In vivo quantification of endotoxin‐induced nitric oxide production in pigs from Na¹⁵NO₃‐infusion

Abstract

1 In this investigation the NO production rate is quanti®ed in the pig during normotensive endotoxininduced shock with increased cardiac output and during subsequent treatment with the NO synthase inhibitor N o -monomethy-L-arginine (L-NMMA). NO production rate was derived from the plasma isotope-enrichment of 15 N-labelled nitrate ( 15 NO 3 7 ). 2 Three groups of animals (control, n=5; endotoxin, n=6; endotoxin+L-NMMA, n=6) were anaesthetized and instrumented for the measurement of systemic and pulmonary haemodynamics. Each animal received a primed-continuous infusion of stable, non-radioactively labelled Na 15 NO 3 (bolus 30 mg, infusion rate 2.1 mg h 71 ). Arterial blood samples were taken 5, 10, 15, 30, 60 and 90 min later and every 90 minutes until the end of the experiment. 3 Continuous i.v. infusion of endotoxin was incrementally adjusted until mean pulmonary artery pressure (PAP) reached 50 mmHg and subsequently titrated to keep mean PAP &35 mmHg. Hydroxyethylstarch was administered as required to maintain mean arterial pressure (MAP)460 mmHg. Six hours after the start of the endotoxin continuous i.v. L-NMMA (1 mg kg 71 h 71 ) was administered to the endotoxin+L-NMMA group. Haemodynamic data were measured before as well as 9 h after the start of the endotoxin. 4 After conversion of NO 3 7 to nitro-trimethoxybenzene and gas chromatography-mass spectrometry analysis the total NO 3 7 pool, basal NO 3 7 production rate and the increase per unit time in NO 3 7 production rate were calculated from the time-course of the 15 NO 3 7 plasma isotope-enrichment. A two compartment model was assumed for the NO 3 7 kinetics, one being an active pool in which newly generated NO 3 7 appears and from which it is eliminated, the other being an inactive volume of distribution in which only passive exchange takes place with the active compartment. 5 Although MAP did not change during endotoxin infusion alone, cardiac output (CO) increased by 42+40% (P50.05 versus baseline) by the end of the experiment due to a signi®cant (P50.05 versus baseline) fall in systemic vascular resistance (SVR) to 65+25% of the baseline value. L-NMMA given with endotoxin did not change MAP, and both CO and SVR were maintained close to the pre-shock levels. 6 Baseline plasma NO 3 7 concentrations were 43+13 and 40+10 mmol l 71 in the control and endotoxin animals, respectively, and did not di er at the end of the experiment (39+8 and 44+15 mmol l 71 , respectively). The mean NO 3 7 pool and basal NO 3 7 production rate were 1155+294 mmol and 140+32 mmol h 71 , respectively, without any intergroup di erence. Endotoxin signi®cantly increased NO 3 7 production rate (23+10 mmol h 72 , P50.05 versus control (6+7 mmol h 72 ) and endotoxin+L-NMMA groups). L-NMMA given with endotoxin (71+2 mmol h 72 , P50.05 versus control and endotoxin groups) had no e ect. 7 Analysis of the time course of the 15 NO 3 7 plasma isotope enrichment during primed-continuous infusion of Na 15 NO 3 allowed us to quantify the endotoxin-induced increase in NO 3 7 production rate i...

Minor involvement of nitric oxide during chronic endotoxemia in anesthetized pigs

“…Furthermore, plasma NOx concentrations observed in anesthetized pigs and dogs might not reliably reflect overall NO production. In endotoxic pigs, Santak et al (32) showed that the absence of NOx increase did not reflect the increase of NO production measured by the 15 NaNO 3 kinetic method. Similarly to our study, baseline plasma NOx concentrations did not differ 9 h after an endotoxin bolus, whereas the direct measurement of NO production increased significantly over time.…”

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the l-arginine/NO pathway in health and disease

“…One approach makes use of the inhalation of a stable-isotope labeled oxygen ( 18 O 2 ), which is subsequently converted to N 18 O and in addition to N 18 O 3 − [26]. Alternatively, the infusion of labeled nitrate [27] or labeled l-arginine [28][29][30][31][32][33][34] is applied with subsequent measurement of labeled nitrate in blood or urine.…”

“…In another pig model of sepsis (9-h continuous LPS infusion with colloid infusion to maintain a normotensive state), whole body nitrate appearance rate measured with labeled nitrate infusion as an indicator of NO production was nearly doubled [27].…”

Microdialysis-evaluated myocardial cyclooxygenase-mediated inflammation and early circulatory depression in porcine endotoxemia

“…In three of the animals, half of the dose of endotoxin was needed to titrate the MPAP until the doubled value. Using MPAP to titrate the administration of endotoxin to ensure that a severe shock condition develops has previously been used (21).…”