title: Measuring nitrate reductase activity from human and rodent tongues.
abstract: Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, United States.
authors.forename: Khandaker A
authors.initials: KA
authors.lastname: Ahmed
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, United States.
authors.forename: Alexandria L
authors.initials: AL
authors.lastname: Nichols
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, United States.
authors.forename: Jaideep
authors.initials: J
authors.lastname: Honavar
authors.affiliation: Department of Medicine, Pulmonary, Allergy and Critical Care, University of Alabama at Birmingham, United States.
authors.forename: Mark T
authors.initials: MT
authors.lastname: Dransfield
authors.affiliation: Anesthesiology and Pulmonary Injury Repair Center, University of Alabama at Birmingham, United States.
authors.forename: Sadis
authors.initials: S
authors.lastname: Matalon
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, United States. Electronic address: rakeshpatel@uabmc.edu.
authors.forename: Rakesh P
authors.initials: RP
authors.lastname: Patel
mesh_terms: D000328:Adult; D000818:Animals; D001966:Bromine / Q000633:toxicity; D002710:Chlorhexidine / Q000494:pharmacology; D015169:Colony Count, Microbial; D005260:Female; D006801:Humans; D019570:Inhalation Exposure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D050901:Nitrate Reductase / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014028:Tobacco Smoke Pollution; D014059:Tongue / Q000187:drug effects / Q000201:enzymology* / Q000382:microbiology; D055815:Young Adult
chemical_list: D014028:Tobacco Smoke Pollution; D009569:Nitric Oxide; D050901:Nitrate Reductase; D002710:Chlorhexidine; D001966:Bromine
keywords: Antiseptic; Chlorohexidine; Halogen; Microbiome; Nitric oxide; Smoking