S-nitrosothiols, and other products of nitrate metabolism, are increased in multiple human blood compartments following ingestion of beetroot juice

Abstract

Ingested inorganic nitrate (NO₃⁻) has multiple effects in the human body including vasodilation, inhibition of platelet aggregation, and improved skeletal muscle function. The functional effects of oral NO₃⁻ involve the in vivo reduction of NO₃⁻ to nitrite (NO₂⁻) and thence to nitric oxide (NO). However, the potential involvement of S-nitrosothiol (RSNO) formation is unclear. We hypothesised that the RSNO concentration ([RSNO]) in red blood cells (RBCs) and plasma is increased by NO₃⁻-rich beetroot juice ingestion. In healthy human volunteers, we tested the effect of dietary supplementation with NO₃⁻-rich beetroot juice (BR) or NO₃⁻-depleted beetroot juice (placebo; PL) on [RSNO], [NO₃⁻] and [NO₂⁻] in RBCs, whole blood and plasma, as measured by ozone-based chemiluminescence. The median basal [RSNO] in plasma samples (n = 22) was 10 (5-13) nM (interquartile range in brackets). In comparison, the median values for basal [RSNO] in the corresponding RBC preparations (n = 19) and whole blood samples (n = 19) were higher (p < 0.001) than in plasma, being 40 (30-60) nM and 35 (25-80) nM, respectively. The median RBC [RSNO] in a separate cohort of healthy subjects (n = 5) was increased to 110 (93-125) nM after ingesting BR (12.8 mmol NO₃⁻) compared to a corresponding baseline value of 25 (21-31) nM (Mann-Whitney test, p < 0.01). The median plasma [RSNO] in another cohort of healthy subjects (n = 14) was increased almost ten-fold to 104 (58-151) nM after BR supplementation (7 × 6.4 mmol of NO₃⁻ over two days, p < 0.01) compared to PL. In conclusion, RBC and plasma [RSNO] are increased by BR ingestion. In addition to NO₂⁻, RSNO may be involved in dietary NO₃⁻ metabolism/actions.

Keywords: Beetroot (BR); Nitrate; Nitrite; Plasma; Red blood cells (RBCs); S-nitrosothiols (RSNO).

Graphical abstract

Fig. 1

Typical GSNO standard curve obtained from the depicted ozone-based chemiluminescence traces. Panel (a): serial dilutions of a GSNO solution treated with a solution of 5% acidified sulfanilamide, were injected in duplicate into tri-iodide solution to obtain the chemiluminescence signal peaks which were quantified by calculating the area under the curve. The inset panel shows the traces from the lowest GSNO concentration, on an expanded y-axis scale for clarity. ↓ indicates the time-point at which the analysed sample (500 μl) was injected. Panel (b): an example of the resulting standard curve. The results shown are typical of those obtained in >20 experiments.

Fig. 2

Scatter plot showing the concentrations of total RSNO in plasma, RBCs and whole blood in healthy volunteers at baseline. The graph shows that the median levels of total RSNO in healthy volunteers were higher (***p < 0.001) in RBCs and whole blood compared to plasma. The long horizontal bar represents the median value in each group, whilst the short horizontal bars represent the interquartile range.

Fig. 3

Scatter plots showing the correlations of baseline total [RSNO] between blood compartments (plasma, whole blood and RBCs). Panel (a) shows that the baseline [RSNO] in plasma was inversely correlated (r_s = − 0.53, p = 0.029) with whole blood [RSNO] from the same subjects. Panel (b) shows the observed trend towards a negative correlation between the baseline [RSNO] in plasma and in RBCs, but this was not statistically significant (r_s = − 0.44, p = 0.07). Panel (c) shows that the baseline [RSNO] in whole blood was not correlated (r_s = 0.001, p = 0.99) with RBC [RSNO]. The solid line indicates the estimated regression line. Spearman's rank correlation coefficient (r_s) was calculated from n = 17 subjects.

Fig. 4

Representative chemiluminescence time-traces forRBCRSNO detection, and a scatterplot showing the calculated concentrations of the total RSNO in RBCs of healthy volunteers. Panels (a) and (b) show examples of chemiluminescence signal peaks from the ozone-based chemiluminescence during the measurement of RSNO in RBC samples from healthy volunteers, following sample injections (one with 5% sulfanilamide in 1 M HCl only (“Acid Sulf ") and another one with “Acid Sulf " and 0.2% HgCl₂) after individuals had ingested beetroot juice (BR) or at baseline (BL). ↓ indicates the time-point at which the analysed sample (500 μl) was injected. The final RSNO levels were obtained by subtracting the values of samples treated with HgCl₂ from those without HgCl₂. Panel (c) shows that the median concentration of RSNO in RBCs of healthy subjects was significantly higher (**, p < 0.01) after ingesting BR juice compared to the median baseline RSNO concentration without supplementation (n = 5).

Fig. 5

Representative chemiluminescence signal traces for plasma RSNO detection, and a scatterplot showing the calculated concentrations of the total RSNO in the plasma of healthy volunteers at baseline, after ingesting the placebo juice, or after ingestingNO₃⁻-richbeetroot juice. The figure shows (in panels a and b) examples of the time courses of the chemiluminescence signal peaks when measuring the NO release from plasma RSNO in volunteers who had received NO₃⁻-rich beetroot juice. Panel (a) shows typical results from injecting samples treated with 5% acidified sulfanilamide in 1 M HCl only (“Acid Sulf"), and panel (b) shows the signal traces for the same injected samples treated with 5% acidified sulfanilamide and 0.2% HgCl₂ in 1 M HCl (“Acid Sulf HgCl₂″). The acid sulfanilamide and sulfanilamide/HgCl₂ solutions were diluted 1/10 when added to biological samples. In panels (a) and (b), ↓ indicates the time-point at which the analysed sample (500 μl) was injected. The final RSNO concentrations were calculated by subtracting the concentrations of the samples treated with HgCl₂ from the concentrations of the samples without HgCl₂. Panel (c) shows: the baseline levels of RSNO for a group of individuals (n=7) before administration of placebo (NO₃⁻-depleted) beetroot juice (BL-PL), the levels of RSNO for the same group after ingesting placebo beetroot juice (PL), the baseline levels of RSNO for another group of individuals (n=7) before administration of NO₃⁻-rich beetroot juice (BL-BR), and the levels of RSNO for the group after ingesting NO₃⁻-rich beetroot juice (BR). Panel (c) shows that the median level of total RSNO in the plasma of healthy volunteers was significantly higher (**, p<0.01) after ingesting BR, compared to both the BL-BR and PL groups. However, there were no statistically significant differences between BL-PL, PL and BL-BR in relation to the median levels of total RSNO.