Abstract
BackgroundIon and metal homeostasis are critical to red blood cell physiology and Inductively Coupled Plasma (ICP) is a decades old approach to pursue elemental analysis. Recent evolution of ICP has resulted in its coupling to mass spectrometry (MS) instead of atomic absorption/emission.MethodsHere we performed Inductively-coupled plasma mass spectrometry (ICP-MS) measurements of intra- and extra-cellular Na, K, Ca, Mg, Fe, and Cu in red blood cells undergoing ionic, heat, or starvation stress. Results were correlated with Ca measurements from other common platforms (e.g., fluorescence-based approaches) and extensive measurements of red blood cell metabolism.ResultsAll stresses induced significant intra- and extracellular alterations of all measured elements. In particular, ionomycin treatment or hypertonic stress significantly impacted intracellular sodium and extracellular potassium and magnesium levels. Iron efflux was observed as a function of temperatures, with ionic and heat stress at 40°C causing the maximum decrease in intracellular iron pools and increases in the supernatants. Strong positive correlation was observed between calcium measurements via ICP-MS and fluorescence-based approaches. Correlation analyses with metabolomics data showed a strong positive association between extracellular calcium and intracellular sodium or magnesium levels and intracellular glycolysis. Extracellular potassium or iron were positively correlated with free fatty acids (especially mono-, poly-, and highly-unsaturated or odd-chain fatty acid products of lipid peroxidation). Intracellular iron was instead positively correlated with saturated fatty acids (palmitate, stearate) and negatively with methionine metabolism (methionine, S-adenosylmethionine), phosphatidylserine exposure and glycolysis.ConclusionIn the era of omics approaches, ICP-MS affords a comprehensive characterization of intracellular elements that provide direct insights on red blood cell physiology and represent meaningful covariates for data generated via other omics platforms such as metabolomics.
Highlights
Red blood cells (RBCs) are the most abundant cell in the human body and play an essential role in oxygen transport and many additional functions relevant to systems physiology (Nemkov et al, 2018a)
Ion homeostasis, membrane potential and signaling triggered by the intra- and extracellular levels of these ions are critical to RBC function in health and disease, and hold relevant implications for life-saving iatrogenic interventions such as storage in the blood bank for transfusion purposes (D’Alessandro et al, 2015; Kuhn et al, 2017)
To maximize the oxygen-transport function, RBCs are loaded with iron (Fe), with ∼two thirds of bodily iron accumulated in mature RBCs (Nemkov et al, 2018a)
Summary
Red blood cells (RBCs) are the most abundant cell in the human body and play an essential role in oxygen transport and many additional functions relevant to systems physiology (Nemkov et al, 2018a). The loss of nuclei and organelles maximizes the RBC hemoglobin content, and results in a cell devoid of de novo protein synthesis capacity In this view, ion homeostasis, membrane potential and signaling triggered by the intra- and extracellular levels of these ions are critical to RBC function in health and disease, and hold relevant implications for life-saving iatrogenic interventions such as storage in the blood bank for transfusion purposes (D’Alessandro et al, 2015; Kuhn et al, 2017). Extravascular hemolysis by means of RBC clearance prevents extracellular accumulation of damageassociated molecular pattern biomolecules, such as hemoglobin, heme, and iron in blood vessels—by diverting them to splenic catabolism (Antonelou et al, 2010) These processes are impaired in the context of stresses that accelerate aging of RBCs in vivo (e.g., sickle cell disease, thalassemia) (Buehler et al, 2021) or in vitro (blood storage) (D’Alessandro, 2021). Recent evolution of ICP has resulted in its coupling to mass spectrometry (MS) instead of atomic absorption/emission
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