Detection Of Congenital Intrahepatic Vascular Shunt In C57BL/6 Mice

Portosystemic shunt (PSS) is an abnormal vein that leaks blood from portal vein into systemic circulation, thus bypassing the liver. We previously reported its sporadic occurrence as a congenital defect in C57BL/6 mice as well as in humans. PSS results in vascular, hepatic and metabolic anomalies. Therefore, in experimental protocols using C57BL/6 mice, ones with PSS should be identified as outliers. Accordingly, we sought methods to discern 8-weeks-old male C57BL/6 mice (n=6) as non-PSS or PSS mice. Serum total bile acids (TBA) is a bona fide biomarker of PSS in mice (non-PSS: 6.1±1.0 vs PSS: 80.6±8.2 μM; p<0.001), but utility of this biomarker is limited by its cost and invasiveness, particularly if large numbers of mice are to be screened. This led us to investigate whether use of urine might serve as a simple, inexpensive, non-invasive means of PSS diagnosis. Since urinary TBA were comparable between non-PSS (4.1±1.3 μM) and PSS mice (4.8±1.7 μM; p=0.45), we asked whether there are other urinary metabolites that could be exploited as biomarkers. Nuclear magnetic resonance-based metabolomics uncovered that Krebs cycle intermediates, i.e. , citrate (non-PSS: 1.0±0.1 v s PSS: 1.6±0.04; p<0.001), α-ketoglutarate (non-PSS: 1.0±0.1 v s PSS: 3.6±0.2; p<0.001), and fumarate (non-PSS: 1.0±0.2 v s PSS: 3.7±0.4; p<0.001) were strikingly elevated (expressed in fold change) in the urine of PSS mice. We leveraged the pH-lowering properties of such metabolites as the basis for two urine-based screening tests. Firstly, using pH test strips with a 5.5-8.0 range, we detected the differences in their urinary pH (non-PSS: 6.3±0.1 v s PSS: 5.6±0.1; p<0.001). Secondly, the difference in urinary pH was additionally quantified using the phenol red assay (non-PSS: 0.9±0.1 v s PSS: 0.2±0.03 at OD 560nm ; p<0.001). Using a larger cohort of non-PSS (n=44) and PSS (n=27) mice, we determined that the urinary phenol red assay had an accuracy of 95.8%, sensitivity of 96.3%, and specificity of 95.5% in identifying PSS mice. Taken together, these results demonstrate the feasibility of using urine as a liquid biopsy to screen for murine PSS. Application of our urinary PSS screening protocols importantly enables stratification of PSS mice, which confound cardiovascular and metabolic studies.