**3. Glutathione and sphingosine-1-phosphate in the erythrocyte**

Glutathione and sphingosine-1-phosphate are two compounds synthesized by and exported from erythrocytes. Glutathione is mainly a protector against oxidative damage, whereas sphingosine-1-phosphate is an immune cell communicator. Glutathione is synthesized by the enzymes glutamate-cysteine ligase and glutathione synthetase which are magnesium-dependent enzymes. Glutamate-cysteine ligase uses magnesium-ATP as substrate, and glutathione synthetase crystals show two magnesium ions in addition to ADP, glutathione, and sulfate [21]. Glutathione is the main reducing

agent in erythrocytes and other cells. Supplementation with the glutathione prodrug N-acetylcysteine or glycine has been shown to improve several health aspects, including insulin resistance and cognition [22]. Erythrocytes and other tissues like liver and brain export glutathione to plasma [23]. Erythrocyte export of glutathione takes place by multi-drug resistance proteins [24]. Extracellular glutathione is of relevance in inflammation and disease [25–27] and can directly regulate immune components in plasma [28]. Intracellular glutathione concentration is usually three orders of magnitude higher than the extracellular glutathione concentration, and an energy-consuming active glutathione uptake mechanism seems not to be known [29]. Therefore, extracellular glutathione is not expected to be taken up by cells. Erythrocyte glutathione synthesis is dependent on the availability of the substrates glutamate and cysteine. Cysteine is imported as cystine through the glutamate/cystine antiporter, also known as SLC7A11 or system Xc-. Glutamate is acquired as glutamine through the ASCT2, also known as SLC1A5 transporter.

Sphingosine-1-phosphate (S1P) is a multifunctional molecule synthesized, stored, and exported to plasma by erythrocytes, platelets, and endothelial cells [30]. The erythrocyte is considered as the main contributor to plasma S1P. The two enzymes sphingosine-kinase-1 and sphingosine-kinase-2 are responsible for the synthesis of S1P. Targeted deletions of the genes coding for these enzymes suggest that erythrocytes and platelets are redundant for S1P synthesis under normal conditions, but necessary in systemic anaphylaxis [31]. Erythrocytes obtain sphingosine from plasma for S1P synthesis, although the precise import mechanism has not been elucidated. S1P is then synthesized by sphingosine-kinase-1, which is dependent on magnesium as shown by the presence of magnesium in the crystal structure of sphingosine-kinase-1 [32]. Evidence for intra-erythrocytic sphingosine synthesis has not been found [33]. The S1P-degrading enzymes sphingosine lyase and sphingosine phosphohydrolase are not present in erythrocytes, leading to some erythrocytic S1P storage capacity [34]. S1P is exported from erythrocytes by the major facilitator superfamily domain 2b (Mfsd2b), whereas export from endothelial cells is mediated through protein spinster homolog 2 (Spns2). S1P also needs apolipoprotein M in complex with high-density lipoprotein for effective export from erythrocytes [35].

S1P has several known effects in plasma. S1P forms a gradient where high levels are found peripherally and low levels are found within lymph nodes. Maturing lymphocytes expressing the S1P-receptor-1 proceed along this gradient, thereby leaving the lymph nodes. S1P1 receptor expression and further downstream signaling in endothelial cells are necessary for blood vessel integrity and vascular tone. Some of these effects are thought to be brought about by nitric oxide signaling [30]. Increased erythrocyte S1P levels have been found in COVID-19 patients [36]. S1P can reprogramme erythrocytes of chronic kidney disease patients to glucose metabolism through the Rapoport-Luebering shunt [37].

### **4. Erythrocytes, Alzheimer's disease, and other types of dementia**

Several studies have indicated changes in erythrocytes of demented patients, including Alzheimer's disease. Binding of amyloid-beta to erythrocytes has been reported, and fibrils supposedly containing amyloid-beta have been visualized on erythrocytes [38]. A metabolomics study of Alzheimer's disease patients and controls identified 750 metabolites of which 7 increased and 24 decreased in erythrocytes of Alzheimer's disease patients [39]. The increased metabolites included argininate,

#### *Erythrocytes as Messengers for Information and Energy Exchange between Cells DOI: http://dx.doi.org/10.5772/intechopen.108321*

2-oxoarginine, and N-acetylarginine, all of which are known to form as a result of deficiency of the enzyme arginase. Erythrocyte arginase activity in Alzheimer's disease patients needs to be investigated to follow up on these results. All the 24 decreased metabolites were lipids, or related to lipid metabolism, whereof 10 were in the sphingolipid group like sphingomyelin. A metabolomic study of plasma in Alzheimer's disease similarly found differential presence of many lipids, but in this study sphingomyelin was higher in plasma of Alzheimer's disease patients [40]. Low blood hemoglobin levels and anemia have been associated, possibly in a causal relationship, with Alzheimer's disease and cognitive function [41].

Genome-wide association studies have found association between Alzheimer's disease and complement receptor 1 (CR1), clusterin, complement component 1 s (C1s), and in some ethnic groups also complement factor H [42–44]. CR1 is a transmembrane protein with a short cytoplasmic part, and many extracellular short consensus repeats (SCR) that bind complement component 3b (C3b) in complex with an antibody and its antigen, also referred to as an immune complex. The bound immune complex is then delivered to macrophages for internalization and degradation. CR1 has been localized to the membrane of erythrocytes where 80–90% of all CR1 in the body is estimated to be localized [45]. C3b binds to immune complexes with amyloid-beta, the main suspected protein in Alzheimer's disease. C3b-antibodyamyloid-beta complexes then bind to CR1 on erythrocytes that carry their load for delivery to the Kupffer macrophages in the liver. This process is sometimes referred to as immune adherence [46]. The anti-amyloid-beta antibody Aducanumab is a recent addition to Alzheimer's disease therapy. Aducanumab showed reduction of amyloid-beta in the brain, but no improvement of cognition or functional ability of patients [47]. Since antibodies have low ability to penetrate the blood-brain barrier, the effect of immunotherapy against Alzheimer's disease may involve amyloid-beta clearance from blood via erythrocytes and CR1 [48]. It should be noted that brain expression of CR1 is controversial, and therefore the erythrocyte-based explanation for CR1 involvement in Alzheimer's disease is preferred [45]. Complement has been shown to be active in normal brain function, for instance by performing synaptic pruning during development [49, 50]. The other complement components or complement factors that have been implicated in Alzheimer's disease may therefore be active in the brain. For instance, the C3b-binding complement factor H is present in both brain and plasma and has been shown to protect erythrocytes and other cells against complement-mediated damage [51]. As mentioned, complement factor H has shown association with Alzheimer's disease in some population studies [43]. A functional association between complement factor H and CR1 on erythrocytes in the pathology of Alzheimer's disease can therefore not be excluded. Inspection of CR1 alleles shows that CR1\*2 is associated with increased risk for Alzheimer's disease. CR1\*2 has an extra short consensus repeat in the extracellular domain of the protein. This length polymorphism is almost always associated with lower expression of CR1\*2 on the erythrocyte surface [52]. The patients also show high levels of soluble CR1. The significance of soluble CR1 seems to be largely unknown. Clusterin, also known as apolipoprotein J, and ATP-binding cassette A7, also known as ABCA7, are two additional erythrocyte proteins [53, 54] that are known to be associated with Alzheimer's disease from genome-wide association studies.

Erythrocyte levels of the omega-3 fatty acids docosahexaenoic acid and eicosapentaenoic acid were lower in subjects with dementia in a longitudinal study population [55]. Possibly omega-3 fatty acids offer some protection against dementia as is further discussed in the section on cholesterol and lipids.

Erythrocytes have also been suggested to be a potential link between Alzheimer's disease and diabetes [56]. For instance, erythrocytes in Alzheimer's patients express more glucose transporter 1 (GLUT1) and insulin receptor than control subjects [57]. It is possible that these results are indications of a dysregulated metabolism that influences the availability of oxygen for the brain which might lead to cognitive impairment [58]. It should also be noted that Alzheimer's is more frequent in patients with type 2 diabetes.

### **5. Magnesium, cognition, and the erythrocyte**

Body magnesium levels can be assessed through measurement of erythrocyte or plasma magnesium levels. Since only about 1% of total-body magnesium is found in blood, further tests may be necessary for more thorough evaluation of whole-body magnesium levels. A correlation between intra-erythrocyte magnesium levels and cognition was found in rats [59], and a similar association could be found in a human study involving patients with vascular cognitive impairment [60]. Erythrocytes show significantly diminished intracellular magnesium levels in patients with vascular cognitive impairment, although plasma magnesium levels are normal. This could be interpreted as a measure of whole-body magnesium levels, including the brain. Low magnesium levels in the central nervous system are known to be associated with complications like depression. An explanation for this may be the excitotoxicity caused by the *N*-methyl-d-aspartate (NMDA) receptor, a glutamate-regulated calcium channel present in the plasma membrane of nerve cells. The NMDA receptor requires magnesium as a "gate-keeper" to prevent opening of the channel. Small areas of blood-brain contact can occur in dementia as microbleeds or microhemorrhages, also known as blood extravasations [61]. Communication between brain and the immune complement and blood coagulation systems has been suggested to be part of the pathology in vascular dementia [62]. A meta-analysis of dementia trials showed that vascular dementia was associated with increased levels of fibrinogen, activated factor VII, factor VIII, von Willebrand factor, D-dimer, and homocysteine [63]. When displaying phosphatidylserine on the surface, erythrocytes contribute to coagulation by interacting with the gamma-carboxyglutamyl (Gla) domains on coagulation factors, initiating the formation of thrombin from prothrombin. Phosphatidylserine exposure is regulated by intracellular calcium level and by the enzymes flippase and scramblase. Flippase moves phospholipids from the exoplasmic to the cytoplasmic side of the plasma membrane. Flippase activity in human erythrocytes is performed by ATP11C, a P4-ATPase [64] that has been crystallized together with its interacting protein CDC50, which is also known as TMEM30A [65]. The structure is similar to other human or yeast P4-ATPase flippase structures [66, 67]. Reduction in ATP11C activity leads to increased phosphatidylserine exposure on erythrocytes [68] and in case of deficiency may lead to mild hemolytic anemia [69]. Flippase activity is dependent on ATP and magnesium, and some of the 3D structures also show magnesium ions at the phosphorylation site [66, 67]. Lower magnesium levels in erythrocytes can therefore lead to lower flippase activity and more exoplasmic exposure of phosphatidylserine.

Scramblase moves lipids between the two monolayers of a membrane in either direction, thus evening out differences in lipid composition between the two sides. Human scramblase comes in three different protein families [70]. The scramblase activity of erythrocytes is performed by transmembrane protein 16F (TMEM16F, also known as anoctamin6) [71, 72]. TMEM16F is a calcium-dependent homodimeric *Erythrocytes as Messengers for Information and Energy Exchange between Cells DOI: http://dx.doi.org/10.5772/intechopen.108321*

structure with 10 transmembrane alfa-helices and a large amino-terminal cytosolic domain in each subunit [73]. The hydrophilic head of the lipid substrate proceeds in a cavity on TMEM16F, similar to the swiping of a credit card [74]. Evidence for TMEM16F scramblase activity also comes from patients with Scott syndrome [75, 76], a condition involving low coagulation ability of platelets and erythrocytes [77]. Three calcium-binding sites can be seen in TMEM16F high-resolution structures, but so far, crystal structures of the TMEM family seem not to have included magnesium. Since TMEM16F is negatively regulated by magnesium [78], low intracellular magnesium levels, as was found in erythrocytes of vascular dementia patients, may lead to more TMEM16F scramblase activity, phosphatidylserine exposure, and blood coagulation. Magnesium binding to TMEM proteins could involve a magnesium-calcium competition similar to some other calcium-dependent proteins often involving the regulatory protein calmodulin [79]. A direct interaction between TMEM proteins and calmodulin has been suggested but is still controversial [80].

## **6. Iron and the erythrocyte**

Iron is necessary for oxygen transport by hemoglobin in the erythrocyte. In addition, iron is stored in ferritin, which has been shown to occur in the erythrocyte and may be necessary for binding excess erythrocyte iron. Excess erythrocyte iron may be a consequence of hemoglobin oxidation and degradation and may be a normal part of erythrocyte aging. The erythrocyte also contains the iron export protein ferroportin, which may likewise protect the erythrocyte from toxic effects of excess iron. Ferroportin is regulated by the iron hormone hepcidin, which in turn is regulated by interleukin-6. Conditional deletion of ferroportin in mice leads to build-up of intracellular erythrocyte iron and may result in hemolysis [81]. The ferroportin Q248H mutation protects ferroportin from degradation caused by hepcidin and seems to have been selected in African populations possibly due to some protection against malaria conferred by the mutation. Ferroportin functions in erythrocytes and erythroid cells as an export gate for the regulation of total-body iron homeostasis [82, 83]. Iron is dysregulated in ferroptosis, a form of regulated cell death distinct from apoptosis [84]. Ferroptosis is characterized by iron overload, peroxidation of lipids and low activity of the selenium-containing enzyme glutathione peroxidase 4. Ferroptosis has been associated with several pathological conditions including neurological diseases such as Alzheimer's disease [85]. Transcriptomic analysis reveals changed expression levels of many ferroptosis-related genes in Alzheimer's disease patients [86], including the selenium-containing enzyme glutathione peroxidase 4. Neuron ferroportin needs the amyloid precursor protein for stability and localization. Degradation of amyloid precursor protein to amyloid-beta may lead to increased iron levels in neurons and pave the way for ferroptosis-induced neuron death [87]. Some amyloidbeta will reach the vascular compartment by way of the glymphatic system. Amyloidbeta can then bind to erythrocytes in the vascular compartment. Amyloid-beta has been shown to induce morphological changes in erythrocytes [88], affect signal transduction [89], and inhibit production and release of ATP from erythrocytes [90]. Amyloid-beta could potentially affect erythrocytes leading to dysregulation of totalbody iron homeostasis, potentially worsening the prodromal phase of Alzheimer's disease, although this remains to be tested.

Hereditary hemochromatosis is another condition also characterized by iron overload in several tissues of the body. Particularly the liver, heart, pancreas, and skin are affected in hereditary hemochromatosis. Erythrocytes of patients with hereditary hemochromatosis may show morphological changes [91], and the patients can be affected by secondary diseases that affect erythrocytes, such as polycythemia vera or hemolytic anemia [92].
