NO adducts in mammalian red blood cells: too much or too little?

T Rassaf, NS Bryan, RE Maloney, V Specian, M Kelm… - Nature medicine, 2003 - nature.com
T Rassaf, NS Bryan, RE Maloney, V Specian, M Kelm, B Kalyanaraman, J Rodriguez…
Nature medicine, 2003nature.com
LETTERS TO THE EDITOR introduced together with these reagents. Attempts to detect
SNOHb in human hemolysates with DAF-2 using the protocol of McMahon et al.(detection
limit of 50 nM) did not show measurable amounts. Quantification of nitrite in aliquots of the
same RBC lysates by chemiluminescence corroborated the lack of SNOHb. In agreement
with two earlier studies6, 7, no HbNO was detected in human whole-blood and RBC lysates
by EPR spectroscopy, sensitivity limitations notwithstanding. Despite contrasting levels of …
LETTERS TO THE EDITOR introduced together with these reagents. Attempts to detect SNOHb in human hemolysates with DAF-2 using the protocol of McMahon et al.(detection limit of 50 nM) did not show measurable amounts. Quantification of nitrite in aliquots of the same RBC lysates by chemiluminescence corroborated the lack of SNOHb. In agreement with two earlier studies6, 7, no HbNO was detected in human whole-blood and RBC lysates by EPR spectroscopy, sensitivity limitations notwithstanding. Despite contrasting levels of NO adducts in RBCs, plasma nitrite and S-nitrosoalbumin concentrations were consistent between rodents and primates (198±33, 205±21 and 413±94 nM nitrite and 1.2±0.3, 7.2±1.1 and 25.2±5.1 nM S-nitrosoalbumin in rats, humans and guinea pigs, respectively). Experiments in animals and human subjects were approved by the Animal Care and Use Committee and the Institutional Review Board, respectively, of the Louisiana State University Health Sciences Center in Shreveport. An important finding of the present translational study is the drop in NO adducts observed from rodent to primate RBCs, which closely mirrors the differences in hemoglobin thiol reactivity for these species8, 9 (r= 0.99 for human, rat and guinea pig data). The near-micromolar nitroso levels found in RBCs of rats and guinea pigs may therefore be a result of the presence of a population of fast-reacting thiols (Cysβ-125) in rodent hemoglobin whose concentration and reactivity exceeds that of glutathione8, 9. In contrast, the much lower concentration of NO adducts in primate RBCs (< 1 nM), in spite of exposure to comparable levels of plasma nitrite and S-nitrosoalbumin (and presumably NO), may be a consequence of the lower thiol reactivity of the Cysβ-93 in this hemoglobin. An equally important and perplexing finding from our study is the vastly different (≥ 3 orders of magnitude lower) level of NO adducts in human RBCs as compared with that reported by Stamler’s group1. Although disputes over the true levels of NO adducts in human RBCs are not new (summarized in Table 1 of ref. 10), the sub-nanomolar values reported here heighten this controversy to a new level that raises the question of whether the regulatory role proposed by McMahon et al. could function at such vanishingly low concentrations. It might be argued that our methodology simply lacks sensitivity or that it destabilizes NO adducts, but this is not consistent with our ability to detect significant NO signals from rodent and spiked human RBCs, or to obtain comparable signals between unprocessed wholeblood aliquots and those analyzed after sample processing. It is equally difficult to dismiss McMahon et al.’s micromolar levels as artificial nitrosation by nitrite, as our own data show that this substance is present only at much lower concentrations in plasma (see above) and RBCs (200–300 nM). What makes the present disagreement about the true concentration in human cells even more perplexing is the fact that Stamler’s methodologies and ours appear to agree fairly well on the SNOHb and HbNO levels in rat RBCs. So, is our method detecting too little NO, or is there indeed little there and Stamler’s method detecting something artificially produced? Or does human hemoglobin possess a subtle mechanism that allows it to assume both high and low NO levels without an immediate physiological consequence? Given the compelling evidence provided here for low levels of NO adducts across the arterio-venous oxygen gradient, and the high levels reported by McMahon et al. in support of the proposed physiological role and therapeutic applications related to such adducts in …
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