Sumbalova 2019b MiP2019

The decline of mitochondrial respiration in isolated platelets after short-term storage.

Link: MiP2019

Sumbalova Z, Gvozdjakova A, Kucharska J, Rausova Z, Vancova O, Mojto V (2019)

Event: MiP2019

In the past years, the use of blood cells for evaluation of mitochondrial function in human studies is more frequent. The methodological aspects of the work with blood cells are still under discussion. Here we evaluated how the short-term storage of isolated platelets (PLTs) affected their respiration.

16 samples of isolated PLTs from patients with chronic kidney disease were included in the study. PLTs were isolated from the whole blood by differential centrifugation, washed and resuspended in DPBS with 10 mM EGTA [1]. 200 million PLTs were introduced into the chamber of O2k-Respirometer (Oroboros Instruments, Austria) with mitochondrial respiration medium MiR05 and 20 mM creatine at 37°C. Respiration was measured folowing substrate-uncoupler-inhibitor titration protocol RP1 for permeabilized cells [2]. The remaining suspension of PLTs was stored at room temperature slowly tilting on a minishaker. After 2 hours the PLTs were introduced into the chamber of O2k-Respirometer and the respiration was measured again. The contents of O2k chambers were used for determination of mitochondrial marker – the activity of citrate synthase (CS).

The respiration per cell count as well as per CS activity was significantly lower (-20% and -21% in median respectively) in multiple respiratory states in samples stored for 2 h vs fresh samples.

For evaluation of PLT respiration the use of freshly isolated PLTs is required. The use of PLTs stored up to 2 h is possible, however, the mitochondrial function could be significantly affected. Our findings are important for quality control in human studies, particularly for evaluation of results when repeated or delayed measurements of respiration are necessary for technical reasons.

• Bioblast editor: Plangger M • O2k-Network Lab: SK Bratislava Sumbalova Z

Labels: MiParea: Respiration, Patients  Pathology: Other 

Organism: Human  Tissue;cell: Platelet  Preparation: Intact cells 

Coupling state: LEAK, OXPHOS  Pathway: F, N, S, Gp, CIV, NS, ROX  HRR: Oxygraph-2k 

Affiliations

Sumbalová Zuzana(1), Gvozdjáková A(1), Kucharská J(1), Rausová Z(1), Vančová O(1), Mojto V(2)

1. Comenius Univ Bratislava, Medical Fac, Pharmacobiochemical Lab 3rd Dept Internal Medicine
2. Comenius Univ Bratislava, Medical Fac, 3rd Dept Internal Medicine; Bratislava, Slovakia. – [email protected]

References

1. Gvozdjáková A, Kucharská J, Sumbalová Z, Nemec M, Chládeková A, Vančová O, Rausová Z, Kubalová M, Kuzmiaková Z, Mojto V (2019) Platelets mitochondrial function depends on CoQ10 concentration in winter, not in spring season. Gen Physiol Biophys 38:325-34.
2. Doerrier C, Sumbalova Z, Krumschnabel G, Hiller E, Gnaiger E (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06:1-12.