Harsha K. Garg

Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction

The nitrite anion is an endogenous product of mammalian nitric oxide (NO) metabolism, a key intermediate in the nitrogen cycle in plants, and a constituent of many foods. Research over the past 6 years has revealed surprising biological and cytoprotective activity of this anion. Hypercholesterolemia causes a proinflammatory phenotype in the microcirculation. This phenotype appears to result from a decline in NO bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide, or both. Since nitrite has been shown to be potently cytoprotective and restore NO biochemical homeostasis, we investigated if supplemental nitrite could attenuate microvascular inflammation caused by a high cholesterol diet. C57Bl/6J mice were fed either a normal diet or a high cholesterol diet for 3 wk to induce microvascular inflammation. Mice on the high cholesterol diet received either nitrite-free drinking water or supplemental nitrite at 33 or 99 mg/l ad libitum in their drinking water. The results from this investigation reveal that mice fed a cholesterol-enriched diet exhibited significantly elevated leukocyte adhesion to and emigration through the venular endothelium as well as impaired endothelium-dependent relaxation in arterioles. Administration of nitrite in the drinking water inhibited the leukocyte adhesion and emigration and prevented the arteriolar dysfunction. This was associated with sparing of reduced tetrahydrobiopterin and decreased levels of C-reactive protein. These data reveal novel anti-inflammatory properties of nitrite and implicate the use of nitrite as a new natural therapy for microvascular inflammation and endothelial dysfunction associated with hypercholesterolemia.

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Nutritional epidemiology in the context of nitric oxide biology: A risk–benefit evaluation for dietary nitrite and nitrate

The discovery of the nitric oxide (NO) pathway in the 1980s represented a critical advance in understanding cardiovascular disease, and today a number of human diseases are characterized by NO insufficiency. In the interim, recent biomedical research has demonstrated that NO can be modulated by the diet independent of its enzymatic synthesis from l-arginine, e.g., the consumption of nitrite- and nitrate-rich foods such as fruits, leafy vegetables, and cured meats along with antioxidants. Regular intake of nitrate-containing food such as green leafy vegetables may ensure that blood and tissue levels of nitrite and NO pools are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis. However, some in the public perceive that dietary sources of nitrite and nitrate are harmful, and some epidemiological studies reveal a weak association between foods that contain nitrite and nitrate, namely cured and processed meats, and cancer. This paradigm needs revisiting in the face of undisputed health benefits of nitrite- and nitrate-enriched diets. This review will address and interpret the epidemiological data and discuss the risk-benefit evaluation of dietary nitrite and nitrate in the context of nitric oxide biology. The weak and inconclusive data on the cancer risk of nitrite, nitrate and processed meats are far outweighed by the health benefits of restoring NO homeostasis via dietary nitrite and nitrate. This risk/benefit balance should be a strong consideration before there are any suggestions for new regulatory or public health guidelines for dietary nitrite and nitrate exposures.

All-natural nitrite and nitrate containing dietary supplement promotes nitric oxide production and reduces triglycerides in humans

There is an emerging paradigm that certain foods promote nitric oxide (NO) production from the stepwise reduction of nitrate to nitrite to NO, providing an endothelium independent source of bioactive NO. We hypothesize that a unique formulation containing nitrate-rich beetroot along with Hawthorn berry shown to have a robust nitrite reductase activity would improve NO status in humans and modify cardiovascular risk factors. The trial was conducted at the Houston Institute for Clinical Research in Houston, Texas. Inclusion criteria for this double-blinded, placebo-controlled study were patients older than 40 years with 3 or more of the following cardiovascular risk factors: hypertension, obesity, hyperlipidemia, smoking, sedentary, family history of cardiovascular disease, and diabetes. Subjects were instructed to take either the NO dietary supplement called Neo40 Daily® or placebo twice daily on an empty stomach for 30 days. Patients taking the NO dietary supplement twice a day for 30 days led to a significant increase in both plasma nitrite (P < .01) and nitrate (P < .0001), indicating an increase in systemic NO availability. There was a statistically significant reduction in 72% of patients with elevated triglycerides (>150 mg/dL) after 30 days compared with their starting levels before taking the NO dietary supplement (168 ± 17 mg/dL vs 232 ± 19 mg/dL, P = .02). The strategy of formulating a combination of natural products and botanicals chosen specifically for their NO activity shows promise in restoring NO homeostasis in human subjects at risk for cardiovascular disease for use as a dietary supplement.

Dietary nitrite improves insulin signaling through GLUT4 translocation.

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.

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, cows, 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 Organizations 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.

Survey of residual nitrite and nitrate in conventional and organic/natural/uncured/indirectly cured meats available at retail in the United States.

A survey of residual nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in cured meats available at retail was conducted to verify concentrations in conventional (C) products and establish a baseline for organic/natural/uncured/indirectly cured (ONC) products. In this study, 470 cured meat products representing six major categories were taken from retail outlets in five major metropolitan cities across the United States. Random samples representing both C and ONC type products were analyzed for NO(2)(-) and NO(3)(-) content (ppm) using an ENO-20 high-performance liquid chromatography system equipped with a reverse phase column. Generally, there were no differences in NO(2)(-) concentrations between C and ONC meat categories, but a few ONC products surveyed in certain cities were lower in NO(3)(-) content. Pairwise comparisons between cities indicated that NO(2)(-) and NO(3)(-) contents of all C type products were not appreciably different, and the same was true for most ONC products. Numerical NO(2)(-) values were less variable than NO(3)(-) concentrations within each meat product category. NO(2)(-) concentrations were similar to those previously reported by Cassens ( Cassens , R. G. Residual nitrite in cured meat . Food Technol. 1997a , 51 , 53 - 55 ) in 1997. Residual NO(2)(-) and NO(3)(-) values in this study were numerically lower than those reported by NAS ( National Academy of Sciences . The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds ; National Academy Press : Washington, DC , 1981 ) in 1981. Data from this survey provide a benchmark of NO(2)(-) and NO(3)(-) concentrations for ONC products available at retail.

Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications.

Traditional Chinese medicine (TCM) has been used for centuries to treat and prevent certain ailments and diseases. Although TCM has served as mainstream medical care throughout Asia for many generations, it is considered an alternative medical system in much of the Western world. Because many TCMs are used primarily for cardiovascular indications characterized by a nitric oxide (NO) insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. We tested a group of convenience samples of TCMs obtained in the United States for endogenous nitrite, nitrate, nitroso, and nitrite reductase activity as well as their ability to relax isolated aortic rings. The results from this study reveal that all of the TCMs tested reveal NO bioactivity through their inherent nitrite and nitrate content and their ability to reduce nitrite to NO. Many of the TCM extracts contain a nitrite reductase activity greater by 1000 times that of biological tissues. Repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs.

A survey of nitrate and nitrite concentrations in conventional and organic-labeled raw vegetables at retail.

A national survey of the nitrate ( NO3(-)) and nitrite ( NO2(-)) concentrations in raw and highly consumed vegetables available at retail in the United States was conducted. A total of 194 samples of fresh broccoli, cabbage, celery, lettuce, and spinach categorized as conventional or organic by label were collected from 5 major cities in different geographic regions of the United States and analyzed to determine NO3(-) and NO2(-) concentrations. There were no differences in the mean NO2(-) values of conventional compared with organic vegetables taken from the 5 metropolitan areas. However, significant differences in mean pairwise comparisons between some conventional and organic vegetables for NO3(-) content were observed. The mean NO2(-) concentration of both conventional and organic vegetables ranged between 0.1 and 1.2 mg/kg of fresh weight (FW) with the exception of conventional spinach that contained 8.0 mg/kg FW. Mean NO3(-) contents of conventional broccoli, cabbage, celery, lettuce, and spinach were 394, 418, 1496, 851, and 2797 mg/kg FW, respectively, while their organic-labeled counterparts averaged 204, 552, 912, 844, and 1318 mg/kg FW. In most cases, organic vegetables were numerically lower in NO3(-) content than their conventional counterparts. Based on survey results, the finding that low NO3(-) levels were observed in some organic vegetables in different cities may warrant further study to determine if true differences exist, due to production practices, seasonal differences, and the magnitudes of those differences. Furthermore, the geographic differences in NO3(-) content of vegetables may flaw estimates of daily NO2(-) and NO3(-) exposure.

Urinary nitrate might be an early biomarker for pediatric acute kidney injury in the emergency department.

NO is involved in normal kidney function and perturbed in acute kidney injury (AKI). We hypothesized that urinary concentration of NO metabolites, nitrite, and nitrate would be lower in children with early AKI presenting to the emergency department (ED), when serum creatinine (SCr) was uninformative. Patients up to 19 y were recruited if they had a urinalysis and SCr obtained for routine care. Primary outcome, AKI, was defined by pediatric Risk, Injury, Failure, Loss of function, End-stage renal disease (pRIFLE) criteria. Urinary nitrite and nitrate were determined by HPLC. A total of 252 patients were enrolled, the majority (93%) of whom were without AKI. Although 18 (7%) had AKI by pRIFLE, 50% may not have had it identified by the SCr value alone at the time of visit. Median urinary nitrate was lower for injury versus risk (p = 0.03); this difference remained significant when the injury group was compared against the combined risk and no AKI groups (p = 0.01). Urinary nitrite was not significantly different between groups. Thus, low urinary nitrate is associated with AKI in the pediatric ED even when SCr is normal. Predictive potential of this putative urinary biomarker for AKI needs further evaluation in sicker patients.