No abstract

There exist reaction products of nitric oxide (NO) with blood that conserve its bioactivity and transduce an endocrine vasomotor function under certain conditions. Although S-nitrosated albumin has been considered the major species subserving this activity, recent data suggest that additional NO species, such as nitrite, nitrated lipids, N-nitrosamine, and iron-nitrosyl complexes, may contribute. We therefore examined the end products of NO reactions in plasma and blood in vitro and in vivo by using reductive chemiluminescent assays and electron paramagnetic resonance spectroscopy. We found that NO complexes in plasma previously considered to be S-nitrosated albumin were <10 nM after elimination of nitrite and were mercury-stable, consistent with ironnitrosyl or N-nitrosamine complex. During clinical NO gas inhalation protocols or in vitro NO donor treatment of human plasma, S-nitroso-albumin did not form with NO exposure <2 M, but plasma methemoglobin was detectable by paramagnetic resonance spectroscopy. Consistent with this formation of methemoglobin, human plasma was found to consume Ϸ2 M NO at a rate equivalent to that of hemoglobin. This NO consumption was mediated by the reaction of NO with plasma haptoglobin-hemoglobin complexes and limited slower reaction pathways required for S-nitrosation. These data suggest that high-affinity, metalbased reactions in plasma with the haptoglobin-hemoglobin complex modulate plasmatic NO reaction products and limit S-nitrosation at low NO flux. The studies further suggest that alternative NO reaction end products in plasma, such as nitrite, N-nitrosamines, iron-nitrosyls, and nitrated lipids, should be evaluated in blood NO transport along the vasculature. N itric oxide (NO) is a diatomic free radical molecule that plays a principal role in basal blood flow regulation and vascular homeostasis. Although NO is inactivated by dioxygenation reaction with oxyhemoglobin, a perierythrocytic unstirred layer (1, 2), membrane diffusion barrier (3), and cell-free zone in laminar flowing blood (4) reduce NO-hemoglobin reactions Ϸ600-fold, permitting tonic NO-dependent vasodilation and NO reactions with plasmatic proteins and other gas molecules (recently reviewed in ref. 5). The reaction of NO with plasma has been the subject of intense study after initial reports that human plasma contains 7 M S-nitroso-albumin (SNO-albumin), effectively creating a major paradigm that S-nitrosothiols serve as a stable circulating storage form of NO (6). Such a model has been supported by observations of the vasoactivity of infusions of SNO-albumin in animal models, distal vasodilation during infusions of NO solutions into the brachial artery, and the in vitro and in vivo formation of SNO-albumin with micromolar NO exposure to plasma (7-16).However, over the last 10 years the measured levels of plasma S-nitrosothiol and SNO-albumin have varied widely with different analytical methodologies and different species (Table 1 and Supporting Text, which are published as supporting information on the PNAS ...