Muscle Expression Of Glucose-6-Phosphate Dehydrogenase Deficiency in Different Variants
Paolino Ninfali a , Luciano Baronciani b , Alessandra Bardoni c and Nereo Bresolin c . a
Institute of Biological Chemistry "G. Fornaini", University of Urbino, Italy b
Research Institute of Scripps Clinic, La Jolla , CA, USA. c
Institute of Clinical Neurology, D.Ferrari Center, University of Milan., Italy
[From Clinical Genetics 1995, 48:232-237]
Key Words: G6PD deficiency, genetic variants, red blood cell, muscle.
Abstract. Muscle expression of G6PD deficiency has been investigated in Mediterranean, Seattle-like and A- variants. G6PD activity was detected on samples obtained from biopsies on the quadriceps muscle of 8 males and 1 female G6PD deficient subjects. The type of genetic variant was determined by molecular analysis of DNA extracted from blood samples.
All variants showed the enzyme defect in muscle. A statistically significant relationship was found in the activity of G6PD between erythrocyte and muscle ( r= 0.965 ; p<0.00002 ). The equation for the best fit line was: Y=0.371X + 0.188. The results suggest that, for a given variant, the extent of the enzyme defect in muscle may be determined by this equation from the G6PD activity of RBC.
Glucose-6-phosphate dehydrogenase (G6PD ; D-glucose-6-phosphate: NADP 1-oxidoreductase, E.C.126.96.36.199 ) catalyzes the convertion of glucose-6-phosphate to 6-phosphogluconate with NADPH production. The enzyme is an important site of metabolic control in the hexose monophosphate shunt which provides reducing power (NADPH) and pentose phosphates. The former is involved in lipid biosynthesis and plays a major role in maintaining sulfhydryl groups in the cell, thus contributing to the detoxication of free radicals and peroxides; the latter are precursors of nucleic acids and of all nucleotide coenzymes (Luzzatto & Metha, 1989).
G6PD is coded by a gene located on the long arm of the human X-chromosome in the band Xq28, which is one of the best mapped in the human genome (Pai et al, 1980; Szabo et al, 1984). More than 380 variants have been found in the past by means of the biochemical characterization of the enzyme protein. With the techniques of DNA analysis it is now emerging that several variants once considered unique are instead the phenotypical expression of the same mutated gene and that new mutations produce indistinguishable mutant enzymes (Beutler, 1991).
Three main areas with a high incidence of G6PD deficiency are present in Italy: Sardinia with 13% of male population affected (Siniscalco et al, 1966), southern Italy and the Po delta area with a proportion of males ranging from 2.2 to 2.5% (Colonna-Romano et al, 1985; Gandini et al, 1969). The wide diffusion of this genetic trait in our country and the ability to deduce quickly the aminoacid substitution continuously stimulate the research of new mutant forms in these areas
together with the identification at molecular level of those determined in the past by phenotyping the enzyme activity (De Vita et al, 1989; Viglietto et al, 1990; Ninfali et al, 1993).
Usually people deficient in G6PD are clinically asymptomatic unless challenged by oxidative stress which may be secondary to ingestion of Vicia Faba beans, drugs, infections or acidosis. Rare variants leading to extremely low residual activity are associated with chronic nonspherocytic hemolytic anemia ( Luzzatto & Metha, 1989). It is commonly believed that in G6PD deficient individuals, the enzyme defect is much more severe in erythrocytes than in other tissues (Luzzatto & Battistuzzi, 1985; Beutler, 1990a). This has been attributed to a unique proteolytic environment in erythrocytes (Beutler, 1983). Muscle has seldom been investigated, since it is in mammals one of the tissues with the lowest G6PD activity (Luzzatto & Battistuzzi, 1985; Ninfali & Palma, 1990) and it has generally been regarded as particularly insensitive to genetically determined G6PD deficiency. Only recently, we suggested that symptoms like myalgias, cramps and myoglobinuria in G6PD deficient subjects may be due to the enzyme defect in muscle tissue (Bresolin et al, 1987; Bresolin et al, 1989; Ninfali et al, 1991). If this view is correct, the assay of the enzyme activity in muscle tissue assumes primary importance in the laboratory tests particularly when the subject is exposed to oxidative stress due to physical activity. Moreover the existence of a correlation between muscle and erythrocyte activity would be very useful for an indirect determination of muscle G6PD which avoids painful and troublesome biopsies.
In this paper we present a quantitative comparison of the G6PD activity in RBC and muscle of patients bearing three different variants. The results indicate a significant correlation between the enzyme activities of the two tissues, while the clinical symptoms of the patients allow speculations on the G6PD role in muscle metabolism .
Materials and Methods Patients and laboratory findings
Eight males and one female admitted to the Neurologic Clinic, University of Milan, for episodes of cramps, myalgias and hyposthenia were investigated for clinical history, underwent physical examination and were studied with the following laboratory tests: blood analysis (RBC, Hb, CPK, LDH), urine analysis (myoglobinuria and hemoglobinuria), instrumental investigations (EMG) and muscle biopsies. No signs of cronic hemolysis were present at rest. Individual glycolitic and mitochondrial enzymes, as well as carnitine palmityl transferase and carnitine, were measured by previously described spectrophotometric and radiochemical assays (Angelini et al, 1981; Bresolin et al, 1985).
All patients were found to be affected by G6PD deficiency. All other enzyme activities tested in muscle and RBC were in the normal range. The female subject was found to be Duchenne Muscular Dystrophy carrier.
Table 1 shows a semiquantitative analysis of the muscle symptomatology and of the most significant clinical signs of our patients.
Table 1. Symptomatology and clinical signs of our G6PD deficient patients
|Patient ||Sex |
|Age ||Hb ||LDH ||CPK ||EMG ||Hypo- |
|Cramps ||Myal- |
|a Hemo- |
lytic crises and Myoglo-
|T.A. ||M ||36 ||dd ||++ ||+++ ||- ||+ ||+ ||+ ||+ ||+ |
|S.S. ||M ||19 ||- ||- ||- ||- ||- ||++ ||+ ||+ ||+ |
|S.E. ||M ||14 ||dd ||+ ||- ||- ||- ||++ ||- ||- ||+ |
|P.L. ||M ||11 ||- ||++ ||+++ ||- ||+ ||+ ||- ||- ||+ |
|G.S. ||M ||21 ||d ||- ||- ||+ ||- ||+ ||+ ||+ ||+ |
|A.M. ||M ||23 ||- ||- ||- ||+ ||+ ||+ ||+ ||++ ||++ |
|B.G. ||M ||24 ||- ||- ||- ||+ ||- ||+ ||- ||+ ||++ |
|F.F. ||M ||17 ||d ||- ||- ||- ||- ||+ ||- ||+ ||- |
|C.A. ||F ||36 ||- ||+ ||+++ ||- ||- ||+ ||- ||- ||- |
a Hemolytic crises and/or myoglobinuria were triggered by ingestion of Vicia Faba beans, drugs, infections, except for T.A. and B.G., who presented these symptoms after intense physical exercise.
LDH: (++) Values higher than 500 IU/l, (+) values between 300 and 400 IU/l.
CPK: (+++) values higher than 1000 IU/l.
Hb: (d) values between 12 and 11 g/dl; (dd) values in the range 10-11 g/dl.
Cramps: (+) present after moderate physical exercise; (++) present at rest.
Hyposthenia: (+) slight mild symptoms and (++) marked symptoms according to MRC scale.
Myalgias: (+) mild symptoms.
EMG: (+) presence of neurogenic signs.
(-): Values in the normal range and no symptoms.
G6PD activity assay. Venous blood from patients and controls was collected in EDTA. The RBC were prepared by centrifuging whole blood at 2, 000 g for 10 min at 4°C. Plasma and buffy coat were carefully removed as described by Morelli et al, (1981) and RBC were hemolyzed with 4 vol. of cold distilled water. The stroma were then removed by centrifuging the lysate for 15 min at 15, 000 g. Muscle biopsies were obtained from the quadriceps muscle of patients and controls, frozen immediately and stored in liquid nitrogen. Control muscle was obtained by diagnostic biopsies from patients who were ultimately deemed to be free of muscle disease. Homogenates were prepared with 9 vol. of 50 mmol/l Tris-HCl buffer pH 7.5 containing 0.15 mmol/l KCl in an all glass motor driven homogenizer. The G6PD activity in muscle homogenates and red cell lysates was measured according to the WHO recommendations (1967). The activity was expressed in micromoles of NADPH formed per minute per gram of hemoglobin in hemolysates or per gram of proteins in muscle homogenates. Proteins were determined by the method of Bradford (1976).
DNA extraction and analysis. High molecular weight DNA was extracted from peripheral blood by standard techniques. All DNA samples were screened for the C->T mutation at nucleotide 563, that is characteristic of G6PD Mediterranean (G6PD Med) mutation. Exon 6 amplification by polymerase chain reaction (PCR) was performed as reported by Beutler et al, (1991). The amplified fragment was analyzed by MboII restriction endonuclease. Samples which were not G6PD Med were analyzed for the A->G mutation at nucleotide 376, characteristic of G6PD A+, A- and Santamaria (Beutler, 1992), by digestion with Nla III endonuclease. Positive samples were analyzed for the G->A mutation at nucleotide 202 that is characteristic of the most common A- variant, by Fok I restriction endonuclease. The G->C mutation at nucleotide 844 on exon 8, characteristic of the G6PD Seattle-like, was found by screening all exons of the G6PD gene (Beutler 1991). Mutations were confirmed by sequencing the reverse strand. Table 2 summarizes the progressive steps of our protocol and the method used for searching mutations by molecular analysis of DNA.
Statistics. Linear regression analysis was performed with STATQUIK computer program, version 4.
Table 2. Protocol followed in searching the DNA mutations of our G6PD deficient subjects
| ||Variant |
|Mutation ||Exon ||Endonucl. |
|Step 1 ||Med ||563 C->T ||6 ||Mbo II |
|Step 2 ||A+ ; A- ||376 A->G ||5 ||Nla III |
|Step 3 ||A- ||202 G->A ||4 ||Fok I |
|Step 4 ||Seattle-like ||844 G->C ||8 ||Screen 13 exons |
Primers used for exon amplifications are those reported by Beutler et al (1991).
The patients were found to bear three different variants: G6PD Med, A- and Seattle-like. Among these, G6PD Med and A- are very common in Italy, while G6PD Seattle-like is quite rare. It was first described at molecular level by De Vita et al, (1989) in a Sardinian patient and subsequently found by us in another subject from northern Italy (Ninfali et al, 1991).
Table 3 shows the variant type and G6PD activity in erythrocyte and muscle of the 9 patients. Among the G6PD Med patients, the subject G.S. had a G6PD activity so low to be undetectable in both cell types. G6PD Seattle-like had nearly the same activity in RBC and muscle. The G6PD A- male subject had a percent of activity which was markedly higher in the muscle than in RBC, while in the female the activity of the two tissues was almost the same. The two A- subjects had however the highest muscle G6PD activity of all samples.
Figure 1 shows that there is a positive correlation between G6PD activity of RBC and muscle in the three variants and in the G6PD B type. The equation for the best fit line was: Y=0.371 X + 0.188; the correlation coefficient r was 0.965 and p value was less than 0.00002.
Table 3. G6PD activity in erythrocyte and muscle of patients bearing different variants
|Patient ||Sex ||Age ||Variant ||RBC activity ||Muscle activity |
| || || || ||IU/g Hb |
|% ||IU/g prot. ||% |
|Contr. || || ||B ||8, 5 ± 1.5 ||100 ||3.5 ± 0.5 ||100 |
|T.A. ||M ||36 ||Med ||0.08 ||0.9 ||0.05 ||1.5 |
|S.S. ||M ||19 ||Med ||0.22 ||2.6 ||0.14 ||4.0 |
|S.E. ||M ||14 ||Med ||0.18 ||2.1 ||0.66 ||18.8 |
|P.L. ||M ||11 ||Med ||0.13 ||1.5 ||0.07 ||2.0 |
|G.S. ||M ||21 ||Med ||n.d ||n.d ||n.d ||n.d |
|A.M. ||M ||23 ||Med ||0.12 ||1.4 ||0.10 ||2.8 |
|B.G. ||M ||24 ||Seattle-like ||1.40 ||16.4 ||0.50 ||14.3 |
|F.F. ||M ||17 ||A- ||0.98 ||11.5 ||1.10 ||31.4 |
|C.A. ||F ||36 ||A- ||5.20 ||61.1 ||1.83 ||52.3 |
Control values were mean ± SD of ten subjects.
n.d. = not detectable.
Fig.1. Relationship between glucose-6-phosphate dehydrogenase
activities in skeletal muscle and erythrocytes from deficient
subjects bearing three different variants and from controls.
The G6PD activity measured on muscle biopsies of patients bearing three different G6PD variants, demonstrated that the enzyme deficiency is always expressed in muscle. The extent of the defect in this tissue results proportional to the defect of the erythrocyte and the correlation between the two enzymatic activities results highly significant. The importance of our results lies in the fact that muscle G6PD activities reflect the true defect in vivo, whereas most of the published studies were performed on cultured myoblasts or fibroblasts of G6PD deficient patients (Kennedy et al, 1983; Maciera-Coelho, 1988). The extent of a G6PD defect in "in vitro" conditions may be very different from "in vivo" (Bresolin et al, 1989; Ninfali et al, 1991) . This is probably due to the lack of neural regulation which causes a mutated equilibrium between the rates of enzyme synthesis and degradation in the cultured cells (Max et al, 1981; Luzzatto & Battistuzzi, 1985).
The highly significant correlation, found between G6PD activity of muscle and RBC, if confirmed on a wider number of variants, represents a useful tool for indirect determination of muscle G6PD activity. In fact, this may be drawn simply by including the value of the RBC activity in the equation of the best fit regression line. By plotting our values, we also included the G6PD activity of the female subject, since it seems justified to assume that in an etherozigous female, RBC and muscle have the same mosaic. Both tissues originated from the mesoderm and at the stage of the formation of the germ layer, the preferential activation of the X chromosome is already defined (Migeon & Kennedy, 1975). A speculative interpretation of the proportionality of the G6PD activity in the two tissues may arise from the common mesodermal origin. In fact, if the level of the mutated enzyme were determined by its vulnerability to specific proteases, as hypothesized by Beutler (1983), there would be similar types of proteases in the two tissues by virtue of their common origin.
By comparing the three variants considered in this study we can infer that the enzyme defect of muscle is less pronounced in the A- variant. This is in agreement with previous studies indicating the A- variant as that which has the enzyme defect very poorly expressed in tissues other than erythrocytes (Beutler, 1990a).
On the base of recent acquisitions on the metabolism of muscle cell, it is possible to speculate on the G6PD role in this tissue and to consider G6PD deficiency as the most probable candidate able to determine the symptomatology and the clinical signs of our patients. They were, in fact, free from other pathologies or enzymophenias and mainly presented symptoms like hypostenia, myalgia, cramps, hemoglobinuria and/or myoglobinuria, high values of CPK and LDH after ingestion of drugs or physical stress. An interesting paper by Martensson & Meister (1989) demonstrated that marked glutathione (GSH) depletion induced skeletal muscle degeneration associated with mitochondrial damage. Since G6PD plays a key role in the production of NADPH utilized by the glutathione reductase to maintain GSH in the reduced form, one may deduce that, in G6PD deficient muscle, a lower level of NADPH leads to a decrease of intracellular GSH, which in turn increases the cell vulnerability to the reactive oxygen compounds and free radicals formed in the aerobic metabolism. With reference to this, it should be emphasized that heart and skeletal muscle have low levels of catalase and superoxide dismutase as compared with other tissues and therefore might be expected to be dependent on GSH linked reactions, for detoxication of reactive oxygen species (Ji and Fu, 1992). Oxo-radicals are in fact responsible of myofiber disruption and loss of intracellular proteins, which cause post-exercise soreness (Armstrong, 1990).
In this study we reported a different symptomatology of our patients. The problem is why patients bearing the same variant do not share the same symptoms? As observed by Luzzatto & Battistuzzi (1985) the clinical symptoms of G6PD deficiency are very erratic, because of the different extent of the deficiency in a patient population and of a peculiar kinetic control of the enzyme activity which is not completely clarified.
In conclusion our data suggest that: i) G6PD activity in muscle cells significantly correlates with the enzyme activity in erythrocytes; ii) subjects who have a marked defect in the erythrocyte should be considered at risk also for muscular pathologies. In our opinion, beyond natural or artificial drugs causing oxidative stress (Beutler, 1990b), G6PD deficient subjects should avoid high intensity physical exercises, since the high amount of oxygen radicals produced by aerobic muscle metabolism may induce skeletal muscle degeneration and myoglobinuria.
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