Photometric flow analysis system for biomedical investigations of iron/transferrin speciation in human serum

Highlights

• Analytical procedures for estimation of iron/transferrin parameters has been proposed under flow analysis conditions.

• MCFA system for serum iron and unsaturated iron binding capacity determination has been developed.

• Two different analytical approaches for determination of each parameter have been proposed.

• The proposed MCFA system is useful for analysis of human serum samples.

Abstract

The Multicommutated Flow Analysis (MCFA) system for the estimation of clinical iron parameters: Serum Iron (SI), Unsaturated Iron Binding Capacity (UIBC) and Total Iron Binding Capacity (TIBC) has been proposed. The developed MCFA system based on simple photometric detection of iron with chromogenic agent (ferrozine) enables a speciation of transferrin (determination of free and Fe-bound protein) in human serum. The construction of manifold was adapted to the requirements of measurements under changing conditions. In the course of studies, a different effect of proteins on SI and UIBC determination has been proven. That was in turn the reason to perform two kinds of calibration methods. For measurements in acidic medium for SI/holotransferrin determination, the calibration curve method was applied, characterized by limit of determination and limit of quantitation on the level of 3.4 μmol L⁻¹ and 9.1 μmol L⁻¹, respectively. The determination method for UIBC parameter (related to apotransferrin level) in physiological medium of pH 7.4 forced the use of standard addition method due to the strong influence of proteins on obtaining analytical signals. These two different methodologies, performed in the presented system, enabled the estimation of all three clinical iron/transferrin parameters in human serum samples. TIBC corresponding to total transferrin level was calculated as a sum of SI and UIBC.

Introduction

It already has been proved that iron is involved in numerous of different biological processes within the body like electron transport, DNA synthesis, erythropoiesis or even cell-division cycle [1], [2], [3], [4]. Such a ubiquity of iron ions is caused by its ability to achieve two oxidation states as ferrous (Fe²⁺) and ferric (Fe³⁺) ions. Therefore, iron is crucial component in the specific oxidation-reduction systems. It is also well confirmed that such a redox activity is unfortunately double-edged sword, which also determine toxic character of iron ions in the course of the Haber-Weiss-Fenton sequence of reactions [1], [5], [6]. The prevention of such unwanted mechanism of radicals forming depends on complex homeostasis system, composed of many regulatory proteins [7], [8].

Nowadays, there are many literature reports which connect metabolism of iron with biochemical processes, important from medical diagnostics point of view. Especially, investigations on iron metabolism have gained importance in the field of neurodegeneration diseases [9], [10] or even tumor cell metabolism investigations associated with higher need for iron ions than in the case of normal cells. The number of researches has demonstrated that iron is a crucial factor in enzymatic activity of ribonucleotide reductase responsible for DNA synthesis [11], hypoxic mechanism [12], [13] and even in metastasis [14], [15], [16], [17]. Moreover, also a link between specific Fe-dependent proteins and cancer diseases in the case of transferrin [18], melanotransferrin [19], [20], [21], transferrin receptors [22], [23] and ferritin [24], [25] has been reported recently. This is the reason of attempts to develop cancer therapies based on iron metabolism [26], [27].

According to the issues mentioned above, the analysis of iron distribution and homeostasis is useful for determination of well-known disorders of the iron metabolism (iron deficiency and overload) [28], [29], as well as perspective for the modern medical diagnostics. In clinical analysis there are a few relatively easy to determine but significant iron distribution parameters which seem to be crucial for anticancer research, such as Serum Iron (SI), Unsaturated Iron Binding Capacity (UIBC) and Total Iron Binding Capacity (TIBC). All of these factors are connected with transferrin - a protein responsible for transport Fe³⁺ ions between organs throughout the body. It is characterized by its ability to bond ferric ions in molar ratio of 1:2 with high stability constants of the order of 10²⁰, which indicate the powerful stabilization of the ions transition [30]. SI, the first mentioned parameter, gives clinical information about the concentration of ferric ions complexed to the transferrin molecules. In physiological conditions only one third of transferrin's active sites are used and, because of very high stability constant of Fe-transferrin complex, iron ions do not occur in any other form in serum. The entire pool of iron binding sites in transferrin molecules are defined by TIBC parameter, wherein the free active sites are described by UIBC parameter [31]. The relationship between SI, UIBC and TIBC as well as between forms of transferrin is depicted in Fig. 1.

In this contribution, the MultiCommutated Flow Analysis (MCFA) system for clinical iron parameters determination is proposed. In this simple indirect way, the developed MCFA system based on photometric detection of free iron with ferrozine as chromogenic agent [32], [33] enables the transferrin speciation study - estimation of total as well as apo- and holo-forms of this protein in serum. In the course of these investigations, construction and optimization procedure of flow analysis system was performed. Moreover, the effect of different reaction conditions (pH, level of proteins) on analytical signals were examined. Finally, the developed MCFA system was used for estimation of mentioned parameters in control and real human serum samples to confirm its analytical and clinical usefulness.