Applications of bioelectrochemistry in medicine

Bioelectrochemistry and Bioenergetics, 19 (1988) 191-205 A section of J. Electrotanai. Clrem., and constituting Vol. 253 (1988) Elsevicr Sequoia S.A., Lausanne - Printed in The Netherlands

Review

Applications

of bioelectrochemistry

in medicine

Giulio Milazzo Presidext of ihe Bioe~ectrochemical Society, Piaxa

G. Verdi, 9, 00198 Rome (Italy)

(Received 24 September 1987)

ABi3TRACT

During the last half century, bioe!ectrochemistry has enjoyed a very lively development in very different directions, among which the medical applications are still extremely important. _ TICS review covers: (a) diagnostics using electrophysiological and electrochemical methods; (b) therapeutics using low-frequency alternating pulsing electromagnetic fields, hyperacidification and other auxiliary means: (c) corrosion studies to identify the metal best suited for implants in iiving bodies; and (d) electrochemical techniques to modify the genetic content of appropriate micro-organisms to produw at relatively low cost products which are extremely important in therapy such as insulin, interferon, etc. iNTRODUCTION

Considering the enormous progress realized in the field of medical applications of bioelectrochemistry, this review should give a general overview summarizing and illustrating as far as possible, on the basis of various examples, the different subfields in which electrochemical methods have proved to be very useful in medical diagnostics and therapy. Two centuries have elapsed since Gaivani’s discovery of strong interactions between electrical phenomena and biological objects. During this time, our knowledge about the essence and mechanism of these interactions has shown remarkable progress in very different directions (even in ones not suspected in Galvani’s time), so that -.ve may think of possible applications of bioclectrochemistry inkmedicine for the benefit of mankind. The differentiation in the development of bioelectrochemistry makes several such applications possible, even if for some of them we are still not able to offer a definitive interpretation of the mechanism of action. Some of the more important applications are reviewed below. 0302-4598/88/‘$03.50

0 1988 Elsevier Sequoia S.A.

192

DIAGNOSTICS

.

A first gross classification of medical applications in general can be made bY dividing them into those developed for diagnostic purposes and those developed for therapeutic purposes. In the first class, it is further possible to identify two groups, the first of which is based on electrophysiological phenomena. It has been observed that the transmission of information in living organisms occurs, at least partly, via coded electric impulses sent through the organized network of the nervous system from the peripheral terminals to the central computer (i.e. the central nervous system) and back again, after elaboration, to the peripheral system in order to stimulate specific organs for action. Not only is the response to certain slimuli (i.e. second-hand information) characteristic of living functions, but also the complete working of living organisms is based on this kind of electrophysiological transmission of information, as demonstrated by the regulation of the cardiac pulsating rhythm and by the regulation of the respiratory rhythm of aspiration and expiration by the lungs. Of course, information is also transmitted along other lines, and particularly a chemical one by elaboration of chemical species which, acting as messengers at specific sites, carry the coded information to the designated point utilizing specific transport mechanisms. But it must be stressed that the synthesis of these chemical messengers is again a consequence of coded information transmitted to the synthesizing organ. On the other hand, many functions produce electric phenomena, mainly po;cntial differences, which can be collected by appropriate external sensors. These sensors qualitatively reveal and quantitatively determine the arriving signals by means of suitable amplifiers which select them and make them readable. This is the basis of electrocardiography and eiectroenccphalography, already developed for a long time, even before the principles of bioclectrochemistry were established and investigated as a separate discipline. Thus, it could be stated that some electric signals produced by sound organisms at certain body sites and captured by appropriate sensors are different from the same signals elaborated by the same organisms under pathological conditions, thereby permitting accurate diagnosis of illnesses, malformations, malfunctioning, etc. The very first origin of these signals and the reasons why they are different in the physiologically sound and in the pathological states are not always fully understood, so that it will be the job of young researchers to investigate these phenomena more deeply in order to improve these kinds of diagnostic techniques. Another widespread application of electrophysiological phenomena - really not diagnostic but therapeutic developed more recently is represented by the pacemakers used to corrrect abnormal cardiac rhythms by sending regularly-timed electric pulses to the heart’s contraction musculature to obtain a regular cardiac rhythm. The second group of diagnostic apparatus is based on the properties of certain component chemical species of living bodies which may be electrochemically qualitatively detected and quantitatively determined, directly or indirectly, through

193

suitable

Electrode reactions

and corresponding electrodes. The number of such biosensors has gmwn enormously in the last few decades, thus making possible a very large number of qualitative and quantitative analyses using different electrochemical techniques. An analytical biosensor consists in principle of two or three components: a receptor, possibly (really mostly) an intermediate membrane, and a detector (electrode). ‘Without considering the classical apparatus (diicctly electrochemically sensing the component to be analysed, as, for example, first kind and pW electrodes), it is interesting to look at the most recent developments in bioanaiytical sensors almost all of which consists of a receptor (i.e. mediator iucorporated in a membrane) which, by chemically reacting with the species to be .nnalysed, produces an electrochemically active species in a quantity linearly proportional to the amount of the original species to be determined. The thus produced species is finally determined electrochemically by using mainly potentiometric or voltammetric techniques, and (possibly) other techniques such as coulometry, chronopotentiometry, etc. The properties of the receptors must be: (a) a high selectivity towards the component to be analysed, thus making possible direct determinations in complex matrices, like the biological ones, avoiding time-consuming, often inefficient, probably dangerous and expensive procedures for sampling and species separations; and (b) a high sensitivity, so that the sample size can be remarkably reduced or the lower limit of detection can be further lowered. Under these conditions, a large number of enzymes have been utilized, after immobilization in suitable supporting membranes, thus taking advantages of the high selectivity and sensitivity of enzymatic reactions. Even membranes with immobilized living bacteria have been utilized for some specific purposes, and artificial membranes with pores of certain diameters have been designed and produced to improve selectivity against charge and molar masses. Another very important advantage of the electrochemical apparatus and techniques comes from the possibility of miniaturizing and sterilizing, thus enabling non-destructive analyses to be made in viva, which are extremely important for knowledge in real time of physiological and of pathological processes. Some electrochemical techniques present the very important advantage that they can reveal and possibly quantify components present in the sample being analysed, producing a recorded result of the analysis which can be cc;nsulted any time later. This is especialiy the case of scaur&ng voltammetry and. electrophoresis. The appearance or disappearance, or even the change of intensity of the signal produced by one or more components can reveal a pathological situation which must be investigated further. Figtire 1 show;; a dramatic modification of the voltammetric recording of the urine of a patient, suffering from digestion troubles, in comparison with the recording of the urine of a sound subject, which is remarkably constant in healthy individuals. The evidence of pathological state does not need any further comment.

Fig. 1. Voltammetric record of the urine of a patient suffering from digestion troubles. ( -) Typical recording from the urine of a sound subject for comparison; (- - --) recording from the urine of a patient suffering from digestion troubles. (Current intensity is given in relative units.)

Figure 2 shows a voltammetric recording of the urine of a patient suffering from acute lymphoblastic leukaemia before, during and after irradiation. Voltammetric peak No. 10 appears much more strongly 1 day after irradation, showing the more abundant presence of the corresponding chemical species. The chemists must identify this chemical species, and the physician must then judge whether this chemical species which appears so strongly during irradiation is dangerous, harmless or useful. An important comment is that these relevant diagnostic results were obtained without any inconvenience to the patient. Other electrochemical techniques which provide a permanent record of several components present in a sample are electrophoretic techniques, either free or on

Fig. 2. Vollammetric recording of the urine of a patient suffering from acute lymphoblastic leukacmiz. (1) Recording from the urine of a lcukaemic subject 1 day before irradiation; (2) during irradiation; (3) 1 first sweep; (- - -) second sweep. (Cwrent day after irradiation; (4) 2 days after irradiation. ( -) intensity in relative units.)

paper, gels, etc. Figure 3 presents an electrophoretic recorc1in.g of the plasma of two patients showing pathological hypogammaglobulinaemia and hypergammaglobulinaemia, respectively, in comparison with the electrophorectic recording of a sound plasma. The much less intense and much more intense gamma-globulin band, respectively, are evident and self-explanatory and do not need any further comment. Some examples of the most recent developments in the field of medical bioelectrochemical analyses were presented at the Bioelectroanalytical Symposium organized by the Electroanalytical Committee of the Hungarian Academy of Sciences [l]. They included voltammetric determination of hydroxyindole, uric acid, catecholamine, indolamine, dopamine in vivo, heterogeneous immunoassay utilizing antigen antibody reaction, adrenal medulla, etc. Also potentiometric determination of urea, choline, lecithine, oxygen, pH and K+ in the brain during transient ischaemia, and the determination of Pb, Ca and Zn at very low levels were discussed. THERAPY

Several applications by means of low-frequency alternating pulsing electromagnetic fields are already used regularly. Among these, tissue stimulations for better

1% HvPdaammaalobu~inemia

t

Gamma-globulin Hypergammagfobulinemia

1

3

(1) Fig. 3. (A) Electrophwograms of the serum of a patient !iuffering from hypogammaglobulinaemia. (2) recording from the patient’s serum. (B) Typical recording from the swum of a sound __. subject; _ Electropherograms of the serum of a patient suffering from hypcrgammaglobulinacmia. (1) the same as (Al); (2) recording from from the patient’s serum: (3) the same as (B2) but after dilution of the serum 1:2.

cicatrization, psoriasis treatment and healing of bone fractures (even of those in very bad conditions, because of infections developed in addition to the fracture) are ‘worth mentioning. According to statistics of some years ago for bone fracture recovery p], more than 75% of the cases treated resulted in a perfect recovery; this figure has probably increased during the last decade (up to 80% has been mentioned). Some extreme cases of very bad fractures healed using the above treatment are listed in Table 1. The result also depends on oscillation and pulsing frequencies and on the field strength. The action mechanism of this therapy is still not fully understood, but it certainly involves electrochemical membrane- and other phenomena. Two more recent interpretations of the interaction between biological objects (tissues and/or cells and electromagnetic fields) are given in refs. 3 and 4, and were discussed by D. Astumian at the 9th. International SymposiuGl on Bioelectrochemistry and Bioenergetics (Szeged, l-5 September 1987). Wider overviews on therapeutic applications

TABLE 1 Extreme cases of bone fractures treated with application of low-frequency alternating pulsing c!;?ctromagnctic fields, followed by complete recovery Patient

Age/ Y-

1

3

2 3 4 S 6 7

12 19 34 38 47 62

Bone

Type of fraclure

Tibia Tisa lz&?~:us ?;‘ihis Shoulder Fibula Femur

Congen. Congen.

No. 01 operations 4 12

COligEL

3

Traum. Trclum. Traum. Traum.

4 3 0 I

Infection

Amputation recommended

Treatment months

NO

Yes

No No ‘1’s YeS No YeS

YeS No YCS No No Yt?.S

2 3 4 7 4 4 6

of low-frequency alternating pulsing electromagnetic fields are given in ref. 5 and in the Proceedings of the annual meetings of the Bioelectromagnetic Society. Another type of therapy for localized cancerous tumours (developed by Professor M. von Ardenne in his Institute at Dresden) has been announced 8s apparently very promising, according to the results obtained so far. It is based on massive administration of glucose (up to eight times the normal content of glucose in blood), followed by hyperthermia up to 42.5 o C and “oxygen multistep immunostimulation” [6]. The resulting hyperglycaemia produces the following effects (of which the second can be considered of electrochemical origin): (1) Synergistic selective thermosensibilization and radiosensibilization of malignant cancerous cells. (2) Strong stimulation of the metabolism within the cancerous tissue with large local production of lactic acid, which cannot diffuse away at the same rate as it is produced. This provokes local acidification within the cancerous tissues, observed for the first time by Fulci 171. This acidification in turn produces occlusion of the capillaries distributing blood within the cancerous tissue (because of the loss of elasticity and flexibility of the erythrocytes [8]), thus stopping any blood circulation within the cancer tissue, and the elimination of locally accumulated lactic acid (see Fig. 4), so that the pH in the stationary state decreases within the cancerous tissue from about 7.2 down to about 6. The therapeutic effect of this hyperacidification and successive occlusion of the capiliaries can be interpret.ated in two ways: cells as a result of them no 60 progressive death of the malignant c&erous longer receiving either food (glucose) or oxygen, both transported by the blood [8]; and retention of accumulated anticancer drugs (for example, Methotrexate) after its massive administration followed by administration of an antidote (for example, Leucoverine). The latter can no longer enter the cancerous tissue (because of stopping of the carrier, i.e. bload flow), so that the action of the anticancer drug remains loczly unaffected while the anticancer drug is

w

198

Transitional mesarteriole

Venule

Capillary

Late stage: artark!

Swelling of endothelial cells (cross section

vesseL dilated and

Decelerated flow pH 57.4

DH Decrease

’ Hyperthermia

lncreasino tendencv

Erythycyte

to aggregate

aggregation

+ lrrewrslble hemstasis

pH 56.7

Incressed hematocrit Fig. 4. Schematic representation of the gfuccse-lactic acid effect on blood capillaries (set text).

being eliminated from the sound tissues, where the blood flow continues as before. The selective action of anticancer drugs will thus be enhanced inside the cancerous tissue only [6].

Uetastasi diswpeat ; \

Fig. 5. X-ray lymphography of the lymph nde weeks after treatment (l3).

metastasis of a clear cell sarcoma before (A) and three

199

(3) The third step (oxygen multistep immunostimulation) has the sole aim of strengthening the resistance of the living organism against cancer it&f and against distress provoked by cherniotherzpy and radiotherapy. Table 2 summarizes the first results of this multistep therapy applied to a group of female patients with cervical carcinoma. Figures 5 and 6 show the result of the aboy+described therapy i?. two typical TABLE 2 First results of cancer multistep therapy according to statistics published in the 1970s a

Recovery after 5 years/% Appearance of mctastases/% Death/%

Comparison group b
Tested group (n-23

72 30 28

92 16 8

a From statistics kindly provided by Professor M. van Ardcnnc, Dresden. b Treated with usual therapy. n: Number of patients.

hrr pawed

Fig. 6. X-ray artcriography of a clear cell sarcoma. A: before treatment: tumor rich in blood -1s. B: after treatment: tumor vessels no more demonstrable (tumor destroyed, normal tissue UndamagecJ, uenr~ femur&s clearly visible).

200

. .

a bone metastasis and a clear cell sarcoma. In both eases, the cancerous tissue disappeared after the glucose-hyperthermia-oxygen multistep therapy. Another case of therapeutic applications is electroanaesthesia, which can be considered as a macroscopic consequence of alterations of transmembrane potentials. It is highly probable that in this case the drugs used selectively modify the electric conductance of cellular membranes for charged species, i.e. for ions and possibly for some other kinds of larger molecules carrying ions, thus producing specific material in balance and the observed anaesthetic phenomena. This field is, however, in its very early stages 193.

ca!%es-

OTHER APPLICATIONS

There are still further fields where electrochemistry is extremely useful for biomedical applications. While it is obviously not possible 20 give details of all of them, two of them are particularly worthy of a short description. corrosion

The first one is concerned with the corrosion of metallic prostheses implanted in living bodies and in contact with biological fluids, as used, for example, in dentistry and orthopaedics. Corrosion has two important detrimental consequences: progressive attack, which can lead to the destruction of the implanted prostheses, and the appearance of toxic effects, due to the general toxicity of the corrosion products. Thus, corrosion in living bodies should be avoided in metallic materials intended for implantable prostheses. Corrosion is the consequence of a typical electrochemical heterogeneous reaction [lo]. A simplified formulation, in words, of the definition of corrosion states that “any element whose electrode potential, under the given experimental conditions, is more negative than the electrode potential, under the same experimental conditions, of another element, displaces this latter from the solution in which it is present in ionic form”. In biological systems, the solvent is water and therefore corrosion can be considered as the behaviour of the metallic material under investigation contacting an aqueous phase containing, of course, H+ and OH- ions. The theoretical tool to investigate and predict possible corrosion is represented by the potential+H diagrams describing the behaviour of the investigated material contacting an aqueous phase where H+ and a simple non-complexing anion are present. The construction of such diagrams is the result of a series of electrochemical and, possibly, chemical reactions. To understand the construction and use of these diagrams, Ni can be considered as a very simple and clear example. The starting point for the construction of this diagram, and others of the same type for other metallic materials, is given by the reactions of Ni with H+ and H,O, their energetics and their electrode potentials at equilibrium. The equations collected in Table 3 describing the reactions and the solubility equilibria of the Ni-H,O system are relevant for the potenti&pH diagram of nickel [ll].

201 TABLE 3

Relevant reaction for the potential-pH

d&ram

of the Ni-Hz0

system.

Reaction

TkTmodynamiccq#ation

Ni * Ni’* Ni+2H,O#Ni(OH),+2H++2 tNi+2H,Or;tHNiOT+3H++2e’ 3NiZ++4H10#Ni,0,+8 H++2 c3NiO+HzO*Ni,0,+2H++2 t3HNi0,+H+#Ni~0,+2Hz0+2 e’ 2Ni’*+3 H,O#Ni,O,+6H++2 c2Ni,0,+HzO#3NizO~+2H*+2eNiz++2H,0skNi0,+4H*-k2 cNi,0,+HzOrt2NiO~t2H++2 e-

U - -0.2!50+0.0295

2 3 4 5 6 7 8 9 10

11 12

Solubihy of Ni-containhg ions in the presence of solid Ni(OH)I Ni2++HzO#Ni(OH)2+2H+ h@Ni2’ ] = 1218 - 2 pH NiO+;ii20 ip HNiO,’ +H+ laHNi0; ] - -18.22+pH

NO.

l&Nit+ 1 -0.110 - o.os91 pl U - 0.648 -0.0886 pl I+O.O295lo&HNiO; J -0,0886 lo&Nil+ 1 U-r.977-0.23Gapr U - 0.897 - 0.0591 pll 1 U - - 0.718 f 0.0295 4-0.0886 to&HNiO; ] u - 1.753 - 0.1773 p1 .-0.0591 h@iz+] u - 1.305 - 0.0591 pl ] U - 1.593 -0.1182 pl I- 0.0295 ,dNi2’ U - 1.434 - 0.0591 p1 ,

u-

p All numerical data arc given for T- 25 * C. On the basis of the equations giving the values of the potential U as a function of the concentration of the ionic species present in solution (Ni2’, HNiOy and H+) for each one of the chemical equations (1) through (10) and considering also the solubility equations (11) and (12), the equ~tibrium dkgram for the system M-H20 can be drawn at 25°C. It is given in Fig. 7 for a Ni2+ concentration of 10W6 JW. The diagram also contains the equilibrium line of water (a) representing the theoretical equilibrium values of the potential of a hydrogen electrode under a pressure of 1 mBar (0.001 atmosphere) of gaseous H2 at 25” C.

6

. Fis 7. Potential-pH

8

LO

diagram of nickel (recalculated for [Ni2+ 1 - 10m6 M and PHz = 1 mBu).

202

Following Nernst’s equation and taking the value

-0.257 V for the standard potential of the Ni JNi z+ electrode, the potential of metallic Ni contacting an aqueous solution of Ni2+ at 10 -6 M concentration will be equal to -0.434 V @H-independent) while the potential of the HJH+ electrode at PHt = 0.081 Bar varies from 3-0.088 V at pH 0 to -0.434 V at pH 8.82, remarkably less acidic than any physiological pH. Only in stU more basic solutions does the potential of the Ni (Ni2+ electrode become more negative than that of the hydrogen electrode, and metallic Ni can no longer be attacked by H+ ions, for thermodynamic reasons. In other words, beyond this potential metallic Ni, under the above-specified conditions, becomes immune. This means that pure Ni at any physiological pH and in almost complete absence of gaseous hydrogen cannot be used as material for prostheses to be implanted in living bodies because it would corrode. Further inspection and consideration of this diagram (for simplicity at pHI = 1 mBar) shows that, at stiil higher pH or at more positive potentials, solid hydroxides and oxides may become so appear as reaction products, which under certain conditions compact that any electrode reaction is completely inhibited: the metallic material is no longer corrodible, it has become passive. From the diagram given in Fig. 7 a second diagram can be derived (Fig. 8 in which the domains where Ni cannot be corroded (because of immunity or passivity) arc clearly &dent. In the particular case of biological liquids containing molecular species capable of producing complexes with Nit+ (organic acids, amino acids, etc.), thus altering the electrode potential of the Ni (Nt.2+ electrode (because of equilibria between the Ni2+ complexing agent and the resulting complex), the potenti&pH diagram will be modified by contributions originating from these complexing and complexed species.

Fig. 8. SimpMcd pH-potential

showing immunity, passivity and corrosion domains.

Fig. 9. Influence of an impurity grab.

203

These considerations are purely thermodynamic and represent the necessary but, possibly, not yet sufficient basis for a reliable conclusion concerning the behaviour and suitability of a given material for use in biological implants. Kinetic effects, which can remarkably alter the values shown by potential-pH diagrams, must also be considered. They are particularly relevant in the case of the hydrogen electrode reaction, since these effects produce overpotentials and slow down the rate of the hydrogen electrode on the metal being tested. The best way to investigate the kinetic effects in corrosion is represented by the polarization diagrams showing the dependence of the potential of a given material on the current density toad when it is connected as the auode and subsequently as the cathode in a working galvanic cell. To give a practical example, the overtension for discharge of H+ ions on a Ni electrode at pH 0 at 25 OC under a current density of lOa3 A/cm2 is - 0.33 V, i.e. the steady-state potential of the HJH+ electrode under these conditions becomes more negative than the equilibrium potential of the Ni .ei%rode under the same conditions at a pH equal to about 4. Ni can no longer be corrosively attacked under these real conditions since its immunity domain has been extended towards lower pH values, and could therefore be used for prostheses in vivo, if there are no other opposing conditions. Similar potential-pH and polarization diagrams can be constructed for any other metallic material (including suitable alleys) to investigate its corrosion properties. In the case where the metallic material to be implanted is a simple metal showing immunity under biological conditions, great care is needed to guarantee its purity. If a metallic impurity is present such as a grain with a potential more positive than that of the bulk metal, a galvanic element is formed, as illustrated in Fig. 9. This element will show an extremely tow resistance, so that a very small potential can produce relatively high current densities, i.e. a strong local corrosion of the bulk metal. The bulk metal acts as a negative pole and therefore goes into solution even if its standard potential is more positive than that of the hydrogen electrode under the same conditions. This consideration is very importz for alloys of the eutecticum type. Here, a real biphasic material is present with grains of both metals; corrosion is then the unavoidzbte consequence. If a particular alloy for whatever reason is preferable, it should be of the solid-solution type. If this type of alloy cannot be found and another type of alloy must be used, then special measures must be taken to improve the occurrence of passivity. The complementary and mutually integrated use of electrochemical thermodynamics (potential-pH equilibrium diagrams) and electrochemical kinetics (polarization curves) provide the best means of formulating sufficiently approximate predictions concerning the corrosion behaviour of metallic mate@ contacting biological liquids. A very good review on the corrosion of metallic materials implanted in living organisms was recently published by Pourbaix 1121. For further details on corrosion see ref. 10.

BIOTECHNOLOGY

Another domain of electrochemical phenomena of biomedical relevance is con- ’ cemed with biotechnology. Electric pulses from outside are capable of producing long-lived yet transient pores in membranes, so+alled “electropermeabition” or The electrically induced porous patches of two membranes may “electroporation”. fuse to form one membrane if two cells are brought into close contact: cell “electrofusion”. On the other hand, DNA double-helices, like plasmid DNA, can penetrate the porous membrane and become part of the cell’s genome: “electroporative gene uptake” or “gene transfer” resulting in “electrotransfection” or “electrotransformation”. Both electric cell fusion and electric gene transfer are being applied more and more in cell biology and biotechnology to produce genetically modified cells, useful for large-scale production at lower costs of such pharmaceutically very important products as insulin, interferon, peptides, proteins, etc. These studies are at present in an active phase of development. Two specialized symposia were organized in I986 by the Bioelectrochemical Society, the first together with the Interdisciplinary Center of the University of Bielefeld [13] and the second with the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the German Democratic Republic at Erfurt 11.41.To get an idea of the problems of this medically interesting domain of bioelectrochemistry and of the progress of our knowledge it will be useful to read the Proceedings of these meetings [13,14]. This review, even if still incomplete and by no means exhaustive, tries to give a clear idea of the importance of bioelectiochemistry in medicine. It wilI be the job of young people to develop these ideas further and even to find new ones to help and alleviate the suffering of mankind. ACKNOWLEDGEMENTS

Figures 1 and 2 were kindly provided by Dr. M. Moutet, University Paris Val de Mame, Crete& France. Figure 3 was kindly provided by Dr. SM. Karandikar, Head of the Department of Pharmacology, Seth GS. Medical College Parel, Bombay, India. The x-ray pictures (Figs. 5 and 6) were kindly provided by Professor M. von Ardenne, Dresden. BEFERENCES 1 E. Pun&or (Ed.), Bioelectroanalysis, F’roceedings of the Bioekctroanalytical Symposium, Riatrafbed, Hungary, 6-8 October 1986, Hung&m Academy of Sciences, Budapest, 1987: see also J. Haves, Ion-and Mokcule-Selective Electrodes in Biological Systems, Springer, Heidelberg, New York, Tokyo, 1985. 2 C.A.L. Bassett, A.A. Pilla and RJ. Pawluk, Clin. Grthop., 124 (1977) 128. 3 G. Co~acicco and A.A. Pilla, Bioelectrochem. Bioenerg., 12 (1984) 259. 4 R. Goodman and A.S. Henderson. Bicnbtrochem. Bioenerg., 15 (1986) 39. S G. Milazzo and L. Zecca (Eds.), I&c. 1st Int. Meeting ~‘Biological Effects and Therapeutic Applications of Electromagnetic Fields**, Bioelectrochem. Bioenerg., 14 (198s) Nos. l-3.

6 M. van Ardcnnc, Inaugural opening lecture to the Dresden Hypcrthennia Sympmium, Dresden, G.D.R., 22-24 January 1986 and rcfs. cited therein. From the manuscript by courtesy of Professor

von Ardennc. 7 F. F&i, Boll. Sot. Lancisiana Gspedali Roma, 30 (1) (1910) 88. 8 M. von Ardenne and P.G. Rtitnaucr, Jpn. J. Clin. Gncol., 10 (1980) 31. 9 A. Limogcs. An Introduction to Elcctroancsthcsia, University Park Press. Baltimore, London, Tokyo, 1975. 10 0. Milazzo, Elektrochemie, Vol. 2, Birkhtluscr, Bascl, 1980. pp. 82-99. 11 R. Dcltombc, N de Zoubov and M. Pourbaix in M. Pourbaix (Rd.), Atlas dTquilibrcs Ekctrochimiques, Gauthier Villars, Paris, 1963. 12 M. Pourbaix, Biomaterials, 5 (1984) 122 and refs. cited thcrcin. 13 E Neumann (Ed.), Proceedings of the Meeting on Biok+al Significance of Conformational Changes in DNA and DNA-Protein Comp!excs, Bielcfcld, F&G.. 21-26 October 1985. University of Bidefeld, Physical and Biophysical Chemistry, P,O. Box 8640, D-4800 Bielafeld 1, F&G. 14 H. Berg and H.-E. Jacob (Eds.). Prongs of the Xlth Jcna Symposium on Biophysical Chemistry, “Bioclcctrochcmistry in Biotechnology”, Erfurt, G.D.R, 22-27 September 1986, Stud. Biophys., 119 (1987). See also H. Berg in G. Milazzo and M. Blank (Eds.). Biockctmchcmistry II, Plenum Press. New York. London, 1987, pp. 135-166.