Quantitative trace element mapping in liver tissue from patients with Wilson`s disease determined by micro X-ray fluorescence

Abstract

Aims: of this investigation were to quantify copper (Cu), iron (Fe) and zinc (Zn) along with sulphur (S) and phosphorus (P) in hepatocytes and connective tissue in liver section from patients with Wilson´s disease (WD) by micro Synchrotron X-ray fluorescence (μ-SRXRF). Secondly to establish two-dimensional μ-SRXRF element mappings for comparison with histologically prepared slices, and thirdly to assess whether elemental distributions are associated.

Methods: Archival liver tissues from twelve patients with end-stage cirrhosis or fulminant WD were investigated. Mutations in ATP7B have been classified before. For control seven archived normal liver tissues were investigated. μ-SRXRF measurements were performed at the DORIS III storage ring at HASYLAB/DESY (Hamburg, Germany). Two-dimensional element distribution were compared with histologically prepared slices about 20-30 μm apart from those investigated by μ-SRXRF.

Results: Elementary copper (Cu) could be demonstrated in all investigated liver sections simultaneously with Fe, Zn, P and S. In WD mean Cu was 20 fold increased in hepatocytes and threefold in fibrotic areas in comparison with controls. In regeneration nodules Cu was strikingly inhomogeneous distributed. Cu concentrations measured by μ-SRXRF correlated with those measured by atom absorption spectroscopy. Strong associations in their regional distribution existed between Zn and Cu or Fe and S. Moreover, differences in Cu/S were found between hepatocytes and fibrotic areas. An increase of Fe could only be documented in hepatocytes compared to fibrotic areas. With a beam size of 15 x 15 μm two-dimensional distributions of these elements are morphologically comparable with histological section with a magnification of about 25x optic microscope.

Conclusions: μ-SRXRF investigations are a valuable tool for quantifying element concentrations in biological tissues and further provide 2-dimensional information of element distribution and elemental association in a biological tissues, thus speeding up basic knowledge in a synopsis with biological and clinical data.

Keywords: Atomic absorption; Copper; Iron; Phosphorus; Sulphur; Synchrotron X-ray fluorescence; Trace element mapping; Wilson’s disease; Zinc.