Nitric Oxide

In the first of this four part series, we will begin to describe and explain the complex biochemistry and physiology of nitric oxide (NO) and why this molecule should be on the radar for optimal health and wellness. Strategies to measure, correct and monitor nitric oxide status will be introduced as well as simple explanations to explain why L-arginine may not be the answer. In the subsequent series, we will describe the effects of NO on specific systems and health conditions. Throughout each series we will introduce nutritional and natural strategies proven to restore NO production/homeostasis and enhance health parameters that begin to restore normal physiology.

Aging is the basic principle of living and the part of life that we all dread and try to resist. As we age, we lose the energy we had when we are young, we begin to forget things, our sexual health and performance declines and we begin to develop aches and pains and symptoms that over time manifest as specific health issues. Research over the past 20 years had revealed that the loss of one of the most important molecules our body produces may be responsible for many of these symptoms and is at the root of poor cardiovascular health. That single molecule is nitric oxide or more simply NO.

The science of NO is relatively new, although NO active drugs such as nitroglycerin have been used for hundreds of years. However, it was not realized they were working through NO. NO was first discovered by Dr. Robert Furchgott in 1980 as a vasodilating substance produced by the endothelial cells in our blood vessels. He first described the substance as “endothelium derived relaxing factor” or EDRF.¹ It was later discovered that EDRF is nitric oxide by Dr. Lou Ignarro in 1987.² Ten years prior, Dr. Ferid Murad discovered that the mechanism of action of nitroglycerin was through the release of NO3 and subsequent activation of the enzyme soluble guanylyl cyclase (sGC).⁴ Collectively, these three U.S. scientists were awarded the Nobel Prize in Medicine or Physiology in 1998 for their discovery of NO. The Swedish Nobel Assembly sagely noted, “The signal transmission by a gas that is produced by one cell, penetrates through membranes and regulates the function of another cell, represents an entirely new principle for signaling in biological systems.” It was shocking to realize that NO, a colorless, odorless gas, was able to perform such important biochemical functions in selective and specific cell signaling events. Dr. Valentin Fuster, then president of the American Heart Association, noted in a 1998 interview that “the discovery of NO and its function is one of the most important in the history of cardiovascular medicine.” Now more than 10 years after the Nobel Prize and more than 120,000 scientific papers published on NO, we are beginning to appreciate its vital role in health and disease.

First, it is important and critical to understand the history of the science behind NO and the metabolic pathways for its production. After EDRF was discovered to be NO, it was quickly realized that L-arginine is the substrate necessary for NO production.⁵ However, in 1982, prior to the identification of EDRF, the endogenous activator of sGC in neuroblastoma cells was identified as L-arginine ⁶ and later it was recognized that NO is formed from L-arginine in the central nervous system and acts as a neurotransmitter.⁷ Even earlier however, nitrogen oxides were emerging as a central participant in the immune response. The discovery of NO in the immune system began with the observation that high concentrations of urinary nitrates were excreted from a patient with infectious diarrhea.⁸ The source of these nitrogen compounds remained unclear until Stuehr and Marletta⁹ demonstrated that serum and urinary nitrates were increased in normal mice after immunostimulation, but not in C3H/HeJ mice, which have a genetic alteration rendering their macrophages resistant to endotoxin. This observation led to the discovery that NO is produced as a cytotoxic molecule to kill off invading pathogens by the immune system. It was through this model that NO was identified as the intermediate compound in the L-arginine to nitrite/nitrate pathway. These observations, together with the discovery of the L-arginine:NO pathway in the vasculature, led to the investigations and subsequent discovery of the existence of this ubiquitous pathway in mammalian physiology. It was really the collective efforts of many research groups working in completely different fields that converged on a single pathway in multiple systems. Although NO has been shown to be involved in every biological system, it is best characterized in the cardiovascular, immune and nervous system.

Although the L-arginine pathway was the first to be discovered for the endogenous production of NO, it may only provide part of the total body NO production. In physiological systems, we know there is enormous redundancy or compensatory systems that kick in when a primary pathway becomes dysfunctional or inefficient. For the past 25 years, that redundant system was poorly explored. The conversion of L-arginine to NO may be one of the most complex and complicated reactions in our body. The reaction carried out by a group of enzymes called nitric oxide synthase (NOS) is a 5-electron oxidation of the guanidine nitrogen of L-arginine to produce NO and L-citrulline requiring many co-factors and substrates. Two moles of O2 and 1.5 moles of NADPH are consumed per mole of NO formed.¹⁰ NOS enzymes are the only enzymes known to simultaneously require six bound cofactors/prosthetic groups: flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide phosphate (NADPH), heme, tetrahydrobiopterin (BH4) and Ca2+-calmodulin (CaM). One can now begin to appreciate the logistics and kinetics for such a reaction to take place. To complicate matters even more, once NO is produced it has a biological half-life of around 1 second. The spatial and temporal efficient production of NO is critical for normal cell signaling events and maintenance of homeostasis.

One of the most predictive indicators for insufficient NO production is age. As we age we lose our ability to produce NO through the L-arginine pathway. The inability to generate sufficient NO is the earliest event that leads to the onset and progression of poor health outcomes. Being able to diagnose and intervene early on is the key to optimal health and disease prevention.

NO and Aging

Aging and hypertension are well-documented cardiovascular risk factors.^11-12 Most of the functional and structural vascular alterations that lead to cardiovascular complications are similar in aging and hypertension.¹³ Moreover, these vascular changes associated with essential hypertension are generally considered to be an accelerated form of the changes seen with aging.¹⁴ When we are young and healthy, the endothelial production of NO through L-arginine is efficient and sufficient to produce NO; however, as we age we lose our ability to synthesize endothelial derived NO. Most of the studies on the activity of NO in cells and tissues agree that the bioavailability or the generation of NOS derived NO decreases with aging. It has been proposed that superoxide can scavenge NO to form peroxynitrite and thereby reduce its effective concentrations in cells.¹⁵ It has also been reported that there is decreased NOS expression with aging both in constitutive and inducible isoforms.^16-17 Berkowitz et al.¹⁸ observed the upregulation of arginase (an enzyme that degrades the natural substrate for NOS, L-arginine) in aged blood vessels and the corresponding modulation of NOS activity. Taddei et al.¹⁹ have shown that there is a gradual decline in endothelial function due to aging with greater than 50 percent loss in endothelial function in the oldest age group tested as measured by forearm blood flow assays. Egashira et al.²⁰ reported more dramatic findings in the coronary circulation of aging adults whereby there was a loss of 75 percent of endothelium-derived nitric oxide in 70-80 year old patients compared to young, healthy 20 year olds. Vita and colleagues²¹ demonstrated that increasing age was one predictor of abnormal endothelium-dependent vasodilation in atherosclerotic human epicardial coronary arteries. Gerhard et al.,²² concluded from their 1996 study that age was the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analysis. Collectively, these important findings illustrate that endothelium-dependent vasodilation in resistance vessels declines progressively with increasing age. These data are illustrated in Figure 1. This abnormality is present in healthy adults who have no other cardiovascular risk factors, such as diabetes, hypertension, or hypercholesterolemia. Most of these studies found that impairment of endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast, endothelium-independent vasodilation does not change significantly with aging, demonstrating that the responsiveness to NO does not change only the ability to generate it. These observations enable us to conclude that reduced availability of endothelium-derived NO occurs as we age, and to speculate that this abnormality may create an environment that is conducive to poor circulation and other vascular health issues, and contribute to brain health and cognitive concerns associated with aging. It appears that aging interrupts NO signaling at every conceivable level, from production to inactivation. Given that NO is a necessary molecule for maintenance of optimal health and wellness, restoration of NO homeostasis may provide a new treatment modality for age and age related health concerns.

NO Diagnostics

The major pathway for NO metabolism is the stepwise oxidation to nitrite and nitrate. For years, both nitrite (NO2-) and nitrate (NO3-) (collectively, NOx) have been used as surrogate markers of NO production in biological tissues, but there have not been any new developments in the use of NO biomarkers in the clinical setting for diagnostic or prognostic utility. In fact, NO status is not part of the standard blood chemistry routinely used for diagnostic purposes. This is simply unacceptable given the critical nature of NO for the maintenance of health in several diverse areas. The only true measure of endothelial NO production (endothelial function) is through flow-mediated dilatation (FMD). FMD is a non-invasive ultrasound-based method where arterial diameter is measured in response to an increase in shear stress, which causes release of NO from the endothelium and consequent endothelium dependent dilatation. FMD has been shown to correlate with invasive measures of endothelial function, as well as with the presence and severity of the major traditional vascular risk factors.²³ Nitrite and nitrate have recently been shown to be biomarkers for cardiovascular and other parameters from both diagnostic and therapeutic aspects.²⁴ However, it is not known if levels of nitrite and nitrate correlate with FMD. A report by Kleinbongard et al.²⁵ demonstrated that plasma nitrite levels in humans progressively decrease with increasing cardiovascular risk load. Risk factors considered included age, hypertension, smoking, and hypercholesterolemia, conditions all known to reduce the bioavailability of NO. Although a correlation exists in the plasma, it is not known whether the situation is mirrored in the heart or other tissue of interest. The recent recognition of a human nitrogen cycle whereby nitrate and nitrite are reduced to NO by an enterosalivary circulation of nitrate²⁶ now opens up the potential for using saliva as a potential biomarker for NO status. Salivary NO test strips are now available as an easy to use non-invasive colorimetric measure of total body NO availability. This provides the only available measure of NO status and can provide physicians and their patients a way to diagnose NO insufficiency and monitor NO levels as strategies are implemented to restore NO production. These strips are a powerful tool and give your patients the power to monitor their progress and they give the physicians a measure of compliance.

NO Restoration

Understanding the normal biochemical pathway for NO production and the effects of aging on NO production, we can begin to implement therapeutic regimens to overcome NO insufficiency and monitor the effectiveness with the test strips.

There are currently only 3 FDA approved products on the market directly related to NO: 1) organic nitrates, such as nitroglycerin for the treatment of acute angina (these have been used for centuries long before the discovery of NO); 2) inhaled NO therapy for neonates for treatment of pulmonary hypertension due to underdeveloped lungs; and 3) phosphodiesterase inhibitors, such as sildenafil, which do not directly affect NO production but act through affecting the downstream second messenger of NO, cyclic guanosine monophosphate (cGMP).

There also are a number of over-the-counter products designed to enhance NO production using supplemental L-arginine. We will discuss the limitations of L-arginine in a subsequent issue, but suffice it to say, these L-arginine based products are not an effective measure for enhancing NO levels in a majority of individuals. Recent innovations in natural product chemistry has led to the commercialization of a safe, effective and clinically proven product for NO restoration in the over 40 crowd. Neo40® Daily was recently tested in a double-blinded, placebo-controlled clinical trial in patients over the age of 40 with known cardiovascular risk factors. Patients taking the Neo40 Daily twice a day for 30 days saw a significant increase in NO production, a significant reduction in triglycerides, a modest reduction in blood pressure and a reduction in C-reactive protein.²⁷ The technology developed out of the University of Texas School of Medicine in Houston is available as an over-the-counter dietary supplement through Complementary Prescriptions™. This technology provides an alternate source of NO to compensate for insufficient NO production associated with aging and other risk factors.

Conclusion

This first in the NO series hopefully gives you an appreciation for the importance of NO in health maintenance and a fundamental appreciation for its production in the human body. In the subsequent series, we will focus more on practical applications of NO-based therapies in specific conditions and introduce diet and lifestyle modifications proven to enhance and restore NO homeostasis.

Reference

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