Marcella M. Hallemeesch

Metabolic flux measurements across portal drained viscera, liver, kidney and hindquarter in mice

A method was developed to measure metabolic fluxes across either portally-drained viscera (PDV) and liver or kidney and hindquarter (HQ) in anesthetized mice. The method includes a primed-constant infusion of ketamine-medetomidine anaesthesia to stabilize the mice for the surgical procedures. For measurement of metabolic fluxes across PDV and liver, blood sampling catheters were inserted in the carotid artery, portal vein and hepatic vein and infusion catheters in the jugular vein and mesenteric vein. For measurement of metabolic flux across kidney and HQ, blood sampling catheters were inserted in the carotid artery, renal vein and caval vein and infusion catheters in the jugular vein and abdominal aorta. 14C-PAH was infused to enable plasma flow measurement using an indicator dilution method. In addition, we developed a blood sampling procedure without waste of blood. We measured plasma flow and metabolic fluxes across PDV, liver, kidney and HQ. Mean plasma flow in post-absorptive mice was: PDV: 0.9 ± 0.2, liver: 1.2 ± 0.3, kidney:1.0 ± 0.1, HQ: 1.1 ± 0.3 ml/10 g body weight (b.w.)/min. Significant glutamine release by the HQ and uptake of glutamine by the kidney and PDV was observed. In PDV, citrulline is produced from glutamine and is in turn used by the kidney for the production of arginine. In conclusion, the described model enables measurement of metabolic fluxes across PDV, liver, kidney and HQ in mice. The availability of such a small animal model allows the potential for measuring metabolic parameters in transgenic and knockout mice, and therefore may lead to an important refinement in metabolic research.

Quantitative in vivo assessment of arginine utilization and nitric oxide production in endotoxemia

BACKGROUND Until recently no methods were available to quantitate nitric oxide (NO) production in vivo. The advent of stable isotope techniques has allowed quantitation of NO production in different animal models and human disease states. METHODS In vivo NO production was assessed with the use of stable isotope labeled arginine. Enrichments of metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Knock-out mice were used to assess the influence of knocking out inducible NOS (iNOS) or constitutively expressed NOS (cNOS) on arginine-NO metabolism. Pig models were used to assess the role of individual organs on arginine-NO fluxes. RESULTS In mice under basal conditions cNOS mediates half of the NO production. After endotoxin challenge NO production doubles as a result of iNOS induction and cNOS-mediated NO production is downregulated. In larger animal models (pig) whole body NO production is augmented after endotoxin challenge, largely resulting from NO production in liver, intestine and kidney. Arginine supplementation increases NO production in pigs in liver, intestine and kidney both in the basal state and after endotoxin challenge. CONCLUSIONS Stable isotope techniques employing LC-MS allow in vivo assessment of NO production in small and large animal models and in patients. This allows definition of the role that iNOS and cNOS-mediated NO production play in several disease states.

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production.

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.