Defence against oxidative stress the production of reducing power

As with all cells, but perhaps more urgently, the red blood cell needs to be protected against the effects of free radicals, hydrogen peroxide and other highly oxidative material in order to maintain the membrane integrity and functional activity. In addition, Hb has to be maintained in its functional state, and the steady production of MetHb reversed back to deoxy-haemoglobin, which is able to combine reversibly with oxygen. The major generator of reducing power within the red cell is the pentose phosphate pathway (hexose monophosphate shunt), which generates reducing power in the form of NADPH from NADP+ coupled to the oxidation of G6P to 6-phosphogluconate and the subsequent oxidation of that compound to ribose 5-phosphate, the two reactions catalysed by the enzyme G6PD and 6-phosphogluconate dehydrogenase (6PGD) (Figure 9.9).

Glutathione (GSH) is important for the protection of cells from oxidative damage by these free radicals, protection against the effects of infection, the maintenance of protein sulphydryl groups in the reduced state and the maintenance of membrane transport. A constant supply of GSH is provided by the gluta-thione cycle, linked to the pentose phosphate pathway through the action of glutathione reductase (Figure 9.10).

An excess of oxidative stress over the reducing power of the red cell leads to intravascular haemolysis following denaturation of the Hb and precipitation as Heinz bodies and peroxidation of the red cell membrane. Methaemoglobinaemia may occur. The clinical features that emerge, acute intravascular haemolysis, Heinz body haemolytic anaemia or methaemoglobinaemia, depend upon the nature of the oxidative stress and the size of the imbalance between the stress and the redox potential.

Glucose

NADP

G6PD

Pyruvate

NADP

6PGD

Transketolase

Ru5P PKE^ \PRI

Xy5P R5P

Transketolase

Ga3P

Transalkolase

Figure 9.9 The pentose phosphate pathway. Substrates: G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Ga3P, glyceraldehye-3-phosphate; F6P, fructose-6-phosphate; Xy5P, xylose-5-phosphate; S7P, septulose-7-phosphate; E4P, erythrothrose-4-phosphate. Enzymes: G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; PKE, phosphoketoepimerase; PRI, phosphoribose isomerase.

The reactions of the pentose phosphate pathway are shown schematically in Figure 9.2 and in more detail in Figure 9.9. In most cells, the pentose phosphate pathway is an essential pathway for the production of ribose and incorporation into RNA. In the red cell, its only function is the production of reducing power in the form of NADPH. The first step of the pathway, catalysed by G6PD, utilizes G6P as the substrate. G6P is also a substrate for the glycolytic pathway. Under normal circumstances, about 10% of glucose is metabolized by the pentose phosphate pathway, the activity of that pathway being determined by the availability of NAD+ and feedback inhibition by ATP. Under conditions of oxidative stress, the flux through the pentose phosphate pathway can be greatly increased. The products of the pathway re-enter the glycolytic pathway through F6P or Ga3P.

From the point of view of haematological disorders, G6PD deficiency is by far the most important step in the pathway. However, other enzymes of the pathway can be important in acquired disorders, some steps being inhibited by drugs, including oestrogen-progesterone contraceptive pills.

Glucose-6-phosphate dehydrogenase

G6PD catalyses the first step of the pentose phosphate pathway and is the enzyme controlling flux through that pathway. The activity is controlled by the availability of NADP+. Conversion of G6P to 6PG is accompanied by the reduction of NADP+ to NADPH, and the second step in the pathway, the oxidation of 6PG to ribose 5-phosphate, produces a second molecule of NADPH.

The gene for G6P is located on the X chromosome at Xq28. The gene has 13 exons and 12 introns. The gene is transcribed as

Glutamic acid

Cysteine

ATP.

ADP<""'' Y-Glutamylcysteine .•■' synthetase

Glycine

Y-Glutamylcysteine

Hb Fe2+

Glucose-6-phosphate dehydrogenase

NADP+

MetHb

NADPH MetHb reductase

Glutathione . ATP synthetase /;

RSSR

Glucose-6-phosphate dehydrogenase

Glutathione reductase

NADPH

Glutathione S-transferase^

Glutathione — peroxidase

GSSG

GSSG

H2O2

Catalase

Pentose phosphate pathway (hexose monophosphate shunt)

Figure 9.10 The glutathione cycle and synthetic pathways. GSH is synthesized in two linked steps. The redox control is exercised by the glutathione cycle linked to the NADPH of the pentose phosphate pathway by glutathione reductase.

Structure G6pd
Figure 9.11 Structure of G6PD in dimer form, showing active sites for G6P and NADP+. Mutations that affect interactions of the monomers lead to CNSHA.

MS A

ÎT flftftf 9? 9m

N 2 3 4 5

6 7 8

9 10 11 12 13 C

à 1 1 à 1

! 1 !! 'I!1!!!1 1 T 1

Figure 9.12 G6PD gene map showing mutation sites (courtesy variants; red circles, polymorphic variants; green circles and of Tom Vulliamy, Hammersmith Campus, Imperial College, squares, class I variants caused by amino acid substitutions and

London). Numbered boxes refer to the location of G6PD exons: small in-frame deletions, respectively. yellow circles, class I and II variants; yellow ellipses, class IV

Figure 9.12 G6PD gene map showing mutation sites (courtesy variants; red circles, polymorphic variants; green circles and of Tom Vulliamy, Hammersmith Campus, Imperial College, squares, class I variants caused by amino acid substitutions and

London). Numbered boxes refer to the location of G6PD exons: small in-frame deletions, respectively. yellow circles, class I and II variants; yellow ellipses, class IV

a monomer of 514 amino acids that come together to produce an equilibrium of dimers and tetramers (Figure 9.11). Each monomer has an NADP+-binding domain and a large domain, with the active site between the two. The gene is a household gene, active in all cells, with an essential role in the production of RNA in nucleated cells. Not surprisingly, the gene is highly conserved in evolutionary history. Complete inactivity of the enzyme in nucleated cells would not be compatible with life. The clinical consequences of G6PD deficiency are virtually confined to the red blood cell, with occasional evidence of leucocyte malfunction in some variants. The majority of mutations affect the stability of the transcribed enzyme so that there is a rapid decline in activity in the mature enucleate red cell as it ages.

In the red blood cell, G6PD catalyses the first reaction of the pentose phosphate pathway, the conversion of G6P to 6-phosphogluconate through the reduction of NADP+ to NADPH (Figure 9.9). The availability of NADP+ is determined by the activity of the glutathione cycle, which is linked to the pentose phosphate pathway through the activity of the enzyme glutathione reductase (Figure 9.12). The availability of NADP+ is the major rate-limiting step for the pentose phosphate pathway.

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  • jill
    What is the reducing power of red blood cells?
    6 months ago

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