Energy dispersive X-ray fluorescence quantitative analysis of biological samples with the external standard method

Highlights

• Variations of elemental concentration in soft tissues related to carcinogenesis;

• Elemental concentration in soft tissues assessed by EDXRF analysis;

• Accuracy of quantification methods in EDXRF was compared and improved;

• Fe, Cu, and Zn accurately quantified in paired samples of normal and tumour tissue;

Abstract

Trace elements are present in minute amounts in the human body but contribute to its proper functioning, by participating in several biological processes. Imbalance of the concentrations of these elements can lead to the development of pathologies, including cancer. As such, the determination of trace element content in tumour tissues and its comparison with normal ones may be helpful for a better understanding of carcinogenesis.

In this work, we address the collection and preparation of biological samples for Energy Dispersive X-Ray Fluorescence (EDXRF) analysis, and present a model for the quantification of trace elements, based on the external standard method of quantification.

The model was used for the quantification of iron, copper, and zinc in a set of paired samples (normal and tumour tissues). The obtained results show the validity of the method and variations of the elemental concentrations in the different tissues.

Introduction

The total percentage of trace elements (Mn, Fe, Cu, Zn, Se, Co, Mo, I) in the human body does not exceed 1%. Their concentration ranges from tenths to hundreds of μg/g, but they play crucial roles in the normal functioning of the organism, by participating in many essential processes like the activation, inhibition, and promotion of enzymatic reactions. Excess or deficiency of these elements may lead to the development of pathologies, including cancer. For example, copper and zinc are cofactors of the superoxide dismutase enzyme that if not regulated causes cell damage; iron is responsible for the formation of reactive oxygen species that trigger oxidative stress and consequently, cell damage [1]. Therefore, it is relevant to study trace element content in different tissues, both normal and tumour, in order to establish possible correlations between trace elements and factors like age, sex or cancer stage, leading to a better understanding of carcinogenesis.

The characteristics of the Energy Dispersive X-ray Fluorescence (EDXRF) technique, such as its non-destructive character (i.e., samples are unaltered for further analysis/treatments), sensitivity at ppm levels and high detection limits, make it a suitable option for these analyses. T. Magalhães et al. analysed carcinoma tissues with EDXRF and reported increased or constant levels of Fe and Cu and decreased levels of Zn. [2] showed increased concentrations of Fe, Cu, and Zn in breast cancer tissues analysed with EDXRF [3].

EDXRF quantitative analysis of samples requires the conversion of the intensity of the measured characteristic radiation to the concentration of the analytes present in the samples. Many methods, both empirical and theoretical, have been develop for quantitative analysis, because of the complexity of the issue. Several factors must be considered, namely the sample characteristics (e.g., composition, shape, thickness) and the characteristics of the spectrometer system (e.g., geometrical setup, spectral distribution of the excitation radiation) [[4], [5], [6]].

The fundamental parameter (FP) method is a theoretical method, based on the equations derived by Sherman [7] and later improved by Shiraiwa and Fujino [8]. It calculates the theoretical fluorescence intensities and compares them with the measured ones, iteratively, until a match is obtained. Even though the FP method can be used to analyse a multitude of samples, using any reference material for calibration or none at all (standardless analysis), it does not consider all physical processes in the sample (e.g., tertiary fluorescence, radiation scattering) and its accuracy is reduced by the uncertainties of the atomic parameters needed for the calculation (e.g., mass attenuation coefficients, cross-sections). Moreover, when the sample is composed of undetectable low-Z elements (H, C, O, N) quantitative analysis is hampered.

When studying biological tissues, the major difficulty in the quantification process is dealing with the aforementioned undetectable low-Z elements, i.e., the dark matrix of the tissues. As such the FP method is not the best option for the quantification. To compensate for the dark matrix effects, it is best to employ methods relying on the scattering of the primary radiation, such as the external standard method [4]. It is a compensation method that consists on the determination of the elemental concentration of an unknown specimen by comparing its fluorescence intensity with one reference specimen whose elemental concentration is accurately known [9]. A set of certified reference materials (CRM) with matrices similar to the unknown samples is analysed and for each element of interest, a calibration curve of concentration versus fluorescence intensity is built. It is essential that the chosen CRMs have a matrix similar to the unknown samples, and that the elements of interest are present in its constitution. Furthermore, the CRMs and the unknown samples must be prepared in similar ways and analysed under the same exact experimental conditions.