## 4 Discussion
In order to achieve the main research goal, mice with vitamin D deficiencies induced by dietary cholecalciferol restriction were inhaled for week with five different doses of calcitriol (1, 5, 10, 25, and 50 pg/g) or its physiologic precursor calcidiol (100, 250, 500, 750, and 1000 pg/g). The effectiveness of inhalations was checked by determining the 1,25(OH)\({}_{2}\)-vitamin D3 level in lung tissue and serum. Performed studies revealed the lack of a simple linear relationship between the dose of vitamin D3 metabolites and the calcitriol concentration in the lungs and blood of inhaled animals. Changes induced by calcidiol at the lower doses (100, 250, and 500 pg/g) significantly improved the calcitriol status in lung compartments, while doses over 500 pg/g did not cause a further significant increase in the concentration of the tested metabolite. Serum results revealed quite a similar pattern of changes; however, the plateau stage was reached just after administration of calcidiol at a concentration of 250 pg/g. On the contrary, murine exposure to the lowest concentration of calcitriol (1 pg/g) induced only a weak improvement in bioactive vitamin D3 status in both examined compartments, while evident enhancement associated with restoration of physiological calcitriol level was observed in animals exposed to 1,25(OH)\({}_{2}\)-vitamin D3 at the concentration of 5 pg/g. It has to be noted that calcitriol doses over 5 pg/g provoked diametrically different effects depending on the tested compartments. Calcitriol at the concentration of 10 pg/g additionally increased its endogenous level in lung tissue, while the higher doses of tested compound provoked only discrete improvement. On the contrary, 1,25(OH)\({}_{2}\)-vitamin D3 doses, starting from 10 pg/g, caused a gradual decrease in serum calcitriol concentration, but the observed changes were not statistically significant compared to untreated mice on the standard diet. The presented data clearly demonstrated that only the lowest doses of vitamin D3 metabolites (calcidiol at the concentration of 100 pg/g and calcitriol at concentrations of 1 and 5 pg/g) administered via inhalation provoke similar patterns of changes in the pulmonary and serum concentration of calcitriol. Consequently, the calcitriol serum level cannot be used to predict this metabolite concentration in the respiratory system. Results obtained from lung and serum revealed that restoration of the physiological calcitriol level required 1-week daily inhalation with 25(OH)-vitamin D3 at the concentration 100 pg/g or 1,25(OH)2-vitamin D3 at the concentration of 5 pg/g. In order to verify this data, as well as check the impact of chronic inhalation on pulmonary function, the time of murine exposure to selected doses of mentioned metabolites was extended to 14 and 28 days. Performed studies revealed that tested compounds in the above-mentioned concentrations administered directly to the respiratory system of mice with vitamin D deficiencies allowed neutralization of the effect of dietary cholecalciferol restriction and increased the endogenous calcitriol concentration in both lung tissue and serum to physiological levels. Moreover, results of plethysmography have shown that murine chronic exposure to vitamin D3 metabolites did not affect most of the examined respiratory parameters, including F, TV, Ti, Te, IFR, EFR, as well as duty cycles of inspiration and expiration. Nevertheless, 14 days of vitamin-D-deficient murine treatment with calcidiol at the concentration of 100 pg/g significantly increased MV, compared to untreated mice with physiological calcitriol levels. Similarly, vitamin-D-deficient mice exposed for 2 weeks to 1,25(OH)2-vitamin D3 at the concentration of 5 pg/g demonstrated elevated levels of PIF, PEF, and EF50, compared to healthy untreated mice.