Selection of markers of reperfusion injury
P-Selectin, Myeloperoxidase and TNF-alpha denote key elements in endothelial-leukocyte interaction, polymorphonuclear leukocyte activity, and pro-inflammatory status during IRI.
P-Selectin, a member of the adhesion molecule family expressed in platelets and endothelial cells, is up regulated during IRI and mediates the initial adhesion of leucocytes to the surface of the endothelium, i.e ‘leucocyte rolling’. Adhered leucoyctes transmigrate through the endothelium and are activated6,7. Leucocytes thus activated release myeloperoxidase. MPO catalyzes the formation of hypochlorite and reactive oxygen species during reperfusion of ischaemic tissue.
TNF-alpha is a pro-inflammatory cytokine released by a variety of cells under stressful conditions. Colletti et al first reported the role of TNF-alpha in IRI in 1990, in a murine model subjected to hepatic ischaemia reperfusion8. Subsequent studies have demonstrated that TNF-alpha increases the expression of many cell adhesion molecules as well as releases many cytokines leading to further exaggeration of the neutrophil activation process.
The role of P-selectin and myeloperoxidase during IRI in skeletal muscle has been demonstrated in animal models. Singbarti et al demonstrated the attenuation of IRI-induced acute renal failure in P-selectin deficient mice9. Jin-Lian Chen et al demonstrated reduced hepatic/renal insufficiency in mice treated with P-selectin blocking Tetramethylpyrazine10. Li et al demonstrated the protective effects of Phloroglucinol, a MPO inhibitor, against myocardial IRI in rats11 . Preclinical models have supported the hypothesis that local skeletal muscle IRI is mediated in part by TNF-alpha. Studies of Seekamp et al, reported that abrogating TNF-alpha activity with a soluble TNF receptor construct and an anti-TNF-alpha antibody, decreased muscle capillary permeability after IRI in skeletal muscle12.
We therefore postulate that P-selectin, Myeloperoxidase and TNF-alpha are suitable markers of reperfusion injury in skeletal muscle ischaemia reperfusion injury in a clinical setting.
Methodology
An observational study was carried out on patients admitted to the University of Colombo, Vascular Unit at the National Hospital of Sri Lanka with acute lower limb arterial occlusion requiring revascularization.
Patients for whom amputation rather than revascularization was indicated and patients below the age of 18 years, were excluded. A peripheral venous sample was obtained immediately prior to surgery as baseline. Prior to revascularization an indwelling femoral venous catheter was inserted and placed for a period of 24 hours for sampling the venous effluent. Subsequently, loco-regional venous sampling was done using the indwelling femoral venous catheter at 0, +2, +4, +6, +12 and +24 hours post-reperfusion to establish a temporal evolution of the reperfusion injury markers in the venous effluent.
Loco-regional venous sampling with an indwelling venous catheter as opposed to peripheral venous sampling for 24 hours has been shown to be safe and advantageous during previous studies. Rowlands T et al13 used femoral vein catheter blood sampling to study lower extremity ischaemia during abdominal aortic aneurysm repair and Wijeyaratne SM et al14 used jugular venous catheter blood sampling to study cerebral ischaemia during carotid clamping for endarterectomy.
Assay Methodology
All laboratory procedures including assaying and analysis of samples were conducted at the Department of Biochemistry, Faculty of Medicine, Colombo.
Serum concentrations of soluble P-selectin and myeloperoxidase were measured by a quantitative sandwich immunoassay technique using an enzyme-linked immunosorbent assay kit (R&D systems, Minneapolis, MN). Whilst Tumour Necrosis Factor-alpha was assayed in the venous effluent using an immunometric enzyme immunoassay technique (Cayman chemical company, Ann Arbot,MI)
All samples, controls and standards were assayed in triplicate to increase the rigor of the study.