Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Original article Sex differences in the nitrate-nitrite-NO • pathway: Role of oral nitrate- reducing bacteria Vikas Kapil a , Krishnaraj S. Rathod a , Rayomand S. Khambata a , Manpreet Bahra a , Shanti Velmurugan a , Amandeep Purba a , David S. Watson a , Michael R. Barnes a , William G. Wade b , Amrita Ahluwalia a, ⁎ a William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK b Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Newark Street, London E1 2AT, UK ARTICLE INFO Keywords: Nitric Oxide Nitrate Nitrite Oral microbiome Community profiling Nitrate reductase ABSTRACT Oral reduction of nitrate to nitrite is dependent on the oral microbiome and is the first step of an alternative mammalian pathway to produce nitric oxide in humans. Preliminary evidence suggests important sex differences in this pathway. We prospectively investigated sex-differences following inorganic nitrate supplementation on nitrate/nitrite levels and vascular function, and separately examined sex differences in oral nitrate reduction, and oral microbiota by 16S rRNA profiling. At baseline, females exhibit higher nitrite levels in all biological matrices despite similar nitrate levels to males. Following inorganic nitrate supplementation, plasma nitrite was increased to a significantly greater extent in females than in males and pulse wave velocity was only reduced in females. Females exhibited higher oral bacterial nitrate-reducing activity at baseline and after nitrate supple- mentation. Despite these differences, there were no differences in the composition of either the total salivary microbiota or those oral taxa with nitrate reductase genes. Our results demonstrate that females have augmented oral nitrate reduction that contributes to higher nitrite levels at baseline and also after inorganic nitrate sup- plementation, however this was not associated with differences in microbial composition (clinicaltrials.gov: NCT01583803). 1. Background Over the past two decades, a new paradigm for the production of nitric oxide ( • NO) in vivo in mammals has emerged involving the che- mical reduction of the inorganic anions nitrate (NO 3 - ) and nitrite (NO 2 - ) to bioactive • NO [1–5]. This pathway depends upon the host micro- biome for nitrate reduction [6] and the specific activity of mammalian metallo-enzymes for nitrite reduction [7,8] and is thought to comple- ment the classical • NO synthase (NOS)/L-arginine pathway [9]. Im- portantly, • NO derived from nitrite exerts a profile of activity very si- milar to the physiological functions of NOS-derived • NO, including vasodilatation, amongst several other effects. Accordingly, nitrite ad- ministration in humans causes venous and arteriolar vasodilatation with concomitant reductions in blood pressure (BP) [10–12]; responses that do not suffer from tachyphylaxis with repeated application [11]. Circulating levels of nitrite are derived from 2 main sources; the oxidative metabolism of endogenous • NO and the diet. The latter is derived from inorganic nitrite found in processed foods or through the in-vivo bacterial conversion of inorganic nitrate to nitrite; an event particularly occurring within the oral cavity [7]. Once within the cir- culation nitrite is converted to • NO by specific enzymes expressed predominantly within the blood vessel wall and erythrocytes [[10,13–15], and it is this phenomenon that is thought to underlie dietary nitrate-induced reductions of BP in humans [4,5]. The critical importance of the oral microbiome in conversion of nitrate to nitrite is reflected by the fact that the vascular effects of ni- trate supplementation are blocked by avoidance of swallowing of saliva or the use of anti-bacterial mouthwash [5,16,17]. Interestingly, post hoc analysis of BP-lowering efficacy and nitrite levels following dietary nitrate ingestion, in healthy volunteers, revealed potentially important sex differences in the nitrate-nitrite- • NO pathway [18], with suggestions of greater activity of this pathway in women compared to men. How- ever, a prospective study assessing whether sex differences exist and delineation of the exact underlying mechanisms for any apparent dif- ference is still lacking. Herein, we describe our findings from pro- spective investigations assessing whether sex differences in the https://doi.org/10.1016/j.freeradbiomed.2018.07.010 Received 25 April 2018; Received in revised form 15 June 2018; Accepted 18 July 2018 ⁎ Corresponding author. E-mail address: a.ahluwalia@qmul.ac.uk (A. Ahluwalia). Free Radical Biology and Medicine 126 (2018) 113–121 Available online 20 July 2018 0891-5849/ © 2018 Published by Elsevier Inc. T