Abstract
Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn.
Highlights
Mitochondria are intracellular organelles involved in energy production through the process of oxidative phosphorylation (OXPHOS) that have their own genome, distinct from that in the cell nucleus
In the blood preparations supplemented with different amounts of platelet-enriched plasma (46 data points), mtDNA copy number (mtDNAcn)(WB) increased 1.01 (95%CI 0.84, 1.17; p
We built multivariate regression models to evaluate the influence of the platelets and leukocytes on mtDNAcn(WB), and we found a significant interaction between platelets and 1/leukocytes that was the only statistically significant term in that model for mtDNAcn(WB)(Table 3), consistent with the correction term that our formulation identified as determinant of the mtDNAcn(WB)(See Formulation framework)
Summary
Mitochondria are intracellular organelles involved in energy production through the process of oxidative phosphorylation (OXPHOS) that have their own genome (mtDNA), distinct from that in the cell nucleus (nDNA). The mitochondrial mass per cell varies with cell type and metabolic state, each cell type typically contains a fairly constant amount of mitochondria and the number of copies of the mitochondrial genome is constant, as it is linked to mitochondrial mass[1]. High-energy requiring cells, such as muscle and neurons, contain a large number of mtDNA copies, while low-energy requiring cells, such as spleen and endothelial cells, contain fewer copies[3,4,5]. The amount of mtDNA per cell, or mtDNA copy number (mtDNAcn), can be expressed as a ratio of mtDNA to nDNA copies, i.e. using nDNA as reference, assuming that all quantified cells are nucleated and diploid[6]. Quantitative real-time PCR (qPCR) is a suitable method to quantify mtDNAcn[7,8,9]
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