Metabolomics For Urinary Biomarker Discovery In Rats Undergoning CT Scan

Objectives To investigate biological changes in urine metabolites of rats after CT scan,identify noninvasive biomarkers of ionizing radiation.Evaluate the availability of liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)as a sensitive method in studying biological effects of low-level X-ray radiation.Methods Twelve male Wistar rats were randomly divided into two groups:sham and CT scans.The rats were housed individually in metabolic cages for 72 hours with free access to fresh drinking water and food.All animals are under a standard 12-h light,12-h dark cycle.CT scans were applied with adault cerebral protocol with dose of 6000mGy·cm(Siemens SOMATOM Sensation 64 CT Scanner,Siemens Healthcare,Forchheim,Germany).Urine was collected 12 hours before CT scan and 12,24,40,60 hours after scan.Aliquots of urine were immediately frozen at-80°C until analyz.All samples were randomized and analyzed by UPLC-MS/MS in the same continuous session to eliminate instrument bias as a potential confounder.Orthal Partial least square-discrimination analysis(OPLS-DA)was applied for the discriminant analysis between sham and CT scans and for the screening of the potential radiation biomarkers.Students't test was applied to compare the change of the biomarkers in urine.Results The results of this study provide new insight into the effects of CT scan on metabolism and its manifestation in urinary output of winstar rats.OPLS-DA model showed good result for recognition and prediction of different groups.LC-MS/MS based metabolomics allowed effective identification of nine urinary biomarkers of low level X-ray radiation in rats.As expected,metabolisms of rats were perturbed after CT scan.The upregulatedurinarybiomarkersincludedthreonine,taurine,creatinine,N~1-Acetylspermidine,indole-3-carboxylic acid,glyceric acid,and proline.The metabolic pathway analysis showed,citric acid cycle,amino acids metabolism and urea cycle were involved in radiation damage.The results provided the experimental evidence for the low-level X-ray radiation damage and repair mechanisms.Conclusion Today's mass spectrometers have the sensitivity and accuracy to detect and measure even subtle changes in the abundance of a metabolite.This makes mass spectrometry the platform of choice for detecting changes in urinary excretion of metabolites even at low doses.Further studies include analysis of the rat tissues to look for changes in gene transcription and regulation of enzymes to relate the metabolites discovered to molecular mechanisms.