sensitive than the phenanthroline chromogen used in the reference method. However, ferrozine binds copper too, but this positive bias error is small at normal serum concentrations [42,45a], and is eliminated with thioglycolate [45b]. Ferrene and pyridyl-azo chelators have also been utilized as chromogens [45c,d]. 

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