g6pd muscolo

Original investigations Journal of

Neurology © Springer-Verlag 1989

J Neurol (1989) 236:193-198

Muscle glucose-6-phosphate dehydrogenase deficiency

N. Bresolin, L. Bet, M. Moggio, G. Meola, F. Fortunato, G. Comi, L. Adobbati, L. Geremia, S. Pittalis, and G. Scarlato

Istituto di Clinica Neurologica, Universit~ degli Studi di Milano, via F. Sforza, 35, 1-20122 Milan, Italy

Summary. Muscle glucose-6-phosphate dehydrogenase (G6PD) deficiency is described in four clinically heterogeneous patients: an athlete who developed myoglobinuria after physical exercise; a 7-year-old, mildly mentally retarded boy, who had episodes of dark urine and high creatine kinase; and two brothers of Sardinian origin, the elder showing moderate exercise intolerance. Histochemical and biochemical studies showed a lack of G6PD activity in muscle biopsy specimens as well as in erythrocytes. G6PD characterization in erythrocytes classified these mutant enzymes as Mediterranean variant in all the patients. The deficiency was confirmed in the patients ' myotubes and skin fibroblasts, where residual activity was present. Electrophoretic studies in tissue culture extracts showed that the residual muscle enzyme migrated as a single electrophoretic band like normal human muscle G6PD.

Key words: Glucose-6-phosphate dehydrogenase - Myo- globinuria - Myopathy

Introduction

Glucose-6-phosphate dehydrogenase (G6PD) is the X-linked enzyme catalysing the conversion of glucose-6-phosphate to phosphogluconate in the hexose monophosphate shunt (HMP) [24, 26, 39].

The deficiency of G6PD is the best understood and most thoroughly studied red blood cell (RBC) enzymopathy. This defect is widely distributed geographically; it has been reported in over 160 variants and is estimated to affect more than 100 million people [39]. Persons deficient in G6PD are clinically asymptomatic unless challenged by oxidant stress, which may be secondary to a drug or its metabolites, infections and acidosis [31]. Muscle involvement has so far never been investigated in these patients. However, in some cases myalgia, fatigability, hyposthenia and high serum level of lactate dehydrogenase (LDH) have been described during the haemolytic crisis, but not attributed to a muscular origin [1, 8, 28]. We now report muscle G6PD deficiency in four patients: one patient who had loss of consciousness and myoglobinuria after 30 min of intense exercise, a mildly mentally retarded boy, and two brothers of Sardinian origin.

Offprint requests to: N. Bresolin

Case reports

Case 1. A 30-year-old pentathlon-trained athlete suffered from loss of consciousness and pigmenturia during the last metres of a 12-km competitive run. The patient was trans- ported to the emergency room sweating and presenting sub- cyanosis and myosis. Blood pressure was 70/0 mmHg, pulse rate 180 beats/min, body temperature 38.5°C. Hypoglycaemia (31 mg/100 ml) was immediately corrected without improve- ment of consciousness. Blood urea nitrogen, ions and proteins were normal; RBCs were 4,000,000/mm 3, Hb 14.9 g/dl, haematocrit 42,8%, white blood cell count 12,000 and metabolic acidosis was present (pH 7.33, PCO2 38.5 mmHg, PO2 70 mmHg, HCO3 21 mEq/1, total CO2 22 mEq/1, standard bi- carbonate level 23 mEq/1, base excess - 5 mEq/1). The patient manifested jaundice with increased total bilirubin (5.4 mg/ 100 ml) and direct bilirubin (1.67 mg/100 ml); serum glutamic oxalo-acetic transaminase (SGOT) was 5,080 and glutamic pyruvate transaminase (SGPT) 5,650 IU/ml. Creatine kinase (CK) and LDH were 41,000 IU and 4,800 IU respectively.

Haemoglobin, myoglobin and ketone bodies were present in the urine. Plasmaphaeresis was immediately performed, maintaining normal diuresis and renal function. ECG showed sinusoidal tachycardia. CT of the head was normal. The next day the patient recovered full consciousness and denied taking any drug. High haemolysis and decreased haptoglobin were found; haemoglobin was reduced (10.3 g/dl); RBC were 3,000,000/mm 3 and bilirubin was markedly increased (total: 20.6 rag/100 ml; direct: 9.2 mg/100 ml). Bilirubinaemia, SGOT, SGPT, CK and LDH decreased gradually and re- turned to normal a few months later.

Our patient, now serving in the army and leading a normal life, reported a previous similar episode, but the urine was not examined for myoglobinuria. Family history showed two brothers who had pigments in the urine in the past after un- specified drug ingestion and a third brother died at age 7 years after ingestion of Vicia faba beans. For this reason our patient was tested for G6PD in RBCs that was 0.9% of controls. When examined by us, he was found to have normal EMG and EEG and to be neurologically normal. Chest radiography, echocardiography, liver echography and spleen scintigraphy were normal as well. Ischaemic exercise of the forearm was normal.

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Case 2. A 7-year-old boy was admitted to the hospital because of "muscle fatigability" and an episode of "dark urine" 4 months before. Family history showed that his mother (of Sicilian origin) was affected by G6PD deficiency. His delivery was normal, he was able to walk at age 18 months, and at age 2 years he was admitted to the hospital because of an episode of dark urine after ingestion of V.faba beans. At that time he was found to have G6PD deficiency. At age 4 years he was admitted again to the same hospital because his parents noticed that he had difficulties in walking and running and because he suffered frequent episodes of cramps and myalgia.

At that time LDH was 220 IU (normal values up to 240 IU) and CK 586 IU (normal values up to 100 IU). A muscle biopsy was not performed but CK was frequently monitored: 1 year later it was 1635 IU and LDH was 672 IU. Similar high levels were found occasionally in later measurements. On ad- mission to our hospital, CK at rest was in the upper normal range (124 IU), but LDH was 470 IU; routine blood analysis was normal. On examination he was found to be mildly mentally retarded and he was unable to jump on one foot. EMG and CT scan were normal. Diffuse slow wave anomalies were present on the EEG. ECG showed a tendency to tachycardia (103 beats/min). Ischaemic exercise of the forearm produced a normal lactate response.

Cases 3 and 4. Two brothers of Sardinian origin, 14 and 9 years old, were admitted to our hospital. The elder had recurrent episodes of myalgia and muscle fatigability so that he had to give up gymnastics at school. Both brothers were found to have G6PD deficiency in the erythrocytes and ischaemic exercise of the forearm produced a normal increase of venous lactate.

Their mother had the same RBC enzymopathy. On examination the elder boy showed generalized mild hypotonia and weakness of neck muscles and of the lower limb-girdle. On ad- mission to hospital CK was normal and LDH was mildly elevated (304 IU); routine blood analysis was normal. ECG, EMG and CT scan were normal; EEG showed diffuse slow anomalies. The younger brother was clinically asymptomatic and only LDH was moderately elevated (322 IU).

Materials and methods

Muscle biopsy was performed on the quadriceps muscle. In case 1 the biopsy was performed 4 months after the episode of myoglobinuria, when the patient had completely recovered. Control muscle was obtained by diagnostic biopsies from patients who were ultimately deemed to be free of muscle disease.

7.8. Background reaction was performed without substrate (G6P). G6PD histochemical activity was evaluated quantita- tively using a Zeiss microscope photometer computer assem- bly. Thirty scans at 10 × and 25 x with a 2.5 gm diaphragm were taken on different frozen reactions from patients and controls.

Tissue culture. Monolayer cultures of skin fibroblasts and muscle were grown by previously described methods [29, 30]. After selective plating muscle cells were allowed to reach con- fluency and fuse and were harvested 48-72 h after optimal myoblast fusion. All cultures were fed 24 h before harvesting. For cytochemical demonstration of G6PD, muscle and skin fibroblast cultures grown on cover slip were rinsed in Earle's balanced salt solution (EBSS), drained and air-dried. The cells were stained with the G6PD reaction mixture for 50 min at 37°C. For biochemistry, muscle cultures and skin fibroblasts were rinsed three times with cold 10 gM NADP and harvested by brief trypsinization. After centrifugation at 1,500 g for 10 min, pellets were resuspended in 0.5 ml of the same medium, sonicated and centrifuged at 10,000 g for 10 min.

Tissue preparation. Muscle specimens were frozen immediately and stored in liquid nitrogen. For measurement of glycolytic enzymes except phosphofructokinase (PFK) and phosphorylase (PPL), homogenates were prepared in 9 vol. of 50 mM Tris HC1 buffer (pH 7.5), 0.15 M KCI, in all-glass motor- driven homogenizers. Homogenates for PFK and PPL were prepared by previously described methods [13]. All enzymes were measured in supernatants after centrifugation at 10,000 g for 10 min. For mitochondrial enzymes the muscle was homo- genized in 9 vol. of the same buffer and centrifuged at 650 g for 10 min. On the full muscle homogenate, carnitine and carnitine palmityl transferase (CPT) (forward, isotope and reverse reactions) were measured by described methods [30]. Platelets obtained from 20 ml blood were isolated as previously described [3]. Erythrocytes were isolated by centrifugation (1,000 g for 10 rain) from 20 ml heparinized blood after the platelets and leucocytes had been removed; the packed red cells were washed three times with 3 vol. of ice-cold saline solution with 10 gM NADP. After the last centrifugation, cells were resuspended in 1 vol. of saline and 10 gM NADP and stored in aliquots at -70°C.

For measurement of G6PD, cell suspensions were thawed, diluted with ice-cold 10 ~tM NADP and centrifuged at10,000 g for 10 min to eliminate the ghosts. Lymphocytes were prepared by separation with Ficoll Paque (Pharmacia, Uppsala, Sweden). The assays were performed on the supernatant of RBCs, platelets and lymphocytes after sonication for 15 s and centrifugation for 10 min at 23,500 g.

Morphology. For histochemical studies, muscle biopsy specimens were frozen in liquid-nitrogen-cooled isopentane and cross-sectioned at 10 gm thickness in a cryostat. Sections were stained with a battery of histochemical procedures [13]. For electron microscopic analysis, muscle was fixed and processed by previously described techniques [13]. The histochemical reaction for G6PD was performed according to Pearse [9, 33] both on patient's muscle and normal control tissues (muscle, liver, kidney): the incubating medium contained 100 mM G6P, 1 mM MgCt2, 0.6 mM nitro blue tetrazolium (NBT), 1 mM nicotinamide-adenine-dinucleotide phosphate (NADP), 100 mg/10 ml polyvinylpyrrolidone in 0.2 M Tris buffer pH

Biochemical investigations. Individual glycolytic and mitochondrial enzymes, as well as CPT and carnitine were measured by previously described spectrophotometric and radio- chemical assays [2, 5]. Proteins were measured by the method of Lowry et al. [25]. G6PD reaction was measured in 1.0 ml of a reaction mixture containing 0.1 M Tris HC1 (pH 7.6), 0.9 mM ethylenediaminetetra-acetic acid (EDTA), 0.2 mM NADP (pH 7.5), 20 mM MgC12 and 5-10 ~tl of tissue prepared as previously described. After 10 min of incubation the reaction was started by the addition of 3.5 mM of G6PD and the reduction of NADP was spectrophotometrically followed at 30°C [40, 42].

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3 1 2 3 4 5 6

Electrophoresis. Aliquots (0.4 gl) of appropriately diluted tissue extracts were applied on cellulose acetate membranes (4.5 x 15 cm; Cellogel Chemetron Milan, Italy) which had been soaked in running buffer overnight. Electrophoresis was performed in 75 mM Tris citrate buffer (pH 7) with 4 mM Na2EDTA for 1 h at 4°C at 250 V in a Saitron 5003 electrophoresis chamber. The membrane was soaked in 9.4 ml of a solution of 0.1 M Tris HC1 (pH 8), 0.5 mM NADP, 6.4 mM G6P, 0.48 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl- tetrazolium bromide) with the addition of 0.6 ml of 0.1% phenazyna-methosulfate. The membrane was then incubated at 37°C for 5 min and the violet stained band was kept in a 5% solution of formaldehyde [9, 15, 20].

Enzyme characterization. The mutant enzyme was electro- phoretically characterized using three different running buffers (Tris citrate pH 7, Tris E D T A borate pH 8.6 and Tris

Fig. 1. Histochemical reaction for glucose-6-phosphate dehydrogenase (G6PD) in normal human muscle. G6PD, x 290

Fig.2. Histochemical reaction for G6PD in patient's muscle. G6PD, x 290

Fig.3. Cellulose acetate electrophoresis of G6PD in human tissues (see Materials and methods). Lane 1, normal human myotubes (1 : 5 dilution); lane 2, normal human fibroblasts (1:5),; lane 3, patient's muscle homogenate (1:5); lane 4, patient's myotubes (1:5); lane 5, patient's erythrocytes (1:5); lane 6, patient's fibroblasts (1:5)

Fig.4. Muscle cultures (14 days): control myotubes and mononuclear cells with cytochemical staining for G6PD activity, x 180

Fig. 5. Muscle culture (14 days): patient's myotubes and mononuclear cells showing scattered G6PD-positive granules, x 180

phosphate pH 7). The pH curve was evaluated using the same buffers ranging from pH 4.0 to 10.0. To study heat stability enzyme preparations were incubated at 45°C (pH 7.5) for up to 1 h. The affinity of mutant G6PD for G6P and NADP was studied varying the concentration of substrate between 35 gM and 70 gM for G6P and 0.5 ~tM and 20 mM for NADP. Michaelis constants (Kin) were derived from double reciprocal plots of substrate concentration and reaction velocity. Lines were determined by linear regression. Analogue utilization was tested with 2-deoxy-D-glucose-6-phosphate (2-DG-6-P) and galactose-6-phosphate (Gal-6-P) [22, 40, 42].

Results

Muscle specimens were normal both on light and electron microscopic examination, but histochemical stain for G6PD

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Table 1. Glycolytic enzymes in muscle homogenate

Case no. Controls

1 2 3 4 (n = 18)

PPL 340 280 230 200 300 (60) PFK 190 340 400 530 190 (40) Glyc.3PDH 2070 1760 2060 2270 1950 (280) Enolase 170 160 190 210 150 (30) PGM 100 200 400 500 200 (70) PGI 580 890 910 1050 920 (250) PK 1590 890 1370 1660 2130 (690) PGAM 1980 1090 1180 1280 2000 (590) PGK 1320 1160 1060 1360 1400 (710) Aldolase 420 350 440 530 530 (260) LDH 2570 1520 2100 1980 1360 (420) A.Maltase a 77 63 73 100 75 (10)

Values expressed as nmol/min/mg protein (SD) a Values expressed as pmol/min/mg protein (SD) PPL = Phosphorylase; PFK = phosphofructokinase; Glyc.3PDH = glyceraldehyde 3-phosphate dehydrogenase; PGM = phosphogluco- mutase; PGI = phosphoglucoisomerase; PK = pyruvate kinase; PGAM = phosphoglycerate mutase; PGK = phosphoglycerate- kinase; LDH = lactate dehydrogenase; A.Maltase = acid maltase

Table 2. Mitochondrial enzymes in muscle homogenate

Case no. Controls

1 2 3 4 (n = 18)

SDH 8.1 9.3 9.4 13.5 11.9 (2.3) NADH dehydr. 216.1 314.5 3 8 3 . 1 434.8 382.1 (75.7) DPNH cyt.c red. 30.4 36.0 42.9 47.7 49.2 (11.2) Succ.cyt.c red. 10.8 8.8 15.2 17.2 16.8 (3.7) COX 28.6 38.7 51.8 68.9 55.8 (10.1) Citrate synth. 84.1 90.1 1 1 4 . 9 162.2 129.9 (35.8)

Values expressed as nmol/min/mg protein (SD) SDH = Succinate dehydrogenase; NADH dehydr. = NADH dehydrogenase; DPNH cyt.c red. = DPNH cytochrome c reductase; Succ.cyt.c red. = succinate cytochrome c reductase; COX = cytochrome c oxidase; Citrate synth. = citrate synthase

Table 3. Glucose-6-phosphate (G6PD) dehydrogenase activity in blood cells

RBC Whole Whole PTLS WBC blood a blood b

Controls 3.59 (0.1) 301.3 (19) 9.7 (0.4) 145.7 (11) 105.1 (6.9) (n = 4)

Case 1 0.034 5.61 0.17 51.0 17.0 Case 2 0.200 8.87 0.33 28.0 25.6 Case 3 - 11.40 0.42 - - Case 4 - 11.00 0.56 - -

Values expressed as nmol/mirdmg protein (SD) a Values expressed as IU/1012 RBC b Values expressed as IU/g Hb RBC = Red blood cells; PTLS = platelets; WBC = white blood cells

was lacking (Figs. 1,2). Quanti ta t ive evaluations of G6PD activity showed a marked difference of light absorbance between control muscle ( t 9 % , SD 3.4) and patients ' muscle (average 1.3%, SD 0.6). Total and free carnitine in muscle and in serum were normal in all patients. In the same muscle

Table 4. Glucose-6-phosphate dehydrogenase activity in muscle homogenate

Controls (n = 4) 3.325 (0.61) (%) Case i 0.044 1.3 Case 2 0.062 1.9 Case 3 0.120 3.6 Case 4 0.550 16.5

Values expressed as nmol/mirdmg protein (SD)

Table 5. G6PD characterization

Case no. Controls

1 2 3 and 4 (n = 3)

Mobility on: Tris citrate 99.7 100 100 99-100 Tris borate 99.3 99 98 99-100 Tris phosphate 99.25 99 100 99-100

Kin: G6P (gM) 24 26 25 45 NADP (I, tM) 2.6 1.2 1.5 1.5

% Utilization: 2-DG-6-P 45 56 51 6 Gal-6-P 39 48 46 9

K~ NADP (#M) 10 10.3 10.1 9

Optimal pH 6.5 6.5 6.5 9 (biphasic) 9.5 9.5 9.5

Heat resistance Very low Very low Very low Normal

Gradient (mEq KCI) 234 236 242 228

Table 6. G6PD activity in myoblasts, myotubes and skin fibroblasts

Case no. Controls

1 2 3 (n = 4)

Myoblasts 29.4 3.0 14.3 165.2 (12) Myotubes 22.8 30.9 28.4 121.4 (10) Fibroblasts 18.9 29.0 23.2 102.6 (6.7)

Values expressed as nmol/min/mg protein (SD)

homogena tes CPT was measured by isotope, forward and reverse reaction and was normal as well as individual muscle glycolytic and mitochondrial enzymes activities (Tables 1, 2). G6PD activity in RBCs and in muscle was very low compared with controls. Residual activity was present in leucocytes and in platelets (Tables 3, 4). Characterizat ion of the mutant enzyme (electrophoret ic mobility, Km, pH, activity curves, heat denatura t ion and ability of the enzyme to utilize ana- logues) classified this deficiency as a Medi te r ranean variant (Table 5). Cellulose acetate electrophoresis of G6PD in pa- t ients ' tissues showed no detectable band of activity in RBCs and in muscle homogena tes (Figs. 3, 4). No abnormali t ies of growth pat tern were found in monolayer cultures of muscle and skin fibrobtasts. Time course by phase contrast showed in pat ients ' muscle cultures a normal fusion in myotubes and the presence of cross-striations in compar ison with control par-

allel cultures.

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Cytochemical reaction for G6PD performed on patients' myotubes and mononuclear cells showed slight activity and cytoplasmic scattered positive granules while in parallel controls the mononuclear cells had a moderate activity, usually more concentrated near the nucleus, and with some variation among different cells. Myotubes had a strong reaction, but in this case the activity was usually evenly distributed (Figs. 5, 6). G6PD activity in patients' myotubes and skin fibroblasts was about 18% of controls (Table 6). The residual enzymes showed a single electrophoretic band migrating like normal human muscle G6PD (Fig. 3).

Discussion

The hexose monophosphate (HMP) shunt is not the main pathway for obtaining energy from the oxidation of glucose in mammalian tissues. However, it is a multifunctional pathway specialized to carry out three main activities: to generate reducing power in the cytoplasmic form of NADPH; to convert hexose into pentose, required in the synthesis of nucleic acids; and to degrade pentose by converting them into hexose, which can be enter the glycolytic sequence [24, 26, 39]. G6PD activity produces NADPH which is necessary to maintain glutathione in the reduced state (GSH). The GSH reduces hydro- gen peroxide (H202) to HzO in the glutathione peroxidase reaction (GSH pX). Under normal circumstances the HMP shunt is relatively inactive and metabolism proceeds via the glycolytic pathway. G6PD can counterbalance "oxidant stress" by accelerating flow through the HMP shunt [24].

G6PD deficiency in RBCs has been widely investigated in the Mediterranean area as well as among African and Ameri- can black population. In these patients a possible muscle involvement has not yet been considered. All our patients had the Mediterranean variant of the G6PD deficiency in RBCs and in them all we describe for the first time the defect in muscle [6, 7]. Muscle biopsy was normal morphologically in all patients, and glycolytic, mitochondrial enzymes, CPT activity and carnitine levels were found to be normal as well. G6PD deficiency does not affect the main energy supply for muscle; however, other metabolic myopathies, such as acid maltase deficiencies, are due to enzyme defects not related to a glycolytic or lipolytic pathway. Case 1 had an episode of myoglobinuria during a competitive run. At that time metabolic acidosis and hypoglycaemia, which can precipitate haemolysis in a G6PD-deficient subject, were present. During metabolic acidosis, complete oxidative degradation of pentose was impaired, further reducing the supply of energy, and the NADPH necessary to stabilize the membranes was probably lacking [21, 26, 37, 41]. In this patient myoglobinuria pre- ceded the haemolysis: Hb, Ht, reticulocytosis, bilirubinaemia, SGOT and SGPT values markedly changed from normal only 2 days after the episode. We consider the possible higher vulnerability of a G6PD-deficient muscle to heatstroke even when the exercise is performed in normal weather conditions. In most cases acquired myoglobinuria is of unknown origin; however, sometimes it is possible to correlate the presence of dark urine with physical exercise [34, 35]. None of the known contributing factors to exercise-induced myoglobinuria was present in our patient. Moreover, haemolytic anaemia is known to be a common trait of some metabolic disorders in which muscle is also involved as PFK and phosphoglycerate kinase deficiencies [19, 21, 36].

Recurrent myoglobinuria has been described in several in- herited disorders of the glycolytic and lipolytic pathways and in a few cases of systemic carnitine deficiency [2, 4, 10, 12, 14, 18, 23, 32, 36]. Attacks never begin before adolescence and myoglobinuria may appear only in adult life as a single episode. Myoglobinuria has been also reported in athletes affected by CPT deficiency in whom, like case 1, neurological examination and muscle biopsy were normal [2].

Case 2 reported episodes of dark urine not related to intense muscular exercise. We have not evidence whether they were caused by haemoglobinuria, myoglobinuria or both.

In our patient the enzyme defect had different tissue expression: in leucocytes G6PD residual activity was about 16%, in platelets 35% and in tissue cultures the activity was al- most 18% of controls. The high activity of G6PD in normal muscle culture was found also by other authors in undiffer- entiated myoblasts, myotubes and fibroblasts [4, 38]. Never- theless our tissue culture findings (skin fibroblast and muscle cultures) suggest the absence of a fetal form of G6PD and the expression of the defect in early myogenesis. In G6PD-deficient myotubes, the lack of neural regulation can account for the higher residual activity [11, 26, 27, 39].

The frequency of G6PD deficiency, the clinical hetero- geneity of our patients presenting mild or absent muscular symptomatology and the possibility that G6PD muscle deficiency may be a cause of myoglobinuria in particular conditions suggest the usefulness of further extensive investigation on muscle metabolism in other G6PD deficient patients.

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Received August 8, 1988 / Received in revised form November 25, 1988 ! Accepted November 30, 1988

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