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Discuss the biochemistry that takes place within the red blood cell and how this biochemistry relates to the structure and function of the erythrocyte

The primary function of a mature red blood cell (RBC) is the transport of respiratory gases to and from the tissues. To be able to do this effectively, the red cell has to traverse the microvascular system without mechanical damage and also retain its shape. Red blood cells (RBCs) have a tough yet highly flexible cell membrane and are also readily deformable due to their protein cytoskeleton. This allows them to pass through narrow capillaries unhindered. The biconcave disc shape of RBCs allows maximum surface area to volume ratio for efficient gas exchange.

The protein cytoskeleton and its interaction with the membrane lipid bilayer allow it to complete its primary function. 1.

There are specific transport proteins in the cell membrane that facilitate the transport of glucose into the red cell. This is essential as a constant supply of glucose from the plasma is required for energy and reducing potential purposes. The red cell membrane is also selectively permeable and thus this allows retention of phosphorylated sugars that are involved in the glycolytic pathway.

The red cell membrane also contains cationic pumps that actively transport sodium ions (Na+) from the cell into the plasma and vice versa for potassium ions (K+). Their activity is stimulated by extracellular K+ or an increase in concentration of intracellular Na+. Hence these pumps are able to precisely control and balance the movement of these cations across the plasma cell membrane.

Balance of intracellular and extracellular concentrations of Na+ and K+ is maintained by a membrane-bound ATPase pump. ATP is required for this pump to actively drive the process, pumping Na+ out of the cell and K+ into the cell. Without ATP as a source of energy would result in failure of the cationic pump, causing osmosis of water into the cell. This leads to swelling of RBCs and then lysis that can result in haemolytic anaemia and plasma bilirubin levels increasing significantly, causing jaundice.

Energy in the form of ATP is required to drive the pumps, producing ADP that is then recycled by the Embden-Meyerhof pathway to produce ATP.

The red cell membrane also contains channels that allow rapid movement of water and anions, such as chloride and hydrogen carbonate. These channels are essential as they allow the plasma to be electrically neutral.

As mature RBCs do not contain a nucleus, mitochondria or ribosomes, this means that they cannot function like other types of body cells. Energy cannot be produced efficiently by oxidative phosphorylation and proteins cannot be synthesised. The energy requirements of the RBC occur through aerobic glycolysis via the Embden-Meyerhof pathway that produces two moles of ATP per mole of glucose metabolised.

Another pathway, the Hexose Monophosphate pathway, is also involved in glycolytic biochemical processes and protects the cell against oxidative stress. These pathways occur within the RBC and are dependent on enzymatic processes.

Energy is used to fulfil important cell processes. These include maintaining intracellular ion balances and cell shape. Phosphorylation of glucose and fructose-6-phosphate in the initial stages of the Embden-Meyerhof pathway (EMP) also require energy, which without, further ATP would not be synthesised.

The Hexose Monophosphate pathway

In the first step of the Hexose Monophosphate pathway (HMP), dehydrogenation occurs of glucose-6-phosphate to form 6-phosphogluconate, and is produced via the intermediate 6-phosphoglucono-d-lactone. This reaction takes place under the influence of the enzyme glucose-6-phosphate dehydrogenase and is accompanied by the generation of one mole of nicotinamide adenine dinucleotide phosphate (NADPH).

Decarboxylation of 6-phosphogluconate then occurs in the presence of 6-phosphogluconate dehydrogenase to form ribulose-5-phosphate. This process also yields one mole of NADPH and thus, for each mole of glucose that enters the pathway, two moles of NADPH are generated.

Under the influence of phosphopentose isomerase, ribulose-5-phosphate is then converted to ribose-5-phosphate, which itself is then converted to fructose-6-phosphate by transaldose. Production of this substance leads to the Embden-Meyerhof pathway of reactions.

It is the formation of NADPH that is the highlight of the HMP pathway as it is this cofactor that is required for reducing glutathione in the presence of glutathione reductase of the glutathione cycle. Reduced glutathione is the most potent antioxidant within RBCs.

As mentioned previously, the HMP protects the RBC against oxidative stress. About 5% of red cell glucose is metabolised via this pathway. However, if there are increased levels of oxidants, then the rate of formation of glucose-6-phosphate to be utilised by this pathway can be increased.

Reducing power is required by red cells to prevent against membrane lipid oxidation and peroxidation as this causes an increase in membrane rigidity and permeability, resulting in red cells being liable to destruction in the spleen.

Methaemoglobin is the oxidised form of haemoglobin and is formed by oxidation of ferrous (Fe2+) iron in haem to the ferric (Fe3+) form. Methaemoglobin accumulation prevents RBCs from transporting oxygen and causes weakening of the structure of the cell membrane. This renders the red cell to rupture and haemolysis.

Small quantities of methaemoglobin are constantly formed within RBCs. Reduced glutathione maintains haemoglobin in its reduced, functional state and prevents oxidation of sulphahydryl groups in the haemoglobin structure.

Hydrogen peroxide is a powerful oxidant and is formed by highly reactive oxygen radicals such as superoxide and hydroxyl radicals during methaemoglobin production. Drugs such as primaquine also result in formation of hydrogen peroxide. Reduced glutathione allows detoxification of these substances before severe oxidant damage can occur to the cell.

The Embden-Meyerhof pathway (EMP)

In the first stage of the EMP, phosphorylation of glucose occurs, and is catalysed by the enzyme hexokinase to form glucose-6-phosphate. This reaction takes place in the presence of divalent metal ions such as magnesium and requires a mole of ATP for every mole of glucose phosphorylated.

Glucose-6-phosphate production inhibits the catalytic activity of hexokinase and therefore allows the rate of glycolysis to be controlled within the red cell via a negative feedback mechanism.

Phosphoglucose isomerase then converts glucose-6-phosphate to fructose-6-phosphate, that is then phosphorylated to form fructose-1, 6-diphosphate, catalysed by the enzyme phosphofructokinase.

Adequate levels of ATP in the red cell inhibit the activity of phosphofructokinase and once again allow the rate of glycolysis to be controlled. Low levels of ATP would once again stimulate the process of glycolysis.

In a series of reactions catalysed by the enzymes aldolase and triose phosphate isomerase, one mole of fructose-1, 6-diphosphate is cleaved to form two moles of glyceraldehyde-3-phosphate.

The next step of the pathway allows the production of NADH due to glyceraldehyde-3-phosphate being converted to 1, 3-diphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase.

So far two moles of ATP have been used but none produced. This changes when 1, 3-diphosphoglycerate is dephosphorylated to 3-phosphoglycerate that then results in ADP being converted to ATP. This reaction is catalysed by phosphoglycerate kinase and results in overall production of two moles of ATP as one mole of glucose is converted to two moles of 1, 3-diphosphoglycerate.

The enzyme phosphoglyceromutase then catalyses the conversion of 3-phosphoglycerate to 2-phosphoglycerate, and another enzyme called enolase subsequently converts 2-phosphoglycerate to phosphoenolpyruvate.

Another molecule of ADP is then phosphorylated to ATP due to the transfer of a phosphoryl group by phosphoenolpyruvate and results in production of pyruvate. This occurs under the influence of the enzyme pyruvate kinase. Thus two more moles of ATP are produced per mole of glucose.

Lactate dehydrogenase then converts pyruvate to lactate in the last step of the EMP.

As can be calculated, this pathway ultimately results in net production of two moles of ATP per mole of glucose consumed.

The Rappaport-Luebering Shunt

In the main EMP, the enzyme diphosphoglycerate phosphatase converts 1, 3-diphosphoglycerate (1, 3-DPG) to 3-phosphoglycerate. However, a critical reaction takes place in the presence of diphosphoglycerate mutase, converting 1, 3-DPG to 2, 3-DPG. This is known as the Rappaport-Luebering Shunt and is a very important pathway as 2, 3-DPG is required by red blood cells to maintain efficient oxygen control.

2 ,3-DPG has a high affinity for oxyhaemoglobin and therefore causes haemoglobin to release its oxygen to tissues at low partial pressures (of oxygen) by inserting itself between the ß-chains of haemoglobin, thus causing electrostatic interactions to take place that facilitate oxygen release by displacement.

At high partial pressures of oxygen, 2, 3-DPG itself is displaced instead of oxygen and this allows haemoglobin to carry oxygen to where it is required.

Red cell enzymopathies

As a large number of enzymes are involved in biochemical pathways that occur within RBCs, enzymatic deficiencies can lead to red blood cell disorders that can have serious health implications. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is a genetic disorder of the HMP. Likewise, pyruvate kinase and hexokinase deficiencies are two genetic disorders of the EMP.

G-6-PD deficiency is an X-linked recessive disorder affecting RBCs found on the long arm of the X chromosome on band Xq28 and is 18.5 Kb long.

As the deficiency follows an X-linked pattern of inheritance, it is the male offspring of a female carrier that exhibit the symptoms. However, female offspring can also be clinically affected by lyonisation. This can occur through random inactivation of an X chromosome in certain cells, producing G-6-PD deficient RBCs that coexist with normal RBCs.

G-6-PD deficiency affects about 1% of the world's population and is the most common of the RBC enzymopathies. The deficiency is most common in populations of the Middle East, South-East Asia, Mediterranean regions and also parts of Central and Western Africa.

Although more than 350 different variants of G-6-PD deficiency have been reported, only a few cause serious disease.

Defects in the HMP increase the susceptibility of the RBC to oxidative stress. Reduced glutathione is required by RBC to prevent oxidative stress. However, it is the HMP that provides NADPH to drive the glutathione cycle, producing reduced glutathione.

Oxidative damage reduces the lifespan of RBCs in a number of ways. Membrane cytoskeletal proteins cross-link and aggregate, resulting in a decrease in the ability of RBCs to deform. This ultimately leads to their destruction by the liver and spleen through sequestration.

Heinz bodies are inclusion bodies formed through oxidation of thiol and other globin chain monomers. This leads to denaturation of globin molecules forming aggregates and precipitates. These Heinz bodies bind to the inner part of the red cell membrane that are then destroyed during passage through the spleen forming 'bite' cells that cause premature haemolysis.

Chronic non-spherocytic haemolytic anaemia (CNSHA) and episodic haemolysis are the two types of haemolysis that individuals with G-6-PD deficiency can suffer from.

CNSHA is a rare, severe, and life-long form of G-6-PD deficiency associated with possessing extremely unstable or dysfunctional G-6-PD variants. The disease results in haemolytic anaemia occurring in patients that can be of variable severity. Unfortunately a splenectomy is not enough to control the disease and further complications through infection or ingestion of oxidant drugs can exacerbate the condition causing sporadic haemolytic or aplastic crises.

Episodic haemolysis is a common disease of G-6-PD deficiency associated with less severe enzymatic variants of G-6-PD.

Enzymatic activity of RBCs is enough to prevent them from suffering from oxidative stress and therefore their survival is mostly unhindered. However, oxidant drugs, ingestion of common Vicia fava broad beans and pneumococcal infection or viral hepatitis can increase oxidative stress levels resulting in acute intravascular haemolysis.

Pyruvate kinase (PK) deficiency affects the survival of RBCs. Inheritance can be either autosomal dominant or recessive.

PK converts phosphoenolpyruvate to pyruvate and in the process donates a phosphoryl group to ADP that results in the formation of ATP. However, in patients with PK deficiency, these two moles of ATP per mole of glucose cannot be generated and therefore the EMP results in no net production of ATP.

As mentioned previously, without ATP as a source of energy, RBCs are unable to maintain their structure or functioning. This causes them to become distorted, rigid and prone to lysis that eventually leads to their death by the spleen and liver.

Treatment options available include removal of the spleen or having a blood transfusion to reduce severity of the symptoms.

Hexokinase deficiency can have an autosomal dominant, or less likely, an autosomal recessive mode of inheritance. It is an extremely rare disorder but nonetheless can result in severe symptoms of anaemia.

Glucose is phosphorylated to glucose-6-phosphate in the presence of the enzyme hexokinase and divalent metal ions like magnesium. ATP is also required for this reaction to take place as the source of phosphate. Without hexokinase, no glucose-6-phosphate production can take place and this results in disruption of the EMP. The halt in production of 2, 3-DPG that normally occurs through the EMP is the main cause of anaemia as this compound is required to displace oxygen to tissues of the body as required. Lack of 2, 3-DPG increases the haemoglobin molecules affinity for oxygen, thus starving cells (of oxygen).


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