Natural Product Nitric Oxide Chemistry: New Activity of Old Medicines

The use of complementary and alternative medicine (CAM) as a therapy and preventative care measure for cardiovascular diseases (CVD) may prove to be beneficial when used in conjunction with or in place of conventional medicine. However, the lack of understanding of a mechanism of action of many CAMs limits their use and acceptance in western medicine. We have recently recognized and characterized specific nitric oxide (NO) activity of select alternative and herbal medicines that may account for many of their reported health benefits. The ability of certain CAM to restore NO homeostasis both through enhancing endothelial production of NO and by providing a system for reducing nitrate and nitrite to NO as a compensatory pathway for repleting NO bioavailability may prove to be a safe and cost-effective strategy for combating CVD. We will review the current state of science behind NO activity of herbal medicines and their effects on CVD

Analytical techniques for assaying nitric oxide bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO\u27s effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography

Acute effects of hemodialysis on nitrite and nitrate: potential cardiovascular implications in dialysis patients

Cardiovascular mortality in dialysis patients remains a serious problem. It is 10 to 20 times higher than in the general population. No molecular mechanism has been proven to explain this increased mortality, although nitric oxide (NO) has been implicated. The objective of our study was to determine the extent of the removal of the NO congeners nitrite and nitrate from plasma and saliva by hemodialysis, as this might disrupt physiological NO bioactivity and help explain the health disparity in dialysis patients. Blood and saliva were collected at baseline from patients on dialysis and blood was collected as it exited the dialysis unit. Blood and saliva were again collected after 4-5h of dialysis. In the 27 patients on dialysis, baseline plasma nitrite and nitrate by HPLC were 0.21±0.03 and 67.25±14.68 μM, respectively. Blood immediately upon exit from the dialysis unit had 57% less nitrite (0.09±0.03 μM; P=0.0008) and 84% less nitrate (11.04 μM; P=0.0003). After 4-5h of dialysis, new steady-state plasma levels of nitrite and nitrate were significantly lower than baseline, 0.09±0.01 μM (P=0.0002) and 16.72±2.27 μM (P=0.001), respectively. Dialysis also resulted in a significant reduction in salivary nitrite (232.58±75.65 to 25.77±10.88 μM; P=0.01) and nitrate (500.36±154.89 to 95.08±24.64 μM; P=0.01). Chronic and persistent depletion of plasma and salivary nitrite and nitrate probably reduces NO bioavailability and may explain in part the increased cardiovascular mortality in the dialysis patient

Metagenomic Analysis of Nitrate-Reducing Bacteria in the Oral Cavity: Implications for Nitric Oxide Homeostasis

<div><p>The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is dependent upon oral nitrate-reducing bacteria, since humans lack this enzyme activity. This pathway appears to represent a newly recognized symbiosis between oral nitrate-reducing bacteria and their human hosts in which the bacteria provide nitrite and nitric oxide from nitrate reduction. Here we measure the nitrate-reducing capacity of tongue-scraping samples from six healthy human volunteers, and analyze metagenomes of the bacterial communities to identify bacteria contributing to nitrate reduction. We identified 14 candidate species, seven of which were not previously believed to contribute to nitrate reduction. We cultivated isolates of four candidate species in single- and mixed-species biofilms, revealing that they have substantial nitrate- and nitrite-reduction capabilities. Colonization by specific oral bacteria may thus contribute to host NO homeostasis by providing nitrite and nitric oxide. Conversely, the lack of specific nitrate-reducing communities may disrupt the nitrate-nitrite-nitric oxide pathway and lead to a state of NO insufficiency. These findings may also provide mechanistic evidence for the oral systemic link. Our results provide a possible new therapeutic target and paradigm for NO restoration in humans by specific oral bacteria.</p></div

Flow diagram for study design and subject recruitment.

<p>Flow diagram for study design and subject recruitment.</p

The nitrate- and nitrite-reducing capacity of four candidate species grown individually and as a consortium.

<p>Each bar represents the concentration of nitrate and nitrite remaining in the spent medium after 24(<i>A. odontolyticus</i>, <i>V. dispar</i>, <i>F. nucleatum</i>, and <i>S. mutans</i>) or a consortium of all four species at 24 hours after biofilm inoculation. The nitrate concentration, orange; nitrite concentration, green. The data are the average ± SEM of three individual experiments.</p

Top Ten Genera Present in Individual Tongue Scrapings.

<p>Top Ten Genera Present in Individual Tongue Scrapings.</p

Unweighted UniFrac-based PCoA analysis reveals a gradient of samples as nitrate reduction capacity decreases with specific taxa associated with different groups.

<p><b>A</b>) Unweighted UniFrac-based PCoA illustrates samples based on community similarity. Red dots = Best nitrate reducing samples, blue dots = intermediate nitrate reducing samples, green dots = worst nitrate reducing samples, and orange dots = inocula (original tongue scrapings). <b>B</b>) Bi Plot superimposing taxonomic information onto an unweighted UniFrac-based PCoA illustrating how similar the microbial communities of the samples are to one another. Red dots = Best nitrate-reducing samples, blue dots = intermediate nitrate-reducing samples, orange dots = worst nitrate-reducing samples, and gray dots = taxa.</p

The mean relative abundance of genera present in each group of nitrate reducers.

<p>Bar charts with insets depict the mean relative abundance of genera present <b>A</b>) in the best (n = 9), <b>B</b>) intermediate (n = 10), and <b>C</b>) worst (n = 5) nitrate-reduction groups. Inset bars depict all genera detected in each group except <i>Streptococcus</i>, which was the most abundant genus detected in all groups and is depicted in the main bars. The percent abundance and taxonomic classification of the most abundance taxa are noted on the graphs.</p