The patient is an African American recruit to the army. He was given the antimalarial drug primaquine, and suffered a delayed reaction with kidney pain, dark urine, and low red blood cell counts that led to anemia and weakness. Centrifugation of a blood sample showed a low hematocrit, and the plasma was red colored.
Similar acute hemolytic attacks have been observed, predominantly in men of Afro-Caribbean origin, in response to primaquine and a variety of other drugs, including dapsone, the antipyretic acetylphenylhydrazine, the antibacterial bactrim/septrin, sulfonamides, and sulfones, whose only common feature is that they all undergo cyclic nonenzymic reactions in the presence of oxygen to produce hydrogen peroxide and a variety of oxygen radicals that can cause oxidative damage to membrane lipids, leading to hemolysis. Moderately severe infection can also precipitate a hemolytic crisis in susceptible people.
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, γ-glutamyl-cysteinyl-glycine, which readily undergoes oxidation to form a disulphide-linked hexapeptide, oxidized glutathione, generally abbreviated to GSSG. Table 57–3 shows the concentrations of GSH and GSSG in red cells from the patient and 10 control subjects, before and after incubation with acetylphenylhydrazine.
TABLE 57–3The Effect of Incubation With 330 μmol/L Acetylphenylhydrazine on Erythrocyte Glutathione ||Download (.pdf) TABLE 57–3 The Effect of Incubation With 330 μmol/L Acetylphenylhydrazine on Erythrocyte Glutathione
| ||Patient in Case 3 ||Control Subjects |
| ||GSH (mmol/L) ||GSSG (μmol/L) ||GSH (mmol/L) ||GSSG (μmol/L) |
|Initial ||1.61 ||400 ||2.01 ± 0.29 ||4.2 ± 0.61 |
|+ Acetylphenylhydrazine ||0.28 ||1540 ||1.82 ± 0.24 ||190 ± 28 |
How much GSH is oxidized per mol of acetylphenylhydrazine added?
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 hemolysate from 0.5-mL packed cells (Table 57–4).
TABLE 57–4Glutathione Reductase, μmol Product Formed/min ||Download (.pdf) TABLE 57–4 Glutathione Reductase, μmol Product Formed/min
| ||Patient in Case 3 ||Control Subjects |
|NADPH ||0.64 ||0.63 ± 0.06 |
|NADH ||0.01 ||0.01 ± 0.001 |
Since none of the red cell 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 oxidize NADPH; the reduced dye then undergoes nonenzymic 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 six possible positional isomers of [14C]glucose were used, and the radioactivity in (lactate + pyruvate) was determined after thin layer chromatography of the incubation mixture. Each incubation contained 1 mL erythrocytes in a total incubation volume of 2 mL (Table 57–5).
TABLE 57–5Production of [14C]lactate, Pyruvate and CO2 by 1 mL Erythrocytes From Control Subjects Incubated for 1 Hour With 10 mmol/L [14C]glucose at 10 μCi/mmol ||Download (.pdf) TABLE 57–5 Production of [14C]lactate, Pyruvate and CO2 by 1 mL Erythrocytes From Control Subjects Incubated for 1 Hour With 10 mmol/L [14C]glucose at 10 μCi/mmol
| ||Control ||+ Methylene Blue |
| ||Lactate + Pyruvate ||CO2 ||Lactate + Pyruvate ||CO2 |
|[14C-1]glucose ||12680 ± 110 ||1410 ± 15 ||1830 ± 20 ||12260 ± 130 |
|[14C-2]glucose ||14080 ± 120 ||nd ||14120 ± 120 ||nd |
|[14C-3]glucose ||14100 ± 120 ||nd ||14090 ± 120 ||nd |
|[14C-4]glucose ||14060 ± 120 ||nd ||14080 ± 120 ||nd |
|[14C-5]glucose ||14120 ± 120 ||nd ||14060 ± 120 ||nd |
|[14C-6]glucose ||14090 ± 110 ||nd ||14100 ± 120 ||nd |
In further studies, only the formation of 14CO2 from [14C-1]glucose was measured, with the addition of:
Sodium ascorbate (which undergoes a nonenzymic reaction in air to produce H2O2)
Acetylphenylhydrazine (which is known to precipitate hemolysis in sensitive subjects, and depletes reduced glutathione)
Methylene blue (which oxidizes NADPH)
The incubations were repeated with N-ethylmaleimide, which undergoes a nonenzymic reaction with the —SH group of reduced glutathione, and thus depletes the cell of total glutathione. The results are shown in Table 57–6.
TABLE 57–6Production of 14CO2 by 1 mL Erythrocytes From Control Subjects Incubated for 1 Hour With 10 μmol/L [14C-1]glucose at 10 μCi/mmol ||Download (.pdf) TABLE 57–6 Production of 14CO2 by 1 mL Erythrocytes From Control Subjects Incubated for 1 Hour With 10 μmol/L [14C-1]glucose at 10 μCi/mmol
|Additions ||Control ||+ N-Ethylmaleimide |
|None ||1410 ± 70 ||670 ± 30 |
|Ascorbate ||8665 ± 300 ||2133 ± 200 |
|Acetylphenylhydrazine ||7740 ± 320 ||4955 ± 325 |
|Methylene blue ||12230 ± 500 ||11265 ± 450 |
Further studies showed that the patient’s red blood cells contained only about 20% of the normal activity of glucose 6-phosphate dehydrogenase (see Chapter 20). 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 minutes, then cooled to 30°C and the activity of glucose 6-phosphate dehydrogenase was determined. After the preincubation at 45°C, his red cells showed only 60% of their initial activity. By contrast, red cells from control subjects retained 90% of their initial activity after preincubation at 45°C for 60 minutes.
What conclusions can you draw from these results?