INSTRUMENTATION
Advisory Panel J o n a t h a n W. A m y Jack W. F r a z e r G Phillip H i c k s
Donald R. J o h n s o n Charles E . Klopfenstein Marvin Margoshes
I
H a r r y L. P a r d u e Ralph E . Thiers W i l l i a m F. U l r i c h
I
Ion Specific Liquid Ion Exchanger Microelectrodes Miniature ion specific electrodes with liquid ion exchanger membranes provide a convenient means of measuring intracellular ionic activities in living cells JOHN L. WALKER, JR., Department of Physiology, University of Utah College of Medicine, Salt Lake City, Utah 84112 Since the d c ~ d o p m e n rof ion specific e3 for hydrogen nnd the alkali ?:itmicroelectrodes have lieen iabricaret1 f r o m tlieae gln-scs ( 4 . 3 ) . Chloride microdecrrode? l i n w lieell i ~ x t l cby coating :I plntiiiiim nirom the cntl of :I r.1 with -lg--lgCl ( 4 ) or li!. clepo-iting silver cliloricle in-icic the tip of n micro1)lpeT (6). The-c electrockc a11 h v c thr tlr:in.ixick of linviiig :I large ion ,5eiilitivc ,.iirfaec. which I n i i s r lie entirelj. within the cell n n d their ii>e is, tliereforc. limited to cellb the size of skeletn! mnsclc or larger. Vliilc these electrotlcs m n ~linl-c L: tip dinmeter of oiic niirron o r h 5 , tlic scnsitii-e nren is on The order of ten microns i n length n i t h n tlinmeter of five microns or more 3t t h e to11 of thi.5 nren, In :iddiTion t o the problem of size, the!. :irr tlifficiilt t o fnhricntc.
other ion specific liquid ion erclinnger clccrrodcs Iinr-e l x c o m e coiiimerciall!:iv:iilalile, This piper dimisses t h e 1 i i i i i i : i t ~ i r i ~ ~ i t i oof I i tliccc electrodc,~for i l i r c>spi-c+ purpose of iiicnwring intrncellu1:ir ionic activities i n living cells. -4 IicIuicl ion erclinnger i- cornpoaed of :in organic electrolyte tlisdo!vecl in :I n-ntc'r-iiiiIni-cii)!(' .solI.ciit, ii-ii:illj. :in o r g x i i c d v c n t n i t h :i low dielectric Co1i~t:lliT. The organic ion -1iould have ;i Ion- v x t e r 5olubility :inti is often liiglily hra~icliedto prmwit iiiicelle form:ition ( I ? J . Owing i o The low dielrctric coiict:iiit of the esclinnger, inorganic ion.. l i a w :I w r y low diiliility i n the c~scl1:inger n i i t l . convqiientl!,, n nieni1)r:ine made of :I liqiiitl ion cschnnger i- m ~ i c f inigre perincnhle to ions n.liosc v:iIcmce s i g n is opposite t o tlint of The org;miie ion 11i:in to ions of tlir -rime \-nlcncc ;ign liccnuse of ion p i r formnLiquid Ion Exchangers t i o n ivith The orgnnic ion. For eznmple, negatively clinrgctl 1ilioqdi:itc esLiqiiitl ioii esclinnr.cr~-h i - c liccn i i w l ter- :irr cniion cscliaiigcr~~ while posiiii lirliiitl-liquid ioii crtr:iction processe. tively cli:irgetl nminr. :ire miion czi n intiiiqtry for sonic time niid, from c l i a n p c r ~ . Fnri hermore. some csclin11gtimc t o time, 3s niotleli for liioiopicnl rr,< csliii)it mnrkccl sclcc,tivity 17itliin a nicnil)r;iiic's ( 7 , 8 1 . In t h e p n s t decndr, gro\ip of ions of the s i m e valence signI T I F ~ C . ~ : i qreiicwetl intrrcst in liyiiid ioii cscli:iiigcri on the p i r t of hiok)gi~~T,5, tiic zclcctivity lirincr n fiinction of the , - t r c n ~ t hof iiitcrnction hcTWW1 t h e or:iiid t h c thcor!- of tlicir lx4invior n~ ion sclcc t ivc m em1 r,i nc K : L clewlo ~ pcd ( 9, g n ri i (, niI tl i no rpnn ic ions. .\n ion .qmifir ?l?cTrotl? i; made b y 1 0 ) . .\ithe m n c time. :I practicnl ~ i l iorminz ;I liquid ioii cschnngrr memc i u m clcctrotlc ~vn: dewloped n-hirli 1)r:inr i n :I -iiitnhlc lioltlcr PO thnt one iitilizcs :I liqiiid ion csclinnpcr mcmiitlc of the mcmhrnnc is in contact n-ith 1ir:inc tlic w i ~ i t i ~clcmcni -r of tlic n u :i(iiicoii. rcfcrencc mliition of conclectrncle ( I 1 ) . Since TllPIl, m - c r d
Table I. Dimensions and Volumes of Some Electrically Excitable Cells Kind of cell
Cell shape
Diameter
Length
Volume
Molluscan neuron Frog skeletal muscle Frog ventricle
Spherical Cylindrical Cylindrical
1 mm 0.1 mrn 0.01 m m
30 mrn 0.13 rnm
0.52 pI 0.25 pl 1 X 10-j HI
ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971
89A
Instrumentation
stant composition while the ot’her side can be brought into contact wit>lithe solution to be analyzed. Electrical contact is made with tlie internal reference solution by means of an electrode which is reversible to one of the ions in the reference solution. The electrical potential difference across the membrane changes in proportion to the activity in the test solution of the ion(s) for which the liquid ion exchanger is selective. -4 quantitative theory has been devcloped (9. I O ) which describes the electrode potential, but it is cumbersome and contains parameters which ;ire difficult to measure. It is, therefore, more convenient to use the following empirical equation:
E =
E i- tlie electric potential (volts) ; E, is :i constant (volts) : R is the gas constant (5.3 joules deg-lmole-1) ; T is t’he temperature (OK) : F is Faraday’s number (96,500coulomb equivalent-1) ; n is an empirical constant’ (dimensionless) cho,sen so tliiit n R T / F is the slope of tlie line n-lien E is plotted as a function of logt, ai when K i j a j = 0 ; z i and z j are the valences of the ith and jtli ions, respectively; ai is the activity (arbitrary concentration iiiiits) of the ion tlie electrode i.5 espectecl to measure; the aj’s :ire the activities (same units as a, i ) of . intcrfcring ions whose vrilence sign 1s that of the principal ion and the K,j’s are the selectivity constants for tlic jtli ions with respect to tlie ith ion. When K , , < 1 the electrode has a higher eclectivity for the ith ion than for the jtli ion. K i j is an empirical number and slioiild not be assigned a strict physicnl interpretation. The problem, then, is to form a liquid ion eschangcr membrane in a holder smnll enough to insert into a cell so that) one surface of the membrane is in contnct with the intracellular fluid.
~~
ganic liquid membrane will not reniaiii in place in t h e tip of the pipet. Instead, it will lie displaced by one of the nque011s phases with wliicli it is in contact. To prevent this, it is necessary to render hydrophobic the portion of the pipet wliicli is to contain tlie organic liqiiid. The metliod used to do this is to apply a n organic silicone compomici to tlic terminal 200 microns of the pipet tip . 1 buccessfiil method for making potassium and chloride microelectrodes involves the folloring steps, Boro,silicare ghss capillary tubing (1.2 mm od, O..?-mm thick 1v-311) i: clennecl with hot etlianol vapor and dried. Pipets are piilled from the clenn tubing with a commerci:ill\. nvailable pipet puller niid Iinve n tip diameter of 0.5 to 1.0 micron. Immedintely nfter pulling, the pipet t i p are clipped in :i fresh solution of Silicliad (Clny-.kIams) in 1cliloroiiaplitlinleiie i i n t il tliere is a collimn of the solution about ‘200microns long iiisitie tlie tip. This tnkes npprosinintel!. 13 sec. The pipets :ire then p1:iced tip 111) in :I metal lilock and n-lien t h e de3irctl iiumlm (onc to two dozen) linve I~ecnprepnrcd, the!. :ire placed in :I 250°C oven for one lioiir. *\fter hcing removed from the oven anti allonerl t o cool, the pipets ;ire rend!. for filling hut can be held in this condition for :it least one week before being filled. To fill a pipet, the tip is dipped into tlie ion exclinnger (Corning code 477317 potassium exchanger or Corning code 377315 chloride esclinnger) iintil tlie terminal 200 microiis (npgrosimatel!.) of tlie ti11 is filled ivitli the esclianger (one to tn-o minutes). One-half molar KC1 is then injected as far doivii into tlie top of the pipet as possible using a three-in., 30-gauge needle attached to a syringe. The pipet is placed horizontally on the movable stage of n compoiind microscope and n hand-draIvn, solid glass needle monnted 011 a micromanipulator is advanced down the inside of the pipet while view-
~~
ing under lO0X magnification. The tip of tlie glnas needle is brought t o the center of the field and tlie microscope stage is carefiill!. moved until the tip of the needle touches the meniscus of the ion eschanger. The KCl solution flows doivii to the liquid ion eschanger, displacing the air toward the top of the pipet where it can he fluslied out using tlie 30-gauge needle. -4ir bubbles of 100 microns or less in length may be disregarded since they will be quickly absorbed. With practice, electrodes can lie dipped and filled in an average of five min. -4 sninll amount of mineral oil i$ then injected into the top of the pipet to prevent water evaporation, nnd the pipet is stored with the tip in a solution of 0.5.11 KC1. Best, results are obtained when the filled electrodes nre allowed to stnnd for a t least two hr before being used. Figure l shows a scliemntic dingrLiin of an electrode ready ior ii.se. and Figure 2 is a pliotograpli of :I potassium niicroelectrode nitli 125 microns of liquid ion esclianger in the tip. Tn-o tlificulties have been encountered with these electrodes. Sometimes the tips of tlic electrode; will not fill with the ion esclinn vcrj. slo~vly. This : i l ) l ~ . , t o tlic tips being plugged by the Piliclnd. If n pipet doe. not t A e u p the ticsireti niiioiiiit of eschnnger within two inin, it is dikcarded. =in nlternative to this is to break the til) slightly, biit this is onl!. useful if n tip diameter of two micron: or lnrger caii be tolerated. Tlie other difficulty is that the electrodes n-ill sometimes lose the eschanger n-itliin two to three lir after being filled. Tlie cause for this difficulty lins not heen determined. To alleviate tliese problems, other siliconizing procediires wing n variety of organic clilorosilnnes in different solvents are being investigated. .4lthougli both kinds of electrodes csnn be made in the same way, there is another method of siliconiziiig which
Electrode Fabrication -4 standard technique for making intracellular electric potential measurements has been to piill a glass micropipet with a tip diameter of about 0.5 micron and fill it with 311 KC1. Estensive research with this technique has shown that when the tip of such a pipet is inserted into a cell, it does not disrupt the normal functioning of the cell (IS) and, therefore, if a liquid ion exchanger membrane could be formed in the tip of such a pipet, the problem would be solved. The primary difficulty is that a clean glass surface is hydrophilic, so an or90A
Figure 1. Schematic diagram of an ion specific liquid ion exchanger microelectrode
ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971
are presented in Table 111 for the chloride electrode and for potassium electrodes made by both of the methods described above. Making Intracellular Measurements
x
B Figure 2. Photograph of a potassium liquid ion exchanger microelectrode with 125 microns of ion exchanger inside the tip. Magnification, 400X
works better for the potassium electrodes but is not satisfactory for the chloride elcctrodes. After bbing pulled, the pipet tip is dipped in a 5% solution of tri-ii-but~~lclilorosilanein 1ehloronaplitliale~ieuntil there is a collimn about 200 microns long inside tlie tip (about 15 see). The pipet is then :~lloa.edto air dry for a t least 24 hr before being filled as described above. The tips of pipets siliconized in this m y do not plug up ns they sometimes do vith the Siiiclad solution. Electrode Resistance and Selectivity Because of the small size of the tip of the electrodes and the lorn conductivity of tbe liquid ion exchangers, the resist.ance of the electrodes is liigh and a voltage measuring device with an input impedance of a t least 10'3 ohms is required. 4 satisfactory solution has been to use a varact.or bridge operational amplifier (Analog Devices, model 31iK) which has an input impedance of 1014 ohms with an input capacitance of two picofarads. The microelcetrode is connected to the input of the amplifier via a chloridieed silver wire inserted into its top, and the ampiifier output is connected t o a digital voltmeter. The resistance of the microelectrodes, as measured from the charging time of the input a i the operational amplifier,is in the range of 109 to io1" ohms. The response time of the electrodes when the concentration of potassium or chloride is changed has not yet heen measured carefnlly hecause of the technical problems involved. However,
when one of the electrodes is dipped into a solution and the amplifier input is opened as rapidly as possible, the new steady-state potential is reached within five see, which indicates that the time constant of the response is not more than one see. Once the steadystate potential is attained, it remains constant to within -C 1 mV for several hours. I n Table 11, some data from experiments ivitli Aplysia neurons (14) are presented showing the long-term stability of both t.he potassium and chloride electrodes. The selectivities of the electrodes for interfering ions with respect to the principal ion has been determined for some intcrfering ions commonly found in biological preparations. These have been determined by measuring the clect,rode potentials in mixtures of constant ionic strength and finding the K i j which makes the data fit Equ at'ion 1. The results of these measurements
Immediately before making an intracellular measurement with one of the electrodes, the electrode must be calibrated. This is done by measuring its potential with respect to B 3M KCI-filled micropipet in a series of KCI solutions o i known concentrations. The KCI concentrations used should bracket the expected concentration of the unknown solution. The electrode is then moved to the solution bathing the cell in which the measurement is to be made, which normally contains some of the ion to he measured, potassium andlor chloride. The potential of the electrode in that solution should agree with the potential predicted for the electrode from tlie calibration curve, taking into account any interfering ions which may he present in the solution. The tip of the electrode is then inserted into t,lie cell. Since it is not possible to see the tip of the electrode miter the cell, the criterion used to determine cell entry is an abrupt shift in the potential of the electrode as it is s l o r l y advanced toward the cell. This shift in potential is dne to two factors: ( 1 ) the difference in activity of the ion being measured between the extraeellnlar and intracellular fluids; and ( 2 ) the cell membrane potential. The shift of dlie elcct,ricpotential as the electrode tip enters the cell can be written as: AE
=
ai"
at
+
Kijaj.
+ Kija,i
where A l 3 (voltq) 1s the difference in electric potential between the inside :md the outside of the cell; E,,$ (volts) is the cell membrane potential; the
T a b l e II. Data from Experiments on Aplysia Neurons with Chloride and P o t a s s i u m Microelectrodes' (14) Potential in artificial Slope, mV seawater, mV Time in Before' Aftera Before2 After2 cell@), min 58 58 58 58 58 54
58 ~.
34
58
31 48
58 58 58 54
44 35 46
34 30 48 44
36 44
335 486
137 118 347 495
'First five measurements are with potassium electrodes and last five are With chloride eiectrodes. In most experiments, more than one celi penetration was made with the electrode during the indicated time. Wefore and after intracellular measurement(s1.
ANALYTICAL CHEMISTRY. VOL. 43, NO. 3, MARCH 1971
91 A
Only Jeolco's new laser Raman spectrophotometer records depolarization ratio automatically and simultaneously with the normal Raman spectra. 0 Jeolco's compact, table-top JRS-S1 is the first unit to give you this important advantage. The first to record depolarization ratio throughout the measured range, automatically and simultaneously, to aid in the assignment of the Raman band. This all-digital model offers the following high performance features: better than 1 cm-' resolution, less than l o - ' ' scattered light at 25 cm-' shift, 10,000 cm-' spectrometer cover range, with 30mW He-Ne laser as standard equipment and the ability to adopt any available cw laser source. 0 High sensitivity benefits include: special low-noise photomultiplier with extended S-20 photocathode, thermoelectric photomultiplier cooling, low-noise photon counting system, optimum sample chamber system, and double-monochromator (f/6.7). 0 Pushbutton selection of operating conditions, with warning lamps for mis-selection, large sample chamber, two-pen recorder with automatic adjustment of wave scale to monochromator sweep range, safety circuit for photomultiplier protection, and automatic attenuation of laser - to protect operator's vision. 0 The unit's monochromator drive is linked to the recorder drive. With automatic setting of repeated recording. Half of the recording scale is above 2,000 cm-' to match the infrared spectra. 0 For full details call or write Jeolco, 235 Birchwood Avenue, Cranford, N.J. 07016: Tel. (201) 272-8820.
Jeolco. Putting the70's into focus.
Mass Spectrometers/Amino Acid Analyzers/Nuclear Magnetic Resonance SpectrometersiScanningElectron Microscopes/ Electron Microscopes/ Electron Spin Resonance Spectrometers /X-ray Diffractometers/ Laboratory Computers Circle No. 102
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on
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 3 , MARCH 1971
Instrumentation
Table 111.
Selectivities for Interfering Ions with Respect to Principal Ion for Chloride and Potassium Microelectrodes KIJ
Interfering ion Bicarbonate2 I set hiona te2 Propionate2 Calciu r n 3
O.lM1 0.05
Sodiu rn
1.OM1 0.05
0.2
0.2
0.5
0.7
0.002
0.002 Hydrogen'
~~~
0.02 0.025 0.02 0.02
a' b a b a b
0.03 0.03 0.03 0.016 0.02 0.014
1 Ionic strength. .' Ions interfering with chloride. I Ions interfering w i t h potassium. refer t o Siliclad a n d tri-n-butylchlorosilane potassium microelectrodes, respectively.
superscripts on tlie activities, o and i, refer to outside and inside of the cell: xiid j t . tic~tcrminedfrom the calibratioii curve for each electrode, is 1.0 for the iiiiii electrodes :inti in the range of 0.!10 to 0.97 for the chloride electrodes. Ts'liilc )i varies from one cliloritlc electrode t o another, it is constuiit for any one electrode and cmst:iiit over tlic range of :it least 1.0 x 10 :{JIKC1 to 1.0.11 KC1 for liotli pot:i-siuni antl eliloridc clectrodes. I;,,, i i nic:isureti by impaling tlie cell n-ith :I 3.11 KCl-filled micropipet, Klieii Equation 2 is >oIved for tlie dr~ioniiiiator of the 1og:irithmic term, it t:tkc- tlic form of Equation 3.
all
+
fi,,ull
=
It is ion. :I biinple matter, knowing all the fnctors 011 tlie right side of Equation 3, to cdciiltite :i numericnl value for the left d e . The only remaining prolileiii is to determine the 1-due of tlie t m n , Ai,cr,l. If microelectrodes , - p i f i r for the interfering ions are :ivnilable, tlie q i r s cnii be niewured directly. I7nfortumite1yj this is not 11s1i:iil:. po,Gsilile and, therefore, it is necessary to use e 4 m n t c s arrived at by other nicaiii. TYlieii whole timie annlyTS h v e lxen done, the values of the coiiccntratioiis can lie iised by assuming values for tlic intrncellular activity corflicieiits ( 2 ) . 1ntr;icellulnr sodium netivity c n n lie estimated from the overs h o t of t l i p nction yotmtial (?,? 1, and in the c a w of ion.? which appear to be i d y distributed across the cell mpmhranc, tlic intracellular activity can he calculated by iisinp the extracellular activity of t h a t ion a n d assliming a Doiiiiaii distribution (-31. TVlien the cells are large, a,= in tlie ( m e of .4pl,vsin neurons or frog skeletal mmsclc, the IiC1-filled micropipet c a n be srcn i n tlic sninr cell as tlie ioii sgecific electrode, hiit with srn:illPr cell9 t1ii.s
' a and b
i,s iiot po.ssible. K h e n the cells are too small to see if both electrode.5 are i n the sxne cell, tlie membrane potential and ion specific electrode measurements can be made separately in ,m-cr:il cells and the average vnlues of tlie iiien$uremeiits used to calciilate intrace1lul;ir :ictivity. -4 better solution to the problem is to make double-b;irrcl electrodes where one barrel is tlie ion sliecific electrode and tlie otlier bnrrel i-. tlie reference electrode. -4ttempt.s to hliricate fiicli electrodes are currently lieiiig nixle. -I>.slion-n in Table 11, both tlie iiot:iAum :iiid chloride microelectrode. :ire stable over a period of severd lioiirs even n-hen several cell penetrntion+ are m n t l e with the same electrode. Wien an electrode has slionii n DC drift, it has usually been traced to a cliniige in the potentinl of the .lg;IgC1 electrode mnking contnct with the interiinl reference solution of the electrodr. Most important is the fact that t h c slopes of the electrodes do not c1i:iiige during tlie coiirse of tlie esperimeiits. Sirice tlie intracellulnr iiiea.iirenienta ;ire mndc with respect to the estrncellulnr solurion, rvliicli is of know11 composition, a .slow DC drift is of little consequence as long ns the slope of tlie respoiise ci:rve remniii,. c0lldt:lllt. is the case wit11 any new tcchiiiqiic, it i q necessary T O c.tnblisli tliat the dntn obtniiied with the technique are v d i d . In measuring iiitracclliilar :letiTiTir.5 with ion specific electrodes, this is not ency t o do because of the kick of ;icciir:itr information concerning intracc1liil:ir nctil-itirs of ions, both orgnnir nnd inorganic, n-liicli ma:. interfere n i t h tlic meawrcment. This is erpecially true of tlie rhloride electrode lxxiii,=r of the presence of organic anion:: in the intrncelhilnr fluid. Some rsperimeiitril re.sii1ts bcnring o n this point linvc l>eeiipresented :inti di.=cnssetl liy Cornn-all e t al. ( 1 6 ) .
.It tlie present time, oiilj- tlie poirim niid chloride ioii eschanger microelectrodes are of practical use. Attempts to make a calcium microelectrode have not been comiiletely ,-iicce.d'iil, altliougli come progress has heen made. They can be made to esliibit n S e r n > t slope in solutioiis of C:iCl, from 1.0 x 10-iJI to 1.0.11 with :I time coiistant aiici stability comparlible t o tlie potassium and chloride microelectrodes. Ilowever, when potas>iuni> i n concentrations upproximating those found inside cella, is added to tlie CnC1, solutions, it reduces the calcium re;ponse dra-ticall:.. K o r k is continuing on the calcium niicroelectrode and as liquid ion escliniigers for otlier ions of intere-t to biologists become available, the!- nil1 lie u e d to m i k e ion specific microelectrodes or' tlie type described above.
References
( 1 ) ,J. -4, ,Jolinson, Anlei.. J . Physioi. 181, 263 (19%). ( 2 1 E. .J. Conivny, Piiysioi. Rrc. 37, b-1 (1957 1 , ( 3 ) E. Page, J . Gen. Physiol. 46, 201 ( 1962). i 4 i ,J. -1.11.Hinke in ,'GI elcctrotlcs," Laxilee e t Kilt!., S e n - Tork, S.I-.1969. ( 5 ) S. \I-, Carter, F. C. Rector, Jr., D. E. C,impion, and D. W.Seldin, J . C'liii, Z i i z i r s t . 46, 020 (1967 I , ( 6 ) G . -1.Jierkiit antl R . S T . lleecli, LiJC s c i . 5, 453 (19GBi. (7') I?. Brntner, A m p i , . J . Physiol. 31, 34:3 ( l 9 l S I . (Si G. Eisenmnn, ,J, P.Saiidhlom, and .J. L. TT'nlker, ,Jr,, Scieiicc 155, 965 (I9G7). 9 i ,T. P.Sandblom, G.Eisenman, nnd .J. L. JYalker, Jr., J . P h y s . C h e m . 71, 3S62 (1967). 10) ,T. P.Sandblom, G. Eisenman, 2nd ,J, L. Ynlker, Jr., J . P / y s . Cheni. 71, r , -*>\I 1 (1967. c i e ) i c e 156, 1;iTF (10071. 12) I:. liunin a n d -4. G. ITiiiger, d?igcic$.Chem.. Z i t t . E d . Eugi. 1, 149 (1962). (1:; I IT. I,. S;ihtuk n r i d -1.L . Hocigkiii. J . C'cli. C ' o v ) p . Piqsiol. 35, 39 (19501, ( 1 4 ) D. L. Kunzc nnd A. 11.Rroivn, wipiihlislicd rewlts! 1970. (131 ;1. L. Hodgkin and R. I i n t z , J . P / y s i o / . 108, 37 (1949). (16) 11.C . Cornrr-all. D. F. Petereoil, L. Kunze, ,J. L. K n l k e r , ,Ti-,, Lind -1, 11, Brown, Riniu Rcs. 23, 133 (1970).
n.
. \ ~ a & a t i n n Grant #70 887. Yt,nh Heart .\sinciation Giant 1T. and rile Tnitecl S t a t e s Public H m l t l i %r\-ire Grnnr G l I 14328.
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