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 ± 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.

FIG. 1

Plasma citrulline levels in different species. Panel A: Basal plasma citrulline levels in rhesus NHPs (n = 209), C57BL/6 mice (n = 20) and Göttingen minipigs (n = 81). Panel B: Plasma citrulline levels in male and female rhesus NHPs (n = 158 and 56, respectively), and male and female Göttingen minipigs (n = 33 and 38, respectively). Data are presented as mean ± SEM.

FIG. 2

Plasma citrulline levels after irradiation. Plasma citrulline levels decreased after irradiation with partial bone marrow shielding in C57BL/6 mice (panel A) and Göttingen minipigs (panel B). Plasma citrulline levels are also shown after total-body irradiation in rhesus NHPs (panel C). The dash line represents the average basal citrulline levels determined prior to study conduct and across all available animals. Values are presented as mean ± SEM. Individual sample sizes in the radiation dose groups ranged from 4 to 8 for mice, 1 to 20 for minipigs and 2 to 20 for NHPs, not accounting for any mortality during the study.

FIG. 3

Plasma citrulline levels decrease in a dose-dependent manner. Plasma citrulline levels after irradiation in C57BL/6 mice at day 3.5 and 7 (panel A) and Göttingen minipigs at day 5 (panel B). Plasma citrulline levels also decreased as shown after total-body irradiation in rhesus NHPs at day 3, 5 and 7 (panel C). Values are presented as mean ± SEM.

FIG. 4

Effect of feeding on plasma citrulline levels in rhesus NHPs. Plasma citrulline levels were measured in rhesus NHPs either when fasted (n = 88) or postprandially (n = 47). Data are presented as mean ± SEM. *P < 0.05 when compared to the fasted animals.

FIG. 5

Effect of ketamine/acepromazine anesthesia on plasma citrulline levels in rhesus NHPs. Plasma citrulline levels were measured in rhesus NHPs with (n = 16) or without (n = 16) a ketamine/acepromazine anesthetic regimen. “Anesthesia” represents pooled data collected within 3 h of the anesthetic regimen. “No anesthesia” represents pooled data collected in excess of 3 h after the anesthetic regimen or from animals that were not previously anesthetized. Data are presented as mean ± SEM. **P < 0.01 when compared to unanesthetized animals.