Glucose SPhosphate Dehydrogenase Deficiency An Inherited Ailment that Affects 100 Million People N. M. Senoran and C. A. Thielman California State University, Long Beach, CA 90840 When the military personnel sent to Korea in the early '50's were routinely given primaquine, an antimalarial drug, some soldiers developed a severe case of anemia. Characteristically, the victim produced a dark, often hlack, urine and manifested signs of jaundice. The red blood cell count and the hemoglobin level dropped abruptly. The patient usually recovered, hut sometimes the massive destruction of the red cells was fatal. Nearly 15% of black U S . soldiers showed primaquine sensitivity (1-3). Research was soon underway t o discover the cause of the massive hemolytic anemia hrought on by primaquine. Inmates from the Stateville Penitentiary in Joliet, Illinois, provided a pool of volunteers, and the University of Chicago supplied the researchers for the army-sponsored project (4). Before long, evidence pointed a t the red cell as the location of the defect. When cellsfrom a healthy subject weremarked with radioactive chromium and injected into a primaquinesensitive patient, the cells continued their normal life span even after primaquine administration. When labeled cells from primaquine-sensitive volunteers were given to a normal person on primaquine, however, the destruction took
place much as it would have in a drug-sensitive individual. The culprit was unmistakably in the red cell (5,6). The underlying cause of the primaquine-sensitive anemia was apparently shared by a number of other anemias that had been mentioned in textbooks of medicine prior to 1957 under such names as favism and Baghdad anemia (7). Primaquine destroyed the red cells of these anemia patients with a pattern similar to that seen earlier in patients sensitive to the antimalarial drug. Whatever made the red cells intolerant of the pollen in Baghdad and the fava beans in Mediterranean dishes also made them sensitive to primaquine (8,9). How Red Blood Cells Cope wlth the Oxldatlve Stress Since primaquine in medical literature is described as an oxidant drup, sensitivity to this compound implicated the reductive m&hanisms of the red cell and suggested a probable defect in one of the metabolic steps designed to combat oxidative stress within the cell. Primaquine and structurally related compounds can indeed act as oxidizing agents by accepting electrons and hydrogens as shown in Figure 1, hut this does not seem to he their mode of operation in the red cell. Instead, they seem to cause oxidative damage by donat-
~H(CH~)~NH* I CH3
Primaquine
CH3
Pmaquine reduced
-
Dihydroxyacetone L ~lyc&ddehyde phosphate 7 3-phosphate
Figure 1. Slruchlres of some of the compounds mentioned in this article. The formulas for convicine and isouram11are similar, respectively,to vincine and divieine. Theonly difference is in the group substnuted to the carbon between the two nihogens of the aromatic ring. in convicine and isouramil an -OH group. instead of the -NH2 group, is present.
&
Figure 2. Glucose metabolism in me red cell. Antioxidants are indicated in bold. NADPH. which is generated in the first and third steps of the HMP pathway, keeps gluta!hione in reduced state and prevents oxidative damage to the cell through a series of coupled redox reactions describsd in the insen.
Volume 68
Number 1 January 1991
7
ing an electron-a paradoxical situation until one examines the role played by hemoglohin. In hemoglobin iron is in the ferrous state, and the oxygen hound to it is in elemental form (10). There is, of course, a tendencv for the oxveen to receive electrons, hut unless a pair of them are provided simultaueously so that the reduction via ~eroxideformation, the l>e"-O? complex ~ - - oroceeds ~ remains fairly stable. The iron in t h e ferrous state can provide onlv one electron: the polypeptide chains surrounding the hemegroups in hemoglobinbl~ckthe convergence of two irons on a siuele - 0 7- and make peroxide formation difficult. The presence of a compound skch as primaquine, however, supplies the requisite second electron and accelerates the oxidative proc&s. Along with methemoglobin, peroxide forms and oxidative damage gets underway. The damage is exacerbated by free radicals derived from the donor molecule. Thus antimalarial drugs heighten the oxidative stress in the red cell not by accepting electrons as a typical oxidant would, hut by providing the second electron necessary for the formation of free radicals and peroxide (11,12). The red cell has no nucleus and no mitochondrion, and elucose is its primary fuel. All the antioxidants that the cell produces derive their electrons from this simple sugar. Under normal conditions about 90% of the glucose is metaholized along the anaerobic Emhden-Meyerhof pathway shown in Figure 2. Two molecules of ATP are consumed and four molecules of ATP are generated from the breakdown of one glucose molecule; the two ATP's gained are used to nrovide euerw -" for the maintenance of the cell membrane and for pumping sodium ions out and potassium in'. One molecule of NADH, a universal intracellular reducing agent, is generated duringthe conversion of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, hut later, during lactate formation, this is oxidized back to NADC. Thus, there is no net production of a reducing agent along the EM pathunless glycolysis is stopped prior to lactate formation (13,14). About 10% of an unstressed red cell's glucose is metaholized aerobically along the hexose monophosphate pathway. The path begins with glucose 6-phosphate and leads to ribulose 5-phosphate (Fig. 2). This five-carbon sugar then converts to glyceraldehyde 3-phosphate and fructose 6-phosnhate and re-enters the EM route. The first and the third steps of HMP pathway require the oxidizing agent NADPf. The aeent is reduced to NADPH and can be regenerated only h i the action of glutathione, CSSG, as showiin Figure 2. In the process GSSG acquires two hydrogens and breaks into two GSH's. The reduced glutathione is reconverted to GSSG by the final oxidant of the chain, HzOz. Hydrogen peroxide itself is a product of oxygen reduction; hence the pathway is characterized as aerobic or oxidative (15). As the breakdown of glucose proceeds along the HMP pathway, the electrons flow from the sugar to hydrogen neroxide via NADPt and GSSG. The reduced forms of these r - - - - ~ ~ twomolecules are the main antioxidants of the red cell; GSH is res~onsiblefor the immediate reduction of not only H202 but aiso other oxidants that happen to find their way into the red cell (14). ~
A~
~
~
~
.~
~~
I Abbreviations: ATP = adenosine triphosphate, EM = EmbdenMeyerhof, GGPD = glucose 6-phosphate dehydrogenase. GSH = giutathione (reduced form),GSSG = giutathione (oxidized form),HMP = hexose monophosphate. NADt = nicotinamide adenine dinucieotide (oxidized form). NADH = nicotinamide adenine dinucleotide (reduced form).NADPf = nicotinamide adenine dinucleotide phosphate (oxidized form). NADPH = nicotinamide adenine dinucleotide phosphate (reduced form). NADHdependent methemoglobin reductase deficiency, discovered in 1948 by Gibson, is generally regarded as the first enzymatic defect demonstrated in genetic disease. This was followed in 1952 by the discovery of glucose 6-phosphatase deficiency in von Gierke's disease, and in 1953 of phenyialanine hydroxylase deficiency in phenyiketonuria (32).
8
Journal of Chemical Education
Carson's Discovery of the Deflclent Enzyme
A number of observations seemed to suggest that the disorder in primaquine-sensitive persons lies in the HMP portion of red cell metabolism. When Emhden-Meyerhof pathway was inhibited in normal cells by fluoride ion, primaquine sensitivity did not develop. But treatment of the cells with iodoacetate or arsenic, which are both known for their affinity for the sulfhydryl groups, led to drug sensitivity. Furthermore, red cells from sensitive subjects registered an abruot fall in the GSH levels when exposed to primaquine. The efforts to elucidate the defective mechanism, therefore, convereed on elutathione and the metabolic steps associated with its form&on (4). In the early '50's a young physician named Paul Carson had been assigned to duty a t the Stateville Penitentiary and was looking into the function of glutathione reductase, an enzvme to reduce GSSG to GSH. For a while he .--" - reauired ~ ~ thought that primaquine sensitivity might be due to alackof this enzvme. but when the sensitive cells were given 'UADPH; a kbstrate for glutathione reductase, GSH-production resumed. The defect appeared to he in the mechanism for the production of ~ D P H indeed ; i t was soon confirmed that red cells from drug-sensitive patients were deficient in glucose 6-phosphate dehydrogenase, the enzyme reauired for the first step of the hexose monophosphate pathway. Carson published his discovery under the title "Enzymatic deficiency in primaquine-sensitive erythrocytes" in Science, September 1956 (16). It was among the first enzyme deficiencies to he reported in medical literature, and it explained not only primaquine sensitivity hut also the hemolvtic effects of a dozen other drugs, as well as the anemias caused by infectious agents and f&a beans2. Vulnerable red cells were almost always low in GGPD activity (2,6,8). I t is interesting to note that when a refrigeratedcentrifuge was later made available to Carson, he failed to reproduce his earlier results. GGPD deficiency is not an all-or-nonetvne disorder: deficient cells still have some enzyme activity. AGparently
