Nitrosylhemoglobin formation after infusion of NO solutions: ESR studies in pigs q Giancarlo Aldini, a, * Marica Orioli, a Roberto Maffei Facino, a Maria Giovanna Clement, b Mariangela Albertini, b Silvia Mazzola, b Federica Pirrone, b and Marina Carini a a Istituto Chimico Farmaceutico Tossicologico, Faculty of Pharmacy, University of Milan, Viale Abruzzi 42, Milan 20131, Italy b Dipartimento di Patologia Animale Igiene e Sanit a Pubblica Veterinaria, University of Milan, Via Celoria 10, Milan 20133, Italy Received 5 April 2004 Available online 20 April 2004 Abstract A saturated nitric oxide (NO) solution (1.88 mM) infused i.v. in the anesthetized pig at a dose of 68 nmol/kg/min for 24 min resulted in a time-dependent increase of nitrosylhemoglobin [HbFe(II)NO] as determined by electron spin resonance (ESR), reaching a C max of 7.99  0.42 lM at the end of the infusion, compared to 1.13  0.42 lM before (p < 0:01). This indicates that NO i.v. is efficiently bioconserved as HbFe(II)NO (34% of the NO dose) and to a greater extent than by the oxidative pathway (24% of the NO dose), as determined by measuring plasma nitrites/nitrates (chemiluminescence) and Met-Hb (ESR analysis). When the NO infusion was stopped, HbFe(II)NO declined with a t 1=2 of 15 min, indicating that it is a stable storage form of NO, able to deliver NO distally to the site of administration. No significant differences were observed in systemic and pulmonary vascular resistances during and after NO infusion, but PO 2 showed a significant decrease 15 and 30 min after the infusion. Thus, in normoxic/physi- ological conditions, HbFe(II)NO does not induce significant NO-dependent vasorelaxation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide solution; Nitrosylhemoglobin; Electron spin resonance; Hemodynamics; Conservative and oxidative metabolism Since its discovery, nitric oxide (NO) has been con- sidered a paracrine mediator, able to act locally at its site of formation. An emerging theory, however, is that NO additionally functions as a hormone, delivered through the circulation to exert biological effects distal to its site of synthesis [1]. Supporting the endocrine mechanism, the hemodynamic effects occur distally to the site of administration. NO inhalation, for example, induces peripheral vasodilation in regions with phar- macologically reduced NO production [2], restores in- testinal blood flow after ischemia/reperfusion injury [3], increases glomerular filtration [4], and raises aortic cGMP levels [5]. Since the very short half-life of NO in the blood (approximately 2 ms) [6] is mainly due to the reaction with HbO 2 to form Met-Hb and nitrate (HbFe(II)O 2 + NO ! HbFe(III) + NO  3 ), the endocrine effect has been attributed to intravascular NO-derived molecules that might stabilize NO bioactivity and deliver it at a dis- tance from the site of generation. Among the proposed bioactive storage forms of NO in circulation, there is evidence of a pivotal role of HbFe(II)NO generated by an additional mechanism of NO to free heme, where NO is maintained as a radical species (HbFe(II) + NO ! HbFe(II)NO). Gladwin found that NO inhalation markedly raised total nitrosylated hemoglobin, with a significant arterial- venous gradient, supporting a role of hemoglobin in the transport and delivery of NO [7]. In another model, Pawloski and Stamler [8] suggested that NO first reacts with hemoglobin forming HbFe(II)NO which can then intra-molecularly transfer NO to b-cysteine 93. In addition, several papers reported the formation of q Abbreviations: HbFe(II)NO, nitrosylhemoglobin; Met-Hb, met- hemoglobin; NOx, nitrites+nitrates; Pw, pulmonary capillary wedge pressure; SVR, systemic vascular resistances; PVR, pulmonary vascu- lar resistances; CO, cardiac output; MAP, mean arterial pressure; MPAP, mean pulmonary arterial pressure. * Corresponding author. Fax: +39-02-5031-7565. E-mail address: giancarlo.aldini@unimi.it (G. Aldini). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.042 Biochemical and Biophysical Research Communications 318 (2004) 405–414 BBRC www.elsevier.com/locate/ybbrc