Age-Related Differences in PBMC Mitochondrial Bioenergetics and Antioxidant Buffering Capacity

The increase in oxidative stress with aging is an important contributor to vascular endothelial dysfunction. Because peripheral blood mononuclear cells (PBMCs) interact constantly with the vascular endothelium, oxidative stress in PBMCs may be reflective of endothelial oxidative stress. The mitochondria are an important source of reactive oxygen species (mtROS), but an imbalance between mtROS production and antioxidant capacity (AOX) results in oxidative stress, which contributes to numerous pathophysiological processes. However, it remains unknown whether age-related changes in mitochondrial bioenergetics and oxidative stress are reflected in peripheral blood mononuclear cells (PBMCs). Therefore, we assessed mitochondrial bioenergetics and AOX buffering capacity in PBMCs isolated from whole blood of young (n = 12; 21 ± 2 yrs) and older adults (n = 3, 67 ± 1). Immediately after PBMC isolation, high-resolution respirometry and spectrofluorometer were used to simultaneously measure mitochondrial respiration rate ( J O 2 ) and membrane potential (mtMP), respectively, in response to substrate provision (pyruvate/glutamate/malate/succinate; PGMS) and a bioenergetic creatine kinase (CK) clamp at physiological ATP:ADP ratios (i.e., from high to low demand for ATP re-synthesis, termed PCr1, PCr2, and PCr3). MtROS emission was measured as real-time H 2 O 2 emission, and inhibitors of glutathione reductase and thioredoxin/peroxiredoxin allowed the quantification of H 2 O 2 production with an AmplexRed/HRP detection system using a spectrofluorometer. AOX buffering capacity was calculated by measuring the percentage of J H 2 O 2 produced but not emitted (due to buffering by antioxidant enzymes). J O 2 was similar between young and older adults at baseline (p = 0.12) and at lower energetic states (PCr2, p = 0.16; PCr3, p = 0.74), but tended to be lower in older adults at the higher energetic states (PCr1: 15.08 ± 2.62 pmol/min/million cells vs.11.81 ± 1.31 pmol/min/million cells, p = 0.02; PGMS: 22.39 ± 2.49 pmol/min/million cells vs. 19.47 ± 1.72 pmol/min/million cells, p = 0.07). Additionally, electron conductance was lower in older compared to young adults (5.51 ± 1.43 A.U. vs. 3.37 ± 0.70 A.U.; p = 0.01). MtMP was lower in older adults at all energetic states (p ≤ 0.03). Although there were no statistical differences in H 2 O 2 emission (Young, 0.70 ± 0.25 pmol/min/million cells; Older, 0.76 ± 0.21 pmol/min/million cells; p = 0.36) or production (Young, 2.04 ± 0.49 pmol/min/million cells; Older, 1.56 ± 0.19 pmol/min/million cells; p = 0.06), AOX buffering capacity was less in older compared to young adults (Young, 64.97 ± 11.07%; Older, 51.91 ± 9.73%; p = 0.04). These preliminary data support the potential utility of PBMCs to assess age- and/or pathology-related differences in mitochondrial bioenergetics and AOX buffering capacity. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.