Erythrocytes in PET studies
Radiolabelled erythrocytes (red blood cells, RBCs) have been used in quantification of tissue blood volume and oxygen consumption. Labelled RBCs could also be used to detect internal haemorrhage, sites of erythrocyte destruction and spleen (Wang et al., 2017; Matsusaka et al., 2018). 99mTc is used for labelling erythrocytes for SPECT imaging, and [11C]CO, [15O]CO, or [18F]FDG for PET imaging. [68Ga]oxine could also be used for RBC labelling (Welch et al., 1977; Thompson et al., 2018), or [67Ga]oxine for SPECT (Ballinger & Boxen, 1992). In vivo labelling of RBCs can lead to biphasic blood curve (Graham & Nelp, 1980).
In other PET studies the erythrocytes need to be taken in account as part of input function. The rate that PET tracer equilibrates between blood cells and plasma determines whether plasma or whole blood should be used as the input function in modelling. Usually the transport rate between plasma and RBCs is slow enough that its effects on exchange between capillary blood and tissue can be ignored (Knudsen et al., 1994). Even if plasma can be considered as the correct input, blood curve may need to be converted to plasma curve in case blood curve is derived from dynamic PET image or using automatic blood sampling system.
Radioactivity concentration in red blood cells cannot be measured directly from centrifuged blood samples, because RBC preparations always contain some plasma. If plasma is washed away, then part of the radioactivity inside the cells or bound to the surface is also removed. However, if the TACs of whole blood and plasma, and hematocrit (HCT) are measured, then the TAC of RBCs can be calculated using equation (Hagenfeldt & Arvidsson, 1980):
Erythrocyte cell membranes contain many adhesive proteins, which may bind PET tracers, and affect plasma-to-blood ratio, together with membrane lipids and binding to plasma proteins (Paixao et al, 2009). Adherence to RBC membrane and/or intracellular uptake will affect plasma pharmacokinetics of the tracer. Erythrocytes have been used for drug delivery to improve drug pharmacodynamics (Villa et al, 2015); if the drug molecule is labelled with positron-emitting label, then PET could be used to study the drug distribution and kinetics in detail.
Erythrocytes (red blood cells, RBCs) originate from hematopoietic stem cell (HSC) in the bone marrow. HCS divides asymmetrically, resulting in an identical HCS and another cell destined for differentiation steps into a reticulocyte and finally into a red blood cell. There are about 4-6 × 109 RBCs/mL blood in healthy adults, and about 1% of them are reticulocytes. Reticulocytes may still have cellular organelles. Human RBC size is about 7.5 × 1-2 µm, with volume of 84-107 µm3. RBC volume is ∼60% of the maximal volume determined by its surface area, enabling shape deformation for passing through narrow capillaries. At about 100-120 days of age the RBC starts to lose its functions and is removed from circulation in the spleen and liver by macrophages. Mouse erythrocytes are smaller and stay in circulation for about a month.
Human erythrocytes lack cellular organelles, DNA and RNA, and thus also many metabolic pathways such as oxidative phosphorylation, TCA cycle, β-oxidation, and protein synthesis. RBCs produce ATP from anaerobic conversion of glucose via pyruvate to lactate. Alternatively, erythrocytes can produce 2,3-biphosphoglycerate (2,3-BPG, or 2,3-DPG) to reduce the affinity of haemoglobin to oxygen. RSR13 is a synthetic compound that also right-shifts the O2 dissociation curve. Most of the ATP is used to maintain the ion balance, cell volume, and RBC deformability. Pentose phosphate pathway (hexose monophosphate shunt) produces substrates for nucleoside synthesis, and is in RBCs the only source of NADPH, which is needed to regenerate glutathione for scavenging reactive oxygen species.
Haemoglobin (Hb) accounts for >90% of the dry mass of the red blood cells (about 29 pg in each RBC) and about 98% of the cytoplasmic protein content. In an adult man the total amount of haemoglobin in the circulation is about 900 g. Haemoglobin is a tetrameric 65 kDa protein with almost spherical shape. It can bind four O2 molecules, with affinity that strongly depends on local oxygen partial pressure (PO2). Oxygen binding to haemoglobin can be described by the Adair equation, but in Hb-O2 saturation (SO2) range 20-80% the much simpler Hill equation provides an accurate dissociation curve:
P50 and n are dependent on the blood pH, temperature, and concentrations of CO2 and 2,3-DPG (Dash & Bassingthwaighte, 2010). In human subjects, P50 is 3.5±0.1 kPa in arterial blood, and 3.7±0.2 kPa in venous blood (Siggaard-Andersen et al., 1990). In a larger group, arterial P50 was found to vary between 2.15 and 6.44, with median 3.44 kPa (Gøthgen et al., 1990).
Carbonic anhydrase 1 and peroxiredoxin 2 are the most abundant proteins, after haemoglobin.
RBC membrane is composed of proteins (52%), lipids (40%), and carbohydrates (8%). Proteins include anion exchangers and membrane transporters and glycophorins. Inner membrane contains proteins such as actin, myosin and tropomyosin. Glycolipids, phosphatidylcholine and sphingomyelin are oriented towards the plasma, and phosphatidylserine, phosphatidylethanolamine and phosphoinositolphospholipids towards the cytoplasm. Carbohydrates are bound to membrane proteins and lipids, forming the glycocalyx. Sialic (neuraminic) acids are bound to glycophorins, causing the negative charge of the erythrocyte membrane, preventing adhesion to endothelium.
Functions of erythrocytes
The role of haemoglobin in red blood cells in the transport of O2 and CO2 is well known. About 3/4 of CO2 is transported in RBCs, and the rest in plasma; the water solubility of CO2 is ∼20 times higher than that of O2. Transport of HCO3- across RBC membrane coupled to Cl- shift, and facilitated by anion exchanger 1. RBCs also transport NH3, and are involved in the transport of purines and cholesterol from the liver to other organs, and iC3b/C3b-carrying immune complexes.
Red blood cells produce nitric oxide (NO) by RBC-nitric oxide synthase. NO can be bound to haemoglobin or stored as nitrite. In hypoxic conditions NO is released inducing vasodilation and RBC deformability, helping RBCs to pass through the capillaries that are smaller (even 1 µm) than their diameter (7-8 µm). Plasma membrane Ca2+ pump keeps the intracellular free Ca2+ concentration on a very low level (30-60 nM), while free [Ca2+] in plasma is about 1.8 mM. Physical forces in capillaries activate Piezo1 protein which leads to Ca2+ influx, reducing the volume of the erythrocytes and possibly releasing ATP, which could lead to NO release from endothelial cells.
The intimate contact between erythrocyte cell membrane and endothelial cells in the capillaries helps the transport of substances to and from the tissue (Highley and De Bruijn, 1996).
RBCs have a role in metabolism of several drugs and PET ligands, despite having limited metabolic pathways. For example, acetylcholinesterases (AChEs), arylesterases, adenosine deaminase, catechol-O-methyl transferase (COMT), steroid dehydrogenase, glutathione transferases, endopeptidase, and also haemoglobin, metabolize many drugs, including L-DOPA, dopamine, epinephrine, insulin, steroid hormones, and anticancer drugs (Cossum, 1988).
Some drugs bind with high affinity to proteins inside erythrocytes. For instance, thiazides bind to carbonic anhydrase, leading to the chlorthalidone RBC-to-plasma ratio of about 70. Fentanyl is mainly bound to haemoglobin, but as it is also bound to plasma proteins, the RBC-to-plasma ratio was found to be 1.01 (Bower, 1982). Alfentanil is diffused into erythrocytes, but not bound there, and therefore its RBC-to-plasma ratio is only 0.14 (Bower and Hull, 1982).
Transporters on erythrocyte membranes
Since red blood cells are easily available and easy to work with, RBCs have been used as model system for studying membrane transporters, for example the Na+-K+-2Cl- cotransporter. RBC membrane is more permeable to anions than to cations, by many orders of magnitude for Cl- compared to Na+ or K+. Transport of halide anions is very fast (Tosteson, 1959).
Long-chain fatty acids can be quickly transported across erythrocyte plasma membrane even without transporters, but the high affinity to plasma proteins leads to very small intracellular FA concentrations (Kleinfeld et al., 1998).
Some of the transporters in RBCs are available in small numbers, especially in old RBCs, limiting the transport capacity. Medications may lead to saturation or inhibition of the transporters. Diseases and genetic differences in transporters between subjects may also affect the plasma-to-blood ratios.
Erythrocyte membranes contain ATP binding cassette (ABC) proteins, ATP powered transporters ABCB1 (MDR1, P-glycoprotein, Pgp), ABCB6, ABCC1 (MRP1), ABCC4 (MRP4), ABCC5 (MRP5), and ABCG2 (BCRP). Most of the blood ATP resides in erythrocytes, and ABC proteins and pannexin 1 channels are involved in the uptake and release of ATP from the erythrocytes as they pass through the tissue capillaries.
Pgp is associated with transport of lipophilic cations. ABCC1 (MRP1) is associated with transport of glutathione conjugated compounds. ABCC4 and ABCC5 are responsible for the efflux of cyclic nucleotides.
Cystic fibrosis transmembrane conductance regulator (CFTR) is found in RBC membranes, and is involved in ATP release from RBCs.
Human erythrocytes have equilibrative nucleoside transporter (hENT1) in its plasma membrane. Red cell metabolism is dependent on exogenous sources of adenine nucleosides, and hENT1 transports nucleosides, including inosine, rapidly between plasma and RBC cytoplasm. hENT1 also transports adenosine, hypoxanthine, thymine, adenine, uracil, and guanine. Nucleoside analogs are being used as immunosuppressants, antiviral agents, and anticancer drugs, and also as PET tracers.
Erythrocyte plasma membranes contain also small amount of P2 purinoceptors.
Erythrocyte membranes contain urea transporters (UT-B, encoded by SLC14A1 gene), which also carry the blood ABO antigens. UT-B is also present on endothelial cells.
The erythrocyte isoform of anion exchanger 1 (eAE1, band 3) is an essential component of erythrocyte plasma membrane. It exchanges Cl- with CO3-, facilitating transfer of CO2 from tissues to the lungs, and indirectly supporting the release and uptake of O2.
Haemoglobin binds to the cytoplasmic domain of eAE1, deoxyHb significantly stronger than oxyHb.
Organic cation transporters
Erythrocyte membranes contain organic cation transporter OCTN1 and organic anion/cation transporter (URAT1, SLC22A12). URAT1 transports and exchanges urate anion.
Glucose transporter 1 (GLUT1) is abundant in human RBC plasma membranes, facilitating rapid transport of glucose (and its labeled analogues such as [18F]FDG). Glucose concentration is the same in the water space of erythrocytes and plasma (MacKay, 1932). Most mammalian fetal erythrocytes are permeable to glucose, transporting and buffering glucose in the blood (Guarner & Alvarez-Buylla, 1990). In addition to humans, RBCs of some mammalian species retain the glucose transport capability to adult age, but in most species (including pigs, rabbits, guinea pigs, cows, horses) erythrocytes lose this capability, and RBCs of rats and dogs may retain some glucose transport capability (Guarner & Alvarez-Buylla, 1989). GLUT1 expression in erythrocytes is increased in chronic hyperglycemia and in Alzheimer’s disease. GLUT3 and GLUT4 may be of less importance in RBCs. GLUT9 (SLC2A9, URATv1) is a facilitative glucose transporter, but its main function in erythrocytes may be the transport of uric acid.
Sodium/glucose cotransporters 1 and 2 (SGLT1 and SGLT2) are found on RBC membranes.
In humans, about 90% of L-lactate is transported across erythrocyte plasma membrane by monocarboxylate transporter MCT1 (SLC16A1, lactate/H+ symporter), member of SLC16 family of solute carriers. Only at very high lactate concentrations AE1 participates in lactate transport significantly. In some species lactate transport is very slow, because only AE1 and diffusion are responsible for the transport.
MCT1 transports also pyruvate, β-hydroxybutyrate, and acetoacetate.
Erythrocyte membranes contain several P-type ATPases, for example ATP7B which transports Cu+ out of the cells.
Enzymes in erythrocytes
Enzymes in erythrocytes may metabolize PET tracers in the blood samples, leading to underestimation of the parent fraction of the tracer in plasma. This is particularly a problem with AChE tracers, because the erythrocyte membranes possess high AChE activity (Lawson and Barr, 1987). Red blood cells and platelets contain phenolsulfotransferase, which sulphoconjugates many radiotracers. Erythrocytes can metabolize a diverse range of compounds, including amino acids, amines, lipids, and organic acids (Cossum, 1988).
Glutathione (GSH) is synthesized from glutamate, cysteine, and glycine in the RBC by two enzymes, glutamate cysteine ligase and glutathione synthetase. Glutamate concentration in erythrocytes is much higher than in plasma, and as erythrocyte plasma membrane is impermeable to glutamate, it is transported into the cells as glutamine, which is hydrolyzed to glutamate by glutaminase, and as α-ketoglutarate, which is transaminated to glutamate by alanine aminotransferase.
Superoxide dismutase, catalase, and peroxidase in RBCs scavenge reactive oxygen species. Carbonic anhydrase 1 facilitates the equilibration between CO2 and H2CO3, important for the transport of CO2 from the tissues.
Receptors on erythrocyte membranes
Erythrocyte membranes contain many receptor types, including insulin receptors; ATP-binding P2X2, P2X4, and P2X7 receptors; and β-adrenergic receptors, which may enable the central nervous system to affect the blood O2 supply to tissues (Kim et al., 2017).
Basara B, Slijper M. Challenges for red blood cell biomarker discovery through proteomics. Biochim Biophys Acta 2014; 1844: 1003-1010.
Bernhardt I, Ellory JC (eds.): Red Cell Membrane Transport in Health and Disease. Springer, 2003. doi: 10.1007/978-3-662-05181-8.
Bogdanova A, Makhro A, Wang J, Lipp P, Kaestner L. Calcium in red blood cells - a perilous balance. Int J Mol Sci. 2013; 14: 9848-9872.
Buxton DB. Glucose permeability in nonprimate erythrocytes. J Nucl Med. 1999;40(12):2125-2126.
Cossum PA. Role of the red blood cell in drug metabolism. Biopharm Drug Dispos. 1988; 9(4): 321-336. doi: 10.1002/bod.2510090402.
Dumez H, Reinhart WH, Guetens G, de Bruijn EA. Human red blood cells: rheological aspects, uptake, and release of cytotoxic drugs. Crit Rev Clin Lab Sci. 2004; 41(2): 159-188.
Ellison S, Pardridge WM. Red cell phenylalanine is not available for transport through the blood-brain barrier. Neurochem Res. 1990; 15(8): 769-772.
Ellsworth ML, Ellis CG, Sprague RS. Role of erythrocyte-released ATP in the regulation of microvascular oxygen supply in skeletal muscle. Acta Physiol. 2016; 216(3): 265-276.
Graham MM, Nelp WB. Cardiac blood pool activity after in vivo and in vitro red blood cell (RBC) labeling. J Nucl Med. 1980; 21(6): P7.
Highley MS, De Bruijn EA. Erythrocytes and the transport of drugs and endogenous compounds. Pharm Res. 1996; 13(2): 186-195.
Hinderling PH. Red blood cells: a neglected compartment in pharmacokinetics and pharmacokinetics. Pharmacol Rev. 1997; 49(3): 279-295.
Kleinfeld AM, Storms S, Watts M. Transport of long-chain native fatty acids across human erythrocyte ghost membranes. Biochemistry 1998; 37: 8011-8019.
Lawson AA, Barr RD. Acetylcholinesterase in red blood cells. Am J Hematol. 1987; 26: 101-112.
Lee J-S, Su K-H, Lin J-C, Chuang Y-T, Chueh H-S, Liu R-S, Wang S-J, Chen J-C. A novel blood-cell-two-compartment model for transferring a whole blood time activity curve to plasma in rodents. Comput Methods Programs Biomed. 2008; 92(3): 299-304.
Nahmias C, Wahl LM, Amano S, Asselin M-C, Chirakal R. Equilibration of 6-[18F]fluoro-L-m-tyrosine between plasma and erythrocytes. J Nucl Med. 2000; 41: 1636-1641.
Paixão P, Gouveia LF, Morais JAG. Prediction of drug distribution within blood. Eur J Pharm Sci. 2009; 36: 544-554.
Pallotta V, D’Alessandro A, Rinalducci S, Zolla L. Native protein complexes in the cytoplasm of red blood cells. J Proteome Res. 2013; 12: 3529-3546.
Thomas SL, Bouyer G, Cueff A, Egée S, Glogowska E, Ollivaux C. Ion channels in human red blood cell membrane: actors or relics? Blood Cells Mol Dis. 2011; 46(4): 261-265.
Tosteson D. Halide transport in red blood cells. Acta Physiol Scand. 1959; 46: 19-41. doi: 10.1111/j.1748-1716.1959.tb01734.x.
Várady G, Szabo E, Fehér Á, Németh A, Zámbó B, Pákáski M, Janka Z, Sarkadi B, Alterations of membrane protein expression in red blood cells of Alzheimer’s disease patients. Alzheimers Dement. 2015; 1: 334-338.
Wick M, Pinggera W, Lehmann P: Clinical Aspects and Laboratory - Iron Metabolism, Anemias, 6th edition. Springer, 2011.
Yu S, Li S, Yang H, Lee F, Wu JT, Qian MG. A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery. Rapid Commun Mass Spectrom. 2005; 19(2): 250-254.
Updated at: 2019-01-16
Written by: Vesa Oikonen