Part of the response to infection is activation of macrophages to produce a mixture of oxygen and other radicals that are cytotoxic to engulphed micro-organisms. This leads to an increase in whole body radical burden, and increased oxidative damage to cell membranes.

One way of screening for sensitivity to primaquine is based on the observation that the glutathione concentration of erythrocytes from sensitive subjects is somewhat lower than that of control subjects, and falls considerably on incubation with acetylphenylhydrazine.

Glutathione (GSH) is a tripeptide, gamma-glutamyl-cysteinyl-glycine, which readily undergoes oxidation to form a disulphide-linked hexapeptide, oxidised glutathione, generally abbreviated to GSSG

The table shows the concentrations of GSH and GSSG in red cells from 10 control subjects, and Samuel W, before and after incubation with acetylphenylhydrazine.

The effect of incubation with 330 µmol /L acetylphenylhydrazine on erythrocyte glutathione.

Samuel’s red blood cells contain significantly less GSH than normal, and a very high level of GSSG.

The results suggest that acetylphenylhydrazine causes the oxidation of glutathione to GSSG. This is not a simple stoichiometric oxidation of glutathione by acetylphenylhydrazine. In Samuel's erythrocytes, the ratio of glutathione oxidised : acetylphenylhydrazine present = 4.7. This suggests that it is likely that acetylphenylhydrazine undergoes a cyclic redox reaction that results in the production of hydrogen peroxide, which in turn oxidises glutathione.

In control subjects incubation with acetylphenylhydrazine leads to a modest decrease in GSH, and a small accumulation of GSSG; Samuel’s red cells show a very considerable depletion of GSH and a very large accumulation of GSSG on incubation with acetylphenylhydrazine.

This suggests that Samuel cannot reduce GSSG back to GSH as effectively as normal. This could be due to lack of either glutathione reductase or NADPH.

The reported Km of glutathione reductase for GSSG is 65 µmol /L and for NADPH 8.5 µmol /L Erythrocyte lysates were incubated with a saturating concentration of GSSG (1 mmol /L) and either NADPH or NADH (100 µmol /L). Each incubation contained the haemolysate from 0.5 mL packed cells.

Glutathione reductase, µmol product formed /min

Obviously Samuel has no problem with the activity of glutathione reductase, suggesting that his problem lies in providing a source of NADPH for the enzyme.

Since none of the lysates showed any significant activity with NADH, it is unlikely that there is any transhydrogenase activity in erythrocytes, to reduce NADP+ to NADPH at the expense of NADH. This raises the problem of the source of NADPH in erythrocytes.

The dye methylene blue will oxidise NADPH; the reduced dye then undergoes non-enzymic oxidation in air, so the addition of a relatively small amount of methylene blue will effectively deplete NADPH, and would be expected to stimulate any pathway that reduces NADP+ to NADPH.

Erythrocytes from control subjects were incubated with 10 mmol /L [14C]glucose with or without the addition of methylene blue; all 6 possible positional isomers of [14C]glucose were used. The radioactivity in (lactate plus pyruvate) was determined after thin layer chromatography of the incubation mixture, and radioactivity in any carbon dioxide formed was also measured. Each incubation contained 1 mL erythrocytes in a total incubation volume of 2 mL.

Production of [14C] (lactate +pyruvate) and carbon dioxide by 1 mL erythrocytes incubated for 1 hour with 10 mmol /L [14C]glucose at 10 µCi /mmol. Figures show mean dpm (radioactive disintegrations per minute) ± sd for 5 replicate incubations.

ND = not detectable – i.e. below the limits of detection.

Considering the control incubations: only carbon-1 of glucose gives rise to carbon dioxide, and the radioactivity in (lactate + pyruvate) is lower from [14C-1]glucose than the other positional isomers. However, the total radioactivity in (lactate + pyruvate + carbon dioxide) is ~14090 dpm in all cases - from the initial specific activity this gives a rate of glucose utilisation of 640 nmol /mL cells /hour.

There is no reaction in glycolysis in which carbon dioxide is produced, and this suggests that there must be some other pathway of glucose metabolism in the erythrocyte, in which carbon-1 of glucose is released as carbon dioxide. In the control incubations, about 10% of the radioactivity from carbon-1 of glucose appears as carbon dioxide, suggesting that perhaps 10% of glucose metabolism is by way of this alternative pathway.

In the presence of methylene blue there is a very considerable increase in the production of [14C]carbon dioxide from [14C-1]glucose, and a corresponding decrease in the label in lactate and pyruvate, although the total radioactivity in (lactate + pyruvate + carbon dioxide) is the same as in the control incubation. This suggests that the overall rate of glucose metabolism is unaffected by methylene blue, but a greater proportion (87%) is by way of the decarboxylation pathway.

Since the action of methylene blue is to oxidise NADPH, it is therefore likely that the pathway that results in decarboxylation is also the pathway that utilises NADP+ and forms NADPH.

Two reactions involving glucose metabolites catalyse the reduction of NADP+ to NADPH: glucose 6-phosphate dehydrogenase, forming 6-phosphogluconate, and phosphogluconate dehydrogenase, which yields the 5-carbon sugar ribulose 5-phosphate, liberating carbon dioxide from carbon-1 of the substrate.

These reactions are the first two reactions in the pentose phosphate pathway, which provides an alternative to part of the pathway of glycolysis, yielding glyceraldehyde 3-phosphate and fructose 6-phosphate. This pathway is a major source of NADPH, and in addition to its importance in red blood cells, it provides about half the NADPH required for fatty acid synthesis. It is also the pathway for synthesis of the 5-carbon sugar ribose. Click here to see the complete pathway.

Beacuse of its importance as a source of NADPH for fatty acid synthesis, you would expect to find a high activity of the pathway in tissues that synthesise fatty acids: liver, adipose tissue and lactating mammary gland.

In further studies, only the formation of [14C]carbon dioxide from [14C-1]glucose was measured, with the addition of:

• sodium ascorbate (which undergoes a non-enzymic reaction in air to produce hydrogen peroxide)
• acetylphenylhydrazine (which is known to precipitate haemolysis in sensitive subjects, and depletes glutathione)
• methylene blue (which oxidises NADPH)

All incubations were repeated in the presence and absence of N-ethylmaleimide, which undergoes a non-enzymic reaction with the -SH group of reduced glutathione, and thus depletes the cell of glutathione.

Production of [14C]carbon dioxide by 1 mL erythrocytes incubated for 1 hour with 10 mmol /L [14C-1]glucose at 10 µCi /mmol. Figures show dpm, mean ± sd for 5 replicate incubations.

Incubation with ascorbate (as a source of hydrogen peroxide, which will oxidise GSH to GSSG) or acetylphenylhydrazine, which also oxidises GSH to GSSG, leads to a considerable increase in the activity of the pentose phosphate pathway, suggesting that the need for increased activity of the pathway is the need for NADP to reduce GSSG back to GSH – or, more correctly, the presence of a relative excess of NADP+ to be reduced to NADPH.

This is confirmed by the effects of adding N-ethylmaleimide, which depletes the cells of GSH; in the control incubations and in the presence of ascorbate or acetylphenylhydrazine, N-ethylmaleimide reduces the activity of the pentose phosphate pathway, since less NADPH is being oxidised by reaction with GSSG.

By contrast, N-ethylmaleimide has no effect on the increased activity of the pentose phosphate pathway caused by adding methylene blue, which directly oxidises NADPH.

Further studies showed that Samuel’s red blood cells contained only about 10% of the normal activity of glucose 6-phosphate dehydrogenase. In order to investigate why his enzyme activity was so low, a sample of his red blood cells was incubated at 45°C for 60 min, then cooled to 30°C and the activity of glucose 6-phosphate dehydrogenase was determined. After the pre-incubation at 45°C, Samuel’s red cells showed only 60% of their initial activity. By contrast, red cells from control subjects retained 90% of their initial activity after pre-incubation at 45°C for 60 min.

It seems likely that Samuel has an unstable variant of glucose 6-phosphate dehydrogenase, which is denatured more rapidly than normal at 45°C, and therefore presumably is also less stable than normal in vivo, so that his older red blood cells will have very much less enzyme than normal.

Since Samuel’s problem is that the enzyme is unstable, and gradually denatures in vivo, his haemolytic crises will be self-limiting, in that only the older red blood cells will have so little glucose 6-phosphate dehydrogenase that they cannot deal with the oxidative stress. Younger red blood cells will have an adequate activity of glucose 6-phosphate dehydrogenase remaining to permit production of NADPH, and hence recycling of glutathione.

The most likely answer is that this is an X-linked genetic condition, so that women are carriers, but are only affected very rarely. A male has one X and one Y chromosome - the X must come from his mother and the Y from his father. This means that there is a 50% chance that a boy whose mother is heterozygous for the condition will be affected.

Further studies showed that Alessandro’s erythrocyte glucose 6-phosphate dehydrogenase was only 10% of normal and the Km for NADP+ was 80 µmol /L (compared with a normal value of 9 µmol /L).

Unlike Samuel W, his red blood cell enzyme was as stable to incubation at 45°C as that from control people.

Alessandro’s problem is that his glutathione reductase has very low activity because it has an abnormally high Km for NADPH. This means that it will have only low activity at normal intracellular concentrations of NADP+, and all of his red blood cells will have an impaired ability to produce NADPH, and so recycle glutathione in response to oxidative stress, not only the older cells. Therefore his haemolytic crises will be very much more severe than Samuel’s.

This severe variant of the condition (named favism because crises are precipitated by eating fava beans, or indeed walking through a field of plants in flower), occurs mainly in people of Mediterranean origin. The milder, self-limiting variant of favism (as in Samuel’s case) occurs mainly in people of Afro-Caribbean origin.

Glucose 6-phosphate dehydrogenase deficiency is the most common human enzyme defect, being present in more than 400 million people worldwide, especially among people of Afro-Caribbean or Mediterranean origin. In some areas between 2 - 7 % of the male population are affected.About 140 different variants have been reported, varying in severity depending on the residual activity of the mutant enzyme. As the genes have been sequenced, many of the variants that were thought to be distinct have been shown to involve the same point mutation.

Presumably there is an evolutionary advantage in being heterozygous for the condition. The global distribution of the disease is closely similar to that of malaria, and it is likely that carriers of glucose 6-phosphate dehydrogenase deficiency have some degree of resistance against malaria.

For more on glucose 6-phosphate dehydrogenase deficiency, see: Cappellini, M. D. and G. Fiorelli (2008). "Glucose-6-phosphate dehydrogenase deficiency." Lancet 371(9606): 64-74.

Key points from this exercise:

• The tripeptide glutathione is an important part of antioxidant defences of the cell, and glutathione reductase requires NADPH to reduce GSSG back to GSH.
• The only source of NADPH in red blood cells (and a major source in other tissues) is the first two reactions in the pentose phosphate pathway, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.
• The pentose phosphate pathway is especially important in tissues that synthesise fatty acids (liver, adipose tissue and lactating mammary gland) as the source of about half the NADPH required.
• The pentose phosphate pathway provides an alternative to part of the pathway of glycolysis, and is important as the source of ribose.
• Glucose 6-phosphate dehydrogenase deficiency is the commonest genetic disease in human beings; it is an X-linked recessive condition. It is likely that heterozygote carriers have resistance to malaria.