Document 6Rvyvvb9bOaQVa0q71BKqk8Od

! '?6'\tnr^ .&*}* . fCZS' (ve*J&eLCGf..'t vbiaitt 'Hi- Hi-Kcjity j -- t -: - I! l ead s t u d ies -' u; .... -- - . ... - _. XIV. . .EXl'F.!ilME>-.TA!. -STUDIES OF LE.\1> .FAUST * . I | '! j ;" j' | . .1 * - * _ \ PAUL KRZNIKOI'F,. MX). '' ' Rcscurclt Fellow, NatJouat Research Council ; -..... . '^- KD * " JOSEPH -C.. AUB, ;M.D. r ........... _ . - wbSToit . 1 , ,, . . - |' i ' _ i :' . _ j 1i j | j ;. ;: ' The mechanism by" which lead produces paralysis has not been studied thoroughly. Those investigations which have been reported have been almost entirely of a clinical and patijologic nature. Recent 5 contributions to the chemistry-and physiology ofiiHisCulaf contraction1 and of the behavior of lead within the organism-have suggested new methods for further study Uf the locution ;ind mechanism of the action of lead on nerve and muscle tissue, and the experiments described in , this, paper -are the .result of this newer knowledge.- .... - j.: . !; - . .' . . . UTEKATUKIi .- - : -Among the older investigators, Gtisscrow ' was the first to obtain : - evidence suggestive of the direct" action of lead on tmtscle. He reported [: Ithat'relatively large amounts of lead" may be lecoveied from the muscles ; , "h of fatbits with ,fcad poisoning, but his. observation vv.re questioned "by - | Heutei r and has never becii' confirmed. These experiments, however, ' ; : | - : ) 1i -_ t " , " ' "-. -- " _ -_ - I `From the Laboratory of Physiology! Harvard School of Public Health. i 1. Fletcher, IV. M.: The Survhal- Respiration of Muscle, T. Physiol. 23: _ 10. 1898-1899; The-Relation of Oxygen to the Survival Metabolism of Muscle, ibid. 28:474, 1902: The Influence of Oxygen on the Survival Respiration of Muscle, ibid. 28:554, 1902: The Osmotic Propert'cs'of Muscle and the Modi- freations in Fatigue and Rigor, ibid. 30:414, 190.1-1904; Lactic Acid Formation, Survival Respiration and Rigor Mortis in Mammalian Muscle, ibid. 47:301, 1913-1914. Fletcher, W. M., and Hopkins, Y. G: Lactic Acid in Amphibian Muscle, J. Physiol. 85:247, 1900-1907. Hopkins; F. G.: The Chemical Dynamics - of Muscle,'Dull. Johns Hopkins Hosp 32:359 (Nov.) 1921. Hill, A. V,: The Mechanism of Muscular Contraction, Physiol. Rev. 2:110, 1922. Meyerhof?, O.: Die EnerEiewandlungcn im Musket: I-VI. Arch. f. d. gcs. Physiol. IS2:232 and 2S4, 1920: 185:11, 188:114. 191.-12S. 1921; 195:22, 1922. En.bdcn, G., and Laqner, F.: Ucbcr die Chcmic des Lactae'idogcns, Ztschr. physiol. Chan. 93: 1S1, and 113:1.1916-1917. Embdcn, G., and Adler, E.: Ucbcr die physiologische Eedcutung des Weschwls ties Fermcnhilitiitsaustandcs- von Muskelfascrgrcm- schichten, Zuclir. f. Physiol. Chem. 118:1, 1922. 2, Gusser.uv, A.: Vntersnchuiigr-n neber Blcivcrgiftung, Arch. f. path. Anat. 21:443, 1861. ' 3. Fleubel, E.: Pathi'icem sc tin.l Symptomv der chroniscbcn Blcivergiftting, Berlin. A. Hirsclnvald, 1871. . I ! i ! . itII ! -\j Iii i i . FV .I j I -i i ! i ii ' ! j i j * REZSIKOFF-AL'B--LEAD STID1ES . 445 . drew attention to the"possibility that lead might affect muscular activity.- Muson 1 is reported to have immersed frogs in water containing lead and to have produced palsy -of -the hind limbs, as far as could be judged from voluntary effort and reflex response. Harnack* injected lead triethyl acetate into the dorsal sac of frogs-and observed rapid exhaus tion of muscular contractility and, in a short lime, complete loss of- excitability. He thought that muscle rather than nerve or nerve-ending was affected because direct stimulation of muscle evoked no stronger response than did stimulation through the "nerve, and also because similar changes in muscular activity could be obtained with curarized frogs after injection of lead trielhyl acetate. Since ihe toxicity of this compound depends not only on lead, but also on the triethvl group, these experiments cannot be. taken ds criteria of theraction of lead on muscular tissue. - One of the phenomena observed by flaruack in the muscles that he considered poisoned with lead was delay in relaxation after stimulation. The same retardation of relaxation was seen by Cash,* who. treated - frogs with lead acetate. - However, his work was entirely uncontrolled and most of his positive results were obtained, at.a warm temperature , which in itself may affect the activity of- muscles from cold-blooded animals. In well controlled experiments on frogs, Mellon' determined.: flic effect of lead on fatigued muscle. To fatigue the gastrocnemius of one side, lie stimulated it for several days ami then injected lead acetate - inlo the dosal sac.- After excision, the previously fatigued muscle was - able to perform much less work than that of. the oilier side. All these investigators suggested that lead produces a definite physiologic lesion - in muscle tissue. ~ - = . ........ Dozzi *. raised the question whether lead might not affect isolated nerve as well as muscle. He used both nerve-muscle preparations (sciatic-gaslrocnemius) from the. same frog-and immersed one nerve in physiologic sodium chloride solution and-the other in isotonic lead _ acetate solution. In the nerve exposed to-lcad, this excitability, as judged - hv muscular responsc-to minimal nerve stimulation, djraiiiis.ijcil lnirkcdly : " and finally disappeared completely. These experiments, however,"can _ hardly he considered of much value because., the lend solution was so . 4. Muson, J. Jf.: I.c.-.c! Poisoning in Frogs, New York M. J., jiily, 1877;. . <pr-icrl in Centra!!)!, f.- <1. mcd. \Yisscnsch. 16:480, 1878. - -. " 5. TTarnack. E.: _ Ucber die Wirfcurgen ties Elgis aufden tierischcn, OrgniitMnns. Arch. f. esprr. Path. u. Pharm. S: 152,-1878. . , . ... . fi. Cash. J. T.: The Contraction of Frog's.1 Muscle After- Administration T.i-a<l. Arch. f. expt-r. Path. u. Phar.n. 59: (siipp. vcd.); 53:106,- 1908. . .. - ' 7. Mcllnn, R. R".: "The Relation of Fatigue to" Paralysis Localization in' l,hnu,..;,ni, -Arch. Jut. Mcd. 12:399 {Oct.} -1913 ' - . . - : . 8. Pozzl, T..: Sn.u'i sulVazionc dc! piombo; : azsone dc! pioinhn sulia . Tta dti nervs motnri. Spcrinirntali 60:666, 1912.- ' ' :. - 446 ARCIIll'P.S OP -x e u r o l o g y Ax d p s y c h ia t r y ' concentrated (27 mg.. of .lead. 'per cubic centimeter) ; 'llie- acidity, _ ^ although .not mentioned by.-Dozzi, must necessarily have been greater - than is physiologic, ami. finally, tile lead solution-contained nu balancing ~ "ions. This "summary indicates that further knowledge-of the action ' of lead on the function of the neuromuscular system is needed. Some " of the .investigations in -this laboratory liavc suggested a means of. . studying the subject further. In experiments dealing'with the effect of lead on red blood celts,8 it was found that lead alters the surface of the- - corpuscle so that its permeability to water is changed. The question , " arises, therefore, as to whether lead may not act similarly on the surface - _ -of muscle and thus change its permeability. Various workers have _ - ' 'studied the passage of substances through the.surface of muscle in both - - directions under different conditions, and before describing our experi- - " ' - - meats it may.be well to review' briefly some of their results.- Fletcher,10 _ who studied the effect-of fatigue and rigor oh the intake of water by the . - -. . isolated" muscle of frogs, observed the development of marked variations . . of permeability-from the normal. In his early work he also investigated . . the carbon dioxide output of muscle. "- - '. Experiments on the production and diffusion of lactic acid have been , ' perfonned on isolated muscles by Fletcher, Hopkins,11 Hill12 and ` Meyerhoff.13 In dcterminatidns of the diffusion .of inorganic phosphates from muscle during rest and-activity, Embden and his co-workers 14 . have shown that inorganic phosphate is an intermediary product of - glucose metabolism which.diffuses in increased amounts during muscular ' ' " activity. Since the quantity of inorganic phosphate in the muscle does not 'increase, the process of its formation is apparently' reversible; . . " " Embden therefore concluded that the increased diffusion of inorganic - phosphate is due to greater permeability of. the cell membrane during . activity and not to the production of more phosphate. Of the various ' . methods of studying the changes in permeability of muscle, Emlxlpn is ' ' the most satisfactory for our purpose, because of the ease of-determining ' quantitatively the inorganic phosphate in solution ; it has proved valuable _ . in studying the effect of lead oil the permeability of the surviving ." .. - -. muscle.of. the frog. -' - ' - - Y' '- ' f METHOn OF" STL'IIV - . " The technic of the experiments carried out" in this laboratorv was as - - ' : follows':- -- ' ' ' " '' ". ' . 9. AiiK ,T. C.: Reaiitroff; P,, and Smith. R. E.: I.ead Studies. III. The * * ' Effect of Lead on Red Mood Cells. ?. Exper. Med. 40:151, 1T3 ;in"d ISO i'Ang.! ' 1924.' .1 ' = '' _ -' " 1 ...... Jft. FIctchcF (footnote !. sixth reference). -_ - 11. Hopkins (footnote 1. seventh reference!. ' 12. TIill (footnote 1, eighth reference). - l.V Meyerhuff le t. ninth reference!. 14. Embden ct al. (i1, tenth jiiul eleventh references!-: .. DUP050332729 " KHXXlKOTF-.-WB~LK.-W STUDIES 447 Both gastrocnemius muscles were removed from pithed frogs with as' little injury as possible. They were handled only by the bone or tendon; they were treated in exactly the same way, and all manipulations were as nearly simul taneous as the procedure permitted. Each was placed in a beaker of known weight, containing 15 cc. of Ringer's solution," and the weight of' the muscle was determined. Both were kept in the solution-through which oxygen was bubbled constantly to prevent rigor (Fletcher). After standing for one hour at room temperature, the muscles were raised above the fluid, carcfuUy. washed with Ringer's solution, and transferred to a second beaker containing fresh Ringer/s solution. This procedure was repeated hourly until the-phosphate content of the solution became low anc! practically constant. After this stage had been reached, in most cases within two or three hours after excision, one" muscle was placed in Ringer's solution containing lead chloride (0.05 mg. of lead per cubic ccritiinelcr) and allowed to stand dor one hour.at 30 C.; the - other was kept in the regular Ringer's solution under identical conditions. In " a few. experiments the muscle was exposed to lead before the lowest value was reached.- Tlte muscles were then washed and placed in fresh Ringer's solution hourly as before, at room temperature The Ringer's solution in each beaker was analyzed for inorganic phosphate, corresponding solutions from the control and "leaded"-muscles being examined at the same time. In these determina tions, a modification of the Hell-Doisr" method .was used. No preliminary protein precipitation was necessary, and because "the quantity of phosphate - present was "small the results were determined more accurately in Ncssler- tybes than in a colorimeter. The entire volume of fluid was compared with standards whiclt were prepared for each test from -stock "blood standard" phosphate solution! Relatively small differences in "the- phosphate, content of these standards produced such marked differences of color that there was an error of only between 0.0005 anti 0.0010 mg. of phosphate for each- reading. The Ringer's solution containing lead was not analyzed, because the phosphate that diffused ironi the l.miscle.combmed with the lead as an insoluble lead phosphate " ami could not be determined by this method. - RESULTS .- ' Before the results of these experiments-are presented in detail, it is necessary to point Gut that the "tests were made at different times of the - year,-whenever animals could be obtained, and that-therefore frogs of different species and of varying weights had to"be used. Consequently, loo much stress cannot be laid on an exact quantitative interpretation of the results. However, the figures show definitely that the diffusion of inorganic phosphate (mg. of phosphorus per-gram of muscle per hour) increases markedly after "leading." Of thirty-five experiments,-thirtyfour demonstrated this dearly. Control muscles underwent no such15 16 15. The Ringer's solution contained: sodium chloride,, 0.6 per cent; potas- Mum chloride,-0.03 per cent; calcium chloride, 0.02 per'cciit. No phosphate or carbonate was added became lead forms insoluble 'phosphate and carbonate. The p,, was kept at 6.5, since lead hydroxide precipitates in jnore alkaline solutions. ." ' 16. I'vlt. ]{. T1., and Duisy, K. A.: Rapid Colorimetric Methods for .the Bctrrmiiiation of Phosphorus in T.'rinr and Blood, i. ftiol. t hem. Mr55 (Oct.) IMd. - ' '448' ;.<iftciii)'iis\"0F x e mo l q x v .i.x o rsixni.-nny change, except for 'a' relatively slight increase in diffusion following stimulation in soine'experiincnts, but t!ic diffusion curve followed the same course as that in Fletcher's carbon dioxide experiments, which was characterized hr --aji immediate rapid decline succeeded by a gradual decrease Tit the rate of diffusion-. Figures i and 2 show graphically the results of two striking experiments in which the increase in the diffusion of phosphate niter_.'expcsurc to lead is marked. -The data from the h:g. I.--Effect of lead'on the rate of diffusion of inorganic phosphates from muscle. The lieavy line represents the rate from "leaded" inusclc aud-thc light line that from the control muscle. The arrow represents the period of exposure to lead (0.05 mg."of- lead as lead chloride tier cubic centimeter of Kiugcr's solution),' " . ' "- . ng.P ,0200 - .0150 ' .0100 .0050 dl - J L. _J__ 1 J __J_li. _1__ 5Hours otter excision " - ' Fig. 2.'--Effect c:f lead <ni the rate of diffusion of inorganic phosphates from muscle. The lieavy line represents the rate from "leaded" muscle and the light line' that from the control muscle- The arrow represents the period of exposure to lead' (0.05 tug. of lead as lead chloride per cubic centimeter of Kiugcr's solution). - thirty-four positive experiments, collected in table 1. demonstrate variation.-. in the degree of rise in .phosphate diffusion ; in the interval between the addition of lead and-the appearance of the maximal increase; in the duration of the increase- and in the return of the rate of phris]vha!e diffusion p, ih,- minimum level. ' RlLZXlKOVF-AVB-UiAD ST-l'DUlS Taw f. 1.--The Diffusion of Inorganic-Phosphate front Xorvial ami '`Leaded*1 Musete* . 449 Contvni of Killer's Solution la Mf. of Per Cent EurutloJi &f Jvvclop*- . Phoephonu* j k r (5m. ol Muscle i-r Hour VnrfatfoD incrc.iscd meat of . ------ ."- % in Zltos'pbate Diffusion from Maxi- Dura- . Minimum Maximum Dix* fusion from Dlffu s.oa f i'f/aiJcd1 ''Mo s l Is inum lion *- ;----------- ,, 1 - a . DijTu- of ...... . . 3>ifTu- Corr*. '`Lcodc*]" Cerre. . rooiroi Jlofurc Without e'.oo Minch ' - ' Number don siun<)inif Mivcle sponcJlnj - Sfuscie Rctiun Return after 'imjra - - . . . , ` of fl'IMO Value of filler Value of Id Cone- . to to * Lcird- Diflu* ' Expert* 'I.csulcil'* Control ENpOMMC Ccmuol *r.carior* siK-ndiric NoniiAl, Nonnuli inc,*-' ploifci...... - : -.1-1 ment Muscle lluwie tO l-CHit Muscle Muscle Time Houn Hour* Hour* Hour*. . * - : ` -- 1 ' o.e S n.>3 -3 0,(K>5 4 o.wa 5 0.CO3 e 0.001 7 0.003 3~ 0.W3 0 n,r<2 10 o.ow J1 O.GoS 12 . 0.005 13 0/CV? 14 o.o IS 0.W8 J6t 0.003 17 0.tt ist 0.001 fi.M # 0.0*13 2H 0.004 2 l.(3 * c*.nc*2 ?4i 0.003 25J (UH 20* o.on V79 0.006 2S? n.we 0.003 aw rums sn o.e 32* : n.((8 as: 0.010 a*: 0.013 o.cto 0.012 0.(04 n.oi3 0.004 f*.C07 (1,003-. O.0M 0.003 0.00ft 0.00G 0.007 J1.1XO 0.(0* 0.00$ 0.005( .005 0.002 !.* 0.004 0.O0G o.m O.OfiS 0.003 O.an (J.KO 0/.O5 O.OW3 4l.OO 0.(05 0.(C-S o.lo 0.016 0.011 0.011 0.026 0.CO8 0.017 o. 0.015 0.008 0,011 0X08 0.010 0.CO5 0.015 0.001 Wo7 0.004 -O.Of'S 0.003 0.014 0.00ft 0.017 0.004 0.011 0.(08 0.(15 0 .m 0.015 0.0J5 0.010 0.i<!4 0.016 0.003 0.019 0.003 O.Ol 0.-<02 0.023 O.C03 0.020 0.003 ft.S 0.004- 0.016 ' 0.005 0.008 O.OU3 0.011 O.OJS 0.008 0.002 0.024 0,W3 n.cco O.OJ2 0.008 0.(02 O.U*7 0.019 0X08 0.012 0.023 0.004 0.022 0.009 0.(01 - O.oii 0.C27 o.ou +460 +770 +240 +400 +270 +150 +100 +130 +300 +-250 +240 +120 +110 - +130 + SO +430 +630 +700 +-2SQ +570 -#-500 +250 +300 +270 +1300 +120+230 + 60 + 40 +220 +1050 -ISO +140 +110 +10 ^-so 0- 1 a -10 +50 -SO -20 . 0 0 0 -so 9 +10 7 0 +30 --50 i -40 --40 1 1 0 --80 -30 ( --SO -20 ... - 0 7. 0 +100 +40 --40 7% --CO -0 +30 --20- +10 -30 -10 4 4 4 4 4 4 5 5 5 7 7 1 5 4 4 $ S. 8 7 V4 e'A ... 0>.i 9V4 su 514. 0 4 3 4 4 -1 ; - $ 2 -. 2" 2 -. 8 1 - = .. . . 8 4' " 91 3 ' . . 3 -1 - aS .2 1 - 3 -1 _ . 3 1 . ... .- 6'4 1 G;i 1 -- . - 3. 33 41 . -: - 414 2V4 2 ^ 4 -2: . 8 7i - '/* 4& " 7 " e>4 3 . -i 21 TVz 1 ... . ... -- ' .. " 7%. i - SV4 1 " 4 2i 4 _ -= .< . _ ~ ' .. "" . ' : .. -' - - ` * Average iDcrea?* of phosphate uiflusioft from "kaiit-vf" muscle, +320 per cent. Average decrease. ol i+o'+hjiu diflvjiou from control TmtsOe In com-ronOIiif period. --10 per edit. i Still maximal ut Just rktcrmlnutioa. * ftimulatid. Kx^rhiiont stopped while lifTu*inn wits maximal. - 8 Mnsrys sthimhmd. .Still maximal at Inst ricieimln.'itlon. " ;' i Mu?ek# ttimukitcd, - :_ " . - - _ _ .- " - --- - Ta w .k 2,--Iwtrasc of Diffusion of Inorganic Phosphate from n].cadcd, Musne * ' Pi-r Ci-ut icc-rcase ntlri' I.cmlhijj" Nuu:l>r ol Experiment* Less t'liin 1M............................ ........... ......................................... TMuean VO ;uid cun................................... ........... i,., Ucpvc.a 2W sn*l 3TM.................................................... r-i-uvn .; nt! .............................................................. IMtVifU UO ;<;() ;j o ................ ......... ............................................ r-l'ui>n r*o nail cuo........................................................... .......... JM-.vfn C'O uni 7i*>...................................................................... 1% Ke n 7t ><i................. ................................................... i:r:v.*<!i + ' f.............. .............................................. ........... Mric il'.iin i.<v m................................. ...................................... * Avt-r; :< c j pV< c~*ilC>:d*n-from "3c. -<T' w'le, +3:0 per c>nt. Avr.ti:e !!<.:* ;;p 1.{ yKt'uvcn Ir.m L-ontrOl rjr.Tfle, --in )>r centi ' ... DUP050332732 ____ . - 450"-- fUiCMMS- OP Mil 'KOLOHY ASP PSyCUIATKY ,, i A summary of these results (table- 2) shows that the avcragc'rate of : "- - - diffusion iiia-eased,; tffler; exposure to lead, 320 per cent above the - ' -- lowest- value.cmlyflht eg-cases was this increase less than 100 per : -' - cent. In nine exp.criifteivts'it was between 100 and 200 per cent of the . ` : - minimum,-iit ku between-260 and 300 per cent, and in two more than - - 1,000 per cent. -l>nrhig the same period the average rale of phosphate - diffusion from the control muscles decreased 10 per renl. 3n the cum - f . I. . posite curve of till the experiments (fig. 3)", the marked difference in . :- phosphate diffusion between the "leaded" and control-muscles is dear. .. .... . It should be pointed out that in some of the experiments (ten) the low . value, before the hegiuning of the rise caused hy lead,.occurred late and ; ' that therefore "the co.nipositc chart, showing the average diffusion for each hour,, does hot give as good an indication of the degree of change : as can be obtained .from".the study of the individual experiments in ": table 1. The length' of time elapsing after the removal of the muscles , - .. from the lead .solution before the diffusion of phosphate became 'maximal . Eig;. 3.--Composite chart showing effect of lead oil the rate oi diffusion of inorganic phosphates from muscle, tphe heavy line represents the rate from the muscle exposed to lead solution and the light line that from the control. The arrow represents tile period of exposure to lead (0.05 nig. oi lead -as lead chloride per cubic centimeter of Ringer's solution). varied from two (seven cxjieritncnts) to eight hours (one experiment). In the majority of cases (toll.experiments),-the peak was reached within three hours. Of the, twenty;scven experiments which were continued after the peak was reached, eighteen showed a sustained maximal diffu sion for only one hour, and six tor two hours. In the other three, the diffusion remained maximal until the experiment was terminated--three hours ill two cases, and-four and one-half hours in one case.- In only four experiments did the rate of diffusion return to the low control value during the course of the observations. In one-case the curve did .not return to its original low level even nine and one-half hours after exposure to lead. Factors other than expostm to 'end may alter the into-of phosphate diffusion. Injury inciva-es the permeability of muscle to inorganic phosphate; hut in thh scries of-experiments the results obtained in the DUP050332733 KEZX1K01-F-A I'B--LIi.ll) yi lVlES 451 controls indicated that this, was negligible. That stimulation increases the rate of diffusion was shown by Entbdcit-; but the degree of increase is never of tire same magnitude as that caused by lead.' In twenty-nine . experiments with normal muscles, stimulation increased the" diffusion so little that the figures usually, fell-well within the limits of experimental ; ' error. Increases of between 0.001-and 0.002mg. of phosphorus per" - gram of muscle per hour followed only eight of-100 stimulations; and' only once did the output increase as. much ns-from 0.008 to 0.016 mg, ' In this case, stimulation was so frequent that rigor probably resulted. , - In most of our experiments, rigor, which of course increases the .rate - of "diffusion of phosphate markedly, was avoided by providing a'-constant - " supply of oxygen and fey preventing undutpfatigue. Although the rela- _ tionship between rigor and the muscular changes caused by-lead cannot ~ be considered in detail here, it will be of Interest to remember that rigor ~; .is supposed to be due to (lie acid products of fatigue. Examination- of nntscles after exposure to lead demonstrates that they tire shrunken, of rubbery consistency and lusterless---in fact, comparable in appearance to muscles in-rigor. - "' - - Other observations merit consideration. If a Meadcd" muscle'is treated with ammonium sulphide and examiued'-und.eT tlie microscope, alb a the lead appears on the surface and none can be demonstrated deep in the muscle. Another fact is also of interest in this connection. When" a muscle is exposed to lead, there'is a change in the reaction of the sur-. rounding solution, in one case from j>s, <x5 to -1.8; in a second experi- mciit to fa 5.5, and in a third to /> 6.1. Such an increase'in-acidity . agrees with the results of ottr work on the.effect of lead on red blood " cells, which show that "soluble lead salts unite with" the inorganic phos- - - phatc to form insoluble lead phosphate with the liberation of.free acid. - - To determine the effect of acid ou the permeability to inorganic phos- - phatc, acid Ringer's solution was added to muscles. ;In one ease in which _ the fa of the Ringer's solution was 4.5, the permeability of the treated - . muscle remained practically like that of the control, hut in two experiments . - in which the pH of the Ringer's solution was 3.5, the rate of phosphate diffusion increased markedly* at once. The maximum increase was brief, . hut throughout the experiment the rate of diffusion remained constant and did not decrease as did that of the control. Iji one case it remained twice as high ns the.onginal rate before exposure to acid.' The maxi mum increase in diffusion due to acid was 140 per rent in' one of those experiments, and in the other 520 per cent. This'type of experiment, however, is not comparable to those in which the muscle is exposed to . a lead salt, for in.the latter acid is produced in they tissueof the muscle ' ` and the buffer phosphates must he diminished !;>v their interaction with . lead. These acid experiments offer further evidence..that "when lend sal's act oil isolated muscle the reaction tbift probably 'occurs is similar to . that demonstrated with red blon'd cells. ' DUP050332734 n i"i T- -452 ..Jkcuit'iar o t x u c r o l o u y a .x o p s y c h ia t r y __ Slid) astriking clitmge .in the pennuiilulity of die stiriauc of muscle '` V fnovgamc p|icis|>liale suggests that lead may have a demonstrable 1 ; i _ effect oil The activity of isolated muscles. To.determine this, e.Njjcri- " .'uieiuV.. \Y.et c. j*cr formed in which nerve-musclc preparations fsciatie- ' " " gastrocnemius) were removed from pithed frogs and placed in Ringer's ; 'solution in specially constructed dianihers... One nutsde was exposed - 'to lead (.0,05 mg. of lead per cubic centimeter) and the other was Kept . " in Ringer's solution as 'a control. They were stimulated both directly " - and through their nerves, and the rate of onset of fatigue and the degree - Fig. 4.--Nerve-muscle stimulation and diffusion chamber. , The muscle is attached !>y the Achilles tendon to the platinum hook A, which is fastened to the bottom ui the oxygen inlet tube-ft. The chamber itself. - made of glass, is S rum high and 2.5 cm. wide. About 2.5 cm. from the top two ' glass tubes, C, C (4 mm. itt diameter) are seated into the wall of the chamber. ' Through these, platinum wires rim into the chamber so that when the tubes arc . filled- with mercury they may serve as stimulating electrodes ior the nerve. The oxygen intet tube runs through the cork I) which is held firmly against the chamber li by means of a heavy screw clamp F. Wound about the platinum ' ' -hook A..and the- oxygen inlet tube is a fine insulated wire which permits the ' hook to serve as one ui the muscle stimulating electrodes. The other nm-clc " electrode consists of a - platinum S-shaped hook run through tlw knee joint- -. . ; and attached th one" end of a fine copper wire which has been made soil by : heat. The other cud oi this wire is fastened to an ordinary mii-cle level tor " recording muscular emitiactions At the bottom of the muscle chamber a stopeock A is inserted, through which KtngerV solution may be admitted :tnd _ removed by gravity pressure. With siirh au apparatus, the muscle mat be. eon- - ... . - - - " --- DUP050332735 HEZSIKOFF-AVB--LEAD STUDIES 45S initially bathed in Ringer's solution which can be changed at regular intervals and through which ox5rgen may be bubbled constantly, so that both the rate of phosphate diffusion and tile character of muscular contractions can be readily determined. "- ' In each experiment two such chambers _wcre used, oiie lor lltecon- -trol'and the other'Cor the "leaded" muscle. 1'lie .electrodes were cou- ncctcd in series so that both nerves-6r both muscles were stimulated simultaneously by the same current. This permitted direct and con trolled- comparison of the two muscles, aiid made unnecessary-an exact regulation of the stimulating current in the different experiments. The recording levers (the ordinary straw variety) were of equal length; they were always afterloaded, and were usually" weighted with- from 5 to 10 Grn. In any given experiment the same weight was applied to both levers at the same distance from, the fnlcruni. The stimulating electrodes were attached to the secondary post of a standard student's induction coil. -A metronome made and broke the primary current at regular intervals. In the secondary circuit a rocking key, which pery mitted the current to he switched to cither nerve or muscle, was inserted. Tn almost all experiments the stimuli were maximal, and in. every case control records were made before lead was added. Usually nerve and muscle were stimulated alternately. - Thirteen experiments werg-perfornicd with tetanic stimulation, in eight cases the control muscle remained in good condition, although-the -"leaded" muscle became fatigued rapidly. Tn most experiments it did not regain its norma! condition. Figure 5 illustrates one experiment in which the muscle was stimulated directly and the rate of phosphate diffusion was determined hourly (fig, (I). A description of the experiment follows: _ a. m. - '" - S:58 In first Ringer's solution "_ _-9:48 Nerve stimulated, 1 9:50 Muscle stimulated, 2 " . 9:58 In second Ringer's solution. 20.3 C. _ lit 02 Upper muscle in Ringer's solution, tower in lead solution, 31.5 C p.in. _ 12:08 In third Ringer's solution, 20.9 C. 12:12 Muscle stimulated, 3 ' `_ -12:18 Muscle stimulated, 4 " 12:24 .Muscle stimulated, 5 _ 12:38 In fourth Ringer's solution. 20.8 C. _ 12:48 Muscle stimulated, 6 " -` 1:38 In "fifth Ringer's solution. 20.9 C ~ ; - .. 2:03 Muscle stimulated, 7 - ' '' ' - ti.. ' 2:19 Muscle stimulated, 8: ' . "' \ : '. 2:38 In sixth RiitgeUs solution - ' ` " " . 3:02 Muscle stimulated,. 9 ... ' - 3:25 Muscle stimulated. 10 1 .... P .. . 3:31 Muscle stinnilalcil, .1! - ' :r - ' 3:38 Rfmortil to sixth Ringer's solution. 50.7 C. " ' ' ' ' ' c L i- Fig. 5.---Muscular response to intermittent tetanic stimulation bciore and after "leading" :" C, tracing made by the control muscle; L, tracing made by the "leaded" muscle (0.05 nig. of lead'as lead chloride per cubic centimeter of Ringer's solution). The figures indicate the periods ol stimulation; the crosses represent readjustment of the after-loading of the muscles. __ |P n*03 6400 090* asoo - a'tir ' Fig. 6.--rYarialions of.the rate of inorganic phosphate diffusion from normal - and ''leaded1' muscles during tetanic stmutlaliuit. The heavy line represents the rale-from the "leaded'* muscle (0.05 mg. of IcacLas lead chloride per cubic centimeter of Khsgor's solution) and the light line that from the contiol muscle. DUP05Q332737 iHiZSlKOFF-AVB--LEAD STfDIES - 455 - This cx|K.-rimi`t (Union st nitos several interesting facts. yThe "leaded" muscle.became fatigued much more rapidly than the control (3 rtml 4) and failed to recover (5). Apparently lend alone cannot -cause- immediate injury ta the muscle, fur the "leaded" muscle gave excellent initial contractions after removal of 1 he. lead-Ringer's solution (3). lint fatigue followed a series of such contractions -rapidly. This' occurred in all experiments and demonstrates that the action of lead on _ - . fig. -Mmcutar response to make and break stimulation: /.T ike irtlciiig _ made by the "leaded** nuisete (0.05 mg. "of lead as lead cldoride per cubic ccnti- -nielcr-of liinger's solution): C. that of the control. . muscle is manifested only after muscular activity occurs. Comparison of the diffusion curve and the muscle tracing shows that the greatest T tpianlity of phosphate diffuses during the first, second and cspcciallv the ; ; third hour after exposure to leach (third, fourth and fifth Ringer's soht- , lion) wlicnfhe muscle is most fatigued. -The slight return of vontractioh eviilent-at 0, 10 ami 11 occurs sinmltaneously with a decrease, in the rate' 1 of diffusion. - .- _- -- ' DUP050332738 r m, .ikaut us oi- s u v r o l o g y a x u i \s y c ij u t k y ' - ]n live of the thirteen.exjKniments, no difference in' the rale of onset of fatigue iit lltc control and "leaded" muscles could-be seen. In these cases the nhisdes performed an excessive amount of work, and rapid fatigue ensued. In none of these tests, however, did the control muscle fatigue more rapidly than the "leaded'Tnuselc. " ' Since control muscles seemed to be fatigued so readily with tetanic - stimulation, a series of experiments (ten) was performed with make and break stimulation. The results showed definitely that after an initial jxiriod of sustained uniform contraction, the "leaded" muscle became fatigued much more rapidly and completely, and recovered with much greater difficulty than the control in every case. Figure 7 illustrates one of these experiments, in which the levers were weighted with only 2.5 (jin, so that an excessive, amount of work was not performed. These investi gations, therefore, indicate that lead interferes markedly with the fmic tion of isolated muscle. Stimulation -produces fatigue much more rapidly after exposure to lead, while recovery, slight at best, in most cases does not occur at all. - - " - EFFECT OF LEAD OX SERVE -_ ; - Because of the clinical. designation of lead palsy as a peripheral neuritis as well as of the occurrence of pathologic lesions in peripheral ' nerves, the impression is prevalent that lead acts specifically on peri pheral nerve. The only experimental study of this problem reported in the literature was made by.Do/.zi and has already been shown to be of "little value. The action of lead on isolated nerve has been investigated in , this laboratory byi. nicanspf the Adrian 1? narcosis chamber. In these ' - experiments the standard`procedure was to excise life two nerve-musde preparations of a frog, place the sciatic nerve of each in a narcosis chamber, and between tho point of stimulation and the muscle to treat one with normal: Ringer's solution as a control and the other with a ` _ . Ringer's;solution,' of the same hydrogen ion concentration, which con : , tained lead chloride/ The' muscles were placed in moist chambers, and ~ .,,, _ .-. the nerves were stimulated by the electrodes of the two narcosis cham bers connected in series. In four experiments the conductivity,. as . -...... - ; judged by muscular' response, was exactly the same in both nerves. In . . one; of these the' strength Of lead used-was 0.05 mg. of lead (as lead : .. chloride) per cubic cetUrnieter of Ringer's solution; in two, the conceit- ; tration-was-0.T mg.; and In the fourth the nerve was treated with . ' 0.23 mg', of lead per cubic centimeter of Ringer's solution for one hour, -and then for another diour with Ringer's .solution containing 0.46 mg. _ per cubic centimeter, the largest quantity soluble in Ringer's solution of pH 6.5. In none of these experiments could any difference between17 17. Adrian, K. IX, and I-uco, K.: On the Suimnstiim oi Prnra.vatcd Di-- tiirbsme- in Nerve aw! Mu.-cle. J. Piiy>i>>I. 44:<iS. 1012.- "_ - _ V/- = v; DOp o '50332739 ' ' KUZXIKOFF~vt VB--J.H.11) STCMBS ' 457 the conductivity of the "leaded'1 am! control- nerves he "detected,. At the end of the test, the nerves were always placed in. a solution of ammonium sulphide to determine whether there had actually been a deposition of lead; the invariable black discoloration of the "leaded" -nerves indicated that this had occurred. As far as can he judged. Xrora, this work, therefore, lead produces no decrement in.nerve conduction".; , To check these results an attempt was made to determine thc-actitm ' currents of both nerve and muscle as a delicate index' of"; function bythe string galvanometer, lent by Dr.^Alexander Forbes" and Miss Anne Hopkins, -who assisted -us hr'these" experiments. lit otic of a uair of. nerve-muscle-preparations the nerve, and in the other the muscle, was exposed to lead. Because a great number of stimulating and Icading-off electrodes must he placed in a small space for -such work, there wasgreat difficulty in avoiding artefacts. Further complications were intro duced by the necessity of performing all manipulations" simultaneously on both "leaded" and control structures. In one test a-distinct action current was seen in the "leaded" nerve,-hut not in tire "leaded" musde.This experiment was, however, unsatisfactory because both muscles were not fatigued at the same time. Another experiment in which this defect was avoided demonstrated distinctlythaf after "fatigue the action current of the "leaded'1 muscle diminished rapidly and soon disappearedcntirely. while that of the control! muscle returned to-normal .within a short while and persisted until, the end of the experiment. Similar, undiminished action currents were obtained -front- both: normal, and "leaded" nerves. Thus, the distinct inhibitory-action of lead on .isolated muscle and the lack of deleterious action on isolated t>ervcare confirmed. -- ~ p a l s y - ts vivo . ... ", - - Tn the explanation of lead palsy, as it occurs in "fife, tlic"experiments already described serve merely as prelim inafv'Tuid' suggestive observa>_ lions which-nnist be correlated with-the production at palsy in living animals if they arc to har e any important practical significance. The ample evidence that, in life, load paralysis develops in inusdc* that are - fatigued 18 links the problem of lead palsy with the chemistry of mus cular fatigue and suggests a method of investigation in "-intact animals. 18. Mcycr,_M.: Die Flcklrizilit in Hirer Anivcmlmig atif praklisdie Mcdirin, Bt-rliu, 1854. quoted by Weill, .Mnebius:- Tchcr citiige lmgewiibnlichc FSlle von Blcitr-bmung. Ceiitralbl. f. Xcrvcnheitk. 1:6. 1S86; r;l!utcd hy fitieglitz: Mine experiinciitcllc I'ntcrsuchtmg ueber Bldvergiftmig niit.bcSQiiderc Beriick- siditigung dor YoriimUnWgcit am Ncrvctisystein. Arch. f. Psj;rliint.-8`f":4!l. 1802. Weill. H.: Znr Prase von tier I.okalizjition dor fiU-il.ilimiing, hung. DissZ Ftrassljurg. 1892.. Kdingf-r. I..: Per Aiiteil tfirFimkliYin an dir- Kststriuing vrm .Xorwnkr.-iiiklii-iu-ii, Wii-sbadcii, Bc-rgnmnit. lytW. 'p. r,7. Tvlt-ky. l..i.-7.ar - Kasnistik tier Bh-il:il-,miin.e. Boiitsche Ztschr. f.'-Xcrvcnb. 37:2.1-1.. 1009. OliA-.er. 'I'.: bead Poi-uuing. I.nndoii. 11. |\. T.i-wi<. 1914. ]). 148. . " ir n it imp Vi? . 458 V .jKClUlHS 01=- \lil'KOI:Ooy .-I.Y/J I'SVCIUA'IRY - ' Attempts were n&idc to product* j k iIs v in the frog. Lead was injected ' ' frequently imb-lhc'duvsal ww, and one leg. was fatigued by intermittent ; stimulation over the sciatic nerve'tor several days. In must of the - experiments there'were tto differences between, fatigued and control * muscles and wiiemmty* difference "Cunkl be found there was always a - question of injury to tissue. .There-are two ol>jcctiuns-to this method: ' ._. first,1 lead sahs'administm-d-subcutaneously are precipitated at the site - - . of- injection-and are-therefore only slightly and*slowly absorbed ; secottd," - ; die mere-response to`electrical stimulation probably results in-nemo- . _" muscular: junction fatigue -without necessarily cansing fatigue in the - - ; muscle comparable to that obtained by lifting weights. _ .. : - . . Consequently-, ait attempt was made to produce -palsv.in" mammals -by fatiguing the extensor-muscles-of one limb by voluntary contraction, - - At -first rabbits were iked, but they -did not -prove as. satisfactory as cats . - .. - y which later were used -exclusively, A lesyl carbonate suspension .was *** : - injected into the lungs of some animals thro ugh the trachea, and others - - were kept as controls. In order ter produce unilateral fatigue, weights ' : i varying from 70 to 100 Gfn. were attached with adhesive'plaster tu the ' " ' dorsal surface of the right forc-pnw of all animals. These rested on - ' cotton jrad.s;-and since they apparently did not-disturb the animals they ... - were removed only to allow occasional insiiection.of thc limb. Tbc- ahi- - " mats-were exercised daily* "in a revolving drum (lent'by Dr. SI. J. - - Rosenail) to prdclucc a marked degree of fatigue in the extensor muscles of the right forepaw by constant raising of the weights. When a dis- - . tincf difference between, the weighted lint!) and the other became . -- : apparent, the animals were allowed to rest for twenty-four hours with the weights .removed. - Then, under urethane-anesthesia, the tmtsculo- - - spiral nerves (radial) and the extensor muscles were exposed and sub- 1 ' : mitted. toe various, physiologic tests. /The threshold of these musclcs-to ' ' . *- Y nerve stimulation, and in some experiments to direct stinntlatfpn. was I -1 detcrniincd.. -Attempts also were made in some cases.to obtain action * T currents, audio demonstrate differences between flic degree of fatigue _ in the weighted and unweighted .muscles following stimulation, but - " ' because of citfiictihies inherent to the experiments, these usually-proved _ " Unsuccessful. In all experfhients the electrodes were connected in series, _ . . , . so that both sides were stimulated equally and simultaneously. . . .." Hefore the ..experimental, results are considered in detail, a brief : . deseriittiou-fli tjic typical 'behavior of-the animals, may he of-"interest. ` ! Alter:rthe-administration-lead, all-the cats lost'weight rapidly, and* within one or two1 weeks showed:distinct lead lines and- became easily y ; 'fatigued' by' exorcist: 'The' weighted foot showed distinct signs of' - - weakness, after about-two weeks of exercise.-. This was manifested by difficulty in "extending the foot when the weight had been removed for . " twenty-four hours, and was especially .marked if the animal was held - mm iir D U P050332741 _ - ~ KLZXIKOI- F-.l CB--I.E.tD STCDIliS 459 by its neck ami permitted to reach for the top of a window ledge. Some of the animals limped decidedly in walking. 'When the weights were still attached, the contrast "between the "leaded" and- control animals was even more striking. Those suffering front plumbistn could not.lift the weighted paw without great effort and (lien .only .with the aid of the upper leg mnschcs, while the control animals lifted both paws.with equal case. After the animals bad been rim in the drum, these differences were accentuated. The "leaded" cats tired much more quickly and to a greater degree than did the normal animals and exhibited a .striking fatigue of the weighted limb, which usually-eomplctely prevented exten sion of the paw. They took shorter steps witluthe. weighted limb" while- _ Ts s l e 3. --Threshold Di'tt'nniiwlions k./0if>6i(4i9. ' " - - Stimulation . of fJ'JjKlmW StJimihi* ' ; - ^PosiMoa ol SocoDdarX: Coll. _ VoH.iee of - Primary - Stimulation? X-:i ^-Left Side, -liiylit (Weighted) Current with Cm. " ' - Side, Cm`. ,,2 S -3 3 S Break* r : Jirart _ . Break - - Break*. . Brcult" 1 ' 13 . .* 11 13.25--- S.251 . *. 8.5 ' as: ; : 7.75 ' 3.-0 = :. Iljl j _ * role? of woodniy reverted. . ._ - t ITw lover the Attire, the trcAJh-r tlx current. '' ' . ^ T.\w.e 4. --Threshold Pcteniwialiohs ih 'I\`ifbbit-W : ' ' Stlnwlfttlon ' fit SetWt MiiseJe VoltAce of Primary Current S 3_ . Position af Secondary Coil' 1 ~ Stinnitatinn with - - Break - - Break ' . ; 13 ' S.75 flight (tVcfg-lited) . S.icle, Cm. V; - 1 ' 0.25 running and seemed to lean toward the left.. This descnpiipn'ftis nearly alhof the animals, and individual variations will" be mentioned in- the; protocols. The threshold experiments, wqre performed On one ''leaded'' rabbit and its control and on four- "Igpd^d" aiid two contrgl ;ca& Of the "leaded" cats, one showed little liitutation of-extension in. the weighted paw.before the experiment.- - .Ohe;"di'r in cqnvufsidns'aifd three of inanition before differences between the-weighted and unweighted limbs seemed Sufficiently 'marker! to warrant a detcriiiinatiim :nf their thresholds. Two animals died during the operation. : ` . ` - I'KOTOCOI.S- '1 - Exi'JJRtMItXT J. ... ' . . fiobhil "L-oded."--head carbonate vyas, injected Lil.raXi$c'hcSiiy' three inner. Two lunidved inillLa' amr of lernb ;as Ivn.l acetate, wits' givciV. by tbe stomach tulie five .times. The weight attached in' tlic 'right Wejiaw- was 460- _ .ih c u u h x oi: mu 'a v l o c v .'ixi> i\sycm.ra<v . 60 Giii. Tilt' iiiiiniiil was rim in drum im- lifty-uv>_ dnjs (7.3 miles). Xlurkcd stippling id reel blond eclU was presell! during ttii> p, ri. d. .-Tlu- m-ru- was too-;short to pennil the recording i.i action currents. No fatigue record wasxrbutiik'd, )is (lie iinimal was" in too poor condition. . .. Rabbit, 493,-Conlrul.--This iinim.ilwns treated in every way like 4SV except that it received_no.lead. ` 7-The most striking" feature of" this experiment is the smiilariiy'Ix'twecn the lliffsholtl of the unweighted imrt-cle of the -kaded'" anijml amlihm of both-unweighted and weighted muscles uf tlie-vontrol, w 1k .ii stiirmltued by nerve with tuiifniiii-aurent. In the --`leaded" rabbit, the. -threslioUr of" the weighted.muscle to stimulation through the nerve was nntch higher: There is the same relative difference between thresfnilds when stimulation is applied directly to the muscle. In the control, tlxslight differeiicebclwcc-n thcTVvo sides probably' fails within the limits of normal variation. The difficulty"of applying the muscle electrodes, _ - -Taw .f . 5--Thmlwli oj KaiiatZXerves ami IhachhiradiiiUs Loiigus - ." " Munirs in Cal 4X0 _ '' -- ... - . - _ Threshold Stimulus _ ~ rosltfoni>f Sccoudfiry ColJ StJumlutloji et - Voitajioof P/imary Cury-ift:- - Left lifc - Bight ..: StliJiulitticui f~- utlh -- C`jn. Rotut/on Sftle. Cm. Nerve _ - 4- 1 ' Msisrlt1 - ' 4 - ' Breeif. ' Break - 13 STr decrees J2.3 ' - 8..' 8.0 . however, to exactly jciiriieyponding punts of" the muscles detracts from the value of the. results obtained f rom muscle stimulation. Exi'ltKiMENT 2. . ` ! -' .- Cal 4S.0, "headed;'--l.cad -carlxiii.-ite was injected intratracheal!)- once. A 60 Gin, weight r;1' attacked to-, the right fort-paw. The animal was.run in the drumfor"fifteen "day* <2-3 miles). Tile body weight fell from 2.7 to 2.2 Kg. After an interval of "seveii days tile- rrinril was.again exercised for eight days (0.42 fnikes).. The body weight was from 2.2 to 2.3 Kg. -The weight-was changed to .100 Gun and the anwas run lor is\o days, (0.18 miles). The weigh! .was ag/uu cliangird-to CO Gm., artd tin* animal was run for ft-ye days (0.44 miles). T-he.body weight was 2.5 Kg. One hundred ami fifty milligrams of lead waV-givun by stomach tube twice. With a weight mi 80 Cm. attached, the animal was run for five days (0.73 miles). The body weight tva> 2.7 Kg. -The total length of the run-\vaa-t4iivly-fm*_(lays_(4 milts j. ' As the animal became unconscious under-urethane anotbesia. the right foresaw was held comuleuly'flexed; ; ' -. " An,attempt to. record action nirrniU -from the nerve -and muscle jf each ' limb, |iroyetI. uir>xicce'SJiful" hecirti>e;''oi artefact?.; this spiled the preparation for fatigue lc*ts. - '. Coi 192, Control.- -Thi> animal was treated in e-vtry.'Way like except tbj<t "no lead \va>`idinmi>U,ml. l*mier_aih >tht >ia ht.th loripaw' \\iTe_cquiitiy active ;md were held in the >t h j u jwilbin. - - . . HliZSIK Oi:F-AVB-- I.EAD STUDIES 461 Since the threshold for stimulation through the nerve' was the same on both - sides, stimulation was not applied directly to muscle. ' ' . . ' In tVris-experiment the difference between, the rtylit (weighted) and _ - left paws after exposure to lead is similar to that in the experiment ' with the rabbits.. The threshold of the-left, unweighted muscle-is " apparently normal, similar-to those of the control, even after exposure .. to lead. - . . =` . - Ex p er ime n t 3. - '` Cat 115, "/.{titled."--I-cad was sivtirimratracheall.v once, The weight placed on the right forepaw was 70 Gat. The animal was exercised in.the drum for a - - . ' . r. ...... . , ' Tabu . 6. --Threshold of Radial Xcri'Cs and BrtU'fiioriHiialh 'Lottyns-_ , ' - Muscles in Co! 492 .. . ... V: -. , - - - - i ThresholdStimulus " Post km of Secondary Call - : ' . .. _ Stimulation - ' of . Serve Voltage of Primary Current i Stimulation with Break ' Left side' Right OYHgbted) Side ' : ' Cm. Rotation - = IS $0^5 ' . - degrees Cm. 'Rotat'on'13 S0-8S- degrees v ... . ... .. _ _ . - k -- - ." _ -- _ - Tabl e 7-- Threshold Determinations'in Cal 11.5 - . -- - -- -_ Threshold Stimulus " Position o| Secondary Coil ' . . - *' _ I " - *"" -- -- Stimulation of - Nerve .Verve Nerve Nerve Mutcte Mu&le . Muscle ilu.'de - Voltage of Primary Current -4 4 4 4 -4 4 4 'i * Stimulation with " ' Break Break Make ami break .Make mui break Break Make find brenk. Make and break Make nod break Lvft.SWe Might (Weighted) Side Cm. Rotation' " Cm. Rotation - 13 ESdegrm 12.3 IS S3 degrees 13 38 ^ -Sadcgreu : _$.5 23 . 43rfffref? - 10.75 -33 degree* . - - -. . _" . ...-y - il f . - - ', - - = 7.B - S.S 'v - `^isr :?.5 . 3.5 - U* '**'* - 5 ' - - _ . period of forty-two days, during which it- rested for. an .interval !pf fourteen - ` > days -(ran 3.2 miles).- Body weight during this time, fell from 2.8 to 2.5 Kg. and thcn-gradually rose to 3.1. __ ; v, . ; %. , :` Records of action .currents could nyt he taken because the' string gal- ' - : vanometet was out-of order, " - . '- " . ;A: -" - In making the fatigue curves the lever* .'were, loaded with 30" Gut-, and the ; - stimuli. (break shocks) were maximal. Xo difference in the rate of fatigue of the right ami left sides could lie deni'insi rated. N*e11':er side was completely-' '*' fatigued. . ,. . i ` ' - " _ - ' ' Although the difference between (he weighted and unweighted _ muscles is not as striking as in the. previous expeVinVeiirs,- it is' distinct and-emislant. As this animal had been exercised Jnr..only, twenty days. the muscles were pmliablv litltf fatigued; - The f.'iitnw to demonstrate a . .: , _ " DUP050332744 462 ' . IM IJII FS - Or XFUKOLOUV ASl) I'SiXUlATRY difference-in the fatigue curves o{ the two sides may be chic to insnf- - ficient weight on ilie muscles. - - Ex m-k imk n t 4. ' ___ - Cat JQT, "Leaded."--3.cad was injected iulratrachcally once. The animal was run in lilt drum for seventeen days (2.6 miles). One hundred and filly; milligrams of lead was then given by stomach tube on lour successive days " and a weight of 70 Grn. was attached .to the right forepaw. The animal was _ then run again fbr'foiuteeh 'day's (2.7 htTles).-" Alter the first administration of lead, the body weight- of the aninut! fell from 2.5 to 2 Kg. Just before the Second administration the weight had risen to 2-3 Kg., but "it again fall to 1.5 .after, the last day.of exercise. The animal was. weak and in a poor condition. w,.. Tabj j : b.^'fltrcAtcld Dclcnnimdicais in C-ai 107 Threshold Stimulus PosiUui: oi SGCoiitJary Coll Tlmcof Vokapr of l.oft Sidr Right (Wcrt'liU'd) Sid-: Stimulation SStUimmunlit)*. 3l>>rrnnuji;:tiryv '"'SStilwimauLliitlfonji ><---- r~ .1 --... --1 of ttOD Cnrrrat VTth Oil Rotation Cm. Rotation Ken* 2:3* p.ui. 2 Brent " 13 Serve Kcive 3:f 3:1)5 ft2 - Male and brink 13 - Break 13 Kmc tin Xerve ' 4:17 !" 2Q " - Break ilttkf and break 3&r IS Xerrt. - 4:43 2 . Break 1H Jrtrve 5:35 2" Break7 ' 32.2-1 Sene . C:i$, .: - -2 Break 11 Kent* - Break n K*u* - . S:17- i - Break y r,, - . --- ... % drgrecs - 13 - 4u degrees* 45 degree* 13 5 degrees 75 decrees IS -rs^deffrees T decree? 12 25 degrees . 13 45degrees 32.25 12.35 - 11 M 5.75 -: - ' - -Tabl e %--Thri'shohl Dclcni<i<uilions in Cal 119 /j SJiiuuhuion - - of ' N*ve -. - *' - .. "Threshold Stimulus - Po?!titG of Secondary Coil Voltage dl Brimary Current Left Sid* " Right ("Weighted) Sid^ JSihmllolioa ,-- . ulth Cm- Rotation Cm. Rotation- 2 -; . Break _ 13 " 75 degrees 13 . 75 degree^ While walking and while becoming narcotised with urethane, it showed signs of distinct extensor weakness iiiltlie right forepaw. -_ - Because the electrodes could not be placed on corresponding parts of the -muscles,-direct inusolc-stimulation proved unsatisfactory. The animal war in too poor .condition lb give good action currents. Constant stimulation fatigued ;thc extensors of both sides easily;". - .5 - '- Cat 319. Control--Except that this-animal received, no lead, it was treated like cat 107. . .... ............ - - . - , -(Thc ease .tvUlr wItielt .the fiittselcs were fatigued in the "leaded" - animal, and the rapid rise of threshold on both sides are evidences that - the circulation of this animalwas poor. Nevertheless, the threshold of" the right side was somewhat higher tlwn tliat of the left .except in two instances, when fatigue was setting in rapidly. The constant threshold in the control is noteworthy, " "- - - KEZX1KOFF-AVB--LEAD STUDIES_ ' -163 - Ex p e r ime n t s . - '. Cal 131, "Leaded."--Lead was given inlratraclrcafiy. Tlic weight placed on - the-right/orepaw was SO Gni. The animal .was exercised f.vr.-a period of fifty- five days (16 miles'). The body Height-Tell-from 3 to 1.8 Kg. This animal used the forelimbs to a limited extent dn the drum. It sprang across the drum, exerting almost its entire effort with the hind-limbs.- As far as could be determined ironi threshold and action currents, there was no diminution in - the threshold of the weighted side. _ . "' - _ ; This animal showed good extension-of "the weighted limb before the: _ operation, and therefore the. ahsencc of "any itufviVsc iit threshold is not surprising. In fact, it confirms a .certain relationshij)- between.the - Utrcshokl of the nutsde and its elinicaLappearance. ... ' -' ~_ , ` '" ", j j ' i- .; ;1 ' "j *j - - -- .c o mme n t - " _. The original purpose of^these mammalian experiments was Ttot - !( [ accomplished. Our .object in this stud)' was to localize the" lesion of - --- . . palsy by producing paralysis and then to obtain action currents front : : muscle and nerve. Only toward the end of the" investigation, however, - - " ;. _ was technic developed-By which such action currents could be obtained -- " " . , r without artefacts."- At this time the`.'leaded" animals Were in a poor - - .- rr i condition, and none were really fit subjects for such an experiment; It r .i is clear from these "experiments, however, - that extensor weakness -' follows muscular work. Both limbs performed approximately the same '; movements, but the muscles of one "had to do .more work because of the 1. -i - - attached weights; Consequently,"this suggests that lead injury is caused " -j by muscular activity ami fatigue. i Proper appreciation of these experiments requires a brief considers- "- j - tion of the chemical relationship-between lead in the . bod)- and the " ' . products of muscular activity. In this laboratory, both Fairhal! ** rmd " - '. . Minot 30'have shown that lead is deposited in the bonesns insoluble lead . j: phosphate, but when mobilized it is transported by tlic'blood stream as : _ - . ; the triple phosphate. Any bodily change which tends to disturb the 1 acid-base equilibrium--for instance, acidosis--liberates lead from the : ' bones and greatly increases the.quantity in circulation.21 This was illustrated by the increased excretion offcad by both patients and'aiii- ' : - - ' mals when they received a diet low in calcium and were, treated with . . _. either ammonium chloride or acid.'- Acute respiratory infection seemed,.,. ..... to produce the same results in animals, r Lead probably acts in tltis way-; . ' " because (be phosphate is very soluble in'a'tfids,.'especially in lactic acid "19 20 * * * 19. Fairhall, L. T.. and Shaw, Cliarfntte P:i Lead Sttulirs: N. The Deposi ts <n of Lead Salts.-J. Indust. Hyp. G:159 /Aug.) 1921. "... .- 20. Minot, A. S.`; Lead Studies: _.V.' A._B. C. i.ia>T rjyiributitiiTin tlic : ` ' ' _ Organbni, L Tndiist; Hyp, 6:125. 137 and 1-19-('Aug.T1-924: -- : ' 21...Minot, A. S.. ailri-Auh. J- C. ::-T.ohd Similes: XII. lyin' Mechanisms -I--. -for Storage anil Excretion of Lead; to be.'published. -. - .... . -- - , . DUP050332746 ..... - - .464- . tikCHires oK--xwuotOGV'.-iXD-j`SYCtiiATKr _ . .. whi'ch hslibaated in large amounts during muscular activity, particularly - _. ... j . - . during dvttigv,lug .exercise.81 'Thcfrfure, Avhen lead phosphate circulates - .. 7 - IhruttglVinuSde uiitferjgomg vigorous exercise, it must be dissolved to a ~ '1 *-' fcohsidehirble extent Ujr tiicMaetle add'and thus be converted to lead " ' ' " ' - lactate. *" '- - '_ - .vv . - 5 r -- If s(telf a soluble lead salt as the lactate conics into contact with a t 'ceb/it can unite \vith" the inorganic phosphate present at the surface ?s : ami form insoluble lead phosphate and free lactic acid. In work already . : - re)>ortwl.*-it'-'was suggested that the precipitation of this-insoluble lead . - . salt, which is accompanied by the liberation of free acid and the removal" of buffer phosphate, changes the colloidal state and the properties of the : ... - . red cel) surface. There is 'considerable evidence that this also'occurs in _ - - - muscle. `Clinical observation has established the fact that the muscles ; jiaralymLaVe those which arc most used. As far as is-known, only- - .. " ... .tuuscle and not nerve becomes fatigued,-and "cxiitrimenlR fn vitro have : ... .- - . .... . . ... . : . _ .-- demonstrated that dead'docs not interfere with-the function of isolated " nerve, whereas it does greatly affect muscle. This change in muscle .function is preceded by an alteration of tlic surface permeability, and it-is therefore probable that lead-acts on muscle just as-on red blood : ;- . -cells by dwnging the-permeability of the surface. ' ~~ ; Thus these.considerations afford a possible explanation of the devel- . -- opment of lead palsy-: Lead is 'transported by the blood as an insoluble - ' . -. - ", "phospliate in colloidal state.- In regions of muscular-activity this is ' dissolved -hy-the excess lactic acid -which diffuses .from fatigued muscle - . . - - cells and is converted into lead lactate. As the soluble lactate conics in ; ... ... . ;contact witlt inorganicjihospluttcat the surface of muscle cells, the lead ' . ... ... ....... ; , is reprecipitated as insoluble phosphate--a reaction dependent oir the relative concentration of lachiie and phosphate. - ., j ." -_" " - t ' j i` i '' J' ; * .; ` - - COX.CLVSIOXS - ' __ - ` 1. Experiments with isolated ncr've-mnscic preparations from frogs . - have.shown that the onset of fatiguc in "muscles which have been exposed `. ' to lead is much more rapid and complete than normal. " : ' . . . -- : 2; The contractility of "leaded"' muscles is often completely lost, and ' recovery from fatigue is always impaired. . -3. Wlieu muscles are immersed in Ringer's solution containing lead, " '. . the"acidity of the. solution increases'markedly. ` ... , - -4l- A change in the permeability of the surface of muscle cell after -. .. : exposure tsi lead iss evidenced by an increased diffusion of-inorganic ..... . : phosphate frimi tlie,muscle into'the surrounding Ringer's solution. . ': J 1 j j " 22. Rit'clikv Av-J.; The Reaction of Resting and Active Muscle, t. Physiol. _ , 50:53,.1922. Parr, D. P ; Itiinwieli, H. aud-Grrcit, R.-J'.: Studies in the -' " Physiology of Muscular I'.vmsc, .1. Biol. CUvnt. S3:-105. <March ) 1033. ~. ' - - 2X Kml.tlon and I.aqr.ev (footnote 1. tenth reference}. . '' DUP050332747 KEXSIKOFF-.lVB~UwW SICDIES -J65 - 5. As fat as cm be seen f rom" the response of muscle , to nerve stimulation,-lead salts seeiu ter have no deleterious-action on the con ductivity of the nerve of an isolated netvc-imtsde prqtaratiun from a frog. . . _ ..... " 6. In one'experiment, records of action currents taken from both nerve and. muscle also indicate that lead acts on muscle anti not.on nerve. 7. Marked weakness or even palsy develops in fatigued extensor muscles of "leaded" rabbits and cats.: This was manifested by great difficulty in extending-the forepaw. . . . 8. The threshold-of these weak muscles to stninilation.is much higher than that of intfatigued muscles or of the corresponding muscles in control animals. . . ' - :1 "... .. * .. 9. Xcithcr lead nor fatigue alone causespatsy, hut both arc necessary; to.establish the condition. 'This is indicated by experimeats in vitro with isolated nervc-musetc preparations and by-the similarity between the threshold tallies of unfa.tigued muscles-of ``leaded'' ammals'nnd-the muscles-of the controls-- '- 1_ - 10. Such experiments indicate that the physiologic lesion of lead jialsv is in the muscle itself and that the muscles- v.diicii are fatigued are mr>t susceptible to lead paralysis. : ' :- ' ' - 11. An attempt has been made to explain'susceptibility 10 lead -palsy on the hasis of the chemical and physiologic reactions between .the metabolic products formed during muscular activity atul lead as it occurs in the human organism. - . _: