Infrared spectroscopy proves to be a convenient method for analyzing the mineral composition of urinary stones [39]. Using the mineral composition obtained through infrared spectroscopy diffraction, urinary stones can be classified into four distinct mineral groups, including calcium oxalate monohydrate (COM, CaC2O4·H2O), also known as Whewellite, calcium oxalate dihydrate (COD, CaC2O4·2H2O), also known as Weddellite, CA (Ca10(PO4)6CO3·H2O), and UA (C5H4N4O3) [40, 41]. Other types of stones were not collected or were too few in number to be considered in this study (such as hydroxyapatite, struvite stones, etc.). Previous research has demonstrated that Whewellite stones and Weddellite stones are distinct minerals with significant differences in crystal structure [16]. Whewellite crystals exhibit a flat monoclinic prismatic shape, displaying a radial pattern and concentric layers, Weddellite crystals have a shape resembling an eight-faced bipyramid [31, 42]. The variations in crystal structure are related to the adhesive characteristics of their crystal faces [42]. However, due to their high Ca content, they are collectively referred to as calcium oxalate stones. We only collected Whewellite stones and mixed stones containing both Whewellite and Weddellite. Existing studies have demonstrated that the elemental behavior of these stone types is similar [43, 44]. Therefore, we categorize Whewellite stones and mixed stones of Whewellite and Weddellite under the group of CO stones. Representative infrared spectra for this study are shown in Fig S1 of the Supplementary file. In the analysis of the 80 stones examined in this study, due to the relative rarity of stone types other than calcium oxalate stones, we incorporated data from 11 stones in our previous research [17, 41], enhancing the statistical significance of the analytical results, 50 were classified as the CO stone group, including COM and COD. 9 were classified as the CA stone group, 8 as the UA stone group, 5 as the mixed CO and CA stone group, and 8 stones were classified as the mixed CO and UA stone group. Among all samples, the proportion of urinary stones in males reaches 80%. The average age of patients with stones was 55.38. There are only 13 cases of urinary stones in individuals below the age of 40, while the highest number of stone cases, 20 in total, occurs in the age group of 51–60 years.

Urinary stones typically consist of organic and inorganic substances, which can potentially jeopardize the health of biological organs. Identifying the elemental composition of urinary stones can provide valuable information for implementing alleviative measures in the treatment of patients [45]. Table 1 presents the results of the major and trace metal content along with their average values and concentrations for each mineral group of urinary stones. Ca2+ constitutes the primary component of all types of urinary stones, and the detailed data are presented in Table S1. The average concentration of Ca2+ in the CO stone group (27.68%) exceeds that in the CA stone group (18.95%), being the lowest in the UA stone group with an average concentration of 2.64%. It should be noted that the Ca2+ content is influenced by factors such as the types of food and beverages consumed by patients, including dairy products, eggs, tea, and hard water, or pathological conditions such as idiopathic hypercalciuria, absorptive or resorptive hypercalciuria [46, 47]. Mg is an essential element in biomineralization [48], and its detection in urinary stones frequently serves as an indicator of elevated concentration in the body [18]. The average concentration of Mg2+ in the CA stone group is significantly higher than in other stone groups, measuring 38.24 mg/g. Typically, higher levels of Mg are observed in struvite stones and struvite-calcium phosphate mixed stones [49]. However, relevant studies have also reported elevated Mg content in pure calcium phosphate stones [49]. This could be attributed to the crystallization process during the formation of CA stones, where minerals in the urine can combine to form crystals. Mg commonly replaces a portion of Ca, contributing to the crystal structure of the stones along with phosphate and carbonate ions [50]. Due to their porous nature, CA stones can absorb various chemical elements. Yet, the precise role of Mg in urinary stone formation remains not fully understood [51]. Dietary sources and hard water, along with some medications, contribute to variations in Mg content [24]. The CA stones may exhibit a stronger capacity for absorbing alkali metals (Na, K). The average concentrations of Na and K in the CA stones are 7527.38 µg/g and 1218.02 µg/g, respectively. Similarly, elevated levels are observed in the CO stones and the mixed CO and CA stones. Conversely, in the UA stones, the concentrations of Na and K are the lowest, measuring 689.33 µg/g and 158.25 µg/g, respectively. The alkali metal content in the mixed CO and UA stones is also significantly reduced, influenced by the presence of the UA stones. These findings suggest that the CA stones may possess distinctive biochemical characteristics in their interactions with alkali metals. The content of trace elements Zn and Sr exhibits similar characteristics to alkali metals. In the CA stones, their concentrations are relatively high at 1210.35 µg/g and 266.14 µg/g, respectively. Conversely, in the UA stones, their concentrations are lower at 14.26 µg/g and 8.72 µg/g, respectively. In addition, other study has also demonstrated that the elemental composition of UA stones exhibits the presence of various trace elements in small quantities [52]. These elemental characteristics may also be reflected in the microscopic structure of the stones, with the microscopic morphology of UA stones showing lower absorption signals and porosity compared to other types of stones [31]. However, in the CO and CA mixed stones, the influence of the CA stones on the content of Zn and Sr is minimal. In the CO and UA mixed stones, their content is significantly affected by the presence of the UA stones. Pb is a potential toxic element that can cause urinary damage at both low and high concentrations, hindering waste elimination from the body [17]. Notably, our study found an average Pb content of 5892.93 µg/kg in the CO stones, indicating no Pb pollution compared to other regions [53]. Furthermore, the content of other elements not mentioned in this study was also found to be low.

In addition to identifying the chemical composition and elements in urinary stones and their potential roles in aggregate formation, assessing the correlations between different elements becomes essential to understand the process of stone formation in the urinary tract [45]. To investigate the associations between the major and trace elements in urinary stones, Pearson correlation analyses were performed for each type of urinary stone (Fig. 1). Figure 1a and e presents the result of the correlation analysis for the CO, CA, UA, mixed CO and CA, and the mixed CO and UA stone groups, respectively. In the case of the CO stone group, Ca and Na exhibit a positive correlation (r = 0.48), likely attributed to their closely matched ionic radius (116 pm for Ca and 144 pm for Na) [54], This similarity enables Na to substitute for Ca in various rock-forming minerals, such as plagioclase feldspar (sodium-calcium feldspar series NaAl3Si3O8-CaAl2Si2O8) and pyroxene [55, 56]. Ca shows a strong positive correlation with Zn (r = 0.66) in the CO stone group, suggesting that these elements may undergo thermodynamically favorable substitution processes or are easily absorbed into the oxalate crystal structure [41]. In the CO stone group, Mg exhibited positive correlations with Na (r = 0.64), Sr (r = 0.61), and Rb (r = 0.72); In the UA stone group, significant positive correlations were observed with Ba (r = 0.97), Zn (r = 0.97) and Pb(r = 0.94); Similarly, in the mixed CO and CA stone group, Mg showed significant positive correlations with Na (r = 0.99), Sr (r = 0.99), Zn (r = 0.99), and Ba (r = 0.98). In the mixed CO and UA stone group, Mg demonstrated significant positive correlations with Zn (r = 0.86) and Ba (r = 0.96). These positive associations with Mg can be explained by its involvement in several crucial processes in the human body, such as contributing to the synthesis and metabolism of proteins and nucleic acids [43]. Mg also serves as a cofactor in many enzyme-catalyzed reactions [16]. When it comes to ion binding, Mg2+ exhibits a greater affinity for oxalate than Ca2+ because of its smaller size. Early studies indicate that urinary stones exhibit reduced crystallization and growth under high concentrations of Mg2+ [52]. This is attributed to Mg’s ability to easily bind to specific sites, thereby lowering the formation rate of oxalates [57]. Furthermore, the positive correlation between Ca and Sr in the CO (r = 0.4) stone group suggests that the human body processes Sr like Ca, aiding in the substitution of Sr for Ca in biomineralization processes [58, 59]. Sr and Zn display a positive correlation in the CO stone group (r = 0.79), and the mixed CO and CA stone group (r = 0.98). Overall, the correlations between elements in the CA stone group and the mixed CO and UA stone group were not significant, while the correlations of Li, Cu, Se, and Pb with other elements were not significant in the UA stone group and the mixed CO and CA stone group, and the overall correlations between other elements were significant in all stone types. The occurrence of urinary stones is believed to be associated not only with geographic distribution, geological environments, and occupational factors but also, more widely, with the suspected influence of various nephrotoxic elements present in drinking water over prolonged periods [60,61,62]. Due to varying research conclusions, the role of hard water or soft water in urinary stone formation has been a topic of debate [63]. Water hardness is typically determined by the Ca and Mg content in water, with hard water generally containing higher levels of Ca and Mg. A review has concluded that hard water is more conducive to the formation of calcium stones [64]. However, the results of a review survey indicate that 41% of studies suggest using high-Ca hard water to reduce the risk of urinary stone formation [64]. Hard water leads to hypercalciuria, but due to other factors influencing stone formation, the overall impact appears to be a reduction in urinary stone formation [64, 65]. Therefore, hard water does not necessarily promote stone formation; this depends on the type of stones and unique patient factors. In addition, the recurrence frequency of urinary stones also influences their elemental composition. A study on recurrent stones suggests that the distribution of calcium is not uniform, and there are significant concentration differences [66].

Correlation analysis of the chemical components of urinary stones. a. CO; b. CA; c).UA; d. CO + CA; e. CO + UA; ** p < 0.01; * p < 0.05

PCA is a common multivariate statistical analysis method that can be used to reduce dimensionality and analyze relationships among various elements. PCA is used to elucidate the relationships among the major elements and to identify potential combinations of the major and trace elements that can play significant roles in the formation of urinary stones. PCA is conducted with the extraction of the maximum rotated variance. PCA was conducted on five categories of urinary stones in this study (Fig. 2). The relationships between the elements in the CO stone group are illustrated in Fig. 2a. Extracting the three highest eigenvalues as PCs account for 74.58% of the total variance, with the first principal component (PC1) explaining 40.42% of the total variance. Figure 2a displays the major loadings in PC1, which include Mg Zn, Li, Rb, and Ba. The second principal component (PC2) contributes to approximately 26.42% of the total variance, with its major loadings being Ca, Na, K, Sr, Ti, and Pb. The third principal component (PC3) is formed by Cu and Se, contributing to 7.74% of the total variance. Certainly, PCA is also being conducted for the CA stone group (Fig. 2b), the UA stone group (Fig. 2c), the mixed CO and CA stone group (Fig. 2d), and the mixed CO and UA stone group (Fig. 2e). For the CA stone group, the three PCs with the highest eigenvalues were extracted, accounting for 73.28% of the total variance. PC1, PC2, and PC3 explain 25.12%, 24.21%, and 23.95% of the total variance, respectively. The primary loadings for PC1 include Mg, Na, K, Ti, Cu, and Se; for PC2, they are Ca, Sr, Li, Rb, and Pb; and for PC3, they are Zn and Ba. For the UA stone group, the three PCs with the highest eigenvalues have been extracted, representing 79.95% of the total variance. PC1, PC2, and PC3 explain 40.02%, 25.51%, and 14.42% of the total variance, respectively. The major loadings for PC1 include Ca, Mg, Sr, Zn, Ba, and Pb; for PC2, they are K, Li, Ti, Cu, and Se; and for PC3, they are Na and Rb. For the mixed CO and CA stone group, the three PCs with the highest eigenvalues account for 99.69% of the total variance. PC1, PC2, and PC3 explain 50.87%, 25.33%, and 23.49% of the total variance, respectively. The major loadings for PC1 include Ca, Mg, Na, Sr, Zn, Ba, and Ti; for PC2, they are K, Li, and Rb; and for PC3, they are Cu, Se, and Pb. For the mixed CO and UA stone group, the three PCs with the highest eigenvalues account for 80.54% of the total variance. PC1, PC2, and PC3 explain 40.24%, 20.24%, and 20.06% of the total variance, respectively. The major loadings for PC1 include Ca, Mg, Zn, Pb, Ba, and Ti; for PC2, they are Na, K, Li, and Cu; and for PC3, they are Sr, Se, and Rb.

PCA of the chemical components of urinary stones. a. CO; b. CA; c. UA; d. CO + CA; e. CO + UA

The PCA of five types of urinary stones suggests an association between Ca and Sr in four stone categories, excluding the Mixed CO and UA Stone Group. However, the mechanisms by which strontium interacts with calcium in four types of urinary stones still require further investigation. Associations between Pb and Ca were observed in the CO stone group, CA stone group, UA stone group, and the Mixed CO and UA stone group. In the UA stone group and two types of mixed stone group, a potential connection between Ca and trace elements Mg, Zn, and Ba was observed. However, this relationship was not observed in the CO stone group, suggesting that the association between Ca and Mg, Zn, and Ba in the Mixed CO and UA stone group may be predominantly driven by the influence of the UA stone group. Importantly, existing research has indicated that Mg, Zn, Ba, and Sr can substitute for Ca in the formation of urinary stones [49, 67], but the conclusion lacks clarity regarding the differentiation of stone types. In conclusion, the three PCs of the major and trace elements in urinary stones can be explained by potential favorable alternative pathways or preferences in the intake process within the human body, revealing co-precipitation or substitution during the formation of urinary stones. Furthermore, variations in elemental distribution between different types of stone can serve as valuable indicators to tailor nutrient intake based on the specific disease. In future research, we plan to conduct quantitative analysis of mixed urinary stones using advanced techniques such as dual-energy CT and X-ray dark-field tomography. This aims to provide a more comprehensive understanding of the elemental composition and structural characteristics of mixed stones. Similarly, physiological factors influencing the elemental composition of stones may include stone recurrence, metabolic functions in the human body, and the impact of other diseases. These areas are also worthy of in-depth research. This approach will contribute to filling gaps in current research and offer additional insights into the mechanisms of stone formation for further investigation.