the pringle maneuver is a procedure that is frequently adopted in liver transplantation and liver resection [ 1 , 2 ] . it causes hepatic ischemia / reperfusion ( i / r ) injury , which contributes to liver damage and multiple organ failure [ 2 , 46 ] . conventional monitoring approaches can only reflect hepatic changes within several days or even several weeks of injury , and they fail to reflect the instantaneous changes of the liver after hepatic i / r . for example , the specificity and sensitivity of current enzymatic assays of serum alanine transaminase ( alt ) or lactate dehydrogenase ( ldh ) are not high enough to detect early stages of liver parenchymal injury , assess the magnitude of parenchymal cell necrosis , or to predict hepatic recovery . there is a need to explore novel sensitive biomarkers that can detect early stages of liver parenchymal cell injury so that hepatic i / r injury can be identified and managed at an early stage . previous study has tried to identify novel biomarkers of acute hepatic i / r injury using dual - platform proteomic / degradomic approaches , and it was found that hepatic proteins , including argininosuccinate synthase ( ass ) and estrogen sulfotransferase ( est-1 ) , were degraded in the liver and rapidly released into circulation during i / r injury . as novel sensitive and specific biomarkers of acute liver ischemic injury with potential clinical application , ass and est-1 have attracted extensive attention , and an elisa kit for the detection of ass has been developed . proteomics studies the overall expression of proteins , and metabolomics characterizes and quantifies their end products , that is , the entire metabolite pool that exists within a cell , tissue , or biofluid under certain conditions . the study of metabolomics is able to quantitatively detect the dynamic metabolic response of living systems under pathophysiological conditions . metabolomics studies mainly adopt two types of techniques , traditional nuclear magnetic resonance spectroscopy , and newly developed mass spectrometry and chromatography . the metabolomics approach has been applied to study renal i / r injury . in 2005 , used nuclear magnetic resonance spectroscopy to study metabolomics after renal transplant - induced renal i / r injury and found a significantly decreased level of polyunsaturated fatty acids ( pufas ) and elevated levels of allantoin in the renal tissue and increased trimethylamine n - oxide ( tmao ) and allantoin levels in blood after ischemic injury . a high - performance liquid chromatography coupled with mass spectrometry - based metabolomics approach was recently used to explore serum changes after i / r - induced kidney injury and the protective effect of l - carnitine on acute kidney injury . currently , gas chromatography - mass spectrometry ( gc - ms ) is used to detect a variety of inherited metabolic disorders . urine samples are convenient to collect , and urease pretreatment - gas chromatography - mass spectrometry ( up - gc - ms ) can detect as many as 175 metabolites including amino acids , organic acids , monosaccharides , disaccharides , sugar alcohols , porphyrins , pyrimidine , and nucleic acids via a single injection of sample . in addition , plasma levels of volatile organic acids are not stable , and the ms - ms approach may provide false - negative results . gc - ms can detect the urinary metabolites of volatile acids , thereby indirectly reflecting blood levels . in addition , gc - ms has been used to study urinary metabolomics for the prediction of gastric cancer metastasis   and for the discovery of potential cancer biomarkers and early - stage biomarkers for diabetic kidney disease [ 13 , 14 ] . however , a metabolomic approach has not been used to study hepatic i / r injury . this study aimed to detect urinary metabolomics that can reflect early - stage hepatic i / r injury using an up - gc - ms approach . male spf - grade sprague - dawley rats weighing 220250  g ( n = 80 ) were used in this study . this study was approved by the institutional review boards ( irbs ) of nanfang medical university , and all animals were treated in accordance with the declaration of helsinki . rats were randomly divided into 4 groups ( n = 20 per group ) : a sham operation group and days 1 , 3 , and 5 after pringle maneuver groups . rats in the days 1 , 3 , and 5 groups underwent a pringle maneuver , and specimens were collected on days 1 , 3 , and 5 afterwards . the lower edge of the liver was dissociated , and the hepatic artery , portal vein , and common bile duct were located and clipped by vascular clips for 30 minutes . the vascular clips were then removed , and the abdomen was closed . in the sham group , rats underwent laparotomy , and the surgical field was covered by warm saline - soaked gauze for 30 minutes following the closure of the abdomen . samples were collected from the rats in the sham group and days 1 , 3 , and 5 groups on day 0 , 1 , 3 , and 5 , respectively . rats were placed in metabolic cages to collect urine samples , and approximately 4  ml of urine per rat was obtained . the collected urine samples were stored in a refrigerator at 20c , and urine sample detection was completed within one week after sample collection . twelve hours after urine sample collection , the rats underwent laparotomy , and approximately 3  ml of venous blood was collected from the inferior vena cava . the blood sample was centrifuged at 3500  rpm for 15 minutes , and the serum was collected . the samples were stored in a refrigerator at 70c and analyzed within 2 days of collection . the rats were killed after venous blood samples were collected , and the left lobe of the liver was excised and rinsed with 0.9%  nacl solution . the specimen was fixed in 10% neutral formalin for 48 hours , and then sectioning and hematoxylin and eosin ( he ) staining were performed . fixed liver tissue was dehydrated with ethanol , and then the tissue was embedded in paraffin . tissue blocks were cut into 5  m slices , dewaxed with xylene , subjected to gradient alcohol hydration , and then he - stained . the samples were cleared in xylene and mounted in neutral gum for observation under a light microscope ( type dx51 , olympus , japan ) . serum alt and total bilirubin ( tbil ) levels were measured at the department of laboratory , nanfang hospital , using an automatic biochemical analyzer ( au5400 , japan ) . a gas chromatograph / mass spectrometer ( type q1000 , japan electronics co. , japan ) was used for urine sample detection . urease was added to remove urea , and then an internal quality standard heptadecanoic acid was added . after adding anhydrous ethanol , the supernatant was vacuum - dried , and the residues were derivatized with n , o - bis - trimethylsilyl - trifluoroacetamide ( bstfa)-trimethylchlorosilane ( tmcs ) in a constant temperature drying oven at 90c for 1 hour . nonsplit stream sampling was used , and sample injection volume was 1  l . the sample was injected into a small volume sample injection vessel for gc - ms analysis , and the conditions were as follows . the temperature was initialized at 60c for 2 minutes and then gradually raised to 220c at a rate of 17c / min and further raised to 325c at a rate of 15c / min . ionization mode was ei , ionizing voltage was 70  ev , and the quadrupole temperature was 150c . helium was used as the carrier gas with a flow rate of 1  ml / min . scanning mode was full - scan mode , and the mass spectrometry scan range frequency was 50650  m / z . data were presented as median ( interquartile range ) and tested using the kruskal - wallis ( k - w ) test . once a significant result was revealed by the k - w test , the mann - whitney u test was used for post hoc pair wise comparisons . principle component analysis ( pca ) was implemented to verify the distribution of variables for study groups , as well as confirm the time trend of variable changes . all analyses were performed using sas statistical software ( version 9.1.3 , sas inc . , cathy , ny , usa ) . the liver tissues in different groups were scored according to the liver damage evaluation criteria reported in the literature . in the sham group , the liver sinusoids were normal , and no congestion was found ( 0 points ) . a very small amount of vacuolization was observed ( 1 point ) . the total score of the sham group was 1 point . in the day 1 group , a small amount of congestion was present in the hepatic sinusoids ( 1 point ) , and a moderate amount of vacuolization was observed ( 3 points ) . the nuclei of the liver cells were darkly stained and pyknotic , the cytoplasm was lightly stained , and the liver cells were swollen , compressing the expanded hepatic sinusoids . 3 group , the liver sinusoids were obviously dilated , which were surrounded by more normal liver cells . however , necrosis of sporadic liver cells was seen ( 1 point ) . some vacuoles were observed ( 2 points ) ; congestion was present ( 1 point ) , and it could be seen that damaged cells were in recovery . 5 group , the liver sinusoidal expansion was normal , and the liver cell arrangement was cord - like , but a small amount of congestion was still present ( 1 point ) . generally , the liver cells were normal appearing , and necrosis of sporadic liver cells was seen ( 1 point ) . compared with the sham group , the serum alt and tbil levels in the day 1 group were significantly elevated ( p < 0.01 ) . the serum alt and tbil levels in the day 3 group were significantly decreased compared with the day 1 group ( p < 0.01 ) , but their levels were still higher than that in the sham group ( p < 0.01 ) . serum alt and tbil levels in the day 5 group were significantly decreased as compared to the day 1 and day 3 groups ( p < 0.01 ) , and levels in the day 5 group were not significantly different from that in the sham group . gc - ms detected 170 metabolites in the urine samples , and values of 163 were 0 . table 2 shows the levels of the 7 urinary metabolites with meaningful results ( nonzero ) including lactic acid , pyruvic acid , alanine , serine , threonine , glycerol-3-phosphate , and oleic acid . significant differences in lactic acid , pyruvic acid , alanine , serine , and glycerol-3-phosphate were found among the groups ( p < 0.001 ) . lactic acid levels were increased significantly in the day 1 group compared with the sham group ( p < 0.001 ) , declined in the day 3 group ( p < 0.001 ) , and returned to a level that was not significantly different from the sham group at day 5 . pyruvic acid levels were increased significantly in the day 1 group compared with the sham group ( p < 0.001 ) , reached a peak at day 3 , and returned to zero at day 5 . alanine levels were decreased significantly in the day 1 group compared with the sham group ( p < 0.001 ) , increased in the day 3 group , and in the day 5 group were not significantly different from the sham group . serine levels were decreased significantly in the day 1 group compared with the sham group ( p < 0.001 ) and returned to a level that was not significantly different from that in the sham group at day 3 . glycerol-3-phosphate levels were increased significantly in the day 1 group compared with the sham group ( p < 0.001 ) , decreased in the day 3 group ( p < 0.001 ) , and returned to a level that was not significantly different from the sham group at day 5 . post hoc pair wise comparisons revealed that the levels of five urinary metabolites among the seven detected urinary metabolites including lactic acid , pyruvic acid , alanine , serine , and glycerol-3-phosphate were significantly different between the sham group and the day 1 group ( p < 0.01 ) , indicating that the changes in the levels of those five urinary metabolites are related to hepatic i / r injury . rats with hepatic i / r injury at day 1 had increased levels of lactic acid , pyruvic acid , and glycerol-3-phosphate and decreased levels of alanine and serine compared with rats in the sham group ( p < 0.01 ) . furthermore , the post hoc pair wise comparisons showed that the levels of those five urinary metabolites were significantly different between day 1 group and day 5 group ( p < 0.01 ) , the groups with the most severe liver injury ( with a liver damage score of 6 points ) and the least severe liver injury ( with a liver damage score of 3 points ) , respectively . when the liver injury was less severe at day 5 , levels of lactic acid , pyruvic acid , and glycerol-3-phosphate were significantly reduced , and levels of alanine and serine were significantly increased compared with those at day 1 when the liver injury was most severe ( p < 0.01 ) , which indicates a relationship between the levels of those five urinary metabolites and the severity of liver injury . the pca analysis revealed 4 components that provided a good summary of the data , and these components accounted for 35.4% , 17.7% , 13.8% , and 11.5% of the total variance ( table 3 ) . the first component consisted of alanine and serine with positive loading , as well as glycerol-3-phosphate and lactic acid with negative loadings . the second eigenvector had high positive loadings from the level of threonine and high negative loadings from the level of pyruvic acid . a high positive loading from the level of oleic acid was found in the third component . the fourth component shared some variables with the previous three components ( data not shown ) . pca results showed that 7 metabolites distinguished the day 1 group and the day 3 group from the sham group ( figure 2 ) . the pca score plot showed a similar changing trend of urinary metabolites as that presented in table 2 , and measurements in the day 1 group were distinct from that in the sham group . on the contrary , measurements in the day 3 and day 5 groups were closer to that in the sham group . notably , compared to the measurements in the day 3 group , more measurements in the day 5 group overlapped with those in the sham group . this study used gc - ms to assess urinary metabolomics in rat model of hepatic i / r injury induced by the pringle maneuver . in addition , conventional histological observation was used to confirm hepatic i / r injury . gc - ms detected 7 urinary metabolites in rats after hepatic i / r injury , and 5 of them including lactic acid , pyruvic acid , alanine , serine , and glycerol-3-phosphate demonstrated significantly different levels between sham group ( without hepatic i / r injury ) and day 1 group ( with hepatic i / r injury ) and between groups with different liver injury severity ( day 1 group and day 5 group ) , indicating that the changes in those five urinary metabolites are associated with liver injury . pca analysis further confirmed that the 7 variables were enough to describe subsets with similar characteristics , and separated the day 1 and day 3 groups from the sham group . the aforementioned five urinary metabolites account for the leading percentage of total variance in pca analysis , which further support an association between the changes in those five urinary metabolites and liver injury . these results indicate a significant potential of early diagnosis of hepatic i / r injury using noninvasive urinary metabolomic analysis . the five detected urinary metabolites with significantly different levels between rats with and without hepatic i / r injury and between rats with different severity of liver injury play important roles in energy metabolism . pyruvic acid is the first substrate of the gluconeogenic pathway and can then be used to generate glucose . transamination or deamination of alanine and serine enables the entrance of their carbon skeleton into gluconeogenesis directly as pyruvate . we speculate that anaerobic conditions increase glycolysis , which induces the buildup of lactic acid in the muscles . the lactic acid diffuses into the blood stream , and the liver increases its synthesis of glucose by gluconeogenesis , resulting in consumption of alanine and serine . at the same time , increased glycolysis and gluconeogenesis result in an increase of the intermediate , glyceraldehyde 3-phosphate . in this study , we constructed a rat model of hepatic i / r injury induced by the pringle maneuver . currently , there are various surgical approaches for liver transplantation and liver resection , and although the operative time has been shortened and the surgical approach improved [ 1921 ] , the pringle maneuver is still a basic means to reduce intraoperative hemorrhage , and it may induce hepatic i / r injury . this is the reason we constructed a rat model of hepatic i / r injury induced by the pringle maneuver . our results showed that conventional examinations clearly outlined the trend of recovery after hepatic i / r injury . alt and tbil were increased most significantly in the day 1 group , indicating the highest severity of injury . their levels were reduced in the day 3 group , and returned to normal in the day 5 group , showing a trend of recovery . the he staining results of the liver tissue also confirmed hepatic i / r injury and the recovery trend , indicating that our animal model construction was successful . our urinary metabolomic analysis using gc / ms detected a changing trend of urinary metabolomics after hepatic i / r injury , which truly reflects the dynamic hepatic i / r injury process in rats . the hepatic i / r injury process reflected by the urinary metabolomics is consistent with the hepatic injury and recovery process indicated by serum enzymes and pathological findings . urinary metabolomic analysis is noninvasive , sensitive , convenient , and rapid . using gc / ms to detect urinary metabolomics urine sample collection is noninvasive and is not limited by sample collection time or frequency . in addition , the urinary metabolomic analysis is very effective for monitoring the dynamic changing process of the liver . however , based on the results of this preliminary study , no conclusion can be drawn that the urinary metabolomic analysis is superior to conventional examination methods for hepatic i / r injury such as serological examinations . i / r injury detection and monitoring may deepen our understanding of the value of urinary metabolomic analysis in early detection and diagnosis of hepatic i / r injury . gc / ms data exhibit great variation within group , and study with a large sample size is warranted . during gc - ms , the limitation of gc - ms analysis is that it is more useful for detection of volatile substances . in future studies , other metabolomics techniques can be used to examine more metabolites related to hepatic i / r injury , and preoperative and postoperative human urine samples can be studied .
background . 
 metabolomics studies can quantitatively detect the dynamic metabolic response of living systems . objective . to detect urinary metabolomics after hepatic ischemia / reperfusion ( i / r ) injury induced by the pringle maneuver using gas chromatography - mass spectrometry ( gc - ms ) . 
 methods . 
 male sprague - dawley rats ( n = 80 ) were randomly divided into 4 groups ( n = 20/group ) : sham operation , day 1 , day 3 , and day 5 . 
 rats in the day 1 , day 3 , and day 5 groups underwent the pringle maneuver . 
 serum alanine transaminase ( alt ) and total bilirubin ( tbil ) were measured , and hematoxylin and eosin ( he ) staining of the liver tissue was performed . 
 gc - ms was used to detect urinary metabolomics . results . compared with the sham group , 
 the serum alt and tbil levels at day 1 were significantly elevated ( p < 0.01 ) and then decreased and reached close to normal levels at day 5 . 
 gc - ms detected 7 metabolites which had similar changes as those of liver tissue revealed by histological examination . 
 significant differences in lactic acid , pyruvic acid , alanine , serine , and glycerol-3-phosphate were found among the groups ( p < 0.001 ) . 
 principle component analysis showed that 7 metabolites distinguished the day 1 and day 3 groups from the sham group . 
 conclusions . 
 noninvasive urinary metabolomic analysis is a potential means for the early detection and diagnosis of hepatic i / r injury .