Surplus Bypass Graft Segments Reveal Real-Time Mitochondrial Respiration in Intact Human Arteries

For decades, the mitochondria inside blood vessels have been studied mostly in isolation — in cultured endothelial cells, in isolated vascular smooth muscle, or in arteries harvested from laboratory animals. Now a team at the Medical University of Gdańsk has shown that the living energy machinery of a real human artery can be measured directly, in real time, using tissue that would otherwise be discarded in the operating theatre. In a proof-of-concept study published in Acta Biochimica Polonica, researchers captured surplus segments of the left internal mammary artery (LIMA) during coronary artery bypass grafting (CABG) and, for the first time, applied the Seahorse XF Flex 3D Analyzer to intact human vascular tissue to quantify mitochondrial respiration as it happens.

The left internal mammary artery is the gold-standard conduit in CABG surgery, prized for its exceptional long-term patency and natural resistance to atherosclerosis. Yet despite its clinical importance, the bioenergetic profile of this vessel has remained largely uncharted. The reason is technical: assessing mitochondrial function in intact human vascular tissue is notoriously difficult. Most of what science knows about vascular mitochondria comes from simplified models that cannot fully reproduce the structural and cellular complexity of a living human arterial wall — the layered interplay of endothelium, smooth muscle and connective matrix that determines how a graft actually behaves inside a patient.

Mitochondria in blood vessels are far more than tiny power plants. In endothelial cells, they act primarily as signalling organelles rather than major energy producers, modulating vascular tone, inflammation and adaptation to metabolic stress. When mitochondrial function falters, the consequences cascade: excessive reactive oxygen species (ROS) production, endothelial activation and reduced nitric oxide bioavailability, all of which promote vascular stiffness, inflammation and atherosclerosis. These processes are especially relevant for patients undergoing bypass surgery, where the functional integrity of the vascular graft is essential for long-term outcomes. Impaired mitochondrial respiration has even been linked directly to atherosclerotic plaque progression, promoting necrotic core formation and thinning of the protective fibrous cap.

The Gdańsk team, led by Barbara Kutryb-Zając of the Department of Biochemistry, designed a workflow that turns routine surgery into a laboratory opportunity. A 67-year-old male patient with stable two-vessel coronary disease, arterial hypertension and orally treated diabetes mellitus underwent elective CABG. During the standard sternotomy procedure, the LIMA was harvested for grafting to the left anterior descending artery, and a residual segment of the vessel — tissue that would normally be discarded — was collected for ex vivo analysis with written informed consent and approval from the local bioethics committee.

The handling protocol was engineered to preserve the vessel’s living architecture. Immediately after harvesting, the artery was placed in Agilent Seahorse XF DMEM Medium at pH 7.4 and transported to the laboratory, where it was cleaned of surrounding tissue and sectioned into small fragments. The vascular rings were cut longitudinally and gently flattened into planar segments, then immobilized on Seahorse XF capture microplates fitted with islet capture screens — with the luminal surface facing upwards, so that the delicate endothelial layer was directly exposed to the measurement chamber. Histological staining with hematoxylin and eosin on parallel sections confirmed that the vascular structure and endothelial lining remained preserved throughout preparation.

The functional core of the study is the mitochondrial stress test, a sequential pharmacological interrogation of the respiratory chain. Vessel segments of two to three millimetres were loaded into the wells of a Seahorse XF Flex 3D Capture Microplate, and the sensor cartridge — hydrated overnight with calibrant solution at 37 degrees Celsius — recorded the oxygen consumption rate (OCR) in real time. Oligomycin A at 20 micromolar was injected first to inhibit ATP synthase and reveal ATP-linked respiration; FCCP at 15 micromolar followed, uncoupling the proton gradient to expose maximal respiratory capacity; finally, a mixture of rotenone and antimycin A, each at 10 micromolar, shut down complexes I and III to isolate non-mitochondrial oxygen consumption. From these measurements the team derived basal respiration, ATP-linked respiration, maximal respiration, proton leak and non-mitochondrial respiration, normalizing all values to total protein content determined by the Bradford assay and expressing them as picomoles of oxygen per minute per milligram of protein.

In parallel, the researchers quantified the tissue’s chemical energy currency using high-performance liquid chromatography (HPLC). Snap-frozen LIMA sections were homogenized in ice-cold perchloric acid to extract nucleotides, which were then separated and quantified against external standards. The panel covered adenosine triphosphate (ATP), diphosphate (ADP) and monophosphate (AMP), the guanosine analogues GTP and GDP, and the redox cofactors NAD+ and NADH. Because adenosine 5′-diphosphoribose (ADPR) is the major product formed by acidic cleavage of NADH during extraction, the measured ADPR level served as a readout of NADH concentration — a clever analytical shortcut that links the nucleotide profile directly to the tissue’s redox balance.

The results demonstrated that both approaches work in human arterial tissue. Real-time OCR measurements showed that mitochondrial respiration can be reliably assessed in intact human arteries on the Seahorse XF Flex 3D platform, with reproducible capture of basal respiration, ATP-linked respiration and maximal respiratory capacity. HPLC analysis confirmed measurable nucleotide pools in the same tissue, and the profile indicated preserved energy status — evidence that the vascular segments remained functionally intact under ex vivo conditions. By combining the dynamic, functional picture from oxygen flux with the static, metabolic snapshot from nucleotide levels, the workflow delivers an integrated assessment of vascular bioenergetics at both functional and metabolic levels, something neither method achieves alone.

The methodological significance extends beyond the operating room. According to the authors, this is the first application of the Seahorse XF Flex 3D platform to intact human vascular tissue, closing a gap between cell culture and animal models on one side and clinical reality on the other. Because the LIMA is harvested in every CABG operation, the approach creates a renewable pipeline of clinically relevant human vascular tissue for metabolic research — a platform for investigating how diabetes, oxidative stress, ageing and other comorbidities reshape the energy metabolism of the vessel wall itself.

The clinical horizon is equally compelling. Current intraoperative assessment of graft quality relies largely on anatomical and flow-based measurements, which reveal nothing about the metabolic condition of the vessel. Ex vivo bioenergetic profiling could offer a complementary functional readout reflecting mitochondrial integrity and cellular viability within the vascular wall, potentially helping to identify subtle metabolic alterations linked to patient comorbidities such as diabetes. In the longer term, the researchers envision this methodology contributing to biomarkers of graft quality and personalized strategies in coronary artery bypass grafting. The team is careful about scope: this proof-of-concept rests on a single human LIMA, and variability in tissue handling, preparation and positioning within the Seahorse platform will require standardization. Larger patient cohorts will be needed to validate robustness, reproducibility and clinical relevance. But the conceptual barrier has fallen — the respiratory heartbeat of a human artery can now be watched in real time, opening a new window onto vascular metabolism in cardiovascular and metabolic disease.

Subject of Research: Real-time ex vivo measurement of mitochondrial respiration and nucleotide levels in intact human left internal mammary artery grafts from coronary artery bypass surgery

Article Title: Mitochondrial respiration and nucleotide profiling in human left internal mammary artery from CABG: a novel real-time ex vivo approach

Article References: Braczko, A., Łoś, A., Piotrowska, M., Kawecka, A., Walczak, I., Krysiak, M., Hellmann, M., Brzeziński, M., Smoleński, R. T., & Kutryb-Zając, B. (2026). Mitochondrial respiration and nucleotide profiling in human left internal mammary artery from CABG: a novel real-time ex vivo approach. Acta Biochimica Polonica, 73, Article 16877. https://doi.org/10.3389/abp.2026.16877

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16877

Keywords: mitochondrial respiration, left internal mammary artery, coronary artery bypass grafting, Seahorse XF Flex 3D, oxygen consumption rate, HPLC nucleotide profiling, vascular bioenergetics, endothelial function, ex vivo analysis, ATP-linked respiration, graft quality, cardiovascular metabolism