Citrulline as a Biomarker for Gastrointestinal-Acute Radiation Syndrome: Species Differences and Experimental Condition Effects

Abstract

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13–17 Gy and 8–16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72–13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean 6 SEM) in mice and minipigs were 57.8 ± 2.8 μM and 63.1 ± 2.1 μM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 μM, very similar to that of humans (~40 μM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

Time- and radiation-dose dependent changes in the plasma proteome after total body irradiation of non-human primates: Implications for biomarker selection

“…Moreover, in the analysis of the urine from the same animals [3], the higher radiation dose (7.4 Gy) resulted in the most unique proteins on day 4 compared to the lower radiation dose (6.7Gy) in which unique proteins were at the highest level on day 7. These results differ from the radiation dose-dependent kinetics observed with citrulline for which higher radiation doses were associated with more pronounced changes at later time points [14]. The distinct kinetics of proteomic changes reported herein presents potential advantages for dosimetry applications for which an earlier assessment of exposure is critical to guide medical management.…”

Metabolomic applications in radiation biodosimetry: exploring radiation effects through small molecules

“…Although as previously described, reduced levels of circulating citrulline can correlate with GI injury (Jones et al, 2014a, Jones et al, 2014b, Jones et al, 2015, Jones et al, 2015, Wang et al, 2015, Bujold et al, 2016) and particularly small intestinal damage, they provide no direct assessment of the population of microbiota or their direct metabolites. Fecal material can be easily obtained and analyzed in a similar manner as urine, serum, saliva, and sebum.…”

“…A targeted approach was developed in plasma of mice to identify and quantify citrulline (Jones et al, 2014a, Jones et al, 2014b, Jones et al, 2015, Bujold et al, 2016). In doses leading to GI syndrome, the citrulline decrease correlated with decreased numbers of crypts in the small intestine (Jones et al, 2014a, Jones et al, 2015).…”

“…Plasma analysis, with the same experimental conditions as in (Zhang et al, 2014a), also revealed time and dose dependent changes. Of particular importance is citrulline that has also been identified in mice as a biomarker of intestinal injury (Jones et al, 2014a, Jones et al, 2014b, Jones et al, 2015, Kurland et al, 2015, Bujold et al, 2016). This is further proof of cross-species validation and that biomarkers of intestinal injury and therefore GI syndrome can be utilized as predictive markers at early time points.…”

“…In order to study GI injury through blood indicators, citrulline was measured by two groups (Shim et al, 2014, Bujold et al, 2016) (Table 3, Supplementary Table 1). Both groups identified decreased levels in plasma, correlating with small intestinal radiation-induced injury.…”