The preservation of meat has been practiced for thousands of years to prevent spoilage long before there was refrigeration. Historically, salt petre, or potassium nitrate was used. Over the past 40 years, sodium nitrite has become the main curing agent to prevent food spoilage and bacterial growth. Over the past few years, efforts have been made to replace nitrite based on consumer demands for a "no nitrite added" product. However, decades of research have revealed that there is no effective replacement for nitrite. The real issue is education and awareness on the important health benefits of nitrite and nitric oxide. Over the past two decades, it has become obvious and apparent there are profound benefits of sodium nitrite to human health and it is actually considered a therapeutic for many chronic diseases. Nitrite serves as a pre-cursor to nitric oxide (NO) but also has signaling properties of its own that appear to be independent of NO production. This review will provide an update on the therapeutic effects of nitrite at specific doses, highlight doses found in meats and vegetables, and provide guidelines for establishing nitrite as an essential nutrient.

BACKGROUND: The study was initiated in 2020 to test the efficacy of a nitric oxide-generating lozenge (NOL) in outpatients with newly diagnosed COVID-19 to mitigate disease severity. The study enrolled high-risk patients, African American and Latino. METHODS: This was a randomized, double-blinded, prospective, placebo-controlled trial. The primary endpoint was hospitalization, intensive care unit admission, intubation, dialysis, and death. The secondary endpoints were time to symptom resolution and the effect on oxygen saturation. Patients ages 50-85 years with recent COVID-19 diagnosis with at least one risk factor were recruited. Patients were randomized to either active treatment or placebo using block randomization. Blood pressure and oxygen saturation (SpO2) was measured prior to and after the first dose and each morning thereafter. RESULTS: A total of 840 patients was planned, half in each of the lozenge and placebo groups. An interim review of data was prespecified. Of 524 patients, the composite endpoint occurred in 6 patients, 3 (1.1%) in each group. The time to symptom resolution was 1 day shorter on active treatment (8.7 +/- 6.6 vs 9.8 +/- 6.8 days) (P = .3). There was no change in SpO2 on placebo (0.0 +/- 2.0%) and no significant change on treatment (0.14 +/- 0.9%), P = .3. All events occurred in the first year (2020). CONCLUSIONS: This study did not find a benefit of NOL therapy in COVID-19 patients and was terminated for futility. NOL treatment did not reduce mortality, hospitalization, intubation, or a reduction in symptoms duration. The study did find the NO lozenges were well tolerated in high-risk patients, without reported side effects. (c) 2023 Elsevier Inc. All rights reserved. center dot The American Journal of Medicine (2023) 136:1035-1040

It is now more than 35 years since endothelium derived relaxing factor was identified as nitric oxide (NO). The last few decades have seen an explosion around nitric oxide biochemistry, physiology and clinical translation. The science reveals that all chronic disease is associated with decreased blood flow to the affected organ which results in increased inflammation, oxidative stress and immune dysfunction. This is true for cardiovascular disease, neurological disease, kidney, lung, liver disorders and every other major disorder. Since nitric oxide controls and regulates blood flow, oxygen and nutrient delivery to every cell, tissue and organ in the body and also mitigates inflammation, oxidative stress and immune dysfunction, a focus on restoring nitric oxide production is an obvious therapeutic strategy for a number of poorly managed chronic diseases. Since dietary nitrate is a major contributor to endogenous nitric oxide production, it should be considered as a means of therapy and restoration of nitric oxide. This review will update on the current state of the science and effects of inorganic nitrate administered through the diet on several chronic conditions and reveal how much is needed. It is clear now that antiseptic mouthwash and use of antacids disrupt nitrate metabolism to nitric oxide leading to clinical symptoms of nitric oxide deficiency. Based on the science, nitrate should be considered an indispensable nutrient that should be accounted for in dietary guidelines.

Hypertension remains a global health crisis. High blood pressure is the number one modifiable risk factor in the onset and progression of cardiovascular disease. Despite many different classes of drug therapies approved for hypertension, the use of polypharmacy and recommendations on lifestyle modification, many patients still suffer from uncontrolled or unmanaged hypertension. Nitric oxide is a naturally produced vasodilator that controls and regulates vascular tone and therefore controls and regulates blood pressure. Research over the past 40 years reveals that loss of nitric oxide production, termed endothelial dysfunction, is the earliest event in the development of hypertension. Strategies aimed at preventing the loss of nitric oxide production and/or therapeutic strategies designed to restore nitric oxide production will likely have a positive effect on patients' health and lead to better management of blood pressure. This review article will focus on the loss of nitric oxide production as the primary contributor to hypertension and also discuss safe and clinically proven strategies to restore nitric oxide production and recapitulate nitric oxide based signaling in humans.

The human microbiome comprises ∼1013–1014 microbial cells which form a symbiotic relationship with the host and play a critical role in the regulation of human metabolism. In the oral cavity, several species of bacteria are capable of reducing nitrate to nitrite; a key precursor of the signaling molecule nitric oxide. Nitric oxide has myriad physiological functions, which include the maintenance of cardiovascular homeostasis and the regulation of acute and chronic responses to exercise. This article provides a brief narrative review of the research that has explored how diversity and plasticity of the oral microbiome influences nitric oxide bioavailability and related physiological outcomes. There is unequivocal evidence that dysbiosis (e.g. through disease) or disruption (e.g. by use of antiseptic mouthwash or antibiotics) of the oral microbiota will suppress nitric oxide production via the nitrate-nitrite-nitric oxide pathway and negatively impact blood pressure. Conversely, there is preliminary evidence to suggest that proliferation of nitrate-reducing bacteria via the diet or targeted probiotics can augment nitric oxide production and improve markers of oral health. Despite this, it is yet to be established whether purposefully altering the oral microbiome can have a meaningful impact on exercise performance. Future research should determine whether alterations to the composition and metabolic activity of bacteria in the mouth influence the acute responses to exercise and the physiological adaptations to exercise training.

In critically ill children with acute kidney injury (AKI), continuous kidney replacement therapy (CKRT) enables nutrition provision. The magnitude of amino acid loss during continuous venovenous hemodiafiltration (CVVHDF) is unknown and needs accurate quantification. We investigated the mass removal and clearance of amino acids in pediatric CVVHDF. This is a prospective observational cohort study of patients receiving CVVHDF from August 2014 to January 2016 in the pediatric intensive care unit (PICU) of a tertiary children’s hospital. Fifteen patients (40% male, median age 2.0 (IQR 0.7, 8.0) years) were enrolled. Median PICU and hospital lengths of stay were 20 (9, 59) and 36 (22, 132) days, respectively. Overall survival to discharge was 66.7%. Median daily protein prescription was 2.00 (1.25, 2.80) g/kg/day. Median daily amino acid mass removal was 299.0 (174.9, 452.0) mg/kg body weight, and median daily amino acid mass clearance was 18.2 (13.5, 27.9) ml/min/m2, resulting in a median 14.6 (8.3, 26.7) % protein loss. The rate of amino acid loss increased with increasing dialysis dose and blood flow rate. CVVHDF prescription and related amino acid loss impact nutrition provision, with 14.6% of the prescribed protein removed. Current recommendations for protein provision for children requiring CVVHDF should be adjusted to compensate for circuit-related loss.

Background: Infected hernia mesh is a cause of post-operative morbidity. Nitric oxide (NO) plays a key role in the endogenous immune response to infection. We sought to study the efficacy of a NO-releasing mesh against methicillin-resistant Staphylococcus aureus (MRSA). We hypothesized that a NO-releasing polyester mesh would decrease MRSA colonization and proliferation. Materials and Methods: A composite polyester mesh functionalized with N-diazeniumdiolate silica nanoparticles was synthesized and characterized. N-diazeniumdiolate silica parietex composite (NOSi) was inoculated with 104,106, or 108 colony forming units (CFUs) of MRSA and a dose response was quantified in a soy tryptic broth assay. Utilizing a rat model of contaminated hernia repair, implanted mesh was inoculated with MRSA, recovered, and CFUs were quantified. Clinical metrics of erythema, mesh contracture, and adhesion severity were then characterized. Results: Methicillin-resistant Staphylococcus aureus CFUs demonstrated a dose-dependent response to NOSi in vitro. In vivo, quantified CFUs showed a dose-dependent response to NOSi-PCO. Treated rats had fewer severe adhesions, less erythema, and reduced mesh contracture. Conclusions: We demonstrate the efficacy of a NO-releasing mesh to treat MRSA in vitro and in vivo. Creation of a novel class of antimicrobial prosthetics offers new strategies for reconstructing contaminated abdominal wall defects and other procedures that benefit from deploying synthetic prostheses in contaminated environments.

The potential benefits of supplemental nutrients and dietary interventions against cardiovascular morbidity and mortality have been extensively investigated throughout the years. Numerous supplements claim cardioprotection and reduction of cardiovascular risk factors, but the roles of many supplements have not been determined. In the vast number of supplements on the market asserting cardioprotective effects, only 3 have been thoroughly evaluated and consistently reported as effective by our clinic patients. They have used supplements such as fish oil, multivitamins, and calcium, but many had not known of the benefits of resveratrol, curcumin, and nitric oxide as supplements for improving cardiovascular health. The cardioprotective effects of these dietary supplements in both animal models and humans have been explored with proposed mechanisms of action mostly attributed to antioxidant and anti-inflammatory properties. Resveratrol is one of the most studied polyphenols with established cardiovascular benefits. Preclinical studies have demonstrated these effects exerted via improved inflammatory markers, atherogenic profile, glucose metabolism, and endothelial function and are further supported by clinical trials. Curcumin has a well-established anti-inflammatory role by regulating numerous transcription factors and cytokines linked to inflammation. Inflammation is an underlying pathology in cardiovascular diseases, rendering curcumin a potential therapeutic compound. Similarly, nitric oxide supplementation has demonstrated cardiovascular benefits by normalizing blood pressure; enhancing blood flow; and reducing inflammation, immune dysfunction, and oxidative stress. A comprehensive review was performed evaluating the cardioprotective effects of these 3 dietary supplements with hope to provide updated information, promote further awareness of these supplements, and inspire future studies on their effects on cardiovascular health.

To determine the efficacy of inorganic nitrite supplementation on endothelial function in humans and mechanisms of action, we performed (1) a randomized, placebo-controlled, parallel-group clinical trial with sodium nitrite (80 mg/day, 12 weeks) in older adults (N=49, 68±1 year) and (2) reverse-translation experiments in young (6 months) and old (27 months) c57BL/6 mice. In the clinical trial, sodium nitrite increased plasma nitrite ( P <0.05) and was well tolerated. Brachial artery flow-mediated dilation (endothelial function) was increased 28% versus baseline after nitrite supplementation ( P <0.05) but unchanged with placebo. Nitrotyrosine, a marker of oxidative stress, was reduced by 45% versus baseline in biopsied endothelial cells after nitrite, but not placebo, treatment. Plasma from nitrite-treated, but not placebo-treated, subjects decreased whole-cell (CellROX) and mitochondria-specific (MitoSOX) reactive oxygen species in cultured human umbilical vein endothelial cells ( P <0.05). Old mice (old [27 months] control, n=9) had ≈30% lower ex vivo carotid artery endothelium-dependent dilation (EDD) versus young mice (young [6 months] control, n=9) due to reduced NO bioavailability ( P <0.05). Nitrite supplementation (drinking water, 50 mg/L, 8 weeks) restored EDD and NO bioavailability in old mice (n=10) to (6 months) control. Mitochondrial reactive oxygen species suppression of EDD was present in old control (increased EDD with a mitochondrial-targeted antioxidant, P <0.05) but not in young control or old mice supplemented with sodium nitrite. A mitochondrial reactive oxygen species inducer (rotenone) further impaired EDD in old control ( P <0.05); young control and old mice supplemented with sodium nitrite were protected. Markers of mitochondrial health were greater in aorta of old mice supplemented with sodium nitrite versus old control ( P <0.05). Inorganic nitrite supplementation improves endothelial function with aging by increasing NO, decreasing mitochondrial reactive oxygen species/oxidative stress, and increasing mitochondrial stress resistance. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02393742.

Introduction Acute stroke incites an inflammatory reaction in the brain's microvasculature, activating formation of nitric oxide oxidative metabolites, nitrate and nitrite (NOx, collectively), measurable in plasma. Our objectives were to investigate plasma NOx in patients with acute stroke presenting to the Emergency Department (ED) and to determine if it could (i) differentiate between ischemic and hemorrhagic stroke; (ii) predict clinical outcomes. Methods A cross-sectional study was conducted in the ED of Aga Khan University Hospital, from January 1 to December 31, 2016. Participants were enrolled if they had clinical acute stroke with confirmatory brain imaging to differentiate between ischemia and hemorrhage. Clinical demographic information, ancillary blood, and diagnostic specimens were collected as per standard of care since the center follows stroke algorithmic guidelines. Plasma NOx analysis was performed using high performance liquid chromatography. Clinical outcomes were assessed using Barthel Index and Modified Rankin Score. Data was analyzed using SPSS 19 and expressed in medians with interquartile ranges. Nonparametric tests were applied for comparing among groups. Pearson's correlation was used to determine associations with aforementioned stroke severity and disability scales. Results Seventy-five patients were enrolled, with median age of 57 years (IQR 47-66 years), 53 (71%) were males, and 46 (61%) had ischemic stroke. Overall, median NOx was 20.8 μM (IQR 13.4-35.3); there was no statistically significant difference between NOx in ischemic versus hemorrhagic stroke (21.2 μM vs. 17.9 μM; p=0.2). However, there was a significant positive correlation between NOx levels and aforementioned acute stroke scales [r(73)=0.417, p=0.0001], for both. Conclusion Although plasma NOx could not differentiate between ischemia and hemorrhage, higher levels of the biomarker did show associations with poststroke disability scales. Further study with more patients in a multicenter trial is warranted to establish the real biomarker potential of plasma NOx in acute stroke.

Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC) yet chemoresistance is common. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life. We sought to study the effect of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 dependent manner. NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high-pressure nitrous oxide. NO release was measured using chemiluminescence. A SMAD4 negative PAC cell line (SMAD4-) was made using retroviral knockdown of Panc1 PAC cells. Panc1 and SMAD4- cells were treated with gemcitabine (100 nm (hi) to 30 μm (lo)), 30 mg NOSi particles, or both (NOSihi or NOSilo) and cell viability assessed. NoSi reduced cell viability by 25.99% in Panc1 and 24.38% in SMAD4-. When combined with gemcitabine, further reductions were seen in a dose dependent manner for both cell lines. We have demonstrated the in-vitro dose dependent cytotoxic effects of NOSi. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NO is useful chemosensitizing agent but that SMAD4- may play a role in its synergism with GEM.

Nitric oxide (NO)–based supplements and functional foods have gained considerable popularity over the past 25 years since a Nobel Prize was awarded for the discovery of NO as a vasodilator and regulator of blood flow. We now understand that NO is not only much more than a vasodilator but also a regulator of mitochondrial biogenesis and adenosine triphosphate production and a ubiquitous signaling molecular that controls many physiological and metabolic pathways. As a result, it can have a profound effect on exercise and sports performance when used appropriately. There are two pathways in the human body that can generate NO. These two pathways are fundamentally different and distinct in how and when they generate NO. Being able to understand the biochemistry and physiology of both will allow for targeting therapy for exercise performance independent of duration or type of exercise. This chapter will provide the basis for use of NO-based supplement and nutrition for enhancing sports performance.

Nitric oxide (NO) production is essential for normal human health and physiology. NO controls and regulates blood pressure, nervous and immune function along with numerous other fundamental physiological processes. Loss of NO production is recognized as one of the earliest events in the onset and progression of many if not all chronic diseases. Development of FDA approved NO based therapeutics has been slow and largely unsuccessful. Historically, natural product chemistry has been a primary strategy for development of new drug compounds. Through plant based chemistry and bacterial metabolism, NO can be produced and harnessed for restoration of NO based signaling in humans. This strategy may provide opportunity for development of safe and effective NO based therapies.

We previously observed improvements in vascular endothelial function as well as exploratory measures of motor and cognitive function in a pilot study of sodium nitrite supplementation in middle‐aged and older (MA/O) adults. Here, we sought to establish the efficacy of sodium nitrite for improving endothelial function (primary outcome) while also taking advantage of the opportunity to confirm our motor and cognitive function findings in a larger randomized, placebo‐controlled, double‐blind, parallel group study with 12 weeks of sodium nitrite (80 mg/day) vs. placebo in healthy MA/O adults (n = 49, 68±1 yr) with impaired endothelial function (baseline brachial artery flow‐mediated dilation [FMD] <6%). Sodium nitrite increased plasma levels of nitrite acutely (10‐fold, p<0.05 vs placebo) and chronically (p<0.05) and was well‐tolerated over the 12‐week supplementation period. Vascular endothelial function, measured by brachial artery FMD, was increased by 28% vs. baseline (p<0.05) after 12 weeks of supplementation (3.9±1.2 to 5.0±1.8%, p<0.05), but unchanged with placebo (3.8±1.4 to 4.0±1.5%, p>0.05). There were no differences in secondary outcomes of motor or cognitive function assessed with measures of the NIH Toolbox and other standardized tests between the sodium nitrite and placebo group after 12 weeks (all p>0.05). There were no differences in subject characteristics, body composition or circulating markers of inflammation and oxidative stress with sodium nitrite or placebo (all p>0.05). Collectively, these results indicate that sodium nitrite supplementation for 12 weeks improves endothelial function but does not affect motor or cognitive function in healthy, high‐functioning MA/O adults. These findings suggest therapeutic strategies to enhance nitrite levels may hold promise for treating age‐related vascular endothelial dysfunction.Clinicaltrials.gov Identifier: NCT02393742Support or Funding InformationNIH R01 AG013038, K01 DK115524 and NIH/NCATS Colorado CTSA Grant Number UL1 TR002535; American Physiology Society UGREF AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The oral microbiome has the potential to provide an important symbiotic function in human blood pressure physiology by contributing to the generation of nitric oxide (NO), an essential cardiovascular signaling molecule. NO is produced by the human body via conversion of arginine to NO by endogenous nitric oxide synthase (eNOS) but eNOS activity varies by subject. Oral microbial communities are proposed to supplement host NO production by reducing dietary nitrate to nitrite via bacterial nitrate reductases. Unreduced dietary nitrate is delivered to the oral cavity in saliva, a physiological process termed the enterosalivary circulation of nitrate. Previous studies demonstrated that disruption of enterosalivary circulation via use of oral antiseptics resulted in increases in systolic blood pressure. These previous studies did not include detailed information on the oral health of enrolled subjects. Using 16S rRNA gene sequencing and analysis, we determined whether introduction of chlorhexidine antiseptic mouthwash for 1 week was associated with changes in tongue bacterial communities and resting systolic blood pressure in healthy normotensive individuals with documented oral hygiene behaviors and free of oral disease. Tongue cleaning frequency was a predictor of chlorhexidine-induced changes in systolic blood pressure and tongue microbiome composition. Twice-daily chlorhexidine usage was associated with a significant increase in systolic blood pressure after 1 week of use and recovery from use resulted in an enrichment in nitrate-reducing bacteria on the tongue. Individuals with relatively high levels of bacterial nitrite reductases had lower resting systolic blood pressure. These results further support the concept of a symbiotic oral microbiome contributing to human health via the enterosalivary nitrate-nitrite-NO pathway. These data suggest that management of the tongue microbiome by regular cleaning together with adequate dietary intake of nitrate provide an opportunity for the improvement of resting systolic blood pressure.

Discovery of the production of gaseous molecules, such as nitric oxide and hydrogen sulfide, within the human body began a new concept in cellular signaling. Over the past 30 years, these molecules have been investigated and found to have extremely important beneficial effects in numerous chronic diseases. Gaseous signaling molecules that diffuse in three dimensions apparently contradict the selectivity and specificity afforded by normal ligand receptor binding and activation. This new concept has also created hurdles in the development of safe and efficacious drug therapy based on these molecules. Mechanisms involving formation of more stable intermediates and second messengers allow for new strategies for safe and effective delivery of these molecules for human disease. The purpose of this review is to highlight the biologic effects of nitric oxide and hydrogen sulfide, their seemingly indistinguishable effects, and how these molecules can be safely harnessed for drug development and precursors or substrates administered for human consumption through applied physiology.

Aging is associated with increased peripheral chemoreceptor activity, reduced nitric oxide (NO) bioavailability, and attenuation of cardiovagal baroreflex sensitivity (BRS), collectively increasing the risk of cardiovascular disease. Evidence suggests that NO may attenuate peripheral chemoreflex sensitivity and increase BRS. Exogenous inorganic nitrate ([Formula: see text]) increases NO bioavailability via the [Formula: see text]-[Formula: see text]-NO pathway. Our hypothesis was that inorganic [Formula: see text] supplementation would attenuate peripheral chemoreflex sensitivity and enhance spontaneous cardiovagal BRS in older adults. We used a randomized, placebo-controlled crossover design in which 13 older (67 ± 3 yr old) adults ingested beetroot powder containing (BRA) or devoid of (BRP) [Formula: see text] and [Formula: see text] daily over 4 wk. Spontaneous cardiovagal BRS was assessed over 15 min of rest and was quantified using the sequence method. Chemoreflex sensitivity was assessed via ~5 min of hypoxia (10% fraction of inspired O2) and reported as the slope of the relationship between O2 saturation (%[Formula: see text]) and minute ventilation (in l/min) or heart rate (in beats/min). Ventilatory responsiveness to hypoxia was reduced after BRA (from -0.14 ± 0.04 to -0.05 ± 0.02 l·min-1·%[Formula: see text]-1, P = 0.01) versus BRP (from -0.10 ± 0.05 to -0.11 ± 0.05 l·min-1·%[Formula: see text]-1, P = 0.80), with no differences in heart rate responsiveness (BRA: from -0.47 ± 0.06 to -0.33 ± 0.04 beats·min-1·%[Formula: see text]-1, BRP: from -0.48 ± 0.07 to -0.42 ± 0.06 beats·min-1·%[Formula: see text]-1) between conditions (interaction effect, P = 0.41). Spontaneous cardiovagal BRS was unchanged after BRA and BRP (interaction effects, P = 0.69, 0.94, and 0.39 for all, up, and down sequences, respectively), despite a reduction in resting systolic and mean arterial blood pressure in the experimental (BRA) group ( P < 0.01 for both). These findings illustrate that inorganic [Formula: see text] supplementation attenuates peripheral chemoreflex sensitivity without concomitant change in spontaneous cardiovagal BRS in older adults. NEW & NOTEWORTHY Exogenous inorganic nitrate supplementation attenuates ventilatory, but not heart rate, responsiveness to abbreviated hypoxic exposure in older adults. Additionally, inorganic nitrate reduces systolic and mean arterial blood pressure without affecting spontaneous cardiovagal baroreflex sensitivity. These findings suggest that inorganic nitrate may attenuate sympathetically oriented pathologies associated with aging.

Primary hypertension is a major risk factor for ischemic heart disease, stroke, and chronic kidney disease. Insights obtained from the study of rare Mendelian forms of hypertension have been invaluable in elucidating the mechanisms causing primary hypertension and development of antihypertensive therapies. Endothelial cells play a key role in the regulation of blood pressure; however, a Mendelian form of hypertension that is primarily due to endothelial dysfunction has not yet been described. Here, we show that the urea cycle disorder, argininosuccinate lyase deficiency (ASLD), can manifest as a Mendelian form of endothelial-dependent hypertension. Using data from a human clinical study, a mouse model with endothelial-specific deletion of argininosuccinate lyase (Asl), and in vitro studies in human aortic endothelial cells and induced pluripotent stem cell-derived endothelial cells from individuals with ASLD, we show that loss of ASL in endothelial cells leads to endothelial-dependent vascular dysfunction with reduced nitric oxide (NO) production, increased oxidative stress, and impaired angiogenesis. Our findings show that ASLD is a unique model for studying NO-dependent endothelial dysfunction in human hypertension.