The inorganic anions nitrate (NO3-) and nitrite (NO2-) were previously thought to be inert end products of endogenous nitric oxide (NO) metabolism However, recent studies show that these supposedly inert anions can be recycled in vivo to form NO, representing an important alternative source of NO to the classical L-arginine-NO-synthase pathway, in particular in hypoxic states This Review discusses the emerging important biological functions of the nitrate-nitrite-NO pathway, and highlights studies that implicate the therapeutic potential of nitrate and nitrite in conditions such as myocardial infarction, stroke, systemic and pulmonary hypertension, and gastric ulceration

The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics

Nitrate content is an important quality characteristic of vegetables. Vegetable nitrate content is of interest to governments and regulators owing to the possible implications for health and to check that controls on the content are effective. Nitrate itself is relatively non-toxic but its metabolites may produce a number of health effects. Until recently nitrate was perceived as a purely harmful dietary component which causes infantile methaemoglobinaemia, carcinogenesis and possibly even teratogenesis. Recent research studies suggest that nitrate is actually a key part of our bodies' defences against gastroenteritis. In this review are reported: (1) vegetable classification as a function of nitrate accumulation; (2) vegetable contribution to the total dietary exposure of nitrate; (3) European Commission Regulation No. 563/2002 which sets limits for nitrate in lettuce and spinach; (4) the maximum levels set in some countries for beetroot, cabbage, carrot, celery, endive, Lamb's lettuce, potato, radish and rocket; (5) the results of surveys on the nitrate content of vegetables in Italy and other European countries.  2005 Society of Chemical Industry

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Nitrate in vegetables: toxicity, content, intake and EC regulation

Nitrate is generally considered a water pollutant and an undesirable fertilizer residue in the food chain Research in the 1970s indicated that, by reducing nitrate to nitrite, commensal bacteria might be involved in the pathogenesis of gastric cancers and other malignancies, as nitrite can enhance the generation of carcinogenic N-nitrosamines More recent studies indicate that the bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active nitrogen oxides could be beneficial Here, we will consider the evidence that nitrate-reducing commensals have a true symbiotic role in mammals and facilitate a previously unrecognized but potentially important aspect of the nitrogen cycle

Nitrate, bacteria and human health.

Nitric oxide metabolism and breakdown

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to λ phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

https://www.pnas.org/content/pnas/94/25/13997.full.pdf

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase

Dietary intake of nitrate generates salivary nitrite, which is acidified in the stomach, leading to a number of reactive intermediates of nitrogen, among which are the potentially carcinogenic N-nitrosamines. Acidified nitrite, however, also has antimicrobial activity which coincides with the formation of nitric oxide. The present study examines the antimicrobial effect in vitro of acidified nitrite on Salmonella enteritidis, Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, and Escherichia coli O157. First-order regression plots showed a linear inverse relationship of log-transformed proton and nitrite concentrations with MICs and MBCs after 30 min, 2 h, and 24 h of exposure (P S. enteritidis > S. typhimurium = Shigella sonnei > E. coli O157 (P < 0.05). Addition of SCN-, but not that of CI-, increased the antibacterial activity (paired t testing, P < 0.001). Generation of salivary nitrite from dietary nitrate may provide significant protection against gut pathogens in humans.

Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.

In a randomized crossover study, the protein binding and serum bactericidal activities (SBAs) of vancomycin and teicoplanin against Staphylococcus aureus and Streptococcus pyogenes were investigated in six healthy volunteers. Total concentrations in serum 1 h postadministration of vancomycin and teicoplanin were 25.5 +/- 2.7 and 10.8 +/- 8.9 mg/liter, respectively; mean free concentrations were 14.6 +/- 2.0 and 0.6 +/- 0.9 mg/liter, respectively. Protein binding for vancomycin was 36.9% +/- 2.87%, and that for teicoplanin was 97.4% +/- 2.6%. SBA determined in pooled human serum at 1 h against S. aureus ranged from 1:8 to 1:32 for both vancomycin and teicoplanin. Against S. pyogenes SBA at 1 h ranged from 1:16 to 1:128 for vancomycin and 1:256 to 1:2,048 for teicoplanin. In vitro kill curve studies showed that vancomycin is slowly bactericidal and that teicoplanin is bacteriostatic. Despite having less in vitro cidal activity against the study isolates and having low or unrecordable levels of free drug in serum, teicoplanin demonstrated a similar or better SBA than vancomycin. SBA was more closely related to the total drug level (r = 0.77 for S. aureus and r = 0.79 for S. pyogenes) than the free level of teicoplanin (r = 0.59 for S. aureus and r = 0.56 for S. pyogenes). The high level of protein binding of teicoplanin did not seem to impair its antibacterial activity as measured by its SBA.

Protein binding and serum bactericidal activities of vancomycin and teicoplanin.

BACKGROUND: In subjects on a low nitrate diet, plasma nitrate concentration and urinary nitrate excretion are thought to reflect endogenous nitric oxide (NO) production, and have been reported to increase during infective and inflammatory bowel disease. AIMS: To compare the extent of NO production in patients with infective versus non-infective forms of bowel dysfunction. SUBJECTS: Four groups: 20 healthy, volunteer clerical and laboratory staff, 12 patients with irritable bowel syndrome, 19 patients with inflammatory bowel disease, and 20 patients with infective gastroenteritis. METHODS: The plasma nitrate concentration was determined with a copper coated cadmium column and spectrophotometry. Mean and median plasma nitrate concentrations were calculated and compared within the four groups. Mann-Whitney distribution free rank testing was used to compare the median values. RESULTS: Median plasma nitrate concentrations in the four groups were: controls 32.7 mumol/l; irritable bowel syndrome 35.5 mumol/l; inflammatory bowel disease 35.1 mumol/l; and gastroenteritis 117.9 mumol/l (p < 0.001 gastroenteritis v all other groups). CONCLUSIONS: Plasma nitrate concentration could serve as a discriminant between infective and inflammatory or functional bowel disease in patients presenting with diarrhoea. It is not clear why there is considerable difference in endogenous nitrate synthesis in these two conditions, which are both characterised by severe gut inflammation.

https://gut.bmj.com/content/gutjnl/39/3/393.full.pdf

Plasma nitrate concentration in infective gastroenteritis and inflammatory bowel disease

BACKGROUND—There is evidence that endogenous nitrate synthesis is notably increased in patients with infective gastroenteritis.
AIMS—To determine whether this is due to nitric oxide (NO) production via the L-arginine/NO pathway.
METHODS—Seven male patients with community acquired bacterial gastroenteritis and 15 healthy male volunteers participated in this study. All patients had stool culture positive infective gastroenteritis. A bolus of 200 mg L-[15N]2-arginine was administered intravenously after an overnight fast. Urine was collected for the next 36 hours. Urinary [15N:14N]nitrate ratio was assessed by dry combustion in an isotope ratio mass spectrometer.
RESULTS—Mean 36 hour total urinary nitrate excretion in the gastroenteritis group was 5157 (577) µmol compared with 2594 (234) µmol in the control group (p<0.001). Thirty six hour urinary [15N]nitrate excretion was considerably higher in the gastroenteritis group compared with the control group (13782 (1665) versus 1698 (98) ηmol; p<0.001). These values represent 1.129 (0.139)% and 0.138 (0.007)% of [15N]nitrogen administered (p<0.001), respectively. Corrected 36 hour urinary [15N]nitrate excretion for urinary creatinine was also significantly higher in the patient compared with the control group (1934 (221) versus 303 (35) ηmol/mmol; p<0.001).
CONCLUSION—Results show notably enhanced nitrate synthesis due to increased activity of the L-arginine/NO pathway in patients with infective gastroenteritis.


Keywords: endothelium derived relaxing factor; L-[15N]2-arginine; nitrates; infection; diarrhoea

https://gut.bmj.com/content/gutjnl/45/3/355.full.pdf

Nitric oxide synthesis in patients with infective gastroenteritis

Clinical features, diagnosis and treatment of 286 women whose high vaginal swabs (HVS) submitted by their general practitioners showed pure, heavy growth of Staphylococcus aureus, beta haemolytic streptococci groups A, C or G, Streptococcus milleri, Streptococcus pneumoniae or Haemophilus influenzae were analysed. Women with group A, C and G streptococci frequently had clinical vulvovaginitis and although the numbers were too small for statistical confirmation, S. pneumoniae and H. influenzae appeared to cause clinical disease as well. The association of S. aureus or S. milleri with clinical vulvovaginitis was much less convincing. It seems relevant for laboratories to report sensitivities for group A, C and G streptococci. Further research is needed to determine the pathogenicity of S. pneumoniae and H. influenzae.

R S Dykhuizen

Papers

Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.

Protein binding and serum bactericidal activities of vancomycin and teicoplanin.

Plasma nitrate concentration in infective gastroenteritis and inflammatory bowel disease

Nitric oxide synthesis in patients with infective gastroenteritis

The high vaginal swab in general practice: clinical correlates of possible pathogens