Short-term dietary nitrate supplementation clinically significantly improves basal macrovascular function in postmenopausal women: a randomized, placebo-controlled, double blind, crossover clinical trial Provisionally Accepted

Jocelyn M. Delgado Spicuzza^1* Jigar Gosalia² Liezhou Zhong³ Catherine Bondonno^{4, 5} Kristina S. Petersen⁶ Mary Jane De Souza⁷ Elmira Alipour⁸ Daniel B. Kim-Shapiro⁸ Yasina B. Somani⁹ David N. Proctor²

• ¹Integrative and Biomedical Physiology, Huck Institutes of the Life Sciences, Intercollege Graduate Degree Program, The Pennsylvania State University (PSU), United States
• ²Kinesiology, The Pennsylvania State University (PSU), United States
• ³Nutrition & Health Innovation Research Institute, School of Medical Health Sciences, Edith Cowan University, Australia
• ⁴Nutrition & Health Innovation Research Institute, School of Medical and Health Sciences, Edith Cowan University, Australia
• ⁵Medical School, University of Western Australia, Australia
• ⁶Nutritional Sciences, The Pennsylvania State University (PSU), United States
• ⁷Kinesiology Department, The Pennsylvania State University (PSU), United States
• ⁸Physics, Wake Forest University, United States
• ⁹Biological Sciences, University of Leeds, United Kingdom

Introduction: Cardiovascular disease (CVD) is the leading cause of death in women, with increased risk following menopause. Dietary intake of beetroot juice and other plant-based nitrate-rich foods is a promising non-pharmacological strategy for increasing systemic nitric oxide and improving endothelial function in elderly populations. The purpose of this randomized, placebo-controlled, double blind, crossover clinical trial was to determine the effects of short-term dietary nitrate (NO3 -) supplementation, in the form of beetroot juice, on resting macrovascular endothelial function and endothelial resistance to whole-arm ischemia-reperfusion (IR) injury in postmenopausal women at two distinct stages of menopause. Methods: Early-(1-6 years following their final menstrual period (FMP), n=12) and late-(6+ years FMP, n=12) postmenopausal women consumed nitrate-rich (400 mg NO3 -/70mL) and nitrate-depleted beetroot juice (approximately 40 mg NO3 -/70 mL, placebo) daily for 7 days. Brachial artery flow-mediated dilation (FMD) was measured pre-supplementation (Day 0), and approximately 24 hours after the last beetroot juice (BR) dose (Day 8, post-7-day BR). Following, FMD was measured immediately post-IR injury and 15-minutes later (recovery). Results: Results of the linear mixed effects model revealed a significantly greater increase in resting FMD with 7-days of BRnitrate compared to BRplacebo (mean difference of 2.21%, 95 % CI [0.082, 4.34], p=0.042), however, neither treatment blunted the decline in post-IR injury FMD in either postmenopausal group. Our results suggest that 7-day BRnitrate-mediated endothelial protection is lost within the 24-hr. period following the final dose of BRnitrate. Conclusion: Our findings demonstrate that nitrate-mediated postmenopausal endothelial protection is dependent on the chronobiological variations in dietary nitrate metabolism and the timing of supplementation in relation to IR injury.