Morpho-chemical characterization of different human gallstone types in Moroccan patients using ATR-FTIR, SEM-EDX, XRF, and XRD techniques

Gallstone disease is a serious public health problem worldwide. However, studies on gallstone characteristics within the Moroccan community are limited. A detailed investigation of gallstones is essential to identify the causal factors and understand their formation process. This study aimed to classify and characterize gallstones from sixty-five Moroccan patients. Gallstone samples were first analyzed by attenuated total reflectance – Fourier transform infrared spectroscopy (ATR-FTIR) to determine their functional groups and then classified into different types. The morphological, microstructural, and compositional characteristics of each gallstone type were determined using scanning electron microscopy-energy dispersive X-ray (SEM-EDX), X-ray fluorescence (XRF), and X-ray diffraction (XRD) techniques. Based on ATR-FTIR results, gallstones were classified into pure cholesterol stones (n= 20; 30.8%), pure pigment stones (n= 2; 3.1%), and mixed stones (n= 43; 66.1%). SEM-EDX results revealed that each type of gallstone exhibited a distinct microstructure, closely associated with its elemental composition. Using the XRF technique, both pigment and mixed gallstones were found to contain various elements such as Ca, Mn, Fe, Ni, Cu, Zn, Pb, S, Br, and K, while pure cholesterol gallstones contained only Ca, Fe, and Zn. XRD analysis showed that pure cholesterol gallstones were crystalline and mainly composed of anhydrous cholesterol, while pigment gallstones were amorphous. Mixed gallstones exhibited a heterogeneous composition, containing several crystalline phases of calcium salts, including the polymorphs of calcium carbonate (calcite, vaterite, aragonite), calcium phosphate (hydroxyapatite), calcium fatty acids (calcium palmitate hydrate and calcium stearate). These findings reveal the heterogeneity of gallstones, leading to a better understanding of their formation and guiding strategies for prevention and non-surgical treatment. • Moroccan gallstones exhibit marked structural and compositional heterogeneity. • ATR-FTIR classified gallstones into pure cholesterol, pure pigment, and mixed stones. • SEM-EDX, XRF, and XRD revealed stone-specific microstructures and crystalline phases. • External color does not systematically reflect the chemical composition of gallstones.