Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

Kirima, Kazuyoshi; Tsuchiya, Koichiro; Séi, Hiroyoshi; Hasegawa, Takehisa; Shikishima, Michiyo; Motobayashi, Yuki; Morita, Kyoji; Yoshizumi, Masanori; Takahashi, Toshiaki

doi:10.1152/ajpheart.01010.2002

Cited by 34 publications

(48 citation statements)

References 73 publications

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“…However, SNP was not detected by triiodide at concentrations up to 10 M (Fig. 1 A), levels that exceed the FeNO concentrations detected in vivo (Ϸ1-5 M) (35,51,54,55). In the triiodide assay, 1 mM SNP yielded a signal equivalent to Ϸ10 nM GSNO standard, corresponding to a recovery of 0.001%.…”

Section: Resultsmentioning

confidence: 85%

Assessment of nitric oxide signals by triiodide chemiluminescence

Hausladen

Rafikov

Angelo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

101

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Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe-and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures. red blood cell vasodilation ͉ S-nitrosohemoglobin ͉ S-nitrosylationT he biological effects of nitric oxide (NO) are mediated in large part through binding to transition metals and cysteine thiols at active or allosteric sites within regulatory proteins (1), which elicits changes in protein activity, protein-protein interactions, and protein location (1). Within tissues, many dozens of S-nitrosylated proteins have been identified, and signatures of NO bound to nonheme and heme iron have been detected (1, 2). Additionally, NO can be transported in endocrine or paracrine fashion by reacting with heme iron and cysteine thiols in proteins [hemoglobin (Hb) and albumin] and peptides (glutathione and cysteinlyglycine) to form NO adducts with longer biological lifetimes (3-5); release of NO bioactivity from stable adducts is effected by allosteric and redox-based mechanisms that alter FeNO or S-nitrosothiol (SNO) reactivity (5, 6). An updated discussion of the factors influencing reactivity of S-nitrosohemoglobin, S-nitrosoalbumin, and low-molecular-weight SNO in the context of vasoregulation (5-15) can be found in supporting information (SI) Text.The dynamic distribution of protein and low-molecular-weight NO compounds that subserve NO transport and signaling instantiate the variation in both FeNO and SNO reactivities (4,6,13...

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Section: Resultsmentioning

confidence: 85%

Assessment of nitric oxide signals by triiodide chemiluminescence

Hausladen

Rafikov

Angelo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

101

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“…To determine whether the EPR signal of HbNO is derived from L-arginine-dependent NO or not, we infused L-and D-arginine into rats via the femoral vein and measured the EPR signal of HbNO using the EPR-subtraction method. In the case of L-arginine, the HbNO concentration was increased with the increment of administered L-arginine, whereas D-arginine did not modify the HbNO (63). These results imply that the EPR signal for HbNO reflects the endogenous changes in NO dynamics.…”

Section: Epr Measurement and Data Processingmentioning

confidence: 76%

New Methods to Evaluate Endothelial Function: Evaluation of Endothelial Function by Hemoglobin-Nitric Oxide Complex Using Electron Paramagnetic Resonance Spectroscopy

et al. 2003

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Abstract. This minireview describes the practical use of assay systems to detect nitric oxide (NO) by electron paramagnetic resonance (EPR) spectroscopy for evaluation of endothelial functions. The iron(II)-dithiocarbamate complexes, such as iron(II)-(N-methyl-D-glucamine dithiocarbamate), are commonly used in EPR detection of NO both in vivo and in vitro. However, due to their redox activity, these complexes have some drawbacks that limit their usefulness for the detection of NO. On the other hand, the measurement of hemoglobin-NO adduct (HbNO) in whole blood by the EPR method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and an unknown radical species overlapping the same magnetic field as that of HbNO, which makes it physically impossible to measure small amounts of HbNO. Thus, to reveal the EPR spectrum of HbNO, we developed the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of the sample blood using software. Using this technique, we succeeded in measuring the steady blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril reduces abnormalities of NO dynamics in the L-NAME (N w -nitro-L-arginine-methylester)-induced endothelial dysfunction of rats using the EPR HbNO signal subtraction method.

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“…EPR spectroscopy can detect the HbNO because HbNO is a paramagnetic species, although there are still some difficulties in obtaining fine HbNO signals because of the existence of paramagnetic compounds other than HbNO, such as ceruloplasmin, other heme proteins, and molybdenum enzymes, which give a strong EPR signal overlapping the same region of HbNO (32 -37). Therefore, we developed an improved method of detecting the HbNO signal in whole blood by EPR spectroscopy (EPR subtraction method) (27). In addition, we adopted a stable isotope of nitrite ( …”

Section: Hbno Measurement By Epr Spectroscopymentioning

confidence: 99%

“…Typical EPR conditions were as follows: power, 20 mW; frequency, 9.045 GHz; magnetic field, 3200 ± 250 gauss; modulation width, 6.3 gauss; sweep time, 15 min; and time constant 0.3 s. Spectra ware processed using software ESPRIT 432 (JEOL Co.). The EPR signal subtraction was accomplished as reported previously (27). …”

Section: Sample Preparation and Epr Measurementsmentioning

confidence: 99%

“…Therefore, we investigated 1) whether the nitrite-derived NO distributes to the circulation, and 2) whether nitrite-derived NO acts like NOS-derived NO does in vivo. We employed a stable isotope of nitrite ( 15 NO 2 -) in order to distinguish between endogenous nitrite and the exogenously administered one and measured nitrosyl hemoglobin (HbNO) as an index of circulating NO in whole blood using electron paramagnetic resonance spectroscopy (27).…”

Section: Introductionmentioning

confidence: 99%

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Malfunction of Vascular Control in Lifestyle-Related Diseases: Formation of Systemic Hemoglobin-Nitric Oxide Complex (HbNO) From Dietary Nitrite

et al. 2004

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Abstract. Nitric oxide (NO) has many physiological functions. It is believed to be produced from L-arginine by nitric oxide synthase (NOS), and nitrite and nitrate are waste forms of it. By the way, nitrate and nitrite are abundant in vegetables and fruits, especially leafy vegetables and pickled vegetables. Orally-ingested nitrate is changed to nitrite by micro-organelles living in the hypopharynx area, and nitrite is expected to change to NO in the stomach due to its low pH. Indeed, some researchers reported that NO is produced in the gastric cavity, although few reports mentioned the physiological meanings of this NO formation. Therefore, we investigated whether the nitrite-derived NO can shift to the circulation and acts like NOS-derived NO does in tissues. We adopted a stable isotope of nitrite ( 15 NO 2 -) in order to distinguish between the endogenous nitrite and the exogenously administered one and measured nitrosyl hemoglobin (HbNO) as an index of circulating NO using electron paramagnetic resonance spectroscopy. It appeared that the oral administration of 15 N-nitrite formed the Hb 15 NO in rat blood and decreased the blood pressure of chronic L-NAME treated rats. Our findings suggest that the intake of nitrite (or nitrate)-rich foods such as vegetables and fruits would alter the systemic HbNO dynamism, resulting in the improvement of cardiovascular diseases.

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Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

Cited by 34 publications

References 73 publications

Assessment of nitric oxide signals by triiodide chemiluminescence

Assessment of nitric oxide signals by triiodide chemiluminescence

New Methods to Evaluate Endothelial Function: Evaluation of Endothelial Function by Hemoglobin-Nitric Oxide Complex Using Electron Paramagnetic Resonance Spectroscopy

Malfunction of Vascular Control in Lifestyle-Related Diseases: Formation of Systemic Hemoglobin-Nitric Oxide Complex (HbNO) From Dietary Nitrite

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