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Muscle membrane potentials in episodic adynamia
508
ELECTROENCEPHALOGRAIPHYAND CLINICALNEUROPHYSIOLOGY
MUSCLE
MEMBRANE EPISODIC
POTENTIALS
IN
ADYNAMIA
OTTO D. CXEUTZFE~VT, BERNARVC. AeeoTr, WILLIAMM. FOWLEr. AND CARLM. PEARSONz Brain Research Institute and the Departments of Anatomy, Zoology and Medicine, University of California, Los Angeles, Calif. (U.S.A.) (Received for publication: August 17, 1962)
sia and the muscle was exposed for a length of about 5-7.5 cm. After retracting the skin and the subcutaneous fat, and after careful control of bleeding, the fascia externa was opened. Then the inner fascia was removed under microscopic control and the muscle tissue was covered with physiological saline solution. The area of this complete exposure of the muscle was about 1 x 4 cm. After the end of the measurements, which lasted for 30-60 rain, the wound was closed after microscopic exploration of the experimental field for broken electrode tips. in all instances the wounds healed primarily without any complications. 2. The membrane-potentials were measured relative to ground with glass micropipett~s, filled with 3 M KCI solution, The resistances of the electrodes were between 10.-50 Mfl and were monitored during the penetration (see below). The electrodes were positioned with a microdrive fixed rigidly on the table of an electric drill stand, which could be swung over the experimental field. The indifferent ground electrode was a chlorided silver plate wrapped in gauze, soaked with physiological saline, and placed on the skin about 10 cm from the experimental area. METIiODS 3. The recording equipment consisted of an 1. The measurements were performed on the Argonaut Negative Capacitance Electrometer right or left M. vastus reed., with the patient pre-amplifier and a Sanborn 320 pen writer, lying on a hard bed. A nerve block of the N. connected in parallel with a Tektronix oscillofemoralis was applied without general anaesthe. scope 502, This arrangement allowed simultat Present addresses: Dr. B. C. Abbott, Department neous observation and recording of the potenof Zoology, UCLA, Los Angeles 24, Calif. (U.S.A.). tials during the penetration of the micro-elecDr. O. D. Creutzfeldt, Max-Planck-lnstitut for i~y~ia- trode. The capacity of electrode and leads was trie, M0n~en 23, Germany. compensated by the negative capacity of the preDr. W. M. Fowler, Department of Physical Medicine, UCLA Medical School, Los Angeles 24, Calif. (U.S.A.). amplifier. During the experiment the impedance Dr. C. M. Pearson, Department of Medicine, UCLA of the micro-electrode was checked by measuring Medical School, Los Angeles 24, Calif. (U.S.A.). the voltage drop across the electrode which lntracellular measurements of membrane and action potentials of human and mammalian muscles have brought new interesting results concerning myotonia (Norris 1962), myasthenia (Elmquist et ai. 1960)and denervation fibrillation (Li eta/. 1957). They have failed, however, to explain the paralytic attacks of periodic paralysis (Shy et al. 1961). Clinical and electromyographic studies of patients with Episodic Adynamia (Gamstorp 1956) (called henceforth e.A.) have suggested that the paralytic attacks in this disease might be due to temporary depolarization of the muscle membranes (Buchthal et aL 1958 a, b; Creutzfeldt 1961; McArdle 1962). In order to evaluate more critically this hypothesis we have in this study measured intracellularly the muscle membr, ne potentials in a patient with e.A. during a normal interval, and during both a spontaneous and an experimentally induced attack. For comparison measurements have also been carried out in a normal male control and in a patient with periodic paralysis but during normal periods, as well as on anesthetized rats. A preliminary note of the results appeared elsewhere (Creutzfeldt el al. 1962).
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Fig. I Recording of membrane potentials. I : Record of an experiment in an anaesthetized rat. 2+ 3: Continuous record in a healthy young man (case 3), nerve block anaesthesia. Read from right to left. Before a: The electrode tip is within the saline solution covering the muscle and is slowly moved towards the muscle surface. Zero potential. In 2 some damalp~d eclls are penetrated before o. a: Penetration o~"the first fibre. The electrode is slowly advaneed deeper till h, penetrating several cells. Between b and ¢ it was removed. ¢: The electrode tip asain in the saline solution, zero chock. Time: 20 sec. Calibration as indicated (negativity upward).
resulted from a square.wave current injected into the input circuit provided by the Argonaut electrometer input stage. The zero potentials were balanced by a potential in the ground circuit. After zeroing the instruments with the electrode tip submerged in the saline covering the muscle surface, the electrode was introduced into the muscle under microscopic control. Ce!! penetration was visualized by a sudden rectangular change of the d.c. potential. The electrode was kept still for about half a minute and then moved forward. As it left the cell the potential fell back and the next cell was approached. The potential frequently did not reach the zero level when the electrode was between two fibres. A safe exp,~anation for this cannot be given on the
basis of our experiments. Possible reasons may be injury currents from destroyed fibres, incomplete localization of the electrode tip in the intracellular space, or slight changes of the tip potential of the electrode due to bending of the tip. During the whole procedure the direct recorder was kept running, the micrometer ronding~ ,were marked on the paper, and in addition the potentials were read on the oscilloscope. From 3 to 20 different muscle fibre~ down to a depth of about 2 mm could be penetrated before the electrode broke, due to strong bending of its tip or slight movements of the patient. Examples of recordings in a rat and in a human are given in Fig. 1. Materials. As mentioned above, the measure-
Electroenceph. din. Neurophyaiol., 1963, 1.5:508-519
510
o . D . CREUTZFELDT e l
ments were carried out on three persons. In order to check the apparatus and the reliability of the method, control experiments were first performed on five adult rats under general anaesthesia (Penthotai). Subject 1 (A.S.}. Diagnosis: Adynamia episodica hereditaria (e.A.) A 41 year old white married woman suffered from typical attacks of flaccid paralysis almost daily for 36 years. There was a strong family history of similar attacks. (The clinical and genealogical data of this patient will be published elsewhere). Within recent years one to three episodes occurred daily, mostly in the morning. Attacks could be elicited by restriction of food in the morning or by oral or intravenous KCI. An improvement of the attacks could be brought about by injection of Ca +~'. A remarkable reduction in the number and severity of the attacks has been noted during daily oral hydrochlorthiazide therapy. Single attacks began with strange feelingsin the stomach, followed by numbness around the mouth and later by increasing weakness of the limb and body musculature. If the patient had not been working or moving before the attack the weakness uniformly involved all muscles. If exercise preceded the attack the muscles mainly used were more severely involved. Frequently the respiratory muscles were involved, occasionally to such an extent that the patient became cyanotic. The tendon reflexes disappeared at the height of an attack, during complete paralysis. Except for the slight numbness in the face no sensory symptoms were observed. During the attacks the serum K +commonly increased by 2-3 mequiv./I, up to 6-7 mequiv./l. Corresponding electrocardiographic changes occurred in the Twaves (see Fig. 2). During the present investigation the validity of the attack was checked by frequent measurements of the hand grip strength with a dynamometer, by te.~ting the tendon reflexes, by EKG recordings and serum K" measurements at regular intervals. In this patient micro-electrode measurements were carried out during two different sessions. The first time the operation was begun at the onset of a spontaneous attack and the measurements were eatried out during the maximum period of weakness and rise in serum K + to 5.5 mequiv./l.The second time, measurements were made in the absence
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Fig. 2 Amplitude of T-wave in mV, plotted against serum K + in patient A. S. (Adynamia episodica hereditaria). The symbols are measurements from different attacks: open circle: May 20, 1961; cross: May 27, 1961; closed circle: June I, 1961.
of weakness, after which an i.v. infusion of 40 mequiv, of KCi in 120 ml of 5 per cent dextrose was given during a 30 rain period. The serum K '~ rose from 3.82-6.90 mequiv, in this interval and a typical attack was produced. A second sample of potential measurements was then collected.
Subject 2 (J.P.), Diagnosis: Periodic'paralysis A 42 year old Mexican labourer who has had many typical attacks of flaccid paralysis since the age of 18 years. These especially occur in the early morning hours after heavy work and a late supper on the previous day. Episodes of weakness last from 2 h to 6 days, with an average duration of 2 days. He can abort an attack by exercise when the early signs appear, He has had an average of five attacks per year, There is a family history of similar episodes in two brothers, a p s 44 and 46 years, Examination revealed normal musculature, good strength and normal reflexes. The patient was hospitalized and an attempt was made to induce an attack by heavy exercise and a late meal on the night before intra-fibr¢ electrode studies were planned. However, ~;~ following morning he showed only minimal weakness of the pelvic flexors at a time when serum K+ was 4,25 mequiv./l and Na + 152.6 mequtv./l, lntra-fibre electrode studies
Electroes~'el~.eli& Nem'olOtysiol.,1~1, 15:50~519
MEMBRANE POTENTIALSOF HUMAN MUSCLE
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Fig. 3 Membrane potentials of three different rats (muse. soleus, generalanacsthesia). Ordinate: Membrane potential in mV. Abscissa: Succession of impalements of consecutive mu~le ~lls durin$ the penetration with one electrode. Measurements carried out with one electrode are connected with a line, the consecutive penetrations with diff©rcntmicro.electrodes are labeled at each line, Further explanation, ~,~ melhod.~.
were performed in the right quadriceps femoris muscle after preliminary femoral nerve block. Then 390 ml of 30 per cent glucose, which con° tained 34 units of regular insulin, was rapidly infused intravenously over a 40 rain period, after which strength had declined moderately, so that the heel of the extended leg could be raised only 25 cm from the bed. At this time the serum K ÷ was 3.17 mequiv./l and the Na + was 137.4 mequiv./I. A series of repeat membrane potential studies was done at this time. The patient recovered rapidly from the mild weakness and had normal strength 4 h later. Hence, a frank episode of periodic paralysis was pot produced on th~s occasion. ": ~. Subject 3. A healthy 24 year old student volunteered for this investigation. In him, data were collected during one session, In the tables and the description of the results
the mean values with the standard deviations for each experiment have been listed. For statistical comparison of the means in different experiments the calculation paid regard to the gradient of values from "superficial" to "deep" measurements. The special statistical method which was used for these calculations has been developed by Dr. W. J. Dixon, Department of Biostatistics, UCLA Medical School, Los Angeles, Calif., whose help we wish to acknowledge gratefully. In the diagramq of Fig. 3-5 the values of the membrane potential are on the ordinate while the abscissa represents the sequence of readings during the penetration with one micro-electrode. Each line connects the figures obtained during one penetration. However, the sequential numbers of penetrations do not give the exact figure of the depth within the muscle from which the Eleetroenceph. olin. NcurophysM.. 196.3, !$: 508-519
O. D. CREUTZFELDT el a[.
512
TABLE i Membrane potentials in three rats and three human beings. Further explanations, see text. Subject
Date
Series
Number of measuremen~
Condition
Mean value with standard deviation
A June 16, 61 Oct. 28, 61 Oct. 29, 61
Rat Rat Rat
l 2 3
General anaesthesia General anaesthesia General anaesth~i~
5 35 42
---77.6-1-0.9 mV I 79.9 -I---77.4 ~ 7 mV --81.8 -t-8.3 mV 7.3 mV
B Oct, 30, 61
Healthy man
1
Nerve block
26
--87.4 ~ 8.9 mV
C Oct. 31, 61 9.30 am. Oct. 31, 61 i0.30 a.m.
Pat. P. S. with periodic paralysis
l
Nerve block; free interval Nerve block; after insulin
23
--85.6 ~6.1 mV
27
--77.1 28.2 mV
2
J
+ glucose D May 6, 61 9 a,m.
Pat. A.S. with episodic Adynamia
I
Nerve block; free interval
21
--68.5 ~ 5.9 mV
May 6, 61 10. a.m.
2
15
--46.3 2~6.6 mV
Juno I, 61
3
Nerve block durin8 an induced attack Nel~e block; durin8 a spontaneaus attack
15
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Membran• potentialsin patient A. S. with Adynamla®pisodicahercditaria,a: Controlduring a frge interval(~rum K+ =~ 3.75 mequiv./I),b: A half hour later, during an attack indugedby infusionof 40 mequiv. KC! in 120 ml 5 per gent dextrose (serum K+ =~ 6.9 m~uiv.li), c: Moasurements during a spontaneousattack, carriedout one week beforea and b on the other leg (sorum K+ ~ 5.24 m~lulv./I). recordings were made. Usually, a membrane potential stayed almost unchanged for about 80-130/~ of micrometer movement (i.e. presence of the electrode tip within a single fibre), whereas it stayed omside for 30-50/~. Th©s¢ r©lativ¢ly high values, which are larger than one would expect considering the actual fibre diameters and intercellular spaces, may possibly be due to slight displacements of fibres by the micro-electrode. Sometimes, the electrode went quickly through a number of fibres without enabling us to read the potential. Also, the electrode sometimes jumped through the first two or three
superficial muscle cells because of the elastic resistance of the muscle sheath. For these reasons the numbers of the readings do not give a direct, but only an approximate, indication of the depth of recording. RESULTS
Fig. 3 shows the membrane potentials obtained in three different rats under general anaesthesia (Pentothai i.p.). It is obvious that the surface readings are almost always lower than the depth readings. This is probably due to the relatively quick cooling and drying ofthe muscle £1eetroe..neeph. din. NeurophydoL, 1963, 1.5:508--519
o.D. CREUTZFELDT et al.
514
surface. The mean values arc given with standard errors in Table 1. in Fig. 4 are shown the results obtained on Subjects 2 and 3: the curves (a) represent the measurements in the healthy young student, while the ones marked (b) show the control measurements in the patient with periodic paralysis. Both figures lie closely together, theaveragebeing nearly thesame(--87.4 ~ 8.9 mV and--85.6 =[=6.1 mV). The third group of curves (c) are measurements in the patient with periodic paralysis during the attempt to induce an attack. During these penetrations the serum K + was 3.17 mequiv./I having been at 4.25 mequiv./I during the control measurements. Even though the average in the second series (--77.1 ~ 8.2 mV) is significantly lower ( p < 0.01) than in the first the slope of the curves shows clearly that this is due at least partly to the low values in the surface layers. Because the second set of measurements was madeabout ! h after the first, during which time an attempt was made to induce an attack, it is conceivable that the lowering of the membrane potentials in the surface layers may be partly due to injury currents from the superficial fibres (see below). Fig. 5 gives the results obtained in A.S., the patient with e.A. The curves (a) show the measurements taken during a free in.
terml with a serum K + of 3.75 mequiv./l. All figures are below --80 mV, the average being --68.5+ 5.9 mV. This average is considerably lower than the figures obtained in the two control subjects. As will be discussed later this low value can be considered as real and cannot be explained simply by the fact that we were not successful in this patient in collecting many data from deeper ceils,During an induced attack (b) with a serum K ÷ of 6.9 mequiv,/I the membrane potentials were markedly lowered to an average of--46.3 ±6.6 mV and the single figures never reached the lowest values of the control series (a). Accordingly, the difference of the average values is significant ( p < 0.01) in spite of the large distribution during the induced attack. The values collected during a spontaneous attack, with a serum K ÷ of 5.24 mequiv./I, are represented by the lines (c). Here, too, the potentials are remarkably lowered, the average --51.5 ~6~3 mV being again significantly ( p < 0.01) different from the control. The mean values of the induced and the spontaneous attacks, however, are not significantly different. In comparison with other measurements the relatively wide distribution of the values during the attacks should be mentioned.
TABLE I! Membrane potentials in human ,iuscles. Results of differentauthors. Muscle
Preparation
Df~ase
Number of measurement
M. intercostalis
Excised.in
Myasthenia
15
~80.9 ± 1.0 mVxS.E.) Elmq'uist,Johns, T h e f t (1960)
Periodic I~ralysis
82
--71.2 ~ 11.6mV($.D.) Shy,Wanke, Rowley,Easel (1961)
4 healthy
40
~77.8 ± 153 mV
--$7.4 :]=8.9mV($.D.) Creutzfeklt, Abbott, Fowler, Pearson (1962) --86.6 ± 7.7 mV !
vitro
Not known
In situ, anaesthesia not known
M. Bastrocnemius In sltu. and quadric~ps local femoris anaesthesia
Membranepotential
Author
Johns (1961)
men
M. VaStUS rood.
In stlU, nerve block anauthesia
Healthy young man
26
med.
nerve block anaesthesia
Periodic paralysis (free interval)
23
--8&6 d:6.1 mV; (S.D.)
F_./ecu~oe~e~,elat. Newo.~y~'o/.,1963, I$: SO8.519
MEMBRANE POTENTIALS OF HUMAN MUSCLE
515
closely together that it is not probable that different tip potentials really had a great inWe shall discuss briefly two points. The first fluence. It may, however, be possible that the concerns the reliability of the measurements; gradients from superficial to "deep" values may the second some pathogenetic considerations have been caused by changes of the tip potentials concerning e.A. during the penetration of the electrode into !. The mean valuesandthedistribution(standdeeper layers of the muscle. This possibility has ard deviation) found in rats, as well as in LNe been noted by different authors (Nastuk et al. human control subjects, are in good agreement 1950; Del Castillo et al. 1955; Frank et al. 1955). with those found by other authors. In rats values The lower values in the superficial cells might of --72.2 H 5.3 mV (Li et al. 1957) and --88.9 also be explained by their injury. In spite] of ±8.06 mV (Harvey e t a i . 1958) were reported, these minor uncertainties the good agreement of whereas our own average was --79.9 ± 7.3 mV, the control measurements with those reported with averages in different experiments from in the literature, as Nell as the relatively narrow m77.6 to --81.8 mV (Table 1). In other species standard deviations of the means, justifies their higher and lower values were reported (--99.8 acceptance as controls and their comparison ±6.5 in mice, Bennett et aL 1953; --79.5 ± 5.7 with other values of our series. Furthermore, in cats, Trautwein et aL 1953; and --84,5 ± 5.7 the statistical comparion of the values paid in Guinea pigs, Trautwein et al. 1953). Our values regard to the gradient from superficiel to deep obtained in men are higher than those reported measurements. by other authors (Tsble !i). The membrane potential in the patient with There are various possibilities to explain the periodic paralysis was slightly reduced during slight differences between different authors. One the effort to induce an attack (--77.1 ± 8.2 mV is the selection of values, as there are some in- compared with --85.6 ± 6.1 mV), and just vestigators who reject extremely low values, as- significantly different from the control figure suming that they may be due to injury of the ( p < 0.01). Even though no conclusive deducmembranes. This might explain the relatively tions can be drawn from this slight decrease, low values obtained in rats, compared with the especially because the readings were mainly higher values reported by some authors quoted limited to the superficial ceils, it should be noted above. If, in our experiments, the electrode re- that Shy et oL (1961) also show in their figures a sistances were in a satisfactory range (10-50 ML~) slight, and probably not significant, decrease we have not rejected any values, in order to during a full paresis. From the diagrams of avoid any bias in the later comparison. Another Shy et al. (with rounded figures) we estimated an factor may be the different state of the animals average membrane potential of--75,4 mV during or the human subjects. We do not know to the free interval and of --70.5 mV during the what extent light anaesthesia plays a role. Our paresis. Therefore, a slight decrease of the memexperiments have been performed in unanes- brane potential during the attack in periodic thetized subjects. The preparation and the open- paralysis might still be suspected, and further ing procedure were kept as constant as possible measurements under bet~:r conditions should be in order to avoid too greet an error from this. carried out, In the patient with e.A. even the values during One factor, however, which has not been fully excluded in our experiments was the tip poten- the free interval were very low (168.$ ± 5.9 tial of the micro-electrode, whose importance mY). The difference from the normal control has been pointed out by Adrian (1956). We is significant ( p < 0.01). During the spontaneous, tried to reduce this disturbing factor by balancing as well as during the induced, attack the memthe zero potential at the beginning of each pene- brane potential showed a further decrease (Fig. 5 tration by a compensating potential in the ground and 6). This decrease is significantly different circuit (Methods). ~ihe actual values obtained (p<~ 0.01) from the values during the interval, with different electrodes in the same subject though there is no significant difference between under identical conditions, therefore, lay so the values during the induced and during the Electroenceph. din. Neuropkyslol., 1963, 15:508-$19 DISCUSSION
o . D . CREUTZFELDT et al.
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Fig. 6 Membrane potential in mV (ordinate) plotted against external [K+! (abscissa, logarithmic scale). Pat. A. S. with e.A. Averages of measured membrane potentials with standard errors. The numbers above the abscissa show the serum [K +] for the different measurements. The broken line at the top shows the calculated curve of the K + potential for a IK+], of 150 mequiv./I. .eo.,~. K
healthy patients, and even the "deep" values (i.e., the ones obtained from deeper cells) are lower than the corresponding values in the controls. It can be concluded, therefore, that the low membrane potentials in patient A.S., even in the free interval, are real. 2. In spite of these results the pathogenetic mechanism of the low membrane potential and of the paralytic attacks in e.A. still remains somewhat obscure. The serum K ÷ level rises almost twofold during an attack, so that a fall in membrane potential would be expected. But neither the initial low "control" value in the patient nor the further decrease during the attack can be reasonably explained in terms of serum K +. The average level of intracellular K ~ in normal subjects is about 150 mequiv./I of fibre water. In Fig. 6 the dashed line represents the variation of membrane potential with extracellular K + level (Ko) predicted by a simple Nernst equation which states that RT [K]o Membrane Potential ......... log [K]i"
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[*~'],,n r'~u*vl* Fig, 7 Membrane potential (ordinate) plotted a$1inst intra~l. lubr [ g q (abscissa). The lines represent calculated rein. tions for thr~ different external potassium coneentra. tions. These [g+]o are values of the serum K* during the different measurements in patient A.S. with o.A. On these calculated curves the corresponding measured mere. brane potentials (averages) are marked with circles, in order to show the corresponding [K,], the circles are connected by vertical lines to the abscissa. Further explanation see text.
spontaneous attack. The low values during the free interval and during the spontaneous attack were obtained from the beginning of the experi. ment, when cooling and drying cannot have been considerable. Furthermore, in spite of the relatively wide distribution, almost all single values arc lower than the ones obtained in the
The actual values in our patient are obviously so much below the theoretical values, under the assumption of RT/F ~, 60 and [K], ~ 150, that some other factors must be involved, Furthermore, it is not possible to explain the low control values or the further lowering of the potential simply by a decrease of the internal K ~ concentration, though several authors have found a loss of intracellular K ~' during e.A. attacks (Buchthal et al. 1958; Klein et al. 1960; Creutzfeldt 1961: McArdle 1962). The expected values of intracellular K+ concentration with different membrane potentials are shown in Fig. 7 for the three measured serum levels in our patient, The points marked with circles are the actual membrane potentials measured. As can be seen it would be necessary to assume a "normal" [Ki] of 51 mequiv./I (instead of 150) and during the attacks a further decrease to41 or 38 mequb,./I. However, direct measurements of the intramuscular K ' concentration in other cases of e.A. have shown normal values in the free interval (Buchthal et al. 1859; Creutzfddt 1961; Klein et al. 1960). Furthermore, a quantitative calculation of the intracellular/extracellular K + balance shows that the serum K + would have to increase Eleetroe.,~eepk. ¢lin. NeL,,~l~y$1ol., 1963, 15:Y~8-319
MEMBRANE POTENTIALS OF HUMAN MUSCLE
by far more than it really does if one assumed merely a muscalar K + loss as the cause of the attack and the observed decrease of the membrane potential (Creutzfeldt 1961). it is therefore necessary to look at the problem from a more dynamic point e l view. This is possible by taking into account some recent observations on cation permeability of muscle membranes under different conditions of membrane polarization (Hodgkin et al. 1959, Adrian 1960) and assuming a slight change in Na + permeability of muscle membranes in e.A.: the value of membr~,ne potential can be expressed as RT M.P. --=-~
[Klo + ~x INalo In [Kh + ~ [Nah
where the subcripts i and o represent intra- and extracellular ionic concentrations and ,', is the ratio of sodium to potassium permeability in the membrane (Hodgkin and Horowicz 1959). in frogs .~ is about 0.01 according to Hodgkin e¢ al. Applied to normal human muscle we would come to the following: with a serum K ~ level of 3.75 mequiv./I and an intracellular level of 150 mequiv./I of fibre water, the K ' equilibrium potential would be about ~ 9 8 inV. If in the normal human muscle, a value for ~x ..... 0.01 is assumed, then with K I ........ ! 50, Ko ........3.75, Nao 150, Nat =:: 20, a potential would be predicted of ~ ~ mV, which is close to the measured value in normal control subjects. If, in the e.A. patient, a disorder of the sodium exchange exists even in the aormal quiescent state, such that the permeability to sodium is increased only three fold to give a ratio of sodium to potassium permeability of 0.03, then a membrane potential of ~ 7 5 mV would be predicted. This can be compared with the mean value o f - - 6 9 mV in the patient. During a paralytic attack the serum potassium increased to 5.24 mequiv./I while the membrane potential fell to ~51.5 mV with loss of excitability. The increased serum potassium must come from within the cells, but will not noticeably alter the intracellular [K] ~ (of about 150 mequiv./! of fibre water). But it will depolarize the membrane further and may induce a further increase in sodium permeability. If there is a further three fold increase, to ~, = 0.1, with serum K ÷ at 5.24, then the membrane potential should be -.51.1 inV. Thus the events could
517
be explained by a change in the permeability of the muscle membranes to sodium at rest, which would result in a relatively unstable system. Any further increase of the serum K* level, such as would occur in the morning hours or after ingestion of a relatively l~rge amount of K + or a loss of muscular K ÷ after exercise, cold shivering etc., could produce a cumulative effect of depolarisation, further increase in sodium permeability, and further depolarisation. Such a series of events might tend to produce regenerative activity and thus might account for the observations of some authors of fibrillation potentials (Buchthal et al. 1958 a, b: Creutzfeldt 1961) and eveu increased mechanical excit,tbility similar to myotonic phenomena (McArdle 1962) in the beginning of an attack. When further depoiarisation occurs sodium conductance increases, and inactivation occurs such that the tendency to produce regenerative activity disappears. There is as yet no immediate proof that depolarisation is due to irregular sodium permeability, and a similar effect might be produced by a permeability change to certain intramuscular anions, as suggested by Buchthal er al. (1958 a, b). However, the fact that an attack could be very rapidly alleviated by intravenous injection of calcium supports the sodium hypothesis, although there has been no opportunity to study directly the effect of increased calcium on the membrane potential in a patient with e.A. Also, the therapeutic effect of cation exchangers, such as acetazolamid (Creutzfeldt 1961, McArdle 1962) and chlorthiazide, as in our patient, is in favour of such a hypothesis. It seems to be ~,f special interest to compare our results with those obtained by Norris (1962) in myotonic patients. As mentioned above, he found with a low membrane potential of ,-63 :t=3 mV an "instability" ofthe muscle membrane, with a tendency to spontaneous discharges similar to the findings on d~nervated muscles (Li et al. !957, Ltillmann 1960). In our patient, when treated with chlorthiaz|de, we saw myotonic phenomena in the EMG. In some patients with e.A., even without any therapy, myotonic phenomena might be observed (McArdle 1962, Van der Meulen et ai. 19ol). it therefore seem~ to be acceptable to relate myotonia to e.A., at least Electroenceph. clin. Neurophystol., 1963, 15:508-S19
o . D . CREUTZPELDTet al.
SIB
pathogenetically, in assuming in both diseases an instability of the muscle membranes due to a disturbance of the sodium permeability. On the other hand, however, the two diseases should be kept separate because of their obvious clinical differences (Creutzfcldt 1961). SUMMARY
1. In ~i~o measurements of membrane potenrials of human muscles (M. vastus mad,; nerve block anaesthesia) have been performed in a healthy youngman, a patient with periodic paralysis and a patient with episodic Adynamia (e.A.)
Control measurements were performed in rats. 2. The resting potentials of striated muscles of rats under general anaesthesia were--79.9 7.3 inV. In the healthy man (without general anaesthesia) the mean value was --87.4 ± 8.9 mV and in the patient with periodic paralysis it was --8S.6 ~ 6.1 mV in a free interval. During the attempt to provoke a paralytic attack in this patient the average dropped to --77.1 ~: 8.2 mV, whereas the serum K + fell from 4.25 to 3.17 mequiv./i. The questionable significance of this slight drop of membrane potential is discussed.
3. In the patient with e,A. the resting potential, even in the free interval, (serum g*, 3,75 mequiv./I) was only ~68.5 ~ 5.9 mY, During an induced attack, with a ~'ise in serum potassium to 6,9 mcquiv,/I, the average value dropped to ~46.3 ~ 6.6mV and during aspontaneous attack, with a serum potassium of 5,24 mequiv,/l, the mean value was ~51.5 ~ 6.3 mY. 4. The significance of the results is discussed.
The pathogenetic mechanism of e.A. can be explained by a disturbance ofsodium permeabiliy of muscle membranes, P.~Um~ POTENTIELS DE MEMBRANES MUSCULAIRES DANS L'ADYNAMIE I~PISODIQtlE
2. Les potenticls de rapes des muscles stri6s chez le rat sous an~sth6sic g~n6rale 6taient de --79.9 -4-7.3 mV. Chez rhommc en bonnc sant~ (sans an6sth(~sie g6n6rale) la valeur moyenne (~tait de - 8 7 . 4 ± 8.9 mV et chez le malade attaint de paralysie familiale l~riodiquc ella ~tait de - - 8 5 6 ± 6.1 mV pendant un intervalle libra. Au cours d'une tentative pour provoquer une crise paralytique chcz ce malade, la moyenne est tomb(~e A --77.1-;- 8.2 mV, tandis qua le s~rum K +est tomb~ de 4.25 A 3.17 m~quiv./l. Une discussion porte sur le probl6me de savoir si ce 16ger abaissement du potentiel du membrane est significatif OU non.
3. Chez le malade atteint de e.A., le potentiel de repos, mi~me pendant I'intervalle libre (s~rum K + 3.75 m~quiv./l) ~tait seulement de 68.5 5.9 inV. Au cours d'une crise provoqu~e, pendant laquelle ie s~rum K +est mont6 ~ 6.9 m~quiv./l, la valeur moyenne est tombee/L 46.3 ± 6.6 mV, et au cours d'une crise spontan~e, ou le s6rum K + ~tait de 5.24 m~quiv./l, la valeur moyenne ~tait de 51.5 ± 6.3 inV. 4. La signification des r~sultats est cliscut~e. L¢ m~canisme pathog~nique de e.A. peut s'expliquer par un~ modification de ia perm~abilit~ au sodium des membranes musculaires.
We wish to acknowledg0 gratefully the help of Dr. David Sheldon, who patientlycarried out the operations, and of Dr. $, M. Chowdbury and Miss Diane Miller for technical assistance. The work has bee. supported by the MDAA. REFERENCES ADRIAN, R. H. The effect of internal and external potu. sium concentration on the n~..mbrane potential of frog muscle. J. P~siol. (Lo~.), 1956, 133: 631.~)58, ADRIAN, R, H. Potassium chloride movement and the membranepotentialof t~)lt muscle.J. P I ~ . (Load.), 1960, 131: I$~185. kNNS~r, A. L., WARn,F. J., DUNN,A. Land Mc INtYU, A R. The norms! membrane resting I~tentlal of mammalian skeletalmuscle measured/m rive. J. or//. temp./~s/o/,, 1953, 42: 343-$75,
I. Des mesures de potentials des membranes des muscles humains (M. vastus mad.; aries- BUCHTHAL, F,, ENO.EK, L. and GAI~i"Og,P, l, Soma u peels of the pathophysiolow or Adynemia episodka th~sie par bloeage des nerfs) ont ~t~ elTectu~es hereditaria. Dam.mad. hll., Ig~la, 3: 167-169. /~ ~/vo sur un j"eune homme en bonne sa~t~0 BUC~AL, F., ENanA~, L. and G~mTOap, ]. h r - , ~ sur un malade attei~,t de paralysie familiale and hyperexeitability in Adynamla episodica hereditaria. Neurology, 19~), 8: 347-351. p~odique, et sur un malade attaint d'adynamie ~pisodique (e,A,), Des mesures de controle ont DEL C,~TILLO, J. and K~,TZ, B. Local activityat a depolarized nerve muscle jansen. J. ~y~ol. (Lo~l.), ~q~ effectuates sur le rat. 19M, 128: 396-411. F./eetroeneepb. e//n. Newopbys/o/., 1963, 15:~8--519
MEMBRANE POTENTIALSOF HUMAN MUSCLE CREUT:eeLDT, O. Die epLsodische Adynamie (Adynamia episodica hereditaria Gamstorp), nine familiire hyperkalimische Ltihmung. Fortsckr. Neurol. PsycMat., 1961, 29: 529-550. CtX~UTZeelmT, O., A u o r r , B. und P~P.SON, C. Membranpotentiale in menschlichen Muskeln bei gesunden Personen und Patienten mit Adynamia episodica hereditaria und periodischer ~ u n g . Naturwissensctmften, 1962, 49: 284-285. ELMQULST,D., JOHNS, T. R. and TtlesxJeeF, S. A study of some electrophysiological properties of human intercostal muscle. J. Physiol. (Lond.), 1960, 154: 602-607. FRANK, K. and FUORTeS, M. G. F. Potentials recorded from the spinal cord with microelectrodes. J. Physiol. (Lond.), 1955, 130: 625-65t. GAMSTORP, !. Adynamia episodica hereditaria. Act. Paedlat. (Uppsala), 1956, suppl. 108: 1-126. HAKVEV,J. and ZI~aL~R, K. L. The production of very high extracellular potassium concentr~x~ion in the intact rat and its effect on muscle membrane potential. Physiologist, 19dJg, 1 : 3 5 HODGKIN, A. L. and HoRowlcz, P. The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Phytiol. (Lond.), 1959, 148: 127-160. JOHNS, R. J. Micro-electrode studies of muscle membrane potentials in man. Ass. Res. nerv. Dis. Pwc., 1960, 38:704-713.
519
KLFJN, Ft EGAN,T, and Using, P. Changes in sodium, potassium and water in hyperkalemic familial periodic pamPa'sis. Metabolism, 1960, 9: 1005-1025. IJ, C. L., SHY, G. M. and WILLS,J. B. Some properties of mammalian skeletal muscle fibres with particular reference to fibrillation potentials. J. Pkysiol. (Lend.), 1957, 133: 522-535. LOLLMANN,H. (Jber die Ursache spontaner Fibrillationen denorvierter Skelettmuskulatur. Kiin. Wsckr., 1960, 38: 1169-1170. McARDLS, B. Adynamia episodica hereditaria and its treatment. Brain, 1962, 85: 121-148. NASTUK, V. O. and HODGKIN,A. L. The electrical activity of single muscle fibres. J. cell. comp. Physiol., 19rJO. 33: 39-73. NORRISJR., F. B. Unstable membrane potential in human myotonic muscle. Electroenceph. clin. NeurophysioL, 1962. 14: 197-201. SHY, G. M., WANKE, H., ROWLEY, P. T. and ENGEL, A. G. Studies in familial periodic paralysis. Exp. Neuwl., 1961, 3: 53-121. TRAUTWEIN, W., ZINK, K. und KAYSEn, U. Ober Merebran- und Aktionspotentiale einzelner Fasern des Warmblttterskelettmuskeis und ihre Veranderun8 bei der Isch~imie.Pilfers Arch. 8es. Physiol., 1953, 237: 20-34. VAN DEarMEULEN,J. P., GICnEnT,G. J. and KANE,C. A. Familial hyperkalemic paralysis with myotonia. New Enfl. ]. Med., 1961, 264: I-6.
Reference: CREUTZFIBLlYr,O. D., ABBOTT,B. C., ]FOWLER,W. M. and PEarSON,C. M, Muscle membrane potentials in episodic adynamia. Electroencepk, olin. Neurophysiol., 1963, 15: 508-519.
ELECTROENCEPHALOGRAIPHYAND CLINICALNEUROPHYSIOLOGY
MUSCLE
MEMBRANE EPISODIC
POTENTIALS
IN
ADYNAMIA
OTTO D. CXEUTZFE~VT, BERNARVC. AeeoTr, WILLIAMM. FOWLEr. AND CARLM. PEARSONz Brain Research Institute and the Departments of Anatomy, Zoology and Medicine, University of California, Los Angeles, Calif. (U.S.A.) (Received for publication: August 17, 1962)
sia and the muscle was exposed for a length of about 5-7.5 cm. After retracting the skin and the subcutaneous fat, and after careful control of bleeding, the fascia externa was opened. Then the inner fascia was removed under microscopic control and the muscle tissue was covered with physiological saline solution. The area of this complete exposure of the muscle was about 1 x 4 cm. After the end of the measurements, which lasted for 30-60 rain, the wound was closed after microscopic exploration of the experimental field for broken electrode tips. in all instances the wounds healed primarily without any complications. 2. The membrane-potentials were measured relative to ground with glass micropipett~s, filled with 3 M KCI solution, The resistances of the electrodes were between 10.-50 Mfl and were monitored during the penetration (see below). The electrodes were positioned with a microdrive fixed rigidly on the table of an electric drill stand, which could be swung over the experimental field. The indifferent ground electrode was a chlorided silver plate wrapped in gauze, soaked with physiological saline, and placed on the skin about 10 cm from the experimental area. METIiODS 3. The recording equipment consisted of an 1. The measurements were performed on the Argonaut Negative Capacitance Electrometer right or left M. vastus reed., with the patient pre-amplifier and a Sanborn 320 pen writer, lying on a hard bed. A nerve block of the N. connected in parallel with a Tektronix oscillofemoralis was applied without general anaesthe. scope 502, This arrangement allowed simultat Present addresses: Dr. B. C. Abbott, Department neous observation and recording of the potenof Zoology, UCLA, Los Angeles 24, Calif. (U.S.A.). tials during the penetration of the micro-elecDr. O. D. Creutzfeldt, Max-Planck-lnstitut for i~y~ia- trode. The capacity of electrode and leads was trie, M0n~en 23, Germany. compensated by the negative capacity of the preDr. W. M. Fowler, Department of Physical Medicine, UCLA Medical School, Los Angeles 24, Calif. (U.S.A.). amplifier. During the experiment the impedance Dr. C. M. Pearson, Department of Medicine, UCLA of the micro-electrode was checked by measuring Medical School, Los Angeles 24, Calif. (U.S.A.). the voltage drop across the electrode which lntracellular measurements of membrane and action potentials of human and mammalian muscles have brought new interesting results concerning myotonia (Norris 1962), myasthenia (Elmquist et ai. 1960)and denervation fibrillation (Li eta/. 1957). They have failed, however, to explain the paralytic attacks of periodic paralysis (Shy et al. 1961). Clinical and electromyographic studies of patients with Episodic Adynamia (Gamstorp 1956) (called henceforth e.A.) have suggested that the paralytic attacks in this disease might be due to temporary depolarization of the muscle membranes (Buchthal et aL 1958 a, b; Creutzfeldt 1961; McArdle 1962). In order to evaluate more critically this hypothesis we have in this study measured intracellularly the muscle membr, ne potentials in a patient with e.A. during a normal interval, and during both a spontaneous and an experimentally induced attack. For comparison measurements have also been carried out in a normal male control and in a patient with periodic paralysis but during normal periods, as well as on anesthetized rats. A preliminary note of the results appeared elsewhere (Creutzfeldt el al. 1962).
F3eelmet~'¢l~. din. Nem'oi~iystol,, l ~ d , 15:508-$19
MEMBRANE POTENTIALS OF HUMAN MUSCLE
--i
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:+.~+I+
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+
.
~
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Fig. I Recording of membrane potentials. I : Record of an experiment in an anaesthetized rat. 2+ 3: Continuous record in a healthy young man (case 3), nerve block anaesthesia. Read from right to left. Before a: The electrode tip is within the saline solution covering the muscle and is slowly moved towards the muscle surface. Zero potential. In 2 some damalp~d eclls are penetrated before o. a: Penetration o~"the first fibre. The electrode is slowly advaneed deeper till h, penetrating several cells. Between b and ¢ it was removed. ¢: The electrode tip asain in the saline solution, zero chock. Time: 20 sec. Calibration as indicated (negativity upward).
resulted from a square.wave current injected into the input circuit provided by the Argonaut electrometer input stage. The zero potentials were balanced by a potential in the ground circuit. After zeroing the instruments with the electrode tip submerged in the saline covering the muscle surface, the electrode was introduced into the muscle under microscopic control. Ce!! penetration was visualized by a sudden rectangular change of the d.c. potential. The electrode was kept still for about half a minute and then moved forward. As it left the cell the potential fell back and the next cell was approached. The potential frequently did not reach the zero level when the electrode was between two fibres. A safe exp,~anation for this cannot be given on the
basis of our experiments. Possible reasons may be injury currents from destroyed fibres, incomplete localization of the electrode tip in the intracellular space, or slight changes of the tip potential of the electrode due to bending of the tip. During the whole procedure the direct recorder was kept running, the micrometer ronding~ ,were marked on the paper, and in addition the potentials were read on the oscilloscope. From 3 to 20 different muscle fibre~ down to a depth of about 2 mm could be penetrated before the electrode broke, due to strong bending of its tip or slight movements of the patient. Examples of recordings in a rat and in a human are given in Fig. 1. Materials. As mentioned above, the measure-
Electroenceph. din. Neurophyaiol., 1963, 1.5:508-519
510
o . D . CREUTZFELDT e l
ments were carried out on three persons. In order to check the apparatus and the reliability of the method, control experiments were first performed on five adult rats under general anaesthesia (Penthotai). Subject 1 (A.S.}. Diagnosis: Adynamia episodica hereditaria (e.A.) A 41 year old white married woman suffered from typical attacks of flaccid paralysis almost daily for 36 years. There was a strong family history of similar attacks. (The clinical and genealogical data of this patient will be published elsewhere). Within recent years one to three episodes occurred daily, mostly in the morning. Attacks could be elicited by restriction of food in the morning or by oral or intravenous KCI. An improvement of the attacks could be brought about by injection of Ca +~'. A remarkable reduction in the number and severity of the attacks has been noted during daily oral hydrochlorthiazide therapy. Single attacks began with strange feelingsin the stomach, followed by numbness around the mouth and later by increasing weakness of the limb and body musculature. If the patient had not been working or moving before the attack the weakness uniformly involved all muscles. If exercise preceded the attack the muscles mainly used were more severely involved. Frequently the respiratory muscles were involved, occasionally to such an extent that the patient became cyanotic. The tendon reflexes disappeared at the height of an attack, during complete paralysis. Except for the slight numbness in the face no sensory symptoms were observed. During the attacks the serum K +commonly increased by 2-3 mequiv./I, up to 6-7 mequiv./l. Corresponding electrocardiographic changes occurred in the Twaves (see Fig. 2). During the present investigation the validity of the attack was checked by frequent measurements of the hand grip strength with a dynamometer, by te.~ting the tendon reflexes, by EKG recordings and serum K" measurements at regular intervals. In this patient micro-electrode measurements were carried out during two different sessions. The first time the operation was begun at the onset of a spontaneous attack and the measurements were eatried out during the maximum period of weakness and rise in serum K + to 5.5 mequiv./l.The second time, measurements were made in the absence
aL 10"
9: @
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+.1-
E 3.
.too
e o
2.
3 Serum K in meclulV, Jl
Fig. 2 Amplitude of T-wave in mV, plotted against serum K + in patient A. S. (Adynamia episodica hereditaria). The symbols are measurements from different attacks: open circle: May 20, 1961; cross: May 27, 1961; closed circle: June I, 1961.
of weakness, after which an i.v. infusion of 40 mequiv, of KCi in 120 ml of 5 per cent dextrose was given during a 30 rain period. The serum K '~ rose from 3.82-6.90 mequiv, in this interval and a typical attack was produced. A second sample of potential measurements was then collected.
Subject 2 (J.P.), Diagnosis: Periodic'paralysis A 42 year old Mexican labourer who has had many typical attacks of flaccid paralysis since the age of 18 years. These especially occur in the early morning hours after heavy work and a late supper on the previous day. Episodes of weakness last from 2 h to 6 days, with an average duration of 2 days. He can abort an attack by exercise when the early signs appear, He has had an average of five attacks per year, There is a family history of similar episodes in two brothers, a p s 44 and 46 years, Examination revealed normal musculature, good strength and normal reflexes. The patient was hospitalized and an attempt was made to induce an attack by heavy exercise and a late meal on the night before intra-fibr¢ electrode studies were planned. However, ~;~ following morning he showed only minimal weakness of the pelvic flexors at a time when serum K+ was 4,25 mequiv./l and Na + 152.6 mequtv./l, lntra-fibre electrode studies
Electroes~'el~.eli& Nem'olOtysiol.,1~1, 15:50~519
MEMBRANE POTENTIALSOF HUMAN MUSCLE
511
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i
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"6
7
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9
10
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13
14
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"/
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Fig. 3 Membrane potentials of three different rats (muse. soleus, generalanacsthesia). Ordinate: Membrane potential in mV. Abscissa: Succession of impalements of consecutive mu~le ~lls durin$ the penetration with one electrode. Measurements carried out with one electrode are connected with a line, the consecutive penetrations with diff©rcntmicro.electrodes are labeled at each line, Further explanation, ~,~ melhod.~.
were performed in the right quadriceps femoris muscle after preliminary femoral nerve block. Then 390 ml of 30 per cent glucose, which con° tained 34 units of regular insulin, was rapidly infused intravenously over a 40 rain period, after which strength had declined moderately, so that the heel of the extended leg could be raised only 25 cm from the bed. At this time the serum K ÷ was 3.17 mequiv./l and the Na + was 137.4 mequiv./I. A series of repeat membrane potential studies was done at this time. The patient recovered rapidly from the mild weakness and had normal strength 4 h later. Hence, a frank episode of periodic paralysis was pot produced on th~s occasion. ": ~. Subject 3. A healthy 24 year old student volunteered for this investigation. In him, data were collected during one session, In the tables and the description of the results
the mean values with the standard deviations for each experiment have been listed. For statistical comparison of the means in different experiments the calculation paid regard to the gradient of values from "superficial" to "deep" measurements. The special statistical method which was used for these calculations has been developed by Dr. W. J. Dixon, Department of Biostatistics, UCLA Medical School, Los Angeles, Calif., whose help we wish to acknowledge gratefully. In the diagramq of Fig. 3-5 the values of the membrane potential are on the ordinate while the abscissa represents the sequence of readings during the penetration with one micro-electrode. Each line connects the figures obtained during one penetration. However, the sequential numbers of penetrations do not give the exact figure of the depth within the muscle from which the Eleetroenceph. olin. NcurophysM.. 196.3, !$: 508-519
O. D. CREUTZFELDT el a[.
512
TABLE i Membrane potentials in three rats and three human beings. Further explanations, see text. Subject
Date
Series
Number of measuremen~
Condition
Mean value with standard deviation
A June 16, 61 Oct. 28, 61 Oct. 29, 61
Rat Rat Rat
l 2 3
General anaesthesia General anaesthesia General anaesth~i~
5 35 42
---77.6-1-0.9 mV I 79.9 -I---77.4 ~ 7 mV --81.8 -t-8.3 mV 7.3 mV
B Oct, 30, 61
Healthy man
1
Nerve block
26
--87.4 ~ 8.9 mV
C Oct. 31, 61 9.30 am. Oct. 31, 61 i0.30 a.m.
Pat. P. S. with periodic paralysis
l
Nerve block; free interval Nerve block; after insulin
23
--85.6 ~6.1 mV
27
--77.1 28.2 mV
2
J
+ glucose D May 6, 61 9 a,m.
Pat. A.S. with episodic Adynamia
I
Nerve block; free interval
21
--68.5 ~ 5.9 mV
May 6, 61 10. a.m.
2
15
--46.3 2~6.6 mV
Juno I, 61
3
Nerve block durin8 an induced attack Nel~e block; durin8 a spontaneaus attack
15
--51.5 ~6.3 mV
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Fig.4 Membrane potentialsof two differenthumans, a: Healthy youns man. b: Control in patientJ. P. with periodicparalysis,during a free interval (serum K + -----4.25 mequiv./l).¢: The same patient
! h later atitr infusion of 39.0nil 81ucose(30%) and 34 u regular insulin (s~rum K ~ - 3,17 mixluiv.ll).
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SUCCeSsion o f impal@mont~
Fig. $
Membran• potentialsin patient A. S. with Adynamla®pisodicahercditaria,a: Controlduring a frge interval(~rum K+ =~ 3.75 mequiv./I),b: A half hour later, during an attack indugedby infusionof 40 mequiv. KC! in 120 ml 5 per gent dextrose (serum K+ =~ 6.9 m~uiv.li), c: Moasurements during a spontaneousattack, carriedout one week beforea and b on the other leg (sorum K+ ~ 5.24 m~lulv./I). recordings were made. Usually, a membrane potential stayed almost unchanged for about 80-130/~ of micrometer movement (i.e. presence of the electrode tip within a single fibre), whereas it stayed omside for 30-50/~. Th©s¢ r©lativ¢ly high values, which are larger than one would expect considering the actual fibre diameters and intercellular spaces, may possibly be due to slight displacements of fibres by the micro-electrode. Sometimes, the electrode went quickly through a number of fibres without enabling us to read the potential. Also, the electrode sometimes jumped through the first two or three
superficial muscle cells because of the elastic resistance of the muscle sheath. For these reasons the numbers of the readings do not give a direct, but only an approximate, indication of the depth of recording. RESULTS
Fig. 3 shows the membrane potentials obtained in three different rats under general anaesthesia (Pentothai i.p.). It is obvious that the surface readings are almost always lower than the depth readings. This is probably due to the relatively quick cooling and drying ofthe muscle £1eetroe..neeph. din. NeurophydoL, 1963, 1.5:508--519
o.D. CREUTZFELDT et al.
514
surface. The mean values arc given with standard errors in Table 1. in Fig. 4 are shown the results obtained on Subjects 2 and 3: the curves (a) represent the measurements in the healthy young student, while the ones marked (b) show the control measurements in the patient with periodic paralysis. Both figures lie closely together, theaveragebeing nearly thesame(--87.4 ~ 8.9 mV and--85.6 =[=6.1 mV). The third group of curves (c) are measurements in the patient with periodic paralysis during the attempt to induce an attack. During these penetrations the serum K + was 3.17 mequiv./I having been at 4.25 mequiv./I during the control measurements. Even though the average in the second series (--77.1 ~ 8.2 mV) is significantly lower ( p < 0.01) than in the first the slope of the curves shows clearly that this is due at least partly to the low values in the surface layers. Because the second set of measurements was madeabout ! h after the first, during which time an attempt was made to induce an attack, it is conceivable that the lowering of the membrane potentials in the surface layers may be partly due to injury currents from the superficial fibres (see below). Fig. 5 gives the results obtained in A.S., the patient with e.A. The curves (a) show the measurements taken during a free in.
terml with a serum K + of 3.75 mequiv./l. All figures are below --80 mV, the average being --68.5+ 5.9 mV. This average is considerably lower than the figures obtained in the two control subjects. As will be discussed later this low value can be considered as real and cannot be explained simply by the fact that we were not successful in this patient in collecting many data from deeper ceils,During an induced attack (b) with a serum K ÷ of 6.9 mequiv,/I the membrane potentials were markedly lowered to an average of--46.3 ±6.6 mV and the single figures never reached the lowest values of the control series (a). Accordingly, the difference of the average values is significant ( p < 0.01) in spite of the large distribution during the induced attack. The values collected during a spontaneous attack, with a serum K ÷ of 5.24 mequiv./I, are represented by the lines (c). Here, too, the potentials are remarkably lowered, the average --51.5 ~6~3 mV being again significantly ( p < 0.01) different from the control. The mean values of the induced and the spontaneous attacks, however, are not significantly different. In comparison with other measurements the relatively wide distribution of the values during the attacks should be mentioned.
TABLE I! Membrane potentials in human ,iuscles. Results of differentauthors. Muscle
Preparation
Df~ase
Number of measurement
M. intercostalis
Excised.in
Myasthenia
15
~80.9 ± 1.0 mVxS.E.) Elmq'uist,Johns, T h e f t (1960)
Periodic I~ralysis
82
--71.2 ~ 11.6mV($.D.) Shy,Wanke, Rowley,Easel (1961)
4 healthy
40
~77.8 ± 153 mV
--$7.4 :]=8.9mV($.D.) Creutzfeklt, Abbott, Fowler, Pearson (1962) --86.6 ± 7.7 mV !
vitro
Not known
In situ, anaesthesia not known
M. Bastrocnemius In sltu. and quadric~ps local femoris anaesthesia
Membranepotential
Author
Johns (1961)
men
M. VaStUS rood.
In stlU, nerve block anauthesia
Healthy young man
26
med.
nerve block anaesthesia
Periodic paralysis (free interval)
23
--8&6 d:6.1 mV; (S.D.)
F_./ecu~oe~e~,elat. Newo.~y~'o/.,1963, I$: SO8.519
MEMBRANE POTENTIALS OF HUMAN MUSCLE
515
closely together that it is not probable that different tip potentials really had a great inWe shall discuss briefly two points. The first fluence. It may, however, be possible that the concerns the reliability of the measurements; gradients from superficial to "deep" values may the second some pathogenetic considerations have been caused by changes of the tip potentials concerning e.A. during the penetration of the electrode into !. The mean valuesandthedistribution(standdeeper layers of the muscle. This possibility has ard deviation) found in rats, as well as in LNe been noted by different authors (Nastuk et al. human control subjects, are in good agreement 1950; Del Castillo et al. 1955; Frank et al. 1955). with those found by other authors. In rats values The lower values in the superficial cells might of --72.2 H 5.3 mV (Li et al. 1957) and --88.9 also be explained by their injury. In spite] of ±8.06 mV (Harvey e t a i . 1958) were reported, these minor uncertainties the good agreement of whereas our own average was --79.9 ± 7.3 mV, the control measurements with those reported with averages in different experiments from in the literature, as Nell as the relatively narrow m77.6 to --81.8 mV (Table 1). In other species standard deviations of the means, justifies their higher and lower values were reported (--99.8 acceptance as controls and their comparison ±6.5 in mice, Bennett et aL 1953; --79.5 ± 5.7 with other values of our series. Furthermore, in cats, Trautwein et aL 1953; and --84,5 ± 5.7 the statistical comparion of the values paid in Guinea pigs, Trautwein et al. 1953). Our values regard to the gradient from superficiel to deep obtained in men are higher than those reported measurements. by other authors (Tsble !i). The membrane potential in the patient with There are various possibilities to explain the periodic paralysis was slightly reduced during slight differences between different authors. One the effort to induce an attack (--77.1 ± 8.2 mV is the selection of values, as there are some in- compared with --85.6 ± 6.1 mV), and just vestigators who reject extremely low values, as- significantly different from the control figure suming that they may be due to injury of the ( p < 0.01). Even though no conclusive deducmembranes. This might explain the relatively tions can be drawn from this slight decrease, low values obtained in rats, compared with the especially because the readings were mainly higher values reported by some authors quoted limited to the superficial ceils, it should be noted above. If, in our experiments, the electrode re- that Shy et oL (1961) also show in their figures a sistances were in a satisfactory range (10-50 ML~) slight, and probably not significant, decrease we have not rejected any values, in order to during a full paresis. From the diagrams of avoid any bias in the later comparison. Another Shy et al. (with rounded figures) we estimated an factor may be the different state of the animals average membrane potential of--75,4 mV during or the human subjects. We do not know to the free interval and of --70.5 mV during the what extent light anaesthesia plays a role. Our paresis. Therefore, a slight decrease of the memexperiments have been performed in unanes- brane potential during the attack in periodic thetized subjects. The preparation and the open- paralysis might still be suspected, and further ing procedure were kept as constant as possible measurements under bet~:r conditions should be in order to avoid too greet an error from this. carried out, In the patient with e.A. even the values during One factor, however, which has not been fully excluded in our experiments was the tip poten- the free interval were very low (168.$ ± 5.9 tial of the micro-electrode, whose importance mY). The difference from the normal control has been pointed out by Adrian (1956). We is significant ( p < 0.01). During the spontaneous, tried to reduce this disturbing factor by balancing as well as during the induced, attack the memthe zero potential at the beginning of each pene- brane potential showed a further decrease (Fig. 5 tration by a compensating potential in the ground and 6). This decrease is significantly different circuit (Methods). ~ihe actual values obtained (p<~ 0.01) from the values during the interval, with different electrodes in the same subject though there is no significant difference between under identical conditions, therefore, lay so the values during the induced and during the Electroenceph. din. Neuropkyslol., 1963, 15:508-$19 DISCUSSION
o . D . CREUTZFELDT et al.
516 - 100-
~
""~E=~.,g [K*]o
-90-.
_c - ~ 0 - ~
o-?O ~
~-30I~ -~0
3.75 5 2 4 6 9
0'1
I
"
2
1
-"
I I J Ill"'",,
3
4 ~ t5 7 8 9 1 0
[g*]Oin mequtv I'
Fig. 6 Membrane potential in mV (ordinate) plotted against external [K+! (abscissa, logarithmic scale). Pat. A. S. with e.A. Averages of measured membrane potentials with standard errors. The numbers above the abscissa show the serum [K +] for the different measurements. The broken line at the top shows the calculated curve of the K + potential for a IK+], of 150 mequiv./I. .eo.,~. K
healthy patients, and even the "deep" values (i.e., the ones obtained from deeper cells) are lower than the corresponding values in the controls. It can be concluded, therefore, that the low membrane potentials in patient A.S., even in the free interval, are real. 2. In spite of these results the pathogenetic mechanism of the low membrane potential and of the paralytic attacks in e.A. still remains somewhat obscure. The serum K ÷ level rises almost twofold during an attack, so that a fall in membrane potential would be expected. But neither the initial low "control" value in the patient nor the further decrease during the attack can be reasonably explained in terms of serum K +. The average level of intracellular K ~ in normal subjects is about 150 mequiv./I of fibre water. In Fig. 6 the dashed line represents the variation of membrane potential with extracellular K + level (Ko) predicted by a simple Nernst equation which states that RT [K]o Membrane Potential ......... log [K]i"
o8C o ~70 £2
~4C
°--~'~q~:" =I~ Ii IL ;~
3
+-~,, ,,HI
,4 b ¢ ) ] 8 g
111~t1~
[*~'],,n r'~u*vl* Fig, 7 Membrane potential (ordinate) plotted a$1inst intra~l. lubr [ g q (abscissa). The lines represent calculated rein. tions for thr~ different external potassium coneentra. tions. These [g+]o are values of the serum K* during the different measurements in patient A.S. with o.A. On these calculated curves the corresponding measured mere. brane potentials (averages) are marked with circles, in order to show the corresponding [K,], the circles are connected by vertical lines to the abscissa. Further explanation see text.
spontaneous attack. The low values during the free interval and during the spontaneous attack were obtained from the beginning of the experi. ment, when cooling and drying cannot have been considerable. Furthermore, in spite of the relatively wide distribution, almost all single values arc lower than the ones obtained in the
The actual values in our patient are obviously so much below the theoretical values, under the assumption of RT/F ~, 60 and [K], ~ 150, that some other factors must be involved, Furthermore, it is not possible to explain the low control values or the further lowering of the potential simply by a decrease of the internal K ~ concentration, though several authors have found a loss of intracellular K ~' during e.A. attacks (Buchthal et al. 1958; Klein et al. 1960; Creutzfeldt 1961: McArdle 1962). The expected values of intracellular K+ concentration with different membrane potentials are shown in Fig. 7 for the three measured serum levels in our patient, The points marked with circles are the actual membrane potentials measured. As can be seen it would be necessary to assume a "normal" [Ki] of 51 mequiv./I (instead of 150) and during the attacks a further decrease to41 or 38 mequb,./I. However, direct measurements of the intramuscular K ' concentration in other cases of e.A. have shown normal values in the free interval (Buchthal et al. 1859; Creutzfddt 1961; Klein et al. 1960). Furthermore, a quantitative calculation of the intracellular/extracellular K + balance shows that the serum K + would have to increase Eleetroe.,~eepk. ¢lin. NeL,,~l~y$1ol., 1963, 15:Y~8-319
MEMBRANE POTENTIALS OF HUMAN MUSCLE
by far more than it really does if one assumed merely a muscalar K + loss as the cause of the attack and the observed decrease of the membrane potential (Creutzfeldt 1961). it is therefore necessary to look at the problem from a more dynamic point e l view. This is possible by taking into account some recent observations on cation permeability of muscle membranes under different conditions of membrane polarization (Hodgkin et al. 1959, Adrian 1960) and assuming a slight change in Na + permeability of muscle membranes in e.A.: the value of membr~,ne potential can be expressed as RT M.P. --=-~
[Klo + ~x INalo In [Kh + ~ [Nah
where the subcripts i and o represent intra- and extracellular ionic concentrations and ,', is the ratio of sodium to potassium permeability in the membrane (Hodgkin and Horowicz 1959). in frogs .~ is about 0.01 according to Hodgkin e¢ al. Applied to normal human muscle we would come to the following: with a serum K ~ level of 3.75 mequiv./I and an intracellular level of 150 mequiv./I of fibre water, the K ' equilibrium potential would be about ~ 9 8 inV. If in the normal human muscle, a value for ~x ..... 0.01 is assumed, then with K I ........ ! 50, Ko ........3.75, Nao 150, Nat =:: 20, a potential would be predicted of ~ ~ mV, which is close to the measured value in normal control subjects. If, in the e.A. patient, a disorder of the sodium exchange exists even in the aormal quiescent state, such that the permeability to sodium is increased only three fold to give a ratio of sodium to potassium permeability of 0.03, then a membrane potential of ~ 7 5 mV would be predicted. This can be compared with the mean value o f - - 6 9 mV in the patient. During a paralytic attack the serum potassium increased to 5.24 mequiv./I while the membrane potential fell to ~51.5 mV with loss of excitability. The increased serum potassium must come from within the cells, but will not noticeably alter the intracellular [K] ~ (of about 150 mequiv./! of fibre water). But it will depolarize the membrane further and may induce a further increase in sodium permeability. If there is a further three fold increase, to ~, = 0.1, with serum K ÷ at 5.24, then the membrane potential should be -.51.1 inV. Thus the events could
517
be explained by a change in the permeability of the muscle membranes to sodium at rest, which would result in a relatively unstable system. Any further increase of the serum K* level, such as would occur in the morning hours or after ingestion of a relatively l~rge amount of K + or a loss of muscular K ÷ after exercise, cold shivering etc., could produce a cumulative effect of depolarisation, further increase in sodium permeability, and further depolarisation. Such a series of events might tend to produce regenerative activity and thus might account for the observations of some authors of fibrillation potentials (Buchthal et al. 1958 a, b: Creutzfeldt 1961) and eveu increased mechanical excit,tbility similar to myotonic phenomena (McArdle 1962) in the beginning of an attack. When further depoiarisation occurs sodium conductance increases, and inactivation occurs such that the tendency to produce regenerative activity disappears. There is as yet no immediate proof that depolarisation is due to irregular sodium permeability, and a similar effect might be produced by a permeability change to certain intramuscular anions, as suggested by Buchthal er al. (1958 a, b). However, the fact that an attack could be very rapidly alleviated by intravenous injection of calcium supports the sodium hypothesis, although there has been no opportunity to study directly the effect of increased calcium on the membrane potential in a patient with e.A. Also, the therapeutic effect of cation exchangers, such as acetazolamid (Creutzfeldt 1961, McArdle 1962) and chlorthiazide, as in our patient, is in favour of such a hypothesis. It seems to be ~,f special interest to compare our results with those obtained by Norris (1962) in myotonic patients. As mentioned above, he found with a low membrane potential of ,-63 :t=3 mV an "instability" ofthe muscle membrane, with a tendency to spontaneous discharges similar to the findings on d~nervated muscles (Li et al. !957, Ltillmann 1960). In our patient, when treated with chlorthiaz|de, we saw myotonic phenomena in the EMG. In some patients with e.A., even without any therapy, myotonic phenomena might be observed (McArdle 1962, Van der Meulen et ai. 19ol). it therefore seem~ to be acceptable to relate myotonia to e.A., at least Electroenceph. clin. Neurophystol., 1963, 15:508-S19
o . D . CREUTZPELDTet al.
SIB
pathogenetically, in assuming in both diseases an instability of the muscle membranes due to a disturbance of the sodium permeability. On the other hand, however, the two diseases should be kept separate because of their obvious clinical differences (Creutzfcldt 1961). SUMMARY
1. In ~i~o measurements of membrane potenrials of human muscles (M. vastus mad,; nerve block anaesthesia) have been performed in a healthy youngman, a patient with periodic paralysis and a patient with episodic Adynamia (e.A.)
Control measurements were performed in rats. 2. The resting potentials of striated muscles of rats under general anaesthesia were--79.9 7.3 inV. In the healthy man (without general anaesthesia) the mean value was --87.4 ± 8.9 mV and in the patient with periodic paralysis it was --8S.6 ~ 6.1 mV in a free interval. During the attempt to provoke a paralytic attack in this patient the average dropped to --77.1 ~: 8.2 mV, whereas the serum K + fell from 4.25 to 3.17 mequiv./i. The questionable significance of this slight drop of membrane potential is discussed.
3. In the patient with e,A. the resting potential, even in the free interval, (serum g*, 3,75 mequiv./I) was only ~68.5 ~ 5.9 mY, During an induced attack, with a ~'ise in serum potassium to 6,9 mcquiv,/I, the average value dropped to ~46.3 ~ 6.6mV and during aspontaneous attack, with a serum potassium of 5,24 mequiv,/l, the mean value was ~51.5 ~ 6.3 mY. 4. The significance of the results is discussed.
The pathogenetic mechanism of e.A. can be explained by a disturbance ofsodium permeabiliy of muscle membranes, P.~Um~ POTENTIELS DE MEMBRANES MUSCULAIRES DANS L'ADYNAMIE I~PISODIQtlE
2. Les potenticls de rapes des muscles stri6s chez le rat sous an~sth6sic g~n6rale 6taient de --79.9 -4-7.3 mV. Chez rhommc en bonnc sant~ (sans an6sth(~sie g6n6rale) la valeur moyenne (~tait de - 8 7 . 4 ± 8.9 mV et chez le malade attaint de paralysie familiale l~riodiquc ella ~tait de - - 8 5 6 ± 6.1 mV pendant un intervalle libra. Au cours d'une tentative pour provoquer une crise paralytique chcz ce malade, la moyenne est tomb(~e A --77.1-;- 8.2 mV, tandis qua le s~rum K +est tomb~ de 4.25 A 3.17 m~quiv./l. Une discussion porte sur le probl6me de savoir si ce 16ger abaissement du potentiel du membrane est significatif OU non.
3. Chez le malade atteint de e.A., le potentiel de repos, mi~me pendant I'intervalle libre (s~rum K + 3.75 m~quiv./l) ~tait seulement de 68.5 5.9 inV. Au cours d'une crise provoqu~e, pendant laquelle ie s~rum K +est mont6 ~ 6.9 m~quiv./l, la valeur moyenne est tombee/L 46.3 ± 6.6 mV, et au cours d'une crise spontan~e, ou le s6rum K + ~tait de 5.24 m~quiv./l, la valeur moyenne ~tait de 51.5 ± 6.3 inV. 4. La signification des r~sultats est cliscut~e. L¢ m~canisme pathog~nique de e.A. peut s'expliquer par un~ modification de ia perm~abilit~ au sodium des membranes musculaires.
We wish to acknowledg0 gratefully the help of Dr. David Sheldon, who patientlycarried out the operations, and of Dr. $, M. Chowdbury and Miss Diane Miller for technical assistance. The work has bee. supported by the MDAA. REFERENCES ADRIAN, R. H. The effect of internal and external potu. sium concentration on the n~..mbrane potential of frog muscle. J. P~siol. (Lo~.), 1956, 133: 631.~)58, ADRIAN, R, H. Potassium chloride movement and the membranepotentialof t~)lt muscle.J. P I ~ . (Load.), 1960, 131: I$~185. kNNS~r, A. L., WARn,F. J., DUNN,A. Land Mc INtYU, A R. The norms! membrane resting I~tentlal of mammalian skeletalmuscle measured/m rive. J. or//. temp./~s/o/,, 1953, 42: 343-$75,
I. Des mesures de potentials des membranes des muscles humains (M. vastus mad.; aries- BUCHTHAL, F,, ENO.EK, L. and GAI~i"Og,P, l, Soma u peels of the pathophysiolow or Adynemia episodka th~sie par bloeage des nerfs) ont ~t~ elTectu~es hereditaria. Dam.mad. hll., Ig~la, 3: 167-169. /~ ~/vo sur un j"eune homme en bonne sa~t~0 BUC~AL, F., ENanA~, L. and G~mTOap, ]. h r - , ~ sur un malade attei~,t de paralysie familiale and hyperexeitability in Adynamla episodica hereditaria. Neurology, 19~), 8: 347-351. p~odique, et sur un malade attaint d'adynamie ~pisodique (e,A,), Des mesures de controle ont DEL C,~TILLO, J. and K~,TZ, B. Local activityat a depolarized nerve muscle jansen. J. ~y~ol. (Lo~l.), ~q~ effectuates sur le rat. 19M, 128: 396-411. F./eetroeneepb. e//n. Newopbys/o/., 1963, 15:~8--519
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KLFJN, Ft EGAN,T, and Using, P. Changes in sodium, potassium and water in hyperkalemic familial periodic pamPa'sis. Metabolism, 1960, 9: 1005-1025. IJ, C. L., SHY, G. M. and WILLS,J. B. Some properties of mammalian skeletal muscle fibres with particular reference to fibrillation potentials. J. Pkysiol. (Lend.), 1957, 133: 522-535. LOLLMANN,H. (Jber die Ursache spontaner Fibrillationen denorvierter Skelettmuskulatur. Kiin. Wsckr., 1960, 38: 1169-1170. McARDLS, B. Adynamia episodica hereditaria and its treatment. Brain, 1962, 85: 121-148. NASTUK, V. O. and HODGKIN,A. L. The electrical activity of single muscle fibres. J. cell. comp. Physiol., 19rJO. 33: 39-73. NORRISJR., F. B. Unstable membrane potential in human myotonic muscle. Electroenceph. clin. NeurophysioL, 1962. 14: 197-201. SHY, G. M., WANKE, H., ROWLEY, P. T. and ENGEL, A. G. Studies in familial periodic paralysis. Exp. Neuwl., 1961, 3: 53-121. TRAUTWEIN, W., ZINK, K. und KAYSEn, U. Ober Merebran- und Aktionspotentiale einzelner Fasern des Warmblttterskelettmuskeis und ihre Veranderun8 bei der Isch~imie.Pilfers Arch. 8es. Physiol., 1953, 237: 20-34. VAN DEarMEULEN,J. P., GICnEnT,G. J. and KANE,C. A. Familial hyperkalemic paralysis with myotonia. New Enfl. ]. Med., 1961, 264: I-6.
Reference: CREUTZFIBLlYr,O. D., ABBOTT,B. C., ]FOWLER,W. M. and PEarSON,C. M, Muscle membrane potentials in episodic adynamia. Electroencepk, olin. Neurophysiol., 1963, 15: 508-519.