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Home , Acetylcholine, Organophosphate

2902Journal ofNeurology, Neurosurgery, and 1993;56:290-294

SHORT REPORT J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.3.290 on 1 March 1993. Downloaded from

Pathophysiological studies of neuromuscular function in subacute poisoning induced by

J L Good, R K Khurana, R F Mayer, W M Cintra, E X Albuquerque

Abstract suggested a (NMJ) A 51 year old man developed progressive defect of the postsynaptic type, but the exact cranial and proximal muscle weakness, mechanism of the dysfunction was not deter- hyperreflexia and mental change. The mined. 5o disorder progressed over 9 days following We describe the pathophysiological changes the fifth weekly spraying with the organo- in a patient who presented with a subacute (OP) , phosmet, progressive neuromuscular syndrome without with limited symptoms of acute toxicity. marked symptoms of acute toxicity after multi- Marked decremental responses of 50-80% ple OP insecticide sprayings. The defect in this on slow and fast rates of stimulation were neuromuscular syndrome occurred at the improved to 15% by or endplate- sites, is similar to the inter- . Intracellular recordings at mediate syndrome previously reported,10 and the endplate region of intercostal muscle differs from the acute and delayed syndromes. l revealed small miniature endplate poten- These results are important in the diagnosis tials (mepps), reduced mean acetylcho- and management of patients exposed to line sensitivity and normal membrane . A preliminary report of potentials. Electronmicroscopy revealed this study has been published in abstract degeneration and regeneration of the form. " endplates. This study demonstrates that OP poisoning due to phosmet can produce a subacute postsynaptic neuromuscular Case report syndrome without marked symptoms of A 51 year old man was admitted to the hospital acute toxicity. 5 days after the onset of diplopia, light headed- ness and a staggering gait. The patient devel- (7 Neurol Neurosurg Psychiatry 1993;56:290-294) oped these symptoms 18 hours after the fifth weekly exposure to a liquid spray insecticide http://jnnp.bmj.com/ University of Organophosphate (OP) insecticide poisoning containing an organophosphate compound, Maryland School of can acute Medicine, Baltimore, in humans produce: 1) peripheral phosmet, (phosphorodiothioic S-[(1,3-di- Maryland, USA and central block; 2) an inter- hydro-1 ,3-dioxo-2H-isoindol-2-yl) methyl] 0,0- Department of mediate syndrome with weakness, and 3) a dimethylester). He described his face and Neurology delayed distal polyneuropathy. l The acute syn- hands becoming wet from the insecticide and J L Good R K Khurana drome that occurs 12 to 72 hours after he did not bathe until the following morning.

R F Mayer exposure to OP involves inhibition of acetyl- Over the next few days, he noted progressive on September 24, 2021 by guest. Protected copyright. Department of (AChE) by the OP24 and direct unsteadiness of gait, dysphagia, change in and actions at the involving a variety of voice tone and excessive diaphoresis. Five days Experimental Therapeutics chemosensitive receptor ion channels.`- Cen- after exposure, he noted jaw weakness and E X Albuquerque tral and peripheral cholinergic signs occur. The droopy eyelids. Laboratory of neuromuscular block to repetitive nerve stim- The patient's general examination was nor- Molecular ulation shows decremental responses that are mal. Profuse oral secretions, bilateral ptosis, Pharmacology II, increased by edrophonium.8 The delayed neu- and marked facial diaphoresis were noted. He Institute of Biophysics "Carlos Chagas ropathic syndrome occurs 1-3 weeks after was alert, mentation was normal, and speech Filho", Federal exposure to the OP and probably results from was dysarthric. Extraocular movements were University of Rio De inhibition of the neurotoxic .9 A distal limited in all directions with the exception of Janeiro, Rio De Janeiro, Brazil axonopathy usually occurs with sensory and downgaze. Pupils were 3 mm and reactive to W M Cintra motor signs. More recently, an intermediate light. There was moderate symmetrical weak- Correspondence to: syndrome has been described in OP poisoning ness of facial, jaw, tongue and neck muscles. Dr Good, Department of that occurs 1-4 after the acute poison- There was mild weakness distal muscles and Neurology, Room N4W46, days of University of Maryland ing.'0 The patients reported developed pro- marked weakness of proximal muscles, es- Hospital, 22 S Greene weakness that was often severe and muscles that The Street, Baltimore, MD gressive pecially antigravity fatigued. 21201, USA involved cranial, respiratory and proximal limb tendon jerks were brisk throughout. Bilateral Received 17 December 1991 muscles which persisted for up to 18 days. Babinski responses were present. Sensory and in revised form Three of the patients had signs suggesting examination was normal. 11 March 1992. Accepted 16 March 1992 CNS dysfunction. Electromyographic studies The patient was admitted with the presumed Endplate studies in OP poisoning 291

diagnosis of acute OP poisoning. and care was taken to keep the patient warm J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.3.290 on 1 March 1993. Downloaded from (2 mg) and chloride (2-PAM, (rectal temperature of 37°C) during the proce- 1 gm) infusions were started. Within several dure. Recordings were obtained at 9 to 95 days minutes of 2-PAM infusion, the patient had after exposure to phosmet. increased systemic and ventilatory weakness Repetitive stimulation was performed at requiring intubation. After the patient was rates of 2, 5, 10, 20, and 50 Hz for 9 responses stabilised on a ventilator, 3 additional doses of in the median and peroneal nerves. Recordings 2-PAM were given without side effects over the of the median nerve were made before and next 24 hours. Atropine (1 0 mg initially and after 10 mg of intravenous edrophonium at thereafter 0-5 mg) was continued 4 times daily days 11 to 47. Recordings were made 5 to 40 for 6 days. was maximum on day 9. minutes after 1-5 mg of intramuscular neo- At this time, he was observed to have visual on day 12. Measurements of the , disorientation, and myoclonic motor amplitudes were peak-to-peak and per- jerks of his extremities. These signs persisted centage change (decrement) was calculated for for approximately 10 days during which time the fourth and ninth responses compared with the EEC showed diffuse slow wave activity. the initial one. The patient remained unchanged for the Concentric needle and single fibre (SF following 18 days, and supportive care con- EMG) electromyography and fibre densities tinued. By day 28, there was improvement in were performed in the extensor digitorum strength of neck flexion, shoulder and anti- communis and first dorsal interosseus muscles gravity muscles. By day 44, he no longer using established techniques. 14 SF EMG required mechanical ventilation or tube feed- recordings were obtained when the patient was ings. Babinski responses were absent. Exam- improving and able to cooperate, 3-4 months ination on day 64 (time of the muscle biopsy) after exposure. showed that he had mild fatiguable proximal A diagnostic intercostal muscle biopsy was and antigravity muscle weakness in the upper obtained 2 months after exposure because of and lower limbs. Facial and oropharyngeal persistent muscle weakness with informed movements were near normal. There was mild consent by the patient. Muscle was removed subjective diplopia on extreme lateral gaze. from the right sixth intercostal space in the Neuromuscular examination was normal by 5 anterior axillary line using standardised aneas- months. thesia and surgical technique. The muscle was Laboratory studies revealed normal routine immediately placed in oxygenated Krebs- blood and urine studies, creatine kinase, EKG Ringer solution and prepared for intracellular and chest radiographs. MRI of the was recordings and ultrastructure as previously normal. CSF was normal except for a total described.'5 Conventional microelectrode protein of 75 mg%. On admission red blood techniques were used for recording from cell cholinesterase level was within normal human intercostal muscle. High resistance limits (6110 U/L); and follow up levels on days pipettes (approximately 150 MQ) filled with 16 and 23 were 5420 and 4180 respectively 2 5 M (ACh) were used for and at 10 months (5114 U/L) remained nor- microiontophoretic application of ACh at the mal (normal range 6666-3590). Studies to neuromuscular junctions. Results were com- rule out other causes of pared statistically (Student t test) with control were normal. ANA and data that we have obtained from normal adult http://jnnp.bmj.com/ titres were negative. human intercostal muscle biopsied during thoracotomies for cardiopulmonary disease. The muscle fibres were fixed in ice-cold Methods phosphate buffered 2-5% glutaraldehyde for Nerve conduction and repetitive stimulation- thin sectioning and electron microscopic neuromuscular studies were performed in examination of the endplate regions.'5 16

peripheral nerves of upper and lower extrem- on September 24, 2021 by guest. Protected copyright. ities using established techniques.'2 53 Record- ings were performed in the intensive care unit Results Clinical electrophysiology Table 1 Nerve conduction and repetitive stimulation studies in median nerve after Initial recordings were performed 9 days after phosmet exposure exposure at which time the weakness was maximal. Electromyography at this time did % Decrement at 5 Hz not show any spontaneous activity such as Before After Tensilon fasciculations or fibrillation potentials. In Time CMAP DL MCV Days mV MS MIS 4th R 9th R 4th R 9th R median, ulnar and peroneal nerves, distal motor responses were mildly reduced in ampli- 9 90 50 54 48 43 1 1 11-6 55 43 33 19 tude and prolonged in latency. Motor conduc- 12 13-0 58 46 10 7 tion velocities were normal. No nerve 19 190 4-7 56 42 26 7 0 32 18-0 4-2 53 39 27 16 6 conduction block on proximal or distal stimu- 47 22-0 4-2 62 16 9 2 2 lation or repe'titve motor responses were 95 19 0 3-9 55 ±6 ±6 Normal subjects observed following single supramaximal Mean (SD) 17-0 (4 8) 3 5 (0 5) 55 (4) 0 (10) 0 (10) 0 (10) 0 (10) shocks. Antidromic motor F responses were in and Serial studies CMAP-Compound muscle amplitude in millivolts, mV, from thenar muscles. normal amplitude latency. DL-Distal motor latency in milliseconds, ms, from wrist to thenar muscles. were performed in the median nerve- MCV-Maximal motor conduction velocity in meters per second, m/s, between elbow and and these results are shown in wrist. thenar muscles R-Motor responses 4th or 9th on repetitive nerve stimulation. table 1. 2922Good, Khurana, Mayer, Cintra, Albuquerque

Figure I ol Recordinrgs No impulse blocking was observed. Fibre J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.3.290 on 1 March 1993. Downloaded from comtipound muscle acti'on potenltials from the thenar densities were normal. No spontaneous fasci- muscles on day 9 us'ing culations or denervation potentials were surjfice electrodes observed. sti'mulatinlg the medi'an A nerve at the wrist at rates 11tlA A of 50 Hz (A), 20 Hz (B1) JVJ vv1 V V V V ' Endplate and 2 Hz ((C). Note the Intracellular recordings at the endplate region marked decrement by the fourth response with some of intercostal muscle fibres revealed that the recovery by the last resting membrane potentials were similar to response at rates of 2 and controls (table 2). Spontaneous miniature 20 Hz but not at 50 Hz. was Amplitude calibration is endplate potential (mepp) frequency 2 mV ALl reduced (p < 0-01) compared with controls. B - I. I I [ Mean mepp amplitude was reduced (p < 0 01) I compared with control values. The shape ofthe I mepps was slightly altered as the potentials were prolonged by 20%. As many ofthe mepps were small (< 0 5 mV), it is possible that some were not observed above the noise level of the I recording system accounting for the reduced C 1- frqency. Microiontophoretic application of ACh to the endplate region revealed decreased and 1 marked, mean (SD), variability of the ACh sensitivity in different fibres [2730 (533) mV/nC to 448 (128)]. Mean ACh sensitivity was reduced significantly (p < 0 01) compared Repetitive stimulation at slow rates with controls (table 2). Falling phase of the (2-10 Hz) in the median arnd peroneal nerves ACh potential was prolonged by 20%. produced marked decremental responses (44-50%) at the fourth response with some Ultrastructure recovery by the last response (figure 1). At fast Endplate regions at low magnification showed rates of stimulation (50 Hz) marked decre- that the subjunctional myofibrils appeared ment (80%) was observed without recovery. normal without evidence of a myopathy. Neu- Serial recordings were performed in the romuscular junctions contained numerous median nerve and the results are shown in synaptic vesicles, mitochondria and synaptic table 1. Response to intravenous edrophonium clefts with well established basal lamina. The chloride was studied from days 11 to 47 (table endplate soleplasm contained an increase of 1) and showed a marked decrease in the smooth endoplasmic reticulum, ribosomes, decrement (58-42% reduced to 33-7%) at polysomes and invaginated nuclei compared slow and fast rates of stimulation. The decrease with controls suggesting high metabolic and in decrement was associated with transient regenerative activity (figure 2). Some endplates increase in strength on muscle testing. The showed subsynaptic vesiculation and phago- beneficial effect of edrophonium persisted cytic lysosomal activity suggesting degenera- http://jnnp.bmj.com/ throughout the 6 week period of neuromus- tion. At higher magnification some of the cular weakness. Similar beneficial effects were secondary synaptic clefts were widened and obtained with intramuscular neostigmine that filled with debris and junctional folds were reduced the decremental response of 58% (4th simplified suggesting endplate damage. response at 65 Hz) to 10% at 35 minutes after the .

Three months after exposure when the Discussion on September 24, 2021 by guest. Protected copyright. patient had mild proximal muscle weakness The patient described in this report had a and fatiguability, no decremental responses subacute progressive neuromuscular symdrome were observed. However, SF EMG revealed associated with CNS dysfunction following the increased variability in the interpotential inter- fifth weekly spraying with phosmet, an OP vals (jitter) in all 20 potential pairs examined. insecticide. Paralysis became maximum by 9 days, continued for 30 days, but some weak- Table 2 Endplate recordings in intercostal nmuscles in ness persisted for 4 months. CNS signs of patient with phosmet exposure and nornmal subjects visual hallucinations, disorientation and Recordings Patient Normals myoclonic jerks occurred at the peak of the toxicity and most likely reflected central chol- Resting membrane - 75 (2 73) - 76 (2 1) Potential, mV 45a 30 inergic dysfunction. These signs cleared MEPP 0-56 (0-07)* 0-83 (0-22) rapidly without residual effects. Limited symp- Amplitude, mV 40/481 b 31/435 MEPP 0-25 (0-02)* 0-38 (0-08) toms and signs of acute OP toxicity were Frequency, Sec' 40/581 30/870 noted. No myopathy or delayed peripheral ACh sensitivity 1182 (216)* 3730 (102) mV/nC 11 20 neuropathy occurred. The neuromuscular dvs- function was associated with CNS involvement Mean (SE) 'Number of fibres studied which was observed also in patients with the bNumber of fibres/number of potentials counted intermediate syndrome following exposure to *Significance p < 0 01 from normals MEPP-miniature endplate potential OP ."' Acute and delayed CNS ACh-Acetylcholine cholinergic dysfunction is well recognised in -Endplate studies in OP poisoning 293

block. The acute syndrome may benefit from J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.3.290 on 1 March 1993. Downloaded from therapy with pralidoxime chloride, an AChE reactivator that may have varied actions,'8 but it should not be used in the subacute syndrome with postsynaptic dysfunction, since it may increase the neuromuscular block and weak- ness. The endplate physiological findings in this patient with subacute neuromuscular weakness are similar to those reported in that have shown loss of functional postsynaptic ACh receptors.'5 9 The mechan- ism of receptor loss in subacute phosmet toxicity is unknown but is likely to result from endplate degeneration as shown on the ultra- structural studies. The physiological findings of mildly prolonged distal motor latency and reduced mepp frequency and the pathological finding of endplate damage suggest that nerve terminals may be affected by OP toxicity. Experimental studies with chronic AChE inhibitor toxicity in rats have shown similar physiological and morphological changes of the neuromuscular junctions.202' Postsynaptic dysfunction was greater and persisted longer than presynaptic changes, was prevented by ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~- chronic nerve section and was therefore possi- ~~ ~ ~ ~ ~ ~ ~ Av bly due to the prolonged increased activity of synaptic ACh. Prolonged action ofACh at the NMJ has been reported to produce a myopathy that is mediated by activation of a Figure 2 High magnification micrograph of a neuromuscularjunction of intercostal protease and requires intact ACh receptors.22 muscle showing numerous synaptic vesicles and clefts. The soleplasm contains large A myopathy was not observed in our patient amounts of smooth endoplasmic reticulum and polysomes. Calibration bar = 2 um. and this may reflect the endplate degeneration with loss of receptors. Prolonged action of experimental animals exposed to OP.29 synaptic ACh accounts for the acute OP Although the paralysis in our patient did not syndrome, and it is possible that it may induce occur after overt cholinergic toxicity, pro- the subacute neuromuscular syndrome by pro- gressed over a longer period (9 days rather than longed receptor desensitisation and influx of 4) and persisted longer (30 days rather than calcium that damages the NMJ. The endplate 18) than those reported,'0 it is likely that OP damage may be enhanced also by the direct toxicity due to phosmet can produce a delayed effects of AChE inhibitors that act as AChR progressive neuromuscular syndrome in and channel blockers.23 It is possible http://jnnp.bmj.com/ humans. In this syndrome the endplate physio- that similar changes may occur at CNS chol- logical studies clearly document postsynaptic inergic junctions. In addition, unpublished neuromuscular dysfunction. This is charac- data from our laboratory suggest that organo- terised by: mildly reduced CMAP amplitudes, cause release of excitatory and decremental responses reversed by edrophon- inhibitory transmitters in the CNS.24 ium and neostigmine, small mepps and Our patient was exposed to phosmet, an reduced junctional sensitivity. These insecticide recommended for the control of ACh on September 24, 2021 by guest. Protected copyright. results support those of the previous report in fruit tree pests.25 It is less toxic as an AChE which limited electromyographic studies sug- inhibitor than other insecticides such as para- gested a postsynaptic defect.'0 Decremental thion.26 Phosmet has very low water solubility responses at slow rates of stimulation observed and is soluble.26 It is possible that in this patient but not in those reported in the repeated weekly exposure to a lipid soluble OP, intermediate syndrome'0 may reflect greater like phosmet, spray by inhalation and skin neuromuscular blockade, desensitisation and contact resulted in the subacute progressive loss of ACh receptors. This may account for neuromuscular and CNS syndrome in our the prolonged duration of the syndrome. This patient. There are no other reports of subacute subacute OP postsynaptic neuromuscular toxicity due to phosmet but , which block can be differentiated electrophysiolog- also has high lipid solubility, produced 4 of the ically from acute OP cholinergic block that 10 reported cases of the intermediate syn- results in spontaneous repetitive firing of mus- drome.'o Proposed mechanisms by which cle action potentials and decremental-incre- some of the OP may produce subacute effects mental responses."' In acute OP toxicity, range from higher lipid solubility to delayed edrophonium increased the decremental res- conversion to a more active metabolite.227 ponses,8 while in subacute toxicity it reduced Although it is possible that the insecticide the decremental responses toward normal. which the patient used contained a toxic Edrophonium is recommended as a test in OP product of phosmet, there are no reports of toxicity to document the acute cholinergic other toxic substances in the compound.25 294 Good, Khurana, Mayer, Cintra, Albuquerque

Supported in part by the Neuromuscular Fund at the Uni- human muscle. In: Desmedt JE, ed. New development in versity of Maryland School of Medicine (RFM) and a Grant electromyography and clinical neurophysiology. vol 1. Basel: J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.3.290 on 1 March 1993. Downloaded from from the US Army Medical Research and Development Karger, 1973;1 13-29. Command Contract DAMD 17-88-C-8119 (EXA). 15 Albuquerque EX, Rash JE, Mayer RF, Satterfield JR. An electrophysiological and morphological study of the neuromuscular junction in patients with myasthenia gravis. Exp Neurol 1976;51:536-63. 1 Wadia RS, Sadagopan C, Amin RB, Sardesai HV. Neuro- 16 Rash JE, Albuquerque EX, Hudson CS, Mayer RF, logical manifestations of organophosphorus insecticide Satterfield JR. Studies of human myasthenia gravis: poisoning. J Neurol Neurosurg Psychiatry 1974;37:841-7. and ultrastructural evidence 2 Russell electrophysiological compat- RW, Overstreet DH. 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