Sterile inflammation is an important part of the pathogenesis of numerous clinical and subclinical disease conditions
4,6,7. Within these inflammatory disorders, serum 25-D levels are frequently observed to be lower compared to healthy individuals
16-19. While clinical trials offer the opportunity to compare vitamin D levels between patients and healthy controls, they do not ascertain whether the observed disparity precedes or results from the disease. Numerous factors such as dietary habits, disease stage, comorbidities, prior treatments, etc., often confound clinical studies, making precise detection challenging. Furthermore, determining the pre-disease vitamin D status of patients is frequently unattainable
16,17,19. The present study was designed to evaluate the effects of an experimental sterile inflammation induced by TO injection on serum vitamin D levels.
The quantity of TO administered per animal to evoke sterile inflammation in rabbits typically ranges from 3 mL to 6 mL22,23,25. In the current study, 5 mL of TO was administered, and systemic inflammation was confirmed through changes in body temperature, WBC count and serum iron levels.
Body temperature values showed significant increases at 12., 24. and 72. hours in the TOG compared to the CG, as reported in the literature26,27. In the TOG, WBC counts exhibited a decrease at 6, 12, 24, and 72 hours, while there was no significant change observed in the throughout the experiment. This contrasts with the findings of Gossett et al.27, who reported an increase in WBC counts following turpentine oil injection in dogs, contrary to the observations in the present study. Similarly, Colditz et al.26 reported an increase in WBC counts in sheep, both of which differ from the results observed in the present study. As both leukopenia and leukocytosis are recognized as signs of inflammation28, the observed changes in WBC counts in the present study confirm the presence of systemic inflammation. Discrepancies in WBC values between the current investigation and other reports may be attributed to variations in species, dosage, or route of administration.
Reference value of serum iron for rabbits is reported between 97-292 μg/dL29. While mean serum iron levels in the CG were within the reference during the experiment, in the TOG the values were below the reference at the 6th, 12th and 24th hours of the experiment. The findings indicate that a solitary TO injection results in a precise decrease in serum iron levels, at least for the first 24 hours. However, by the 72nd hour following the injection, these levels rebounded and returned to those observed in the CG. In a study conducted on Pony horses, sterile inflammation was induced by a single intramuscular 5 mL TO injection. In this study it was reported that serum iron levels decreased significantly 24 h after the injection, started to increase at 72nd hour and reached to the controls’ level at day 6 after the injection30. In another study performed on rats, 5 mL/kg TO was injected intramuscularly. In this study serum iron reduction reached to the minimum at 24th hour and increased at 36th hour31. The variances in the time taken to decrease and to return to the normal levels between the studies could be attributed to differences in the species or injection method.
Vitamin D exerts its effects by binding to vitamin D receptor (VDR) in target cells. Notably, the affinity of 1.25-D to VDR is significantly higher, approximately 100-200 times greater than that of 25-hydroxyvitamin D (25-D). Moreover, 1,25-D is recognized as the most active form of vitamin D32. However, despite the potency of 1,25-D, serum 25-D levels are utilized to assess individuals' vitamin D sufficiency due to several reasons: i) Many imbalances in vitamin D metabolism are correlated with concentrations of 25-D rather than 1.25-D; ii) The half-life of 1.25-D is relatively short, ranging from 4 to 15 hours, compared to the substantially longer half-life of 25-D, which lasts from 20 to 24 days; iii) The circulating concentration of 1.25-D is approximately 1/1000th of that of 25-D; iiii) Serum 25-D levels are indicative of the total amount of vitamin D acquired from dietary sources and synthesized in the skin32,33.
In the present study serum 25-D and 1.25-D levels were measured by LC-MS/MS method, which is considered as gold standard for measurement of vitamin D levels34. Mäkitaipale et al.35 has been reported that mean serum 25-D levels in healthy rabbits are 25,9 ng/mL and values below 17 ng/mL indicate vitamin D deficiency. In the current study, none of the rabbits exhibited vitamin D deficiency, as the mean serum 25-D levels for both the TOG and CG rabbits at the start of the experiment (0th hour) were 59.5 ng/mL and 57.6 ng/mL, respectively. The study revealed a significant decrease in serum 25-D levels in the TOG at the 72nd hour compared to the CG. Additionally, when changes within the groups over time were examined, a significant decrease was observed in the TOG at the 72nd hour compared to those of 0th hour, whereas no difference was noted in the CG. This decline may be attributed to the utilization of 25-D as a substrate in the heightened synthesis of 1.25-D in the TOG.
As reported by Titmarsh et al.18 and Vieira et al.36, serum 25-D levels commonly decrease in various inflammatory conditions. However, clinical studies are influenced by numerous factors such as patient demographics, diet, disease stage, comorbidities, prior treatments, and the inability to ascertain pre-disease vitamin D levels in most cases, rendering it challenging to discern whether the decrease in serum 25-D levels is the cause or consequence of the disease16,33. Therefore, to investigate the relationship between sterile inflammation and vitamin D levels experimentally can help mitigate these uncertainties observed in clinical studies. The data obtained from this study demonstrate that sterile inflammation leads to a significant decrease in circulating 25-D levels. This finding suggests that sterile inflammation plays a causal role in vitamin D deficiency, a common occurrence in sterile inflammatory diseases.
Although no statistical difference was detected between the TOG and CG in terms of 1.25-D levels throughout the experiment, intra-group statistical analyses revealed a significant increase within the TOG at the 12th and 72nd hours compared to the 0th hour. It has been reported that serum 1.25-D levels increase in many sterile and non-sterile inflammatory diseases37-39. It is known that immune system cells can synthesize sufficient 1.25-D to elevate its circulating levels in certain conditions, facilitated by the CYP27B1 enzyme they contain37-39. Therefore, it is likely that the observed increase in 1.25-D levels in the TOG is attributable to its synthesis in immune system cells activated by inflammation.
In conclusions, vitamin D deficiency or insufficiency is frequently observed in inflammatory conditions, yet due to the inherent limitations of clinical research, the causal relationship remains incompletely understood. However, in the current experimental study, it was demonstrated that sterile inflammation induces a decrease in serum 25-D levels and an increase in 1.25-D levels. Based on these findings, it can be inferred that vitamin D deficiency or insufficiency is at least partially, if not entirely, a consequence of inflammation.
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