Mitochondria-Derived Vesicles and Inflammatory Profiles of Adults with Long COVID Supplemented with Red Beetroot Juice: Secondary Analysis of a Randomized Controlled Trial

Abstract

In a recent clinical trial, beetroot juice supplementation for 14 days yielded positive effects on systemic inflammation in adults with long COVID. Here, we explored the relationship between circulating markers of mitochondrial quality and inflammation in adults with long COVID as well as the impact of beetroot administration on those markers. We conducted secondary analyses of a placebo-controlled randomized clinical trial testing beetroot juice supplementation as a remedy against long COVID. Analyses were conducted in 25 participants, 10 assigned to placebo (mean age: 40.2 ± 11.5 years, 60% women) and 15 allocated to beetroot juice (mean age: 38.3 ± 7.7 years, 53.3% women). Extracellular vesicles were purified from serum by ultracentrifugation and assayed for components of the electron transport chain and mitochondrial DNA (mtDNA) by Western blot and droplet digital polymerase chain reaction (ddPCR), respectively. Inflammatory markers and circulating cell-free mtDNA were quantified in serum through a multiplex immunoassay and ddPCR, respectively. Beetroot juice administration for 14 days decreased serum levels of interleukin (IL)-1β, IL-8, and tumor necrosis factor alpha, with no effects on circulating markers of mitochondrial quality control. Significant negative associations were observed between vesicular markers of mitochondrial quality control and the performance on the 6 min walk test and flow-mediated dilation irrespective of group allocation. These findings suggest that an amelioration of mitochondrial quality, possibly mediated by mitochondria-derived vesicle recycling, may be among the mechanisms supporting improvements in physical performance and endothelial function during the resolution of long COVID.

Keywords: cell quality; cytokine; extracellular vesicles; functional food; inflammation; mitochondrial DNA; muscle; physical performance.

Figure 1

Changes from baseline to day 14 in serum concentrations of inflammatory markers in the placebo (n = 10) and beetroot juice (n = 15) groups. Abbreviations: IFN-γ, interferon gamma; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; and TNF-α, tumor necrosis factor alpha. * p < 0.05 vs. baseline.

Figure 2

Changes from baseline to day 14 in vesicular/mitochondrial markers in the placebo (n = 10) and beetroot juice (n = 15) groups. The lower panels show representative Western blot bands and transmission electron microscope images of extracellular vesicles. Abbreviations: ATP5A, ATP synthase F1 subunit alpha; a.u., arbitrary unit; B, beetroot juice; ccf-mtDNA, circulating cell-free mitochondrial DNA; CD, cluster of differentiation; Ctrl, control; EVs, extracellular vesicles; MTCOI, mitochondrial cytochrome C oxidase I; OD, optical density; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; RPS6, ribosomal protein s6; and SDHB, succinate dehydrogenase [ubiquinone] iron–sulfur subunit.

Figure 3

Changes from baseline to day 14 in inflammatory and vesicular/mitochondrial markers adjusted for pre-intervention values in (A) placebo (n = 10) and (B) beetroot juice (n = 15) groups. Abbreviations: ATP5A, ATP synthase F1 subunit alpha; CD, cluster of differentiation; ccf-mtDNA, circulating cell-free mitochondrial DNA; EVs, extracellular vesicles; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; MTCOI, mitochondrial cytochrome C oxidase I; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; SDHB, succinate dehydrogenase [ubiquinone] iron–sulfur subunit; and vs-mtDNA, vesicular mitochondrial DNA. * p < 0.05 for comparisons between pre- and post-intervention adjusted for baseline values according to analysis of covariance (ANCOVA).

Figure 4

Correlation analyses between inflammatory and vesicular/mitochondrial markers and measures of physical and endothelial function at 14 days in the whole study sample (n = 25). Abbreviations: ATP5A, ATP synthase F1 subunit alpha; CD, cluster of differentiation; ccf-mtDNA, circulating cell-free mitochondrial DNA; EVs, extracellular vesicles; FMD, flow-mediated dilation; IFN-γ, interferon gamma; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; MTCOI, mitochondrial cytochrome C oxidase I; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; SDHB, succinate dehydrogenase [ubiquinone] iron–sulfur subunit; and vs-mtDNA, vesicular mitochondrial DNA. * p < 0.05.

Figure 5

Correlation analyses between pre/post-intervention differences in inflammatory and vesicular/mitochondrial markers and measures of physical and endothelial function at 14 days in (A) the placebo group (n = 10) and (B) participants assigned to beetroot juice (n = 15). Abbreviations: ATP5A, ATP synthase F1 subunit alpha; CD, cluster of differentiation; ccf-mtDNA, circulating cell-free mitochondrial DNA; EVs, extracellular vesicles; IFN-γ, interferon gamma; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; MTCOI, mitochondrial cytochrome C oxidase I; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; and SDHB, succinate dehydrogenase [ubiquinone] iron–sulfur subunit; vs-mtDNA, vesicular mitochondrial DNA. * p < 0.05.

Figure 6

Correlation analyses between pre/post-intervention differences in inflammatory and vesicular/mitochondrial markers and measures of physical and endothelial function at 14 days independent of intervention group (n = 25). Abbreviations: ATP5A, ATP synthase F1 subunit alpha; CD, cluster of differentiation; ccf-mtDNA, circulating cell-free mitochondrial DNA; EVs, extracellular vesicles; IFN-γ, interferon gamma; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; MTCOI, mitochondrial cytochrome C oxidase I; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; SDHB, succinate dehydrogenase [ubiquinone] iron–sulfur subunit; vs-mtDNA, vesicular mitochondrial DNA * p < 0.05.