Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference

Abstract

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CV B ), and analytical imprecision (CV A ) in experimental endpoints. However, no study has quantified the CV A and CV B of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CV B , CV A, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CV B of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CV B in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ± 5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (~12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitratereducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CV B for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CV B for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CV B for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), 3Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CV B in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

Ergogenic Effect of Nitrate Supplementation: A Systematic Review and Meta-analysis

“…The ergogenic potential of NO 3 − supplementation is largely dependent on the reduction of concentrated NO 3 − to nitrite (NO 2 − ), which is regulated by anaerobic bacteria in the oral cavity ( 121 – 123 ). Although the oral microbiome may be disturbed by many oral substances (e.g., antibiotics, antibacterial mouthwash, gum chewing, etc.)…”

“…( 124 ), only about 50% of studies reported controlled environments for the oral microbiome; for example, “subjects were asked to abstain from using antibacterial mouthwash and chewing gum…” ( 115 ) Thus, variability in the oral microbiome may contribute to the observed variability in the efficacy of NO 3 − supplementation. Furthermore, recent studies have demonstrated that under controlled conditions, the reduction of NO 3 − to NO 2 − in biological fluids varies substantially within individuals across repeated visits ( 121 ). The large variability in the performance-enhancing effects of NO 3 − supplementation may be due to the profound biological variability of the oral microbiome ( 121 ), and this postulation warrants future investigation.…”

“…Furthermore, recent studies have demonstrated that under controlled conditions, the reduction of NO 3 − to NO 2 − in biological fluids varies substantially within individuals across repeated visits ( 121 ). The large variability in the performance-enhancing effects of NO 3 − supplementation may be due to the profound biological variability of the oral microbiome ( 121 ), and this postulation warrants future investigation. One approach to reduce the potential impact of the variability of oral microbiome is to provide NO 3 − supplementation for several days before an exercise test, which has been shown to increase abundance of some bacteria capable of NO 3 − reduction ( 123 ), which may optimize the effectiveness of NO 3 − supplementation.…”

Isolation and Characterization of Nitrate-Reducing Bacteria as Potential Probiotics for Oral and Systemic Health

“…In fact, disruption of oral microbial communities by over-use of antiseptics or antibiotics will interfere with the nitrate–nitrite–nitric oxide pathway ( Kapil et al, 2013 ), as well as food habits with low dietary nitrate ( Ashworth et al, 2015 ). As a result of these, humans appear to vary widely in their NRC ( Doel et al, 2004 ; Liddle et al, 2019 ), with important potential consequences for diseases or conditions that are influenced by a deficit in nitric oxide, ranging from cardiovascular diseases to reduced sport performance or diabetes development, among others ( Lundberg et al, 2018 ). The current work shows that, in oral bacterial communities with a slightly reduced NRC, the supplementation of nitrate may suffice to restore and promote efficient nitrate reduction; however, our data also show that in individuals with extremely low NRC, the addition of a nitrate-reducing probiotic could be instrumental for a recovery of the function.…”

“…The amount of nitrate-reducing species varies among individuals and, accordingly, the NRC as well ( Liddle et al, 2019 ). Individuals with low baseline levels of nitrate-reducing species could use nitrate as a prebiotic to increase the levels of these bacteria over time.…”