Michalak 2017 MiP2017
Ethylenediaminetetraacetic acid (EDTA) is widely used in laboratory practice for chelation of divalent ions, particularly Ca2+ and Mg2+. It is applied as anticoagulant in blood samples, but also in media used for mitochondria isolation. It was reported to prevent mitochondria swelling [1,2]. Heparin, a member of the family of polysaccharides, the glycosaminoglycans was found to stabilize mitochondrial membranes . The aim of this study was to analyse mitochondrial respiration in intact peripheral blood mononuclear cells isolated from blood EDTA and heparin blood samples.
We included in the study 40 subjects hospitalized or consulted in Department of Neurology at Poznan University of Medical Sciences in Poznan, Poland. Patients with Parkinsons disease, atypical parkinsonisms, multiple sclerosis, Guillain-Barre syndrome, anti-NMDA antibodies encephalitis, myopathy, optic nerve degeneration, periodic hypokalemic paralysis and healthy volunteers were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood or heparinized blood via density gradient centrifugation (Histopaque, Sigma-Aldrich). The isolated fractions were supplemented with protease inhibitors cocktail (Sigma-Aldrich; 1:200 vol/vol) on ice. The cell number was counted in Bürker’s chamber and the volume corresponding to 1x106 cells was applied for respirometry. Cell viability was tested using the trypan blue method. Mitochondrial respiration was analyzed in intact PBMCs according to the ROUTINE, LEAK, electron transfer pathway (ET-pathway), and residual oxygen consumption (ROX) protocol  using a High-Resolution FluoRespirometer (O2k-FluoRespirometer; Oroboros Instruments, Innsbruck, Austria). Briefly, a total of 1x10sup>6 PBMCs were added to the respirometer chamber after 10 min of stabilization at 37 °C and incubated with continuous stirring at a speed of 750 rpm. The data were subsequently collected with the application of DatLab software 220.127.116.11 (Oroboros Instruments, Innsbruck, Austria).
ROUTINE respiration in PBMCs from heparinized blood was higher (16.85; 13.14 – 22.44 pmol O2/s*10sup>6 cells, median; interquartile range, P = 0.0007) than in EDTA blood (11.73; 9.25 – 16.26 pmol O2/s*10sup>6 cells). LEAK respiration was increased in PBMCs from heparinized blood (7.94; 6.2 – 12.41 pmol O2/s*10sup>6 cells, P < 0.0001) compared to EDTA blood (4.07; 3,00 - 4.99 pmol O2/s*10sup>6 cells). Also, ETS respiration was increased in PBMCs from heparinized blood (31.51 ±9.82 pmol O2/s*10sup>6 cells, mean ± SD; P = 0.0007) in relation to PBMCs isolated from EDTA samples (23.30±11.14 pmol O2/s*10sup>6 cells). No differences in ROX respiration were observed between heparin (3,88; 2.99 – 4.88 pmol O2/s*10sup>6 cells) and EDTA samples (3.92; 1.88 – 5.10, pmol O2/s*10sup>6 cells, P = 0.4441). Passing – Bablock analysis of ROUTINE, LEAK, ETS and ROX respiration analysed in PBMCs isolated from EDTA and heparin blood showed no significant deviation from linearity. No systematic differences between the two methods in ROUTINE, ETS and ROX were evidenced, while LEAK respiration showed systematic lowering of LEAK respiration in PBMCs from EDTA blood. No proportional differences were observed in ROUTINE, ETS and ROX respiration, while proportional difference was found in LEAK respiration. Residual Standard Deviation (RSD) as a measure of the random differences between the two methods showed significant differences between mitochondrial respiration measured in PBMCs from EDTA and heparin blood.
The type of anticoagulant apllied in blood samples used for analyses of respiration in intact cells may significantly affect final results. A possible cause for anticoagulants effect on mitochondrial function can be divalent ions chelation by EDTA or inhibition of phosphorylation processes by heparin.
Labels: MiParea: Respiration, Patients, Pharmacology;toxicology Pathology: Neurodegenerative, Parkinson's, Other
Organism: Human Tissue;cell: Blood cells Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET Pathway: ROX HRR: Oxygraph-2k
- Michalak S(1), Rybacka–Mossakowska J(1), Osztynowicz K(1), Biernacka–Lukanty J(1), Tokarz–Kupczyk E(2), Kozubski W(2)
- Dept Neurochem Neuropathol
- Dept Neurol; Poznan Univ Medical Sciences, Poznan, Poland. – [email protected]
- Lynn WS, Fortney S, Brown RS (1964) Osmotic and metabolic alterations of mitochondrial size. J Cell Biol 23:1-8.
- Parsons DF (1965) Recent advances correlating structure and function in mitochondria. Int Rev Exp Path 4:1-54.
- Dubinina EE, Nadirova T Ya. Effect of heparin on the mitochondrial ATP-ase system of rabbit white muscles. (1973) Bull Exp Biol Med 76:1314–6.
- Gnaiger E. Renner-Sattler K (2014) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondrial Physiology Network Vol 08.09, 1–8.