• U Wachter, M Groeger, K Wagner, P Radermacher, and J V Vogt.
• Department APV, University Ulm, Germany.
• Shock. 2015 Oct 1;44 Suppl 2:19.

IntroductionDue to the limited sample volume (total blood volume is 1.5-2.5 mL), metabolic parameter sets in mice are usually assessed by killing a defined number of animals at selected time points with subsequent analysis of pooled plasma. However, reducing the number of animals used is a cornerstone of the ARRIVE guidelines, and, moreover, may reduce costs and workload. Therefore, we developed a stable-isotope-based gas chromatography/mass spectrometry (GC/MS) method to allow for serial quantification of metabolic pathways in mice.Methods15 hours after cecal-ligation-and-puncture (CLP, n = 7) or sham operation (n = 5) anesthesized and ventilated mice received constant, stable, non-radioactive-labelled glucose, urea, glycerol, and leucine isotope infusions. Plasma substrate concentrations and substrate isotope enrichment were measured together with plasma and urine creatinine levels and total expiratory CO2 and CO2 isotope enrichment. Plasma (sample volume 5 μL) analysis comprised internal standard addition, de-proteinization, solid phase extraction with subsequent compound separation, derivatization of the derived fractions and GC/MS analysis. Urine was treated identically to separate creatinine from creatine and creatine phosphate. Expiratory CO2 concentrations and isotope enrichment were measured directly.ResultsCLP-induced sepsis increased total CO2-release, glycerol production and glucose oxidation, the increased ratio of glycerol production to glucose oxidation indicating preferential fatty acid oxidation. This effect coincided with markedly depressed creatinine clearance.ConclusionThis method enables simultaneous, repetitive in vivo monitoring of kidney function (glomerular filtration), determinants of energy metabolism (glycolysis, lipolysis), and nitrogen balance as a function of hepatic urea production and protein degradation (leucine production).

22 keywords...

hide…