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

The research team assembled a single-center cohort of 157 patients who had undergone coronary artery bypass grafting with intraoperative transit-time flow measurement, or TTFM, the ultrasound-based technique that records mean graft flow, pulsatility index and diastolic filling in real time. From this cohort the investigators initially identified 130 left internal mammary artery grafts, the arterial workhorse of modern bypass surgery, and 171 saphenous vein grafts, the venous conduits typically reserved for targets that arterial grafts cannot reach. Applying an intraoperative mean flow threshold of 20 milliliters per minute or less, they flagged 86 low-flow LIMA grafts and 84 low-flow vein grafts for deeper analysis. This threshold matters: it is the point at which surgeons begin to worry, and it is precisely the population in which the competitive-flow-versus-stenosis question becomes clinically urgent.

To establish what was actually happening inside each graft, the team turned to computed fractional flow reserve, or cFFR, derived from postoperative coronary computed tomography angiography. Rather than relying on a pressure wire pushed into the artery during a second catheterization, the researchers built patient-specific zero-dimensional and three-dimensional models of each patient’s coronary circulation and computed the fractional flow reserve numerically. Values between 0.75 and 0.80 were treated as an equivocal gray zone and excluded from the strict binary analyses, leaving 80 arterial grafts and 80 vein grafts with unambiguous physiology. This modeling approach is elegant in principle: by fusing the anatomy from CT with lumped-parameter representations of the downstream microcirculation, the models yield a physiologic index that behaves like the invasive gold standard without the risks of a repeat procedure.

The results split cleanly along graft-type lines, and that split is the study’s central message. In the left internal mammary artery grafts, the preoperative physiology of the target coronary artery dominated the picture. Each 0.1-unit increase in preoperative cFFR was associated with significantly higher odds that a low-flow graft represented competitive flow rather than a stenotic problem, with an odds ratio of 1.418 and a confidence interval running from 1.086 to 1.851, statistically significant at P equals 0.010. In plain terms, the healthier and less diseased the native vessel downstream of the anastomosis, the more likely it was to compete with the graft and drag its measured flow down. A continuous analysis of cFFR as an outcome confirmed the same relationship, reinforcing that the arterial graft’s low-flow state tracks the native vessel’s preserved physiology.

The hemodynamic fingerprints of the two phenotypes in arterial grafts were equally telling. Unadjusted comparisons showed that grafts in the competitive-flow group had lower mean wall shear stress and lower wall shear stress gradients along the graft wall, together with higher relative residence time, a parameter that quantifies how long blood particles linger in a region and is associated with thrombogenic potential. After the researchers applied Holm correction for multiple comparisons, the differences in mean wall shear stress and relative residence time remained statistically significant, while the wall shear stress gradient difference did not. Biologically, this pattern makes sense: a graft that is being quietly outcompeted carries less flow, and lower flow means lower shear on the endothelium and slower, longer-residing blood, both of which are conditions that in the long run can promote graft failure even when the original anastomosis was technically perfect.

Saphenous vein grafts told a completely different story. In the strict binary analysis of 80 vein grafts drawn from 59 patients, analyzed with a patient-clustered Firth-type penalized generalized estimating equation model to account for the fact that some patients contributed multiple grafts, the preoperative cFFR lost its statistical significance. Instead, two intraoperative TTFM waveform characteristics took center stage: higher pulsatility index and higher diastolic filling fraction were both associated with lower odds of the competitive-flow phenotype, with odds ratios of 0.725 and 0.919 respectively, both statistically significant. The pulsatility index reflects how pulsatile the graft flow waveform is, and the diastolic filling fraction captures how much of the graft’s flow arrives during diastole, the phase in which coronary perfusion naturally dominates. Vein grafts with more coronary-like, diastole-dominant waveforms were therefore less likely to be competing with a vigorous native vessel.

Perhaps the most sobering finding for the computational side of the field was what the CFD parameters failed to show in the vein grafts. After Holm correction, no CFD-derived parameter differed significantly between the two vein graft phenotypes, a null result that contrasts sharply with the significant wall shear stress and residence time differences seen in the arterial grafts. The authors also report the discriminative performance of their models honestly: optimism-corrected areas under the curve were 0.653 for the LIMA analysis and 0.703 for the vein graft analysis, with a cluster-bootstrap fitting success rate of 67.85 percent for the more complex vein model. These are modest discrimination values, useful for framing hypotheses rather than for making bed-side decisions, and the team is explicit that the findings are exploratory and require prospective external validation before any clinical application.

The methodological care underlying these conclusions deserves attention, because low-flow graft studies are notoriously vulnerable to statistical fragility. The gray-zone exclusion of six LIMA and four SVG grafts with cFFR values between 0.75 and 0.80 sharpened the binary contrast but reduced the sample, and the supplementary analyses that treated cFFR as a continuous outcome served as a check that the binary categorization was not driving the associations. Internal validation through 2,000 bootstrap resamples corrected the apparent performance of the logistic and penalized models for overfitting, and the clustering of multiple grafts within the same patient was handled with penalized generalized estimating equations rather than naive pooling. Each of these choices trades statistical power for robustness, and the modest AUCs reflect that trade. The work was supported by the National Natural Science Foundation of China, the Natural Science Foundation of Beijing and an undergraduate research fund at Beijing University of Chinese Medicine, and the ethics approval came from the Medical Ethics Committee of Beijing University of Chinese Medicine.

What does this mean for the surgeon staring at a TTFM console at two in the morning? The study suggests that the answer depends on which conduit is in hand. A sluggish arterial graft attached to a target with good preoperative physiology is more likely to be a victim of its own success, competing with a native vessel that never became severely stenotic, whereas a sluggish vein graft with a low pulsatility index and a low diastolic filling fraction fits the competitive pattern instead. Neither signal is definitive on its own, and the authors are careful not to claim that intraoperative numbers alone can replace angiographic or physiologic confirmation. But by grafting together three layers of evidence—the preoperative coronary physiology, the intraoperative waveform characteristics and the postoperative computational hemodynamics—the study sketches a graft-specific framework for interpreting one of cardiac surgery’s most common intraoperative anxieties. If prospective validation confirms these associations, the low-flow graft of the future may be read not as a single alarming number but as a pattern, with arterial and venous conduits each telling their own hemodynamic story.

Subject of Research: Hemodynamic differentiation of competitive flow from anastomotic stenosis in low-flow coronary artery bypass grafts

Article Title: Competitive flow identification and hemodynamic analysis in coronary artery bypass grafting

Article References: Kai, C., Gaoyang, L., Zhou, Z., Yanxi, C., Bao, L., Yue, F., Shaohui, Y., & Boyan, M. (2026). Competitive flow identification and hemodynamic analysis in coronary artery bypass grafting. BioMedical Engineering OnLine. https://doi.org/10.1186/s12938-026-01622-6

Image Credits: AI Generated

DOI: 10.1186/s12938-026-01622-6

Keywords: coronary artery bypass grafting, competitive flow, transit-time flow measurement, computed fractional flow reserve, computational fluid dynamics, wall shear stress, left internal mammary artery, saphenous vein graft, hemodynamics, coronary computed tomography angiography, pulsatility index, graft failure