A Breakthrough in Respiratory Physiology: Inhaled Nitric Oxide Transported as SNO-Hb

Key Points

• Inhaled nitric oxide (NO) increases circulating levels of SNO-Hb, a bioactive form of NO

• Inhaled NO also increases circulating levels of nitrite, another NO metabolite

• The lungs might act as a reservoir of SNO-Hb, releasing it into circulation as needed

The Breathing Diabetic Summary

Inhaled nitric oxide (NO) has many systemic impacts.  An overview of these effects can be found here and here.  However, it has remained unclear how inhaled NO exerts these effects.  In general, inhaled NO is believed to react and become inactive after reaching the lungs.  Thus, conventional thinking is that the systemic effects of NO are due to improved gas exchange in the lungs, which then has positive downstream impacts.

Interestingly, despite its widespread clinical use, there have been very few studies testing this hypothesis to truly discover how inhaled NO exerts its systemic effects.  This paper sought to fill that gap.

To do this, they recruited 15 healthy volunteers.  They had them inhale NO at concentrations of 40 ppm (the maximum produced in the paranasal sinuses is on the order of ~20 ppm, but typically much less).  They inhaled the added NO for 15 minutes.  Blood samples were collected before inhalation, at the end of the 15 minutes of inhalation, and then at 5, 15, and 30 minutes post-inhalation. 

A Breakthrough in Cardio-Respiratory Physiology

The results were striking.  They found that NO inhalation significantly increased circulating levels of SNO-Hb and nitrite.  This is important because SNO-Hb plays a significant role in whole-body oxygenation.  A 2015 PNAS study discovered that SNO-Hb “senses” areas of low oxygen, and then releases bioactive NO to increase blood flow and oxygenation.  This discovery led to breathing be considered as a three-gas system involving oxygen, carbon dioxide, and NO.  Thus, if inhaling NO increases SNO-Hb, it could be playing a critical role in whole-body oxygenation.  This gets even more intriguing (see next two sections), but first, let’s cover their nitrite finding.

They also observed increases in circulating nitrite.  This is important because, like SNO-Hb, nitrite can also release bioactive NO in regions of hypoxia. However, nitrite can do this independent of the hemoglobin, thus providing a “back-up mechanism” for increasing blood flow in regions of low oxygen.

The Lungs as a Reservoir of SNO-Hb

An interesting finding from this study was that nitrite levels were most significant at the 5-min post inhalation mark.  In contrast, SNO-Hb continued rising throughout the 30 minutes.  This led the authors to believe that the lungs might be acting as an SNO-Hb reservoir, releasing it "as needed." 

Why These Findings Matter

When we breathe through our nose, we carry NO into the lungs (although not at concentrations as high as those studied here).  Based on these findings, we can now be reasonably confident this NO enters the bloodstream and is carried as SNO-Hb and nitrite.  Thus, breathing through your nose might not just improve gas exchange in the lungs.  It might also help make sure oxygen gets delivered where it is needed most throughout the body. 

Additionally, their finding that SNO-Hb levels continued increasing after NO inhalation is intriguing.  It might support the idea that nose breathing provides a baseline level of NO that keeps SNO-Hb in its normal range.  Then, when excess NO is inhaled, the body stores that "just in case."  This is speculative, but interesting to contemplate.

Finally, this is one study, and it’s relatively new.  We’ll need more to confirm/deny that NO inhalation consistently increases SNO-Hb and nitrite across different populations.  In the meantime, let’s keep breathing through our noses.  It may just be the key to whole-body oxygenation.

Abstract

Rationale: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. 

Objectives: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response.

Findings: We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively).

Conclusions: Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature.

Journal Reference:

Tonelli AR, Aulak KS, Ahmed MK, Hausladen A, Abuhalimeh B, Casa CJ, Rogers SC, Timm D, Doctor A, Gaston B, Dweik RA. A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers. PLoS One. 2019 Aug 30;14(8):e0221777. doi: 10.1371/journal.pone.0221777. PMID: 31469867; PMCID: PMC6716644.

A Concise Review of Inhaled Nitric Oxide’s Systemic Impacts

Key Points

• The classical viewpoint that inhaled nitric oxide (NO) only has local effects cannot explain observations.

• For example, inhaled NO has many systemic effects, including the ability to selectively increase blood flow where it is needed most.

• SNO-Hb might be the likely candidate for how inhaled NO is transferred into the blood and transported throughout the body while retaining its bioactivity.

The Breathing Diabetic Summary

This paper presented a concise review of inhaled NO’s systemic effects.  So, I’ll keep the summary brief as well. 

The classical view that inhaled NO only has local effects in the airways and lungs is not supported by observations.  It turns out that inhaled NO has many systemic effects.  Notably, inhaled NO selectively increases blood flow where it is needed most.  Thus, our bodies have a way of using inhaled NO other than just in the airways and lungs.  It can also be transported to distant regions where blood flow is restricted, resulting in vasodilation and increased blood flow.  This was also shown in the Cannon et al. (2001) study.   

Here, as in that study and others, the precise mechanism for how this is done is unknown.  However, there is one pathway that has been brought up repeatedly, which is SNO-Hb.  As we learned in a 2015 PNAS study, SNO-Hb is critical to blood flow regulation and oxygen delivery.  It “senses” regions of hypoxia, releases bioactive NO, and improves blood flow to get more oxygen to the tissues.

The authors suspect that this is also the mechanism by which inhaled NO is selectively improving blood flow, stating that this pathway “likely represents an important mechanism by which inhaled NO can cause systemic effects.”  The difficulty is that SNO-Hb is hard to measure; therefore, there have been no conclusive studies to show that this is the mechanism by which inhaled NO works. 

Altogether, this paper shows that the traditional view of inhaled NO is not adequate to explain its systemic effects.  It’s selective vasodilating effects suggest that SNO-Hb is the mechanism by which inhaled NO is transported throughout the body.  Still, more studies are needed to support this hypothesis.

Abstract

Many effects of inhaled nitric oxide (NO) are not explained by the convention that NO activates pulmonary guanylate cyclase or is inactivated by ferrous deoxy- or oxyheme. Inhaled NO can affect blood flow to a variety of systemic vascular beds, particularly under conditions of ischemia/reperfusion. It affects leukocyte adhesion and rolling in the systemic periphery. Inhaled NO therapy can overcome the systemic effects of NO synthase inhibition. In many cases, these systemic-NO synthase-mimetic effects of inhaled NO seem to involve reactions of NO with circulating proteins followed by transport of NO equivalents from the lung to the systemic periphery. The NO transfer biology associated with inhaled NO therapy is rich with therapeutic possibilities. In this article, many of the whole-animal studies regarding the systemic effects of inhaled NO are reviewed in the context of this emerging understanding of the complexities of NO biochemistry.

Journal Reference:

Gaston B. Summary: systemic effects of inhaled nitric oxide. Proc Am Thorac Soc. 2006 Apr;3(2):170-2. doi: 10.1513/pats.200506-049BG. PMID: 16565427.

Evidence of Systemic Transport and Delivery of Inhaled Nitric Oxide

Key Points

• Inhaled nitric oxide (NO) is traditionally thought to only have local effects in the upper airways and lungs.

• However, this study found that inhaled NO can improve blood flow in distant regions when endothelial NO is suppressed. The measurements were consistent with systemic transport and delivery of inhaled NO.

• The effects of inhaled NO on systemic blood flow might be important in diseases that disrupt endothelial-derived NO (such as diabetes).

The Breathing Diabetic Summary

There are two primary sources of nitric oxide (NO) in the body: inhaled NO and endothelial-derived NO.

Inhaled NO is produced in the paranasal sinuses.  When you breathe through your nose, you bring this NO into your lungs, where it aids in blood flow redistribution and increases oxygen uptake.  However, it is traditionally thought that this NO only affects the airways and lungs; it is said to immediately react and lose its bioactivity.  Although there are many benefits of inhaled NO in the lungs, its journey ends there. 

Endothelial-derived NO, on the other hand, has systemic effects in the body, including improving whole-body blood flow and, especially, blood flow to the heart.  However, it is thought that there is a complete disconnect between these two sources of NO: Inhaled NO does not have systemic effects. 

But several studies suggest otherwise (see review here).  The reported systemic effects of inhaled NO imply it is somehow retaining its bioactivity and being transported throughout the body.  But, it’s now quite sure how. 

This study did something interesting to try to find out. They administered L-NMMA, which inhibits endothelial-derived NO from being produced. Then, they measured what happened to forearm blood flow under several conditions:

1. When participants breathed normal air.

2. During a handgrip exercise (which should increase blood flow).

3. During inhalation of extra NO (at 80 ppm) and repeat the two measurements (sitting still and the handgrip exercise). Note that 80 ppm is much higher than what is produced in the paranasal sinuses, which maxes out around 25 ppm.

4. Lastly, they had participants inhale the added NO without using L-NMMA, which, as we will see, turns out to be a critical measurement.

The results were quite fascinating.  First, when NO was inhaled without the L-NMMA administered, nothing happened to forearm blood flow.  Therefore, under normal conditions, inhaling extra NO doesn’t seem to impact blood flow.  But things got interesting when L-NMMA was administered.  Inhaling NO counteracted the blood flow reduction due to L-NMMA.

Thus, under normal conditions, inhaled NO doesn’t have much impact on systemic blood flow.  But, when endothelial-derived NO is suppressed (the L-NMMA case), the inhaled NO “takes over,” compensating for the missing NO.  This opens up the blood vessels and increases blood flow.  This effect was most marked during the handgrip exercise.

Moreover, by looking at arterial-to-venous gradients in different gases, which show how gases change from when the blood leaves the lungs versus when it returns to the heart, they found evidence of NO transport and delivery.  This led them to conclude:

“The most fundamental and important observation of this study is that NO gas introduced to the lungs can be stabilized and transported in blood and peripherally modulate blood flow.” 

This study was groundbreaking in that it showed, for the first time, evidence of inhaled NO being transported throughout the body while maintaining its bioactivity.  These results might be significant to diabetics because we suffer from reduced endothelial-derived NO and reduced blood flow.  Thus, the results might provide more support for nose-breathing (although again, NO concentrations in the nose are far less than what was administered here).

To conclude, I’ll borrow a line from the abstract, which succinctly states how the findings of this study could be particularly important to diabetics: 

“These results indicate that inhaled NO during blockade of regional NO synthesis can supply intravascular NO to maintain normal vascular function. This effect may have application for the treatment of diseases characterized by endothelial dysfunction.”

Abstract

Nitric oxide (NO) may be stabilized by binding to hemoglobin, by nitrosating thiol-containing plasma molecules, or by conversion to nitrite, all reactions potentially preserving its bioactivity in blood. Here we examined the contribution of blood-transported NO to regional vascular tone in humans before and during NO inhalation. While breathing room air and then room air with NO at 80 parts per million, forearm blood flow was measured in 16 subjects at rest and after blockade of forearm NO synthesis with NG-monomethyl-l-arginine (l-NMMA) followed by forearm exercise stress. l-NMMA reduced blood flow by 25% and increased resistance by 50%, an effect that was blocked by NO inhalation. With NO inhalation, resistance was significantly lower during l-NMMA infusion, both at rest and during repetitive hand-grip exercise. S-nitrosohemoglobin and plasma S-nitrosothiols did not change with NO inhalation. Arterial nitrite levels increased by 11% and arterial nitrosyl(heme)hemoglobin levels increased tenfold to the micromolar range, and both measures were consistently higher in the arterial than in venous blood. S-nitrosohemoglobin levels were in the nanomolar range, with no significant artery-to-vein gradients. These results indicate that inhaled NO during blockade of regional NO synthesis can supply intravascular NO to maintain normal vascular function. This effect may have application for the treatment of diseases characterized by endothelial dysfunction.

Journal Reference:

Cannon RO 3rd, Schechter AN, Panza JA, et al. Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery. J Clin Invest. 2001;108(2):279-287. doi:10.1172/JCI12761

Nasal Nitric Oxide: Nature’s Answer to Gravity?

Key Points

• Nitric oxide redistributes blood flow in the lungs to be more uniform

• Nitric oxide increases gas exchange in the lungs

• Nasal nitric oxide might be an evolutionary adaptation to counter gravity

The Breathing Diabetic Summary

Blood flow in the lungs is essential for gas exchange and defense against infections. However, lung blood flow is not as uniform as we might think. And, although several factors account for this, gravity plays a significant role.  Gravity acts to focus blood flow toward the bottom of the lungs.  

Interestingly, in humans and higher primates, a large amount of nitric oxide (NO) is released in the nasal passages. As we learned, NO is critical for blood flow and whole-body oxygenation. The authors of this study wondered if nasal NO might also redistribute blood flow in the lungs, thus countering the effects of gravity and increasing gas exchange in the lungs. This adaptation would have allowed us to evolve into the bipedal mammals we are today.

To test this, they examined how different breathing protocols affected lung blood flow. Participants were injected with a radionuclide that acted as a passive tracer of blood flow, which could then be imaged to show relative “heat maps” of blood flow in the lungs.

Fourteen participants were broken into three groups. The first group served as a control to ensure the radiotracer imaging technique worked as intended. The second group was used to see how nasally produced NO affected lung blood flow. These participants sat in an upright position and breathed through their mouths for 20 min. The tracer was injected, and their lung blood flow was imaged. Then, they switched to nasal breathing for 10 min. Tracer was again injected imagery was taken.

The final group was used to see if NO was, in fact, the driver of lung blood flow redistribution. These participants breathed through their mouths but were given supplemental NO. If NO was the driver, mouth breathing with additional NO should result in similar blood flow redistribution as nasal breathing.

They found that nasal breathing redistributed blood flow both vertically and horizontally in the lungs, making it more uniform. The same occurred when mouth breathing with supplemental NO. Thus, NO, whether produced naturally in the nasal passages or supplemented, acts to redistribute blood flow and increase gas exchange in the lungs.

The authors hypothesize that the NO produced in the nasal passages is an evolutionary adaptation to walking upright.  The NO acts to make blood flow and gas exchange more uniform, thus countering the effects of gravity.

In summary, nasal nitric oxide counteracts the effects of gravity and makes lung blood flow more uniform in the upright position. Interestingly, this only occurs in humans and higher primates. Thus, NO production in the upper airways might have been a critical evolutionary adaptation that allowed us to walk upright.

Abstract

There are a number of evidences suggesting that lung perfusion distribution is under active regulation and determined by several factors in addition to gravity. In this work, we hypothesised that autoinhalation of nitric oxide (NO), produced in the human nasal airways, may be one important factor regulating human lung perfusion distribution in the upright position. In 15 healthy volunteers, we used single-photon emission computed tomography technique and two tracers (99mTc and 113mIn) labeled with human macroaggregated albumin to assess pulmonary blood flow distribution. In the sitting upright position, subjects first breathed NO free air through the mouth followed by the administration of the first tracer. Subjects then switched to either nasal breathing or oral breathing with the addition of exogenous NO-enriched air followed by the administration of the second tracer. Compared with oral breathing, nasal breathing induced a blood flow redistribution of approximately 4% of the total perfusion in the caudal to cranial and dorsal to ventral directions. For low perfused lung regions like the apical region, this represents a net increase of 24% in blood flow. Similar effects were obtained with the addition of exogenous NO during oral breathing, indicating that NO and not the breathing condition was responsible for the blood flow redistribution. In conclusion, these results provide evidence that autoinhalation of endogenous NO from the nasal airways may ameliorate the influence of gravity on pulmonary blood flow distribution in the upright position. The presence of nasal NO only in humans and higher primates suggest that it may be an important part of the adaptation to bipedalism.

Journal Reference:

Sánchez Crespo A, Hallberg J, Lundberg JO, Lindahl SG, Jacobsson H, Weitzberg E, Nyrén S.  Nasal nitric oxide and regulation of human pulmonary blood flow in the upright position.  J Appl Physiol.  2010;108:181–188.