The Role of Glucose-6-Phosphate Dehydrogenase in the Clearance of Oxidant Stress Products in Muscle During Exercise
Paolino Ninfali a and Nereo Bresolin b .
a Institute of Biological Chemistry "G. Fornaini", University of Urbino, Italy
b Institute of Clinical Neurology, D. Ferrari Center, University of Milan., Italy
Glucose-6-phosphate dehydrogenase (G6PD ; D-glucose-6-phosphate: NADP 1-oxidoreductase, E.C.188.8.131.52 ) catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate with NADPH production. The enzyme is an important site of metabolic control in the hexose monophosphate shunt. The shunt provides NADPH and pentose phosphates: the former is involved in lipid biosynthesis and plays a major role in maintaining sulfhydryl groups in the cell; the latter are precursors of nucleic acids and all nucleotide coenzymes (1).
The deficiency of G6PD is the best understood and most thoroughly studied enzymopathy. This defect is widely distributed geographically and it is estimated to affect 400 million people in the world. More than 380 variants have been found through the biochemical characterization of the enzyme. Most of them have been confirmed at DNA level (2), others have been shown to share the same mutation, while new variant types are still emerging (3-4). They are grouped into four classes depending on the degree of the enzyme deficiency and clinical symptoms.
The major clinical consequence of G6PD deficiency is hemolytic anemia, which is, in most variants, a consequence of ingestion of Vicia Faba beans, drugs, infections or acidosis. Some of the rare types of G6PD deficiency are associated with chronic non-spherocytic hemolytic anemia, which is further exacerbated by oxidant stress (1). It is commonly believed that in all G6PD deficient variants, the enzyme defect is much more severe in erythrocytes than in other tissues (2,5). Other tissues have seldom been investigated, since no convincing proof of an association between G6PD deficiency and other pathologies exists. Among the mammalian tissues, muscle has the lowest G6PD activity (5-6) and it has generally been regarded as particularly insensitive to genetically determined G6PD deficiency. However in the last decade, we had the occasion to study various G6PD deficient subjects, exhibiting symptoms like myalgias, cramps and muscle weakness, under various conditions of stress, including stress caused by physical exercise. We hypothesized that G6PD deficient subjects may be more prone to manifest this muscle symptomatology than normal subjects (7-9) and that G6PD deficiency may be included among those pathologies which restrict participation in high intensity sports. Since the enzyme defect is asymptomatic in most cases, many people do not even know they are affected by G6PD deficiency. Therefore it is conceivable that an individual begins to practise high intensity sports in his youth, without paying any attention to the possible complications associated with this enzyme defect.
This commentary calls attention to this problem.
Patients and laboratory findings
The patients under study were 33 G6PD deficient male patients, admitted to the Neurologic Clinic, University of Milan. Twelve of them had frequent episodes of cramps, myalgias and muscle weakness. In these patients we attempted to correlate the RBC G6PD activity with the extent of fatigability, cramps and myalgias, but these data did not reach statistical significance. On the contrary, we have seen that fatigability and myalgias correlate statistically (p<0.05) with cramps (10).
The clinical history and the severity of the symptomatology, prompted us to perform a more complete laboratory investigation, including muscle biopsy, on 8 out of 33 patients. Biopsies were performed after having received the voluntary consent of the patients or their parents, in conformity with the principles of the Helsinki declaration. The 8 patients had three different variants: 4 were G6PD Mediterranean: 2 were G6PD A-; 1 was G6PD Seattle-like. The G6PD assay on muscle cell homogenates revealed that the enzyme deficiency was always present in muscle and there was a significant correlation (p<0.0002) between G6PD activity in skeletal muscle and its activity in erythrocytes (11).
Four out of eight had severe muscle problems after prolonged aerobic exercise. One was a 30-year-old pentathlon-trained athlete who suffered from loss of consciousness and pigmenturia during the last meters of a 12-km competitive run. In the emergency room, metabolic acidosis and hypoglycaemia, which can precipitate the haemolysis in a G6PD deficient subjects, were detected. But in this patient myoglobinuria preceded the hemolysis by two days, suggesting the possible higher vulnerability of a G6PD deficient muscle. Another patient was a 20-year-old soccer-trained athlete, who suffered from myalgias in the lower limbs and dark urine with myoglobinuria after intense physical exercise, but no signs of anemia or chronic hemolysis were present at rest. He was obliged to stop competitive activity because of the recurrence of these episodes. The other patients were two brothers of Sardinian origin,14 and 9 year-old. The older brother had such frequent and painful episodes of myalgias and muscle fatigability during gym class at school that he had to give it up.
Since all individual glycolytic and mitochondrial enzymes, as well as carnitine palmityl transferase and carnitine were normal in our patients, we attributed these symptoms to G6PD deficiency in muscle.
Possible phatophysiology of G6PD deficient muscle
On the basis of recent discoveries regarding the metabolism of muscle cell, it is possible to speculate on which mechanism the deficiency of G6PD may cause the muscle symptomatology described above. An interesting paper by Martensson and Meister (12) demonstrated that marked glutathione (GSH) depletion induced skeletal muscle degeneration, associated with mitochondrial damage. Considering that G6PD plays a key role in the production of NADPH, utilized to maintain GSH in the reduced form, one may deduce that, in the G6PD deficient muscle, a lower level of NADPH leads to a decrease of GSH, which in turn increases the cell vulnerability to the reactive oxygen compounds and free radicals formed in the aerobic metabolism. As reported above, G6PD activity in normal muscle is the lowest compared to the other body tissues; thus a deficiency of this enzyme may lead to a dramatic fall in the steady-state concentration of NADPH and GSH. Furthermore it should be emphasized that skeletal muscle has also lower levels of catalase and superoxide dismutase, in comparison with other tissues and therefore might be expected to be dependent on GSH linked reactions for detoxication of reactive oxygen species (13). Oxo-radicals are in fact responsible for myofiber disruption and the loss of intracellular proteins, which cause post-exercise soreness (14).
Because of the low number of subjects and variants tested, our results do not allow any definitive conclusion on the relationship between G6PD activity and muscle symptomatology. However, our previous work (7-11) has demonstrated that genetically determined G6PD deficiency is expressed in muscle and that there was a significant correlation between G6PD activity in skeletal muscle and its activity in erythrocytes. The episodes of myalgia and myoglobinuria; observed in our G6PD deficient patients after intense physical exercise, support the hypothesis of a scarce protection against reactive oxygen species, which triggered the metabolic events responsible for myofiber disruption and loss of intracellular proteins.
While waiting for more cases in order to definitely confirm our hypothesis, the widespread diffusion of this enzymophenia in the world suggests that sport physiologists and physicians should pay more attention to young people who practise high intensity sports. When problems arise, doctors should verify, among other things, if the patient is G6PD deficient. In our opinion, G6PD deficient subjects, expressing in RBC an activity value lower than 15 % of the normal, should avoid high intensity physical exercise, which could lead to episodes of skeletal muscle degeneration and myoglobinuria. Moreover , we suggest that brown-red or dark urine, collected during hemolytic crises of G6PD deficient patients after intense physical exercise, be tested not only for the presence of hemoglobin but also for myoglobin.