Assessing the biological variation of nitrate and nitrite concentration in human plasma and saliva following ingestion of nitrate-rich beetroot juice

Introduction

Dietary nitrate (NO3-) supplementation increases circulating nitric oxide (NO) metabolites and has been shown to improve exercise performance in some, but not all, studies To date, the intra-individual biological variation in NO metabolite pharmacokinetics have not been quantified, despite these data being important to determine the thresholds that are sufficient to improve performance. The aim of this study was to determine the biological variation and critical difference (CD) in salivary and plasma nitrite [NO2-] and [NO3-] before and after ingestion of NO3--rich beetroot juice (BR).

Methods
Ten healthy male participants (age 25 ± 5 years and body mass 81 ± 11 kg) completed three identical trials ~7 days apart. Following a 30 min period lying supine, blood and saliva were collected before (PRE) and 2.5 h after (POST) ingestion of 140 ml of BR (~12.4 mmol NO3-). Samples were analysed for [NO2-] and [NO3-] using gas-phase chemiluminescence. The analytical coefficient of variation (CVa) for these measures was ≤4.1%. The within-subject variation (CVi) of measurements for the three visits was calculated and coupled with the CVa to determine the CD. The between-subject variation (CVg) was also calculated for each measurement time point.

Results
Across the three trials, plasma [NO2-] increased (P<0.01) from PRE (124 ± 49 nM, CVi 9%, CD 19%, CVg 40%) to POST (279 ± 94 nM, CVi 19%, CD 45%, CVg 32%) ingestion of BR. Likewise, plasma [NO3-] increased (P<0.01) from PRE (34 ± 10 µM, CVi 5%, CD 10%, CVg 32%) to POST (415 ± 92 µM, CVi 12 %, CD 24%, CVg 23%). Both salivary [NO2-] and [NO3-] also increased from PRE to POST (P<0.01) although the variation in salivary [NO2-] was substantially greater than [NO3-]. Salivary [NO2-] values were 136 ± 100 µM at PRE (CVi 33%, CD 87%, CVg 79%) and 904 ± 268 µM at POST (CVi 24%, CD 58%, CVg 38%). Salivary [NO3-] values were 0.5 ± 0.2 mM at PRE (CVi 15%, CD 37%, CVg 25%) and 8.5 ± 2.1 mM at POST (CVi 12%, CD 30%, CVg 37%).

Conclusion
There is profound biological variation in the measurements of NO metabolites in human saliva and plasma suggesting that a large change in these parameters is required before a biologically relevant mean change can be suggested. Nevertheless, the increases in salivary and plasma [NO3-] and [NO2-] following ingestion of BR consistently exceeds the CD range. Studies measuring [NO3-] and [NO2-] should carefully consider the CD of these measures when interpreting experimental outcomes.