Skip Navigation

Cardiovascular Research 2007 75(2):434-441; doi:10.1016/j.cardiores.2007.04.019
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Rogers, S. C.
Right arrow Articles by James, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rogers, S. C.
Right arrow Articles by James, P. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

NO metabolite flux across the human coronary circulation

Stephen C. Rogersa,1, Afshin Khalatbaria,1, Borunendra N. Dattab, Sue Elleryc, Vince Paulc, Michael P. Frenneauxd and Philip E. Jamesa,*

aDepartment of Cardiology, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff, CF14 4XN, United Kingdom
bDepartment of Medical Biochemistry, School of Medicine, Cardiff University, Cardiff, CF14 4XN, United Kingdom
cDepartment of Cardiology, Ashford and St. Peters NHS Trust, Surrey, KT16 0PZ, United Kingdom
dDepartment of Cardiovascular Medicine, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom

* Corresponding author. Tel.: +44 29 20743512; fax: +44 29 20743500. jamespp{at}cardiff.ac.uk

Received 27 August 2006; revised 16 April 2007; accepted 18 April 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objectives The theory of a red blood cell derived nitric oxide (NO) reserve conserving NO bioactivity and delivering NO as a function of oxygen demand has been the subject of much interest. We identified the human coronary circulation as an ideal model system in which to analyse NO metabolites because of its large physiological oxygen gradient. Our objective was to identify whether oxygen drove apportion between various NO metabolite species across a single vascular bed.

Methods Plasma and red blood cell NO metabolites were assessed from the left main coronary artery, coronary sinus and pulmonary artery (providing cross heart and cross pulmonary analysis) of healthy subjects under resting conditions and following administration of an inhibitor of NO biosynthesis. Physiological parameters and angiographic data were monitored throughout the study.

Results Under baseline conditions we observed significant metabolite flux upon the transit of blood across the coronary and pulmonary vascular beds. Whilst there was no net loss of NO through the coronary circulation (p=0.0759), plasma nitrite/protein NO (excluding nitrate) (p=0.0279) and red blood cell sulphanilamide labile signal (p=0.0143) decreased whereas haemoglobin-bound NO increased three-fold (p=0.005). These changes across the coronary circulation were reversed through the pulmonary circuit with red blood cell sulphanilamide labile signal (p=0.0143) and plasma nitrite/protein NO (p=0.0279) increasing and haemoglobin-bound NO decreasing. Blockade of NO synthesis increased mean arterial blood pressure (p<0.01) and reduced coronary artery diameter (p<0.05), however we observed similar apportion of NO metabolites across the heart and lung with no net loss or gain in total NO metabolites.

Conclusions For the first time in human subjects across the resting coronary circulation we reveal significant re-apportionment of NO between metabolite species which correlate with haemoglobin oxygen saturation. These changes occur even within the transit time of blood across this single vascular bed. We demonstrate no net loss/gain of NO from the total metabolite pool across the coronary circulation even where NO biosynthesis is inhibited.

KEYWORDS Nitric Oxide; Metabolites; Coronary; Pulmonary; Flux


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The theory of a blood derived nitric oxide (NO) reserve of circulating metabolites conserving NO bioactivity has been the subject of much interest. Presently, it is generally agreed that NO metabolites can regulate vascular tone under conditions of low oxygen to mediate hypoxic vasodilatation. Debate in this field has now turned to the exact identity of the major bioactive species and to its importance in relation to other endogenous dilatory factors in the human circulation.

In terms of the total NO metabolite pool (NO+NOx), our interest lies in only a small fraction of metabolite species thought to constitute a physiologically active NO reservoir. Arguments presented in support of and against these species playing a physiological role in hypoxic vasodilatation have focussed on the relative levels measured in human blood samples and the demonstration (or not) of significant arterial–venous (A–V) metabolite gradients [1–6]. We recognise that actual metabolite levels are not the primary concern [4,7] given that only a very small amount of NO related moiety is required to induce vasodilatation via activation of soluble guanylyl cyclase (~20 nM) [8]. A–V gradients in NO metabolites also tell us little about the physiological relevance of individual metabolite species [9] since utilisation could be masked by replenishment from endothelial and other sources. However they do provide an insight into the overall metabolite equilibrium, the compartmentalisation of NO in terms of plasma and red blood cells, and importantly show net NO gain or loss across a given tissue bed when all the major metabolite species are accounted for. Under conditions where NO biosynthesis is inhibited (such as with L-NMMA) A–V NO metabolite gradients directly reflect gain or loss from the blood NO metabolite pool.

The red blood cell derived NO reserve is proposed to regulate vascular tone only at very low tissue oxygen levels. The original metabolite proposed to undertake this function was the S-nitrosylated protein, S-nitrosohaemoglobin (SNO-Hb) [9–16]. More recently, much interest has focussed on the well established direct vasodilator nitrite, following the suggestion that free NO is produced upon nitrite reduction by deoxyhaemoglobin within erythrocytes [1,3,17–24]. Maximal physiological dilatation via this mechanism is proposed to occur at around 50% haemoglobin oxygen saturation (HbO2 sat (%)). Given that normal mixed venous blood has a HbO2 sat (%) of ~70%, the contribution of either process to normal arterial (and possibly venous) dilatation would therefore be minimal. However, the NO reserve may accrue greater importance where HbO2 sat (%) is lower, such as the pre-capillary arteriole or post-capillary venule, or where oxygen delivery is reduced, such as under pathological conditions or during exercise. A second issue that has thwarted the hypothesis of a plasma derived NO metabolite species (such as nitrite) functioning to regulate vascular tone is the fact that such an NO moiety would need to transit the red blood cell membrane, interact with haemoglobin within to produce NO (or a derivative thereof) and escape the red blood cell to affect arterial tone. Calculations based on diffusional and time limitations have suggested this is extremely unlikely over the course of a single A to V transit [25].

Investigations performed to date in humans have never looked across the coronary circulation, which has a much larger A–V oxygen gradient than most other vascular beds. Even at rest (and during exercise) ~70% of arterial oxygen is constantly extracted by the heart. Consequently the haemoglobin oxygen saturation of coronary venous effluent is consistently ~30%. We chose to analyse NO metabolites simultaneously across the coronary (left main coronary artery to coronary sinus) and pulmonary (pulmonary artery to left main coronary artery) vascular beds. At rest in healthy human subjects we found no net loss/gain of NO metabolites across either vascular bed. However, we discovered significant re-apportionment of NO between metabolite species, with haemoglobin-bound NO being inversely correlated with HbO2 sat (%), and red blood cell sulphanilamide-labile signal directly proportional to HbO2 sat (%). Blockade of endothelial derived NO had little effect on these changes. These findings are consistent with the ability of NO metabolites to re-apportion between blood compartments as a function of tissue oxygen level across a single vascular bed, and a role for the pulmonary circulation in normalising the NO metabolite distribution and NO flux.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Clinical study
2.1.1 Subjects
Eight otherwise healthy subjects (4 men and 4 women, mean age 49±15 years) undergoing electrophysiology (EP) testing for paroxysmal supraventricular arrhythmias were studied. All subjects were in sinus rhythm with no past history of ischemic heart disease, smoking, diabetes mellitus, hypertension (blood pressure >140/90 mm Hg) or hypercholesterolemia (total cholesterol >5.2 mmol/L). All gave fully informed written consent approved by the relevant local research ethics committee. The investigation conformed with the principles outlined in the Declaration of Helsinki.

2.1.2 Protocol
Prior to catheterisation subjects underwent an 8 hour fast during which time they were allowed water. Diagnostic left heart catheterisation was performed via per cutaneous right femoral approach. A left heart catheter was positioned in the left main coronary artery and two further catheters were introduced via the right femoral vein and positioned in the coronary sinus and pulmonary artery. The position of the sampling catheters was confirmed with contrast injection. After baseline haemodynamic parameters had been maintained for 5 min, blood samples were obtained from the left main coronary artery (LMCA), the coronary sinus (CS) and the pulmonary artery (PA). LMCA-CS difference was taken to reflect "cross heart" whereas PA-LMCA was taken to reflect "cross pulmonary". We acknowledge that strictly speaking "cross pulmonary" should be accurately measured as PA to pulmonary vein (PV) however it is practically difficult to obtain such samples from healthy human subjects. An infusion of L-NMMA (Clinalfa; loading dose 5 mg/kg) was then introduced for 7 min after which a maintenance dose of 50 µg/kg/min was continued. Blood samples were then taken from the same three anatomical sites (LMCA, CS and PA). Quantitative coronary angiography was undertaken to compare the LAD diameter before and after L-NMMA infusion. (Philips QCA software).

2.1.3 Biological sample collection
Blood samples were collected in 10 ml syringes and transferred after blood gas analysis (OSM3 Hemoximeter, Radiometer) into 6 ml gas tight EDTA vacutainers. These were centrifuged at 600 g for 10 min at room temperature. The red cell fraction and plasma were immediately separated, snap frozen in liquid nitrogen and stored at –80 °C for subsequent analysis. Samples were frozen because during such a time-constrained clinical protocol the instant measurement would have resulted in an inevitable delay in assessing some of the samples, confounding these measurements. Additionally, although others have developed techniques to potentially inhibit in vitro chemistry occurring during sample processing [26] we prefer to adhere to a well validated, strictly timed sample preparation protocol involving a centrifugation followed by separation of plasma/red blood cells and immediate snap freezing. During this time the samples are maintained at the given oxygen saturation in sealed vacutainers. This protocol minimises potential confounding factors including nitrite contamination, chemical modification of the sample and sample processing time.

2.2 Biochemistry
2.2.1 Chemicals and reagents
All chemicals were purchased from Sigma other than glacial acetic acid, HPLC grade nitrite free water and hydrochloric acid from Fisher Scientific. A stock solution of tri-iodide reagent was prepared fresh each day [26]. The tri-iodide reagent, probably the most widely used in the NO metabolite field, has been validated against standards in several laboratories [4,26–29]. In our hands (and other groups) we achieve a near 100% recovery of NO added across a physiological and pharmacological range. The reader is referred to a complete discussion of the relative merits and pitfalls of the assay [3,7,9,30–33]. During the revision of this manuscript, an investigation into the performance of the original tri-iodide assay versus photolysis chemiluminescence has been performed [31]. Hausladen et al. [31] question the ability of the original tri-iodide reagent to measure certain haem NO complexes (such as the hybrid Hb(II)NO+/Hb(III)NO [3,34]) along with the efficacy of various sample pre-treatment protocols (specifically sulphanilamide in acid). In this work we have used a modification of the tri-iodide assay [35] which has been demonstrated to improve the recovery of haem NO species [33] and which we rationalise measures all haemoglobin NO hybrids (as shown by Nagababu et al. [3]). This modified reagent also inhibits the autocapture of NO by cell free haem in the reaction chamber, which is identified as another confounding issue in the original tri-iodide reagent when used to measure red blood cell/haemoglobin samples within the physiological haem:NO range [31,35,36]. It is also stated that Hb(II)NO+/Hb(III)NO species measured by photolysis chemiluminescence are stable to sulphanilamide/acid treatment in the short term (although this data is not shown) [31]. It is likely therefore that the conditions we employed are capable of detecting Hb(II)NO+/Hb(III)NO and HbSNO/Hb(II)NO species.

2.2.2 Ozone-based chemiluminescence
Carried out as described in detail previously [35]. For plasma measurements the original tri-iodide reagent was used, however for red blood cell measurements the modified reagent was used with 7.2 ml of tri-iodide along with 800 µl of potassium ferricyanide (25 mmol/L final).

2.2.3 Sample thawing
Immediately prior to analysis all frozen samples (red blood cell or plasma) were thawed in a water bath at 37 °C for 3 min. Sample freezing was found to have no effect on red blood cell or plasma NO metabolite stability. Samples from 5 healthy subjects were collected as above (see Biological sample collection); one sample was measured immediately (fresh) and the rest were frozen in 300 µl aliquots for future analysis (<7 days) having been stored at –80 °C. All study samples were analysed within 7 days from collection (Fig. 1).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effect of sample freezing and thawing on red blood cell NO, red blood cell haemoglobin-bound NO, plasma nitrite/protein NO and plasma sulphanilamide stable signal (n=5). Plasma samples were measured in the original tri-iodide reagent [26] and red blood cell samples in the modified tri-iodide reagent [35].

 
2.2.4 Red blood cell-contained NO
Once thawed red blood cells were lysed 1:4 in HPLC nitrite free water and injected immediately. The signal from 160 µl water was subtracted from the signal from 200 µl red blood cell lysate to give total red blood cell-contained NO.

2.2.5 Haemoglobin-bound NO
A duplicate red blood cell sample was lysed 1:4 in water to which a 10% volume of 5% acidified sulphanilamide (290 mM stock) had already been added (to remove water nitrite contamination) [26,37]. The sample was incubated for 15 min in the dark and then injected to provide a measure of haemoglobin-bound NO (including all sulphanilamide stable species).

2.2.6 Red blood cell sulphanilamide-labile signal
The difference in signals from total red blood cell NO and haemoglobin-bound NO was defined as red blood cell sulphanilamide labile signal. To assess whether the sulphanilamide-displaced signal was entirely low mass and non-protein related (for example nitrite), red blood cell fractions from five arterial blood samples were assessed for (1) total red blood cell signal, (2) sulphanilamide labile signal (total minus sulphanilamide stable signal), and (3) red blood cell signal minus protein (ethanol precipitation was employed rather than micro-filtration because of the large nitrite contamination observed from filters). As ethanol also produces a small signal itself in tri-iodide this was subtracted from the ethanol supernatant signal following the precipitation of protein. Via this method we found a significant component (50–60%) of the signal removed by sulphanilamide was associated with protein (Fig. 2). This suggests either that acidified sulphanilamide pre-treatment can remove protein bound NO species in addition to nitrite or that a significant amount of nitrite is closely associated with protein. Consequently, the signal removed by sulphanilamide cannot unequivocally be termed nitrite. We therefore use the term sulphanilamide labile signal for this component of red blood cell NO. It is important to point out that our total red blood cell NO measurement and total plasma NO measurements and the changes in these totals we report "cross heart" and "cross pulmonary" are not affected by these arguments.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Origin of the red blood cell signal removed by acidified sulphanilamide. Arterial red blood cell fractions were assessed for total red blood cell signal; sulphanilamide labile signal (total minus sulphanilamide stable signal); and red blood cell signal minus protein (ethanol precipitation) (n=5). All samples were measured in the modified tri-iodide reagent [35]. ** p<0.01.

 
2.2.7 Plasma nitrite/protein NO
Plasma nitrite/protein NO was measured as described previously [26].

2.2.8 Plasma nitrate
Plasma NOx (nitrite plus nitrate) was measured using a fluorometric assay as described previously [38].

2.2.9 Data presentation and statistics
All chemiluminescence signals were smoothed using Origin 7.0 and the area under curve was analysed using Origin peak analysis. Data are presented as means±SEM. Differences between means (i.e., ±L-NMMA) were compared by Students t test. For multiple comparisons (i.e., LMCA, CS and PA) a repeated measures one way ANOVA (Newman–Keuls post hoc test) was performed. A bivariate correlation (Spearman correlation coefficient) assessed the relationship between haemoglobin-bound NO, plasma nitrite, red blood cell sulphanilamide-labile signal and HbO2 sat (%). A two tailed p value <0.05 was considered significant throughout.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
HbO2 sat (%) and blood oxygen content (ml/dl) decreased significantly across the heart (p<0.0001), increased significantly when CS effluent mixed with systemic venous return in the PA (p<0.0001) and increased further across the lungs (p<0.0001) (Fig. 3).


Figure 3
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Baseline blood oxygenation and nitric oxide metabolite measurements in the left main coronary artery (LMCA), coronary sinus (CS) and pulmonary artery (PA). LMCA-CS represents "cross heart" and PA to LMCA "cross pulmonary". Individual data points for each patient are plotted with the mean value (n=8 for all parameters excluding red blood cell haemoglobin-bound NO and red blood cell sulphanilamide-labile signal where n=7; as a result of sample spoilage). * p<0.05. ** p<0.01.

 
Significant changes in NO metabolite levels were observed across the coronary and pulmonary circulations. However, from the total metabolite pool (plasma nitrate, nitrite, protein-bound NO, red blood cell sulphanilamide-labile signal and haemoglobin-bound NO) there was no significant net loss or gain of NO across either the heart or the lung (p=0.0759).

Excluding plasma nitrate, combined plasma and red blood cell NO remained unchanged across the coronary and pulmonary (p=0.2988) circuits. Across the heart total red blood cell NO increased (p=0.0223), mainly reflecting a significant increase in haemoglobin-bound NO (p=0.0005), whilst plasma nitrite/protein NO (p=0.0279) and red blood cell sulphanilamide-labile signal (p=0.0143) decreased. These effects were largely reversed on mixing of systemic venous return with CS blood (to give rise to PA blood) and completely reversed across the lungs where red blood cell sulphanilamide-labile signal (p=0.0143) and plasma nitrite/protein NO (p=0.0279) increased and haemoglobin-bound NO decreased (Fig. 3). A direct correlation was observed between red blood cell sulphanilamide-labile signal and HbO2 sat (%) (p=0.0123; r=0.3743) and an inverse correlation between haemoglobin-bound NO and HbO2 sat (%) (p< 0.0001 r=–0.5985) across all sites sampled (Fig. 4).


Figure 4
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Correlation between haemoglobin-bound NO, red blood cell sulphanilamide labile signal and haemoglobin oxygen saturation (%). r=–0.5985 (p<0.0001) for haemoglobin-bound NO; r=0.3743 (p=0.0123) for red blood cell sulphanilamide labile signal. Individual data points for each patient are plotted including baseline and L-NMMA data.

 
Infusion of L-NMMA resulted in significant increases in systolic and diastolic blood pressure and a decrease in coronary artery diameter (Table 1). Apportion of NO metabolites between blood compartments in samples taken during L-NMMA infusion was similar to that observed in baseline samples (Table 2). The "cross heart" and "cross pulmonary" trends observed at baseline were essentially unaffected during infusion of L-NMMA (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 1 Physiological data for subjects comparing baseline to L-NMMA infusion

 

View this table:
[in this window]
[in a new window]

  		Table 2 Plasma nitrite/protein NO, red blood cell haemoglobin-bound NO and red blood cell sulphanilamide-labile signal pre and post L-NMMA infusion at each sample site (n=8 for all parameters excluding red blood cell haemoglobin-bound NO and red blood cell sulphanilamide-labile signal where n=7; as a result of sample spoilage)

 

View this table:
[in this window]
[in a new window]

  		Table 3 Change in NO ({Delta} NO) for each metabolite "cross heart" (LMCA-CS) and "cross pulmonary" (PA-LMCA) comparing subjects at baseline and with L-NMMA infusion

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We demonstrate in healthy human subjects under baseline resting conditions significant re-apportion of NO metabolite species across both the heart and lung. However, no net loss or gain of NO from the total metabolite pool was observed through either of these vascular circuits. Across the coronary circulation this equilibrium in the NO metabolite pool could be interpreted in one of two ways. In the absence of L-NMMA extraction of NO by cardiac tissue could be balanced by replenishment from endothelial sources giving the overall appearance of no net flux from the circulation into the tissue, but with sufficient NO delivery to contribute to the regulation of coronary blood flow. However following the blockade of endothelial NOS, which eliminates the potential of replenishing the NO metabolite pool, the lack of change suggests no utilisation/consumption of metabolite NO. This latter point is contentious given that even very small changes in NO metabolite levels could provide sufficient NO to dilate the coronary circulation. In this work NOS inhibition was confirmed with the significant increase in blood pressure and decrease in coronary artery diameter, which is consistent with endothelial NO contributing to basal coronary tone.

Oxygen-dependant increases in haemoglobin-bound NO were matched by decreases in plasma nitrite and protein NO and also red blood cell sulphanilamide-labile signal across the heart. This could suggest NO transfer between species resulting from nitrite reduction to NO by deoxyhaemoglobin or nitrite forming haemoglobin bound NO species (such as HbFe(III)NO or HbFe(II)NO+ as described by Nagababu [3,34]). Whilst some groups have reported nitrite gradients in the systemic circulation [1,4,23], others have observed no gradient [39]. Where nitrite gradients have previously been observed they have been taken to signify A–V nitrite consumption eliciting vasodilatation [1,3,4,23], however it has been reported that NO formed from this reaction mechanism cannot leave the red blood cell [40]. For this reaction mechanism to contribute to the regulation of vascular tone secondary NO species capable of traversing the red blood cell membrane must be formed. Nitrite gradients across the circulation have been proposed by others to more likely reflect sites of nitrite production (artery > vein; NOS activity) and consumption (vein > artery; nitrite reductase activity) [25]. Important to note that in our studies similar results were observed in the absence and presence of endothelial NO (±L-NMMA) implying production of NO contributes little in the acute setting to NO metabolite levels or the observed "cross heart" changes. Taken together with our observation of no net loss or gain of total NO metabolites (NO+NOx), the inverse relationship between haemoglobin-bound NO and plasma nitrite/protein NO in conjunction with red blood cell sulphanilamide-labile signal implies either direct exchange between these NO species, or consumption of nitrite-derived NO being replenished from another source (apart from the endothelium) resulting in haemoglobin-NO formation in venous blood.

In previous NO metabolite studies the total red blood cell NO metabolite pool has been demonstrated to remain constant throughout the systemic human circulation [10,12,13]. Only the relative levels of the individual red blood cell metabolite species have been shown to change as a function of oxygen tension (or more precisely HbO2 sat (%)) [12]. In agreement with these findings our measurements across the pulmonary circulation demonstrate no change in total red blood cell NO. However, in this first study to measure NO metabolites across the substantial oxygen gradient of the coronary circulation we observed a significant increase in total red blood cell-contained NO, mainly reflecting an increase in haemoglobin-bound NO. We cannot conclude as to the relative increases in the individual haemoglobin-bound NO metabolite species (HbNO or SNO-Hb) as these were not differentiated. However, we have previously measured "cross pulmonary" gradients in haemoglobin-NO metabolites and demonstrated a clear dependence on HbO2 sat (%) [41]. It must be acknowledged that this earlier work was undertaken utilising a different NO measurement technique and sample pre-treatment protocol. Although the NO metabolite levels do not agree with values quoted herein (which are in fact in very close agreement with recent values reported by other groups using similar methodologies, showing an NO:Hb ratio ~0.00012 [4,23,42,43]), previously observed trends in species, correlation with oxygen, and lack of net NO loss or gain are confirmed [41].

The pattern of NO metabolite interplay observed across the coronary circulation was reversed across the pulmonary circulation, implying a role for the lungs in normalising the NO metabolite equilibrium and adjusting for any systemic change in the balance of metabolite species. As the lungs function to re-oxygenate blood, accounting for oxygen consumption throughout the vascular circuit, the same appears true for NO metabolites with the restoration of the NO metabolite equilibrium prior to the re-transit of blood around the vasculature. In this study we have not quantitatively compared NO uptake or interchange between the coronary and pulmonary vascular beds due to the complications associated with different blood flow through these circuits and the fact that blood was not sampled from the PV.

This work in healthy human subjects with intact endothelium has not addressed the case where endothelium is damaged or dysfunctional. We speculate that under such conditions NO metabolites may play a more significant role in the regulation of vascular tone. We have demonstrated that apportionment of NO metabolites in human blood is maintained remarkably stable across the circulation, in part due to the buffering effect of haemoglobin. Nevertheless this does not preclude the fact that the therapeutic enhancement of specific NO metabolites may have selective beneficial effects on maintenance of vessel patency. We also observed significant re-apportion of NO between metabolites as a function of the substantial oxygen gradient across the heart (and lung). This dynamic interplay between NO species throughout the vascular circuit provides in vivo evidence that a related NO moiety could in principle be transferred between blood compartments as a function of oxygen and contribute to local vasodilatation within the time constraints of blood transit across a single vascular bed.

Time for primary review 23 days


   Acknowledgements
 
We would like to thank the British Heart Foundation for its continued support. We also thank Joan Parton for technical expertise and the clinical staff at St Peters Hospital, Chertsey.


   Notes
 
1 SCR and AK are joint first authors. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Cosby K., Partovi K.S., Crawford J.H., Patel R.P., Reiter C.D., Martyr S., et al. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med (2003) 9:1498–1505.[CrossRef][Web of Science][Medline]
  2. Hunter C.J., Dejam A., Blood A.B., Shields H., Kim-Shapiro D.B., Machado R.F., et al. Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med (2004) 10:1122–1127.[CrossRef][Web of Science][Medline]
  3. Nagababu E., Ramasamy S., Abernethy D.R., Rifkind J.M. Active nitric oxde produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction. J Biol Chem (2003) 278:46349–46356.[Abstract/Free Full Text]
  4. Gladwin M.T., Shelhamer J.H., Schechter A.N., Pease-Fye M.E., Waclawiw M.A., Panza J.A., et al. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc Natl Acad Sci U S A (2000) 97:11482–11487.[Abstract/Free Full Text]
  5. Cicinelli E., Ignarro L.J., Schonauer L.M., Matteo M.G., Galantino P., Falco N. Different plasma levels of nitric oxide in arterial and venous blood. Clin Physiol (1999) 19:440–442.[CrossRef][Web of Science][Medline]
  6. Gladwin M.T., Crawford J.H., Patel R.P. The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radic Biol Med (2004) 36:707–717.[CrossRef][Web of Science][Medline]
  7. Stamler J.S. S-nitrosothiols in the blood: roles, amounts, and methods of analysis. Circ Res (2004) 94:414–417.[Free Full Text]
  8. Bellamy T.C., Wood J., Goodwin D.A., Garthwaite J. Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses. Proc Natl Acad Sci U S A (2000) 97:2928–2933.[Abstract/Free Full Text]
  9. Singel D.J., Stamler J.S. Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin. Annu Rev Physiol (2005) 67:99–145.[CrossRef][Web of Science][Medline]
  10. Jia L., Bonaventura C., Bonaventura J., Stamler J.S. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature (1996) 380:221–226.[CrossRef][Medline]
  11. McMahon T.J., Stamler J.S. Concerted nitric oxide/oxygen delivery by hemoglobin. Methods Enzymol (1999) 301:99–114.[Web of Science][Medline]
  12. McMahon T.J., Moon R.E., Luschinger B.P., Carraway M.S., Stone A.E., Stolp B.W., et al. Nitric oxide in the human respiratory cycle. Nat Med (2002) 8:711–717.[Web of Science][Medline]
  13. Stamler J.S., Jia L., Eu J.P., McMahon T.J., Demchenko I.T., Bonaventura J., et al. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science (1997) 276:2034–2037.[Abstract/Free Full Text]
  14. McMahon T.J., Exton Stone A., Bonaventura J., Singel D.J., Solomon Stamler J. Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin. J Biol Chem (2000) 275:16738–16745.[Abstract/Free Full Text]
  15. Stamler J.S., Simon D.I., Osborne J.A., Mullins M.E., Jaraki O., Michel T., et al. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A (1992) 89:444–448.[Abstract/Free Full Text]
  16. Gow A.J. Nitric oxide, hemoglobin, and hypoxic vasodilation. Am J Respir Cell Mol Biol (2005) 32:479–482.[Free Full Text]
  17. Reutov V.P., Sorokina E.G. NO-synthase and nitrite-reductase components of nitric oxide cycle. Biochemistry (Mosc) (1998) 63:874–884.[Medline]
  18. Doyle M.P., Pickering R.A., DeWeert T.M., Hoekstra J.W., Pater D. Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites. J Biol Chem (1981) 256:12393–12398.[Abstract/Free Full Text]
  19. Gladwin M.T., Schechter A.N. NO contest: nitrite versus S-nitroso-hemoglobin. Circ Res (2004) 94:851–855.[Free Full Text]
  20. Gladwin M.T. Hemoglobin as a nitrite reductase regulating red cell-dependent hypoxic vasodilation. Am J Respir Cell Mol Biol (2005) 32:363–366.[Free Full Text]
  21. Gladwin M.T., Schechter A.N., Kim-Shapiro D.B., Patel R.P., Hogg N., Shiva S., et al. The emerging biology of the nitrite anion. Nat Chem Biol (2005) 1:308–314.[CrossRef][Web of Science][Medline]
  22. Crawford J.H., Isbell T.S., Huang Z., Shiva S., Chacko B.K., Schechter A.N., et al. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood (2006) 107:566–574.[Abstract/Free Full Text]
  23. Dejam A., Hunter C.J., Pelletier M.M., Hsu L.L., Machado R.F., Shiva S., et al. Erythrocytes are the major intravascular storage sites of nitrite in human blood. Blood (2005) 106:734–739.[Abstract/Free Full Text]
  24. Huang K.T., Azarov I., Basu S., Huang J., Kim-Shapiro D.B. Lack of allosterically controlled intramolecular transfer of nitric oxide from the heme to cysteine in the beta subunit of hemoglobin. Blood (2006) 107:2602–2604.[Abstract/Free Full Text]
  25. Allen B.W., Piantadosi C.A. How do red cells cause hypoxic vasodilation? The SNO-hemoglobin paradigm. Am J Physiol Heart Circ Physiol (2006) 291(4):H1507–H1512.[Abstract/Free Full Text]
  26. Yang B.K., Vivas E.X., Reiter C.D., Gladwin M.T. Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res (2003) 37:1–10.[Web of Science][Medline]
  27. Feelisch M., Rassaf T., Mnaimneh S., Singh N., Bryan N.S., Jourd'Heuil D., et al. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J (2002) 16:1775–1785.[Abstract/Free Full Text]
  28. Rassaf T., Preik M., Kleinbongard P., Lauer T., Heiss C., Strauer B.E., et al. Evidence for in vivo transport of bioactive nitric oxide in human plasma. J Clin Invest (2002) 109:1241–1248.[CrossRef][Web of Science][Medline]
  29. Xu X., Cho M., Spencer N.Y., Patel N., Huang Z., Shields H., et al. Measurements of nitric oxide on the heme iron and beta-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation. Proc Natl Acad Sci U S A (2003) 100:11303–11308.[Abstract/Free Full Text]
  30. Rassaf T., Bryan N.S., Maloney R.E., Specian V., Kelm M., Kalyanaraman B., et al. NO adducts in mammalian red blood cells: too much or too little? Nat Med (2003) 9:481–482. [author reply 482–3].[CrossRef][Web of Science][Medline]
  31. Hausladen A., Rafikov R., Angelo M., Singel D.J., Nudler E., Stamler J.S. Assessment of nitric oxide signals by triiodide chemiluminescence. Proc Natl Acad Sci U S A (2007) 104:2157–2162.[Abstract/Free Full Text]
  32. Macarthur PH, Shiva S, Gladwin MT. Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence. J. Chromatogr B (in press). doi:10.1016/j.jchromb.2006.12.012.
  33. Wang X., Bryan N.S., Macarthur P.H., Rodriguez J., Gladwin M.T., Feelisch M. Measurement of NO levels in the red cell: validation of tri-iodide based chemiluminescence with acid-sulfanilamide pre-treatment. J Biol Chem (2006) 281(37):26994–27002.[Abstract/Free Full Text]
  34. Nagababu E., Ramasamy S., Rifkind J.M. S-Nitrosohemoglobin: a mechanism for its formation in conjunction with nitrite reduction by deoxyhemoglobin. Nitric Oxide (2006) 15:20–29.[CrossRef][Web of Science][Medline]
  35. Rogers S.C., Khalatbari A., Gapper P.W., Frenneaux M.P., James P.E. Detection of human red blood cell-bound nitric oxide. J Biol Chem (2005) 280:26720–26728.[Abstract/Free Full Text]
  36. Doctor A., Platt R., Sheram M.L., Eischeid A., McMahon T., Maxey T., et al. Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients. Proc Natl Acad Sci U S A (2005) 102:5709–5714.[Abstract/Free Full Text]
  37. Marley R., Feelisch M., Holt S., Moore K. A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols. Free Radic Res (2000) 32:1–9.[Web of Science][Medline]
  38. Misko T.P., Schilling R.J., Salvemini D., Moore W.M., Currie M.G. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem (1993) 214:11–16.[CrossRef][Web of Science][Medline]
  39. Lauer T., Preik M., Rassaf T., Strauer B.E., Deussen A., Feelisch M., et al. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A (2001) 98:12814–12819.[Abstract/Free Full Text]
  40. Jeffers A., Xu X., Huang K.T., Cho M., Hogg N., Patel R.P., et al. Hemoglobin mediated nitrite activation of soluble guanylyl cyclase. Comp Biochem Physiol A Mol Integr Physiol (2005) 142:130–135.[CrossRef][Medline]
  41. Datta B., Tufnell-Barrett T., Bleasdale R.A., Jones C.J., Beeton I., Paul V., et al. Red blood cell nitric oxide as an endocrine vasoregulator: a potential role in congestive heart failure. Circulation (2004) 109:1339–1342.[Abstract/Free Full Text]
  42. Gladwin M.T., Ognibene F.P., Pannell L.K., Nichols J.S., Pease-Fye M.E., Shelhamer J.H., et al. Relative role of heme nitrosylation and beta-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation. Proc Natl Acad Sci U S A (2000) 97:9943–9948.[Abstract/Free Full Text]
  43. Gladwin M.T., Wang X., Reiter C.D., Yang B.K., Vivas E.X., Bonaventura C., et al. S-Nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function. J Biol Chem (2002) 277:27818–27828.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. A. Vitturi, X. Teng, J. C. Toledo, S. Matalon, J. R. Lancaster Jr., and R. P. Patel
Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation
Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1398 - H1407.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
D. M. Bailey, K. A. Evans, P. E. James, J. McEneny, I. S. Young, L. Fall, M. Gutowski, E. Kewley, J. M. McCord, K. Moller, et al.
Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function
J. Physiol., January 1, 2009; 587(1): 73 - 85.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
N. Marozkina, B. Gaston, and A. Doctor
Transnitrosation Signals Oxyhemoglobin Desaturation
Circ. Res., August 29, 2008; 103(5): 441 - 443.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Rogers, S. C.
Right arrow Articles by James, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rogers, S. C.
Right arrow Articles by James, P. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?