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      A randomized controlled trial of nitrate supplementation in well-trained middle and older-aged adults

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          Abstract

          Purpose

          Nitrate (NO 3 -), through its conversion to nitrite (NO 2 -) and nitric oxide, has been shown to increase exercise tolerance in healthy younger adults and older diseased patients. Nitrate’s effect in well-trained middle to older-aged adults has not been studied. Therefore, the purpose of this investigation was to examine the effects of a NO 3 - rich beverage on submaximal constant work rate exercise time in well-trained middle to older-aged adults.

          Methods

          This was a randomized controlled cross-over trial with 15 well-trained middle to older-aged adults, 41–64 year-old, who received one of two treatments (NO 3 - rich beverage then placebo or placebo then NO 3 - rich beverage), after which an exercise test at 75 percent of the subject’s maximal work rate was completed.

          Results

          The NO 3 - rich beverage increased plasma NO 3 - and NO 2 - levels by 260 μM and 0.47 μM, respectively (p<0.001). Exercise time was not significantly different (p = 0.31) between the NO 3 - rich versus placebo conditions (1130±151 vs 1060±132 sec, respectively). Changes in exercise time between the two conditions ranged from a 55% improvement to a 40% decrease with the NO 3 - rich beverage. Oxygen consumption and rating of perceived exertion were not significantly different between the two conditions.

          Conclusion

          In middle to older-aged well-trained adults, NO 3 - supplementation has non-significant, albeit highly variable, effects on exercise tolerance.

          ClinicalTrials.gov Identifier: NCT03371966

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          Most cited references28

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          Drinking Water Nitrate and Human Health: An Updated Review

          Nitrate levels in our water resources have increased in many areas of the world largely due to applications of inorganic fertilizer and animal manure in agricultural areas. The regulatory limit for nitrate in public drinking water supplies was set to protect against infant methemoglobinemia, but other health effects were not considered. Risk of specific cancers and birth defects may be increased when nitrate is ingested under conditions that increase formation of N-nitroso compounds. We previously reviewed epidemiologic studies before 2005 of nitrate intake from drinking water and cancer, adverse reproductive outcomes and other health effects. Since that review, more than 30 epidemiologic studies have evaluated drinking water nitrate and these outcomes. The most common endpoints studied were colorectal cancer, bladder, and breast cancer (three studies each), and thyroid disease (four studies). Considering all studies, the strongest evidence for a relationship between drinking water nitrate ingestion and adverse health outcomes (besides methemoglobinemia) is for colorectal cancer, thyroid disease, and neural tube defects. Many studies observed increased risk with ingestion of water nitrate levels that were below regulatory limits. Future studies of these and other health outcomes should include improved exposure assessment and accurate characterization of individual factors that affect endogenous nitrosation.
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            Inorganic nitrate is a possible source for systemic generation of nitric oxide.

            Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.
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              Effects of dietary nitrate on blood pressure in healthy volunteers.

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Project administrationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 June 2020
                2020
                : 15
                : 6
                : e0235047
                Affiliations
                [1 ] Health and Exercise Department, Wake Forest University, Winston-Salem, North Carolina, United States of America
                [2 ] Physics Department, Wake Forest University, Winston-Salem, North Carolina, United States of America
                University of Alabama at Birmingham, UNITED STATES
                Author notes

                Competing Interests: This work was supported by a grant from Isagenix International LLC, Gilbert, AZ awarded to MJB. MJB, GDM, DBK-S and SB received funding from Isagenix LLC to conduct the study. Contents of the manuscript are the sole responsibility of the authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The funding source had no involvement with the study design, data collection, data analysis and interpretation or the decision to submit this work for publication. The results of the study are presented clearly, honestly, and without fabrication, falsification or inappropriate data manipulation. Dr. Kim-Shapiro is co-inventor on a patent related to the use of nitrite for cardiovascular conditions, and owns stock in and serves on the scientific advisory board for Beverage Operations LLC, which has licensed Wake Forest University intellectual properties and thus has a financial interest in Beverage Operations LLC. None of the other authors have any financial, non-financial, professional or personal competing interests to declare.

                Author information
                http://orcid.org/0000-0002-5798-1861
                http://orcid.org/0000-0003-0091-1616
                Article
                PONE-D-20-00673
                10.1371/journal.pone.0235047
                7310701
                32574223
                b4b8e029-0e99-4292-b23b-8dbdd653c6d9
                © 2020 Berry et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 17 January 2020
                : 5 June 2020
                Page count
                Figures: 5, Tables: 2, Pages: 17
                Funding
                Funding: This work was supported by a grant from Isagenix International LLC, Gilbert, AZ awarded to MJB. The funding source had no involvement with the study design, data collection, data analysis and interpretation or the decision to submit this work for publication.
                Categories
                Research Article
                Biology and Life Sciences
                Nutrition
                Diet
                Beverages
                Medicine and Health Sciences
                Nutrition
                Diet
                Beverages
                Medicine and Health Sciences
                Public and Occupational Health
                Physical Activity
                Physical Fitness
                Exercise
                Medicine and Health Sciences
                Sports and Exercise Medicine
                Exercise
                Biology and Life Sciences
                Sports Science
                Sports and Exercise Medicine
                Exercise
                Medicine and Health Sciences
                Vascular Medicine
                Blood Pressure
                Biology and Life Sciences
                Biochemistry
                Neurochemistry
                Neurochemicals
                Nitric Oxide
                Biology and Life Sciences
                Neuroscience
                Neurochemistry
                Neurochemicals
                Nitric Oxide
                People and Places
                Population Groupings
                Age Groups
                Elderly
                Biology and Life Sciences
                Anatomy
                Body Fluids
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                Blood Plasma
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Physical Sciences
                Chemistry
                Chemical Compounds
                Nitrates
                Medicine and Health Sciences
                Cardiology
                Heart Rate
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                All relevant data are within the Supporting Information file.

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