Dietary Nitrate from Plant Foods: A Conditionally Essential Nutrient for Cardiovascular Health.

Under specific conditions, such as catabolic stress or systemic inflammation, endogenous nutrient production becomes insufficient and exogenous supplementation (for example, through dietary intake) is required. Herein, we propose consideration of a dietary nitrate from plant foods as a conditionally essential nutrient for cardiovascular health based on its role in nitric oxide homeostasis. Nitrate derived from plant foods may function as a conditionally essential nutrient, whereas nitrate obtained from other dietary sources, such as drinking water and cured/processed meats, warrants separate consideration because of the associated health risks. We have surveyed the literature and summarized epidemiological evidence regarding the effect of dietary nitrate on cardiovascular disease and risk factors. Meta-analyses and population-based observational studies have consistently demonstrated an inverse association of dietary nitrate with blood pressure and cardiovascular disease outcomes. Considering the available evidence, we suggest 2 different approaches to providing dietary guidance on nitrate from plant-based dietary sources as a nutrient: the Dietary Reference Intakes developed by the National Academies of Sciences, Engineering, and Medicine, and the dietary guidelines evaluated by the Academy of Nutrition and Dietetics. Ultimately, this proposal underscores the need for food-based dietary guidelines to capture the complex and context-dependent relationships between nutrients, particularly dietary nitrate, and health.

Accuracy of Parent-Measured Weight and Height of Preschool Children at Home With Increasing Levels of Instruction.

Background: The purpose of this study was to determine how accurately parents measure their preschool child's weight and height with increasing levels of instruction. Methods: Parents measured their child's (n = 30 dyads) weight (own weight scale) and height (soft tape measure) using three levels of instruction: instructional guide (level 1); guide, demonstration video (level 2); and guide, video, and virtual monitoring (level 3), which were compared to researcher measurements (electronic weight scale, Stadiometer). Paired t-tests were used to determine differences between researcher and parent measurements and between the three parent levels. Inaccurate classifications were calculated using parent-measured values for the four categories (underweight, healthy, overweight, obese). Results: Raw mean parent-measured weights (17.4 ± 2.3 kg) differed from researcher by 0.2 kg (level 1), 0.3 kg (level 2), and 0.1 kg (level 3). Raw mean parent-measured heights (104.0 ± 5.9 cm) differed from researcher by 0.9 cm (level 1, p = 0.005), 0.4 cm (level 2, NS), and 0.3 cm (level 3, NS). Across all levels, 48.9% and 65.5% parents overmeasured their children's weights and heights, respectively. Using parent-measured values, 29.4% of children were classified high while 70.5% were classified low. Parents were more likely to make errors if their children were on the borderline between any of the two weight categories. Conclusion: Findings indicate that an instructional guide with demonstration video is helpful in improving the parents' accuracy of their children's weights and heights. More research is needed to determine accuracy in population other than White parents with high education levels and children under overweight and obese category.

A Genetically Heterogeneous Rat Model with Divergent Mitochondrial Genomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Dietary nitrate biochemistry and physiology. An update on clinical benefits and mechanisms of action.

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.

Nitrate exposure reprograms hepatic amino acid and nutrient sensing pathways prior to exercise: A metabolomic and transcriptomic investigation in zebrafish (Danio rerio).

Scope: Nitrate supplementation is a popular ergogenic aid that improves exercise performance by reducing oxygen consumption during exercise. We investigated the effect of nitrate exposure and exercise on metabolic pathways in zebrafish liver. Materials and methods: Fish were exposed to sodium nitrate (606.9 mg/L), or control water, for 21 days and analyzed at intervals during an exercise test. We utilized untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and measured gene expression of 24 genes central to energy metabolism and redox signaling. Results: We observed a greater abundance of metabolites involved in endogenous nitric oxide (NO) metabolism and amino acid metabolism in nitrate-treated liver at rest, compared to rested controls. In the absence of exercise, nitrate treatment upregulated expression of genes central to nutrient sensing (pgc1a), protein synthesis (mtor) and purine metabolism (pnp5a and ampd1) and downregulated expression of genes involved in mitochondrial fat oxidation (acaca and cpt2). Conclusion: Our data support a role for sub-chronic nitrate treatment in the improvement of exercise performance, in part, by improving NO bioavailability, sparing arginine, and modulating hepatic gluconeogenesis and glycolytic capacity in the liver.

Long Term Survivals in Aggressive Primary Brain Malignancies Treated With an Adjuvant Ketogenic Diet.

Aggressive primary brain tumors (APBT) glioblastoma multiforme and grade IV astrocytoma are treated with multimodality treatments that include surgery to remove as much tumor as possible without sacrificing neurological function followed by radiation therapy and chemotherapy usually temozolomide. Survivals in adults are in the range of 8-16 months. The addition of a ketogenic diet (KD) to rodents with transplanted brain tumors increased survival in nine of 11 animals to over 299 days compared to survival in untreated controls of 33 days and radiation only controls of 38 days. We treated humans with APBT with standard of care neurosurgery immediately followed by 6 weeks of an adjuvant ketogenic diet concurrent with radiation therapy and temozolomide. Twice daily measurements of blood ketones and glucose were recorded and the patients' diet was modified toward the goal of maintaining blood ketone levels approaching 3 mM. Of the nine patients who completed the protocol three younger patients age 32, 28, and 22 at enrollment are alive and employed with clinically stable disease and brain images 74, 58, and 52 months since diagnosis. All the six older patients mean age 55 have died with disease progression detected on average 8 months after Dx. In conclusion: 1. It is possible to implement and maintain dietary induced ketosis in patients with APBT; 2. The longer survivals observed in younger patients treated with KD need to be confirmed in larger studies that should be focused on younger patients possibly under age 40.

Nitrate-induced improvements in exercise performance are coincident with exuberant changes in metabolic genes and the metabolome in zebrafish (Danio rerio) skeletal muscle.

Dietary nitrate supplementation improves exercise performance by reducing the oxygen cost of exercise and enhancing skeletal muscle function. However, the mechanisms underlying these effects are not well understood. The purpose of this study was to assess changes in skeletal muscle energy metabolism associated with exercise performance in a zebrafish model. Fish were exposed to sodium nitrate (60.7 mg/L, 303.5 mg/L, 606.9 mg/L), or control water, for 21 days and analyzed at intervals (5, 10, 20, 30, 40 cm/s) during a 2-h strenuous exercise test. We measured oxygen consumption during an exercise test and assessed muscle nitrate concentrations, gene expression, and the muscle metabolome before, during, and after exercise. Nitrate exposure reduced the oxygen cost of exercise and increased muscle nitrate concentrations at rest, which were reduced with increasing exercise duration. In skeletal muscle, nitrate treatment upregulated expression of genes central to nutrient sensing (mtor), redox signaling (nrf2a), and muscle differentiation (sox6). In rested muscle, nitrate treatment increased phosphocreatine (P = 0.002), creatine (P = 0.0005), ATP (P = 0.0008), ADP (P = 0.002), and AMP (P = 0.004) compared with rested-control muscle. Following the highest swimming speed, concentration of phosphocreatine (P = 8.0 × 10-5), creatine (P = 6.0 × 10-7), ATP (P = 2.0 × 10-6), ADP (P = 0.0002), and AMP (P = 0.004) decreased compared with rested nitrate muscle. Our data suggest nitrate exposure in zebrafish lowers the oxygen cost of exercise by changing the metabolic programming of muscle prior to exercise and increasing availability of energy-rich metabolites required for exercise.NEW & NOTEWORTHY We show that skeletal muscle nitrate concentration is higher with supplementation at rest and was lower in groups with increasing exercise duration in a zebrafish model. The higher availability of nitrate at rest is associated with upregulation of key nutrient-sensing genes and greater availability of energy-producing metabolites (i.e., ATP, phosphocreatine, glycolytic intermediates). Overall, nitrate supplementation may lower oxygen cost of exercise through improved fuel availability resulting from metabolic programming of muscle prior to exercise.

Nitrate and nitrite exposure leads to mild anxiogenic-like behavior and alters brain metabolomic profile in zebrafish.

Dietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. We tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We utilized targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the extent to which treatment resulted in changes in nitrate or nitrite concentrations in the brain and altered the brain metabolome. Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2-4 weeks and free swim, startle response, and shuttle box assays were performed. Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment was associated with mild anxiogenic-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted metabolomics analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. Untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment. Overall, the depletion in brain metabolites is plausibly associated with the regulation of neuronal activity including statistically significant reductions in the inhibitory neurotransmitter γ-aminobutyric acid (GABA; 18-19%), and its precursor, glutamine (17-22%). Nitrate treatment caused significant depletion in the brain concentration of fatty acids including linoleic acid (LA) by 50% and arachidonic acid (ARA) by 80%; nitrite treatment caused depletion of LA by ~90% and ARA by 60%, change which could alter the function of dopaminergic neurons and affect behavior. Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health. It is plausible that indirect NO-mediated mechanisms may be responsible for the nitrate and nitrite-mediated effects on the brain metabolome and behavior in zebrafish.

Coffee Consumption and C-Reactive Protein Levels: A Systematic Review and Meta-Analysis.

Coffee contains bioactive compounds with anti-inflammatory properties, and its consumption may reduce c-reactive protein (CRP) levels, a biomarker of chronic inflammation. A previous meta-analysis reported no overall association between blood CRP level and coffee consumption by modeling the coffee consumption in categories, with substantial heterogeneity. However, the coffee cup volume was not considered. We conducted a systematic review and dose-response meta-analysis investigating the association between coffee consumption and CRP levels reported in previous observational studies. A dose-response meta-analysis was conducted by mixed-effects meta-regression models using the volume of coffee consumed as metric. Eleven studies from three continents were identified using the PubMed database, totaling 61,047 participants. Three studies with the largest sample sizes observed a statistically significant association between coffee and CRP levels, which was inverse among European and United States (US) women and Japanese men (1.3%-5.5% decrease in CRP per 100 mL of coffee consumed) and positive among European men (2.2% increase). Other studies showed no statistically significant associations. When all studies were combined in the dose-response meta-analysis, no statistically significant associations were observed among all participants or when stratified by gender or geographic location, reflecting the conflicting associations reported in the included studies. Further studies are warranted to explore these inconsistent associations.

Treatment with Nitrate, but Not Nitrite, Lowers the Oxygen Cost of Exercise and Decreases Glycolytic Intermediates While Increasing Fatty Acid Metabolites in Exercised Zebrafish.

BACKGROUND: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. OBJECTIVES: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. METHODS: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. RESULTS: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., ß-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. CONCLUSIONS: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

Increasing dietary nitrate has no effect on cancellous bone loss or fecal microbiome in ovariectomized rats.

SCOPE: Studies suggest diets rich in fruit and vegetables reduce bone loss, although the specific compounds responsible are unknown. Substrates for endogenous nitric oxide (NO) production, including organic nitrates and dietary nitrate, may support NO production in age-related conditions, including osteoporosis. We investigated the capability of dietary nitrate to improve NO bioavailability, reduce bone turnover and loss. METHODS AND RESULTS: Six-month-old Sprague Dawley rats [30 ovariectomized (OVX) and 10 sham-operated (sham)] were randomized into three groups: (i) vehicle (water) control, (ii) low-dose nitrate (LDN, 0.1 mmol nitrate/kg bw/day), or (iii) high-dose nitrate (HDN, 1.0 mmol nitrate/kg bw/day) for three weeks. The sham received vehicle. Serum bone turnover markers; bone mass, mineral density, and quality; histomorphometric parameters; and fecal microbiome were examined. Three weeks of LDN or HDN improved NO bioavailability in a dose-dependent manner. OVX resulted in cancellous bone loss, increased bone turnover, and fecal microbiome changes. OVX increased relative abundances of Firmicutes and decreased Bacteroideceae and Alcaligenaceae. Nitrate did not affect the skeleton or fecal microbiome. CONCLUSION: These data indicate that OVX affects the fecal microbiome and that the gut microbiome is associated with bone mass. Three weeks of nitrate supplementation does not slow bone loss or alter the fecal microbiome in OVX.

Aging and serum MCP-1 are associated with gut microbiome composition in a murine model.

Introduction. Age is the primary risk factor for major human chronic diseases, including cardiovascular disorders, cancer, type 2 diabetes, and neurodegenerative diseases. Chronic, low-grade, systemic inflammation is associated with aging and the progression of immunosenescence. Immunosenescence may play an important role in the development of age-related chronic disease and the widely observed phenomenon of increased production of inflammatory mediators that accompany this process, referred to as "inflammaging." While it has been demonstrated that the gut microbiome and immune system interact, the relationship between the gut microbiome and age remains to be clearly defined, particularly in the context of inflammation. The aim of our study was to clarify the associations between age, the gut microbiome, and pro-inflammatory marker serum MCP-1 in a C57BL/6 murine model. Results. We used 16S rRNA gene sequencing to profile the composition of fecal microbiota associated with young and aged mice. Our analysis identified an association between microbiome structure and mouse age and revealed specific groups of taxa whose abundances stratify young and aged mice. This includes the Ruminococcaceae, Clostridiaceae, and Enterobacteriaceae. We also profiled pro-inflammatory serum MCP-1 levels of each mouse and found that aged mice exhibited elevated serum MCP-1, a phenotype consistent with inflammaging. Robust correlation tests identified several taxa whose abundance in the microbiome associates with serum MCP-1 status, indicating that they may interact with the mouse immune system. We find that taxonomically similar organisms can exhibit differing, even opposite, patterns of association with the host immune system. We also find that many of the OTUs that associate with serum MCP-1 stratify individuals by age. Discussion. Our results demonstrate that gut microbiome composition is associated with age and the pro-inflammatory marker, serum MCP-1. The correlation between age, relative abundance of specific taxa in the gut microbiome, and serum MCP-1 status in mice indicates that the gut microbiome may play a modulating role in age-related inflammatory processes. These findings warrant further investigation of taxa associated with the inflammaging phenotype and the role of gut microbiome in the health status and immune function of aged individuals.

Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature.

BACKGROUND: Based on the hypothesis that cancer cells may not be able to metabolize ketones as efficiently as normal brain cells, the ketogenic diet (KD) has been proposed as a complementary or alternative therapy for treatment of malignant gliomas. CASE PRESENTATION: We report here our experience in treating two glioma patients with an IRB-approved energy-restricted ketogenic diet (ERKD) protocol as monotherapy and review the literature on KD therapy for human glioma patients. An ERKD protocol was used in this pilot clinical study. In addition to the two patients who enrolled in this study, we also reviewed findings from 30 other patients, including 5 patients from case reports, 19 patients from a clinical trial reported by Rieger and 6 patients described by Champ. A total of 32 glioma patients have been treated using several different KD protocols as adjunctive/complementary therapy. The two patients who enrolled in our ERKD pilot study were monitored with twice daily measurements of blood glucose and ketones and daily weights. However, both patients showed tumor progression while on the ERKD therapy. Immunohistochemistry reactions showed that their tumors had tissue expression of at least one of the two critical mitochondrial ketolytic enzymes (succinyl CoA: 3-oxoacid CoA transferase, beta-3-hydroxybutyrate dehydrogenase 1). The other 30 glioma patients in the literature were treated with several different KD protocols with varying responses. Prolonged remissions ranging from more than 5 years to 4 months were reported in the case reports. Only one of these patients was treated using KD as monotherapy. The best responses reported in the more recent patient series were stable disease for approximately 6 weeks. No major side effects due to KD have been reported in any of these patients. CONCLUSIONS: We conclude that 1. KD is safe and without major side effects; 2. ketosis can be induced using customary foods; 3. treatment with KD may be effective in controlling the progression of some gliomas; and 4. further studies are needed to determine factors that influence the effectiveness of KD, whether as a monotherapy, or as adjunctive or supplemental therapy in treating glioma patients. TRIAL REGISTRATION: ClinicalTrials.gov# NCT01535911.

Immunomodulation by dietary long chain omega-3 fatty acids and the potential for adverse health outcomes.

Recommendations to consume fish for prevention of cardiovascular disease (CVD), along with the U.S. Food and Drug Administration-approved generally recognized as safe (GRAS) status for long chain omega-3 fatty acids, may have had the unanticipated consequence of encouraging long-chain omega-3 (ω-3) fatty acid [(eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] supplementation and fortification practices. While there is evidence supporting a protective role for EPA/DHA supplementation in reducing sudden cardiac events, the safety and efficacy of supplementation with LCω-3PUFA in the context of other disease outcomes is unclear. Recent studies of bacterial, viral, and fungal infections in animal models of infectious disease demonstrate that LCω-3PUFA intake dampens immunity and alters pathogen clearance and can result in reduced survival. The same physiological properties of EPA/DHA that are responsible for the amelioration of inflammation associated with chronic cardiovascular pathology or autoimmune states, may impair pathogen clearance during acute infections by decreasing host resistance or interfere with tumor surveillance resulting in adverse health outcomes. Recent observations that high serum LCω-3PUFA levels are associated with higher risk of prostate cancer and atrial fibrillation raise concern for adverse outcomes. Given the widespread use of supplements and fortification of common food items with LCω-3PUFA, this review focuses on the immunomodulatory effects of the dietary LCω-3PUFAs, EPA and DHA, the mechanistic basis for potential negative health outcomes, and calls for biomarker development and validation as rational first steps towards setting recommended dietary intake levels.

Exploration of a symptoms experience in people with obesity-related nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent condition strongly associated with obesity that can result in premature death. Little is known about the symptoms experience in this progressive disease, preventing health care providers from intervening in the early stages. PURPOSE: This study explicated symptoms in persons with NAFLD at higher risk of disease progression defined as the presence of one or two copies of the PNPLA3 (rs738409)-G allele. METHOD: Guided by the Symptoms Experience Model, 42 persons older than 21 years of age with diagnosed NAFLD were recruited from Western Michigan specialty offices in this cross-sectional descriptive study design. The Memorial Symptom Assessment Scale was used to measure the symptoms experience. DISCUSSION: Participants (97%) experienced 1 or more symptoms (average number of symptoms 12.02, standard deviation = 8.817). There was no statistically significant relationship between symptoms and the PNPLA3 (rs738409) variants. Significant predictors of mean frequency, severity, and distress of symptoms (the Total Memorial Symptom Scale) (F[15, 25] = 2.609, p = .016) were identified. CONCLUSION: People with NAFLD experience an average of 12 symptoms.

Concentration- and stage-specific effects of nitrite on colon cancer cell lines.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM-100 µM and inhibits stage 3 HCT15 proliferation at 100 nM-1 µM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 µM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 µM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 µM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.

Dietary nitrates, nitrites, and cardiovascular disease.

Dietary nitrate (NO(3)), nitrite (NO(2)), and arginine can serve as sources for production of NO(x) (a diverse group of metabolites including nitric oxide, nitrosothiols, and nitroalkenes) via ultraviolet light exposure to skin, mammalian nitrate/nitrite reductases in tissues, and nitric oxide synthase enzymes, respectively. NO(x) are responsible for the hypotensive, antiplatelet, and cytoprotective effects of dietary nitrates and nitrites. Current regulatory limits on nitrate intakes, based on concerns regarding potential risk of carcinogenicity and methemoglobinemia, are exceeded by normal daily intakes of single foods, such as soya milk and spinach, as well as by some recommended dietary patterns such as the Dietary Approaches to Stop Hypertension diet. This review includes a call for regulatory bodies to consider all available data on the beneficial physiologic roles of nitrate and nitrite in order to derive rational bases for dietary recommendations.

Nitrate and nitrite content of human, formula, bovine, and soy milks: implications for dietary nitrite and nitrate recommendations.

BACKGROUND: Estimation of nitrate and nitrite concentrations of milk sources may provide insight into potential health risks and benefits of these food sources for infants, children, and adults. The World Health Organization and American Academy of Pediatrics recommends exclusive consumption of human milk for the first 6 months of life. Human milk is known to confer significant nutritional and immunological benefits for the infant. Consumption of formula, cow's, and soy milk may be used as alternatives to human milk for infants. METHODS: We sought to estimate potential exposure to nitrate and nitrite in human, formula, bovine, and soy milk to inform total dietary exposure estimates and recommendations. Using sensitive quantitative methodologies, nitrite and nitrate were analyzed in different samples of milk. RESULTS: Human milk concentrations of colostrum (expressed days 1-3 postpartum; n=12), transition milk (expressed days 3-7 postpartum; n=17), and mature milk (expressed >7 days postpartum; n=50) were 0.08 mg/100 mL nitrite and 0.19 mg/100 mL nitrate, 0.001 mg/100 mL nitrite and 0.52 mg/100 mL nitrate, and 0.001 mg/100 mL nitrite and 0.3 mg/100 mL nitrate, respectively, revealing that the absolute amounts of these anions change as the composition of milk changes. When expressed as a percentage of the World Health Organization's Acceptable Daily Intake limits, Silk® Soy Vanilla (WhiteWave Foods, Broomfield, CO) intake could result in high nitrate intakes (104% of this standard), while intake of Bright Beginnings Soy Pediatric® formula (PBM Nutritionals, Georgia, VT) could result in the highest nitrite intakes (383% of this standard). CONCLUSIONS: The temporal relationship between the provision of nitrite in human milk and the development of commensal microbiota capable of reducing dietary nitrate to nitrite supports a hypothesis that humans are adapted to provide nitrite to the gastrointestinal tract from birth. These data support the hypothesis that the high concentrations of breastmilk nitrite and nitrate are evidence for a physiologic requirement to support gastrointestinal and immune homeostasis in the neonate.

Food sources of nitrates and nitrites: the physiologic context for potential health benefits.

The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Despite the physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites as healthful dietary components has received little attention. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, we found that nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds the World Health Organization's Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients.