## 3 Results  
As presented in Figure 1 and Table S1, the diets of the mice changed the calcitriol concentration in their lungs. Mice fed the diet with standard vitamin D3 level (0.5 IU/g) revealed a significantly higher calcitriol concentration (30.31 pg/mg) than mice on the diet with a 10-fold reduced vitamin D3 level (18.20 pg/mg). The calcitriol amount in murine lung tissue was also affected by inhalation with vitamin D3 metabolites. Mice exposure to calcidiol in the whole range of tested doses restored the physiological pulmonary level of calcitriol (level recorded in untreated mice on the diet with standard vitamin D3 content). Furthermore, calcitriol content in lung tissues of mice nebulized with calcidiol at  
concentrations of 500, 750, and 1000 pg/g significantly exceeded the indicated physiological concentration (30.31 pg/mg) as follows: 37.73 pg/mg, 38.75 pg/mg, 37.90 pg/mg, respectively. Murine nebulization with exogenous calcitri at the concentration of 1 pg/g was not able to restore the physiological level of the tested compound; on the contrary, this desired effect was observed in mice exposed to 1,25(OH)2-vitamin D3 at a concentration of 5 pg/g. An additional increase in 1,25(OH)2-vitamin D3 doses led to a further increase in endogenous calcitriol amount to the following levels: 33.16 pg/mg (10 pg/g), 33.52 pg/mg (25 pg/g), and 34.00 pg/mg (50 pg/g). As presented in Figure 1 and Table S1, the serum concentration of calcitriol recorded in mice on the diet with the standard amount of vitamin D3 was 132.24 pg/mL, while in mice on the diet with a 10-times reduced vitamin D3 level the serum content of calcidiol was significantly lower at 98.61 pg/mL. Murine exposure to exogenous vitamin D3 metabolites altered the serum level of calcitriol. Calcidiol at the concentrations of 100 pg/g and 1000 pg/g increased the calcitriol concentration to 137.29 pg/mL and 147.37 pg/mL,  
Figure 1: Alterations in pulmonary and serum levels of calcitriol in response to inhalation with vitamin D3 metabolites. The mice were exposed daily to 25(OH)-vitamin D3 (calcidiol) at the following concentrations: 100, 250, 500, 750, and 1000 pg/g or 1,25(OH)2-vitamin D3 (calcitriol) at the following concentrations: 1, 5, 10, 25, and 50 pg/g. Inhalations were performed for 7 consecutive days. As the control, untreated mice from Group 1 (main control) and Group 2 were used. Animals from Group 1 were given the diet with a standard cholecyst level (0.5 IU/g), while the mice from other research groups were given a diet with a reduced cholecyst level (0.05 IU/g). Calcitriol concentration was determined using the ELISA method in both lung homogenates and serum samples collected from all animals. Data are presented as a mean of calcitriol concentration \(\pm\) SEM of at least 4 measurements. * \(p\) < 0.05, ** \(p\) < 0.01, and *** \(p\) < 0.001 vs. main control (Group 1); \(p\) < 0.05, ** \(p\) < 0.01, and *** \(p\) < 0.001 vs. control (Group 2). Analyzed using a one-way ANOVA test and Tukey post hoc test. eliminating the negative impact of a vitamin D3 deficient diet on the examined parameter. The most significant increase in the serum level of calcitriol (recorded concentration 155.39 pg/mL) was noted in response to calcidiol at the concentration of 250 pg/g, while the additional increase in calcidiol doses did not result in a further increase in the concentration of the bioactive form of vitamin D3 in the serum. In the case of calcitriol nebulization, the most significant changes were observed in response to 1,25(OH)\({}_{2}\)-vitamin D3 at a dose of 5 pg/g, where the calcitriol serum concentration amounted to 136.75 pg/mL, reaching the physiological level. It needs to be highlighted that a further increase in calcitriol doses did not result in a further increase in serum concentrations of this metabolite.