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Principles of Bioelectrical Impedance Analysis

Rudolph J. Liedtke (1-Apr-1997)

History In 1940, clinically induced changes in hydration status were rst correlated with total body changes in resistance and capacitive reactance (1). Also in 1940, Dr. Jan Nyboer pioneered work relating bioelectrical impedance changes to dynamic changes in pulsatile blood ow to organs, arterial pulse waveforms and respiration (19). The applications of impedance plethysmography, (the term given to the measurement of electrical impedance changes across limbs, organs and other body sites to detect dynamic blood volume changes), have been extensively validated by numerous investigators (9, 21, 24). A relationship between TBW and electrical impedance was rst reported by Thomasett in 1962 (24) and further delineated by Hoffer, et.al. in 1969 (7). There were no subsequent attempts for over a decade to determine the usefulness of impedance for the analysis of human body composition. In 1983, Nyboer applied the electrical volume resistivity principals of impedance plethysmography to the study of body composition using static total body impedance measurements (15). Since that time, when RJL Systems pioneered BIA sciences, with the help of Dr. Jan Nyboer, until today (4/97) the utility and reliability of whole body impedance measurements to assess body water and composition has been reported by hundreds of peer review papers and thousands of abstracts. This brief essay will try to explain the physics and electrical nature of BIA. Resistance, Reactance and Phase Angle Resistance All substances have resistance to the ow of an electric direct current (DC). For example, metal conductors such as copper have resistance in millions where as insulators have resistances in mega-mega ohms (1 X 1012). Ohms law states that the resistance of a substance is proportional to the voltage drop of an applied current as it passes through a resistive substance, or Resistance(ohms) = appied voltage drop (volts) current (amps)

An ohm is a unit of electrical resistance equal to the resistance of a circuit in which an electromotive force of one volt maintains a current of one ampere. An ampere is a unit representing the rate of charge ow in a conducting medium, whereas a volt is a practical unit of electrical work. Pure electrical resistance (resistors with no reactance) is the same when applying direct current and alternating current at any frequency. In the body, highly conductive lean tissues contain large amounts of water and conducting electrolytes, and represent a low resistance electrical pathway. Fat and bone, on the other hand, are poor conductors or a high resistance electrical pathway with low amounts of uid and conducting electrolytes.

-2Reactance Reactance, also known as capacitive reactance when describing biological tissues, is the opposition to the instantaneous ow of electric current caused by capacitance. Mathematically reactance is expressed by the following equation in alternating current (AC) circuits: Reactance(ohms) = Where: Reactance is expressed in Ohms Frequency is expressed in Hertz Capacitance is expressed in Farads PI = 3.1428 Farads are large units of measurement and are usually expressed in smaller fractions, such as microFarads (1 X 10-6) or pico-Farads (1 x 10-12). The above equation demonstrates that reactance is the reciprocal of frequency and capacitance, therefore, reactance decreases as frequency increases. At extremely low frequencies reactance is virtually innite. A capacitor consisting of two plates separated by a thin air wafer to insulate the plates, would have less reactance than if the plates where larger in surface area. In addition, if the plates where further separated by the air wafer the reactance would increase. In biological conductors for example, the smaller the semi-permeable membrane volume or the smaller quantity of membranes the greater the reactance. This is the opposite of what one would expect. Generally, high reactance values from a bioelectrical impedance measurement indicate better health and cell membrane integrity. The reason for this paradox is in visualizing the proper model of a single resistor and capacitor in a series or parallel combination. Ideally reactance should be expressed in capacitance at a given frequency. Capacitance is independent of frequency and indirectly denes cell membrane volume. The mathematical transformations to resolve this problem will be discussed later. By denition, a capacitor consists of two or more conducting plates separated from one another by an insulating, or non-conductive material known as a dielectric. Capacitors will store a charge of electrons for a period of time depending on the resistance of the dielectric. The amount of charge a capacitor will hold is determined by: Q W2 = 2 C= E E Where: Q = Quantity of electricity C = Capacitance in Farads E = Applied voltage drop (volts) W = Energy in Joules (Watt seconds) In the healthy living body, the cell membrane consists of a layer of non-conductive lipid material sandwiched between two layers of conductive protein molecules. The structure of cell membranes makes them capacitive reactive elements which behave as capacitors when exposed to an alternating current (See image below). Biologically, the cell membrane functions as a selectively permeable barrier separating the intracellular and extracellular uid compartments. It protects the interior of the cell while 1. 0 2 frequency capacitance

-3allowing passage of some materials to which it is permeable. The cell membrane maintains a uid osmotic pressure and ion concentration gradient between the intracellular and extracellular compartments. This gradient creates an electrical potential difference across the membrane which is essential to cell survival. Damage to the cell membrane, and its functions, is as lethal to the cell as direct damage to the nucleus itself. Theoretically, reactance is a measure of the volume of cell membrane capacitance and an indirect measure of the intracellular volume or body cell mass. Whereas, body fat, total body water and extracellular water offer resistance to electrical current, only cell membranes offer capacitive reactance. Since fat tissue cells ARE NOT SURROUNDED BY CELL MEMBRANES, reactance is not affected by the quantity of body fat.
The Plasma Membrane of a Cell and its Electrical Equivalent

extracellular space
pump protein
lipid bilayer

lipid bilayer

Resistor

Capacitor 11111 00000 11111 00000 11111 00000 00000 11111 dielectric

Cytoplasm

protein

intracellular space
Electrical capacitor in PARALLEL with a resistor. Capacitance is analogous to intracellular volume and resistance is analogous to extracellular volume. The outer boundary of the cell is a plasma membrane of All RJL instruments measure the SERIES phospholipid molecules which become a dielectric to form equivalent of impedance an electrical capacitor when a radio frequency signal is directly. These values are introduced to the cells environment.

protein channel

converted to their PARALLEL equivalent to predict intracellular and extracellular volume.

Phase Angle Phase angle is a linear method of measuring the relationship between resistance and reactance in series or parallel circuits. Phase angle can range from 0 to 90 degrees; 0 degrees if the circuit is only resistive (as in a system with no cell membranes) and 90 degrees if the circuit is only capacitive (all membranes with no uid). A phase angle of 45 degrees would reect a circuit (or body) with an equal amount of capacitive reactance and resistance, such as in fresh vegetables.

-4Normal statistics The National Health and Nutrition Examination Survey (NHANES) is a periodic survey conducted by NCHS. The third National Health and Nutrition Examination Survey (NHANES III), conducted from 1988 through 1994, was the seventh in a series of these surveys based on a complex, multi-stage sample plan. It was designed to provide national estimates of the health and nutritional status of the United States civilian, noninstitutionalized population aged two months and older. Normal Phase Angle from NHANES III
Reactance Phase angle of a resistor and capacitor in series

Parameter Mean SD Range

Males (n = 8545) Phase Angle Age 7.80 1.25 6.19 - 8.83 42.56 21.16 12 - 90

Females (n = 9115) Phase Angle Age 7.23 1.07 5.98 - 8.04 42.76 21.06 12 - 90

Impedance Phase angle ( )

Resistance Reactance Phase angle ( ) = arctan Resistance

The average phase angle for a healthy individual is approximately 6 to 9 degrees, depending on gender. Lower phase angles appear to be consistent with low reactance and either cell death or a breakdown in the selective permeability of the cell membrane. Higher phase angles appear to be consistent with high reactance and large quantities of intact cell membranes and body cell mass. Resistance and Capacitance in Parallel and Series The human body consists of resistance and capacitance connected in parallel and in series. A circuit model will be used to illustrate this point. When two or more resistors and capacitors are connected in series, they have an orientation to each other as seen in the series frame. In the series pathway, two or more resistors and capacitors are equal to impedance (Z) as the vector sum of their individual resistance and reactance since both are expressed in Ohms. When two or more resistors and capacitors are connected in parallel, they have an orientation to each other as seen in the parallel frame. In the parallel pathway, two or more resistors and capacitors are equal to impedance as the vector sum of their individual RECIPROCAL resistances and reactance. Series Parallel impedance2 = resistance2 + reactance2 1 1 1 = + 2 2 impedance resistance reactance2

Converting a series model to a parallel model Resistance parallel = Resistance series + Reactance parallel = Reactance series + Reactance2 series Resistance series Resistance2 series Reactance series

-5In vivo, the human body is a mixture of resistance and reactance in both parallel and series orientations. Bioelectrical impedance models which assume the human body consist only of resistance and capacitance in series and would invalidate sciences which depend upon direct current conduction, such as electrocardiography and galvanic skin response (direct current will not ow through a capacitor). With the realization that the human body consists of resistance and capacitance both in parallel and series orientations, the knowledge of reactance quantities becomes essential for the accurate determination of cellular body composition compartments. The simplest biological equivalent model is a single resistor and capacitor in parallel. This network may be analyzed with a single frequency of 50 Khz. There is some degree of validity to this model since it can be conceived that cells and their supporting mechanisms are not a series of extracellular and intracellular components but reside in parallel with a small series effect from the nucleolus of the cells. There is a major effect contributed by electrode length (or stature height) and must always be a variable to correct resistance and/or reactance independent variables used in prediction equations. Typical total body bioelectrical impedance (BIA) measurements display the vectors of resistance and reactance which are intrinsically based on a series network (resistor and capacitor in series). Resistance is indirectly related to the extracellular mass and reactance is indirectly related to the intracellular mass. These measured resistance and reactance values are highly interactive with each other in a simple parallel network, and are obscure when not mathematically transformed to an equivalent parallel model. All BIA measurements assume an equivalent series model and must be transformed to their parallel equivalent. The transformation is shown in the series to parallel transformation frame. The transformed parallel equivalent of Xc and R are the close approximations of the real electrical values of the biological tissue assuming an equivalent parallel model of a single resistor and capacitor. Total Body Measurements The measurement of total body impedance (resistance and reactance) from a macroscopic perspective is the vector sum of resistance and reactance in the limbs and torso. The limbs, because of the smaller circumference and greater length, contribute to most of the impedance. The torso impedance ranges from 15 to 30 ohms depending on the physical size of the subject. This is approximately 5.5 % of the total body when compared to a typical total body impedance of 450 ohms for males. The impedance of a geometrical system, in this case the human body, is related to conductor length and geometrical size, its cross-sectional area and signal frequency (18). Using a constant signal frequency and fairly constant conguration, the bodys impedance to current ow can be related to its volume, since conductor volume equals the cross-sectional area x length or height. Resistance = Length Length Multipling by or 1. 0 Area Length Then Resistance = Length2 Volume Volume = Length2 Resistance

(rho) is determined statistically when applied to whole body prediction equations.

Determining body composition using any method, volume or TBW must be known. Electrically determined TBW is equal to the L2/R, multiplied by a coefcient and constant (y intercept). This coefcient is the relative volume resistivity per cubic liter of ionic water distributed in organized tissues for males

-6and females (23). It is numerically less in adult males as a group. When measuring TBW with impedance the mathematical coefcients of length (or height) L2/R, weight and the associated constants for males and females are the only variables subject to change. A complex impedance measurement assesses both resistance and reactance, including phase shift, or phase angle which develops between the alternating current being passed through the body and the voltage drop across the body. At a xed frequency, such as in the RJL BIA 50 Khz instruments, an increase in the phase angle and reactance represents an increase in the ratio of charge stored per potential difference, or voltage drop across the body. Whole body impedance and muscle resistivity measured by Settle contralaterally (using the tetrapolar method) were both found to have a frequency of approximately 50 KHz. For this reason, muscle, as a large portion of body volume, is considered primarily responsible for the total body impedance measurement (23). Static impedance measurement of any homogeneous conductor describe its absolute electrical volume. When the resistivity of the conductive material is known then the electrical volume equals the physical volume. This general principle is easily applied to simple geometric shapes. When applied to the complex geometry of the human body, some difculties arise. Total body resistance and reactance measured by Settle from the wrist to the contralateral ankle was found to closely resemble the complex impedance properties of muscle tissue (23). In earlier studies by Hoffer (1970), (using a tetrapolar electrode technique), TBW was measured using tritium oxide dilution and total body impedance with a HewlettPackard Model 200CD oscillator at 100 KHz with a vacuum tube volt meter (7) (Impedance not resistance and reactance). A signicant correlation coefcient of -.70 was found between subject height2/Z. This correlation improved to .92 in healthy subjects and .93 in patients with various diseases when a linear regression of TBW versus subject height2/Z was performed. Despite this strong correlation in a group of patients (some with hydration disorders), there was a large standard deviation of 11% or 3.84 liters between predicted TBW and observed TBW (7). In 1996 Kotler reported: "Predictive equations for body cell mass (BCM), fat free mass (FFM), and total body water (TBW) were derived from direct measurements through use of single-frequency bioelectrical impedance analysis (BIA) in 322 subjects, including white, black, and Hispanic men and women, who were both healthy control subjects and patients infected with the human immune deciency virus (HIV). Preliminary studies showed more accurate predictions of BCM when parallel-transformed values of reactance were used rather than the values reported by the bioelectrical impedance analyzer. Modeling equations derived after logarithmic transformation of height, reactance, and impedance were more accurate predictors than equations using height2/resistance, and the use of sex-specic equations further improved accuracy. The effect of adding weight to the modeling equation was less important than the BIA measurements. The resulting equations were validated internally, and race and disease (HIV infection) were shown not to affect predictions. The equation for FFM was validated externally against results derived from hydrodensitometry in 440 healthy individuals; the SEE was < 5%. These results indicate that body composition can be estimated with simple and easily applied techniques, and that the estimates are sufciently precise for use in clinical investigation and practice". Am J Clin Nutr 1996;64(suppl):489S-97S. Reproducibility Electrical impedance prediction equations for assessing TBW and LBM are based on the variables of total body resistance, height and to weight. The variable height is the standing height of the subject and weight is the mass of the subject measured by a medically accepted weight scale. The resistance variable

-7is the resistance measured by the impedance analyzers detecting electrodes in ohms. When protocols are followed, the reproducibility in measuring total body water and lean body mass will approximate the .99 test-retest correlation coefcient as reported by Lukaski, et.al. (14) of the United States Department of Agriculture, Human Nutrition Research Center, in numerous studies using the RJL BIA instruments (12,13,14). "No signicant difference (p > 0.05) was found among resistance values determined on ve successive days. The individual coefcients of variation for these resistance values ranged from 0.9 to 3.4%, and the average precision was 2%. Test-retest correlation coefcient was .99 for a single resistance measurement and the reliability coefcient for a single resistance measurement over 5 days was .99" (13). Dr. Karen R. Segal, et.al. of the Department of Medicine, College of Physicians and Surgeons, Columbia University at St. Lukes-Roosevelt Hospital Center also reported: "The Biological Impedance Analyzer resistance readings were extremely stable. They exhibited virtually no change within the ve measurements when the electrodes were kept in place. The accuracy of the measurement of resistance was checked using 250, 400, 500 and 750 ohm precision resistors. The measured resistance did not deviate from the expected values by more than 2%"(22). BIA is the most reproducible technique available to assess body composition, assuming electrode protocols are followed. It is this characteristic that allows BIA to track change, both long term (days, months) and short term (minutes, hours). If any method of evaluation does not have repeatability, it can not have sensitivity to change. The specicity of BIA has been proven by hundreds of validation studies throughout the world over the past ten years (see abstracts). Recently, the sensitivity of BIA (TBW, FFM, BCM) has been positively shown with long term studies of Kotler (HIV wasting disorders) and Lukaski (controlled weight loss). (papers in publication) These studies had remarkable correlations to criterion methods of dual X-Ray absorptiometry (DEXA) and K40 total body isotope counting (K40). Measurement Technique Whole body electrical impedance is measured by passing a small constant alternating current (I) through the body and measuring the voltage drop (V) produced as a product of R X I, since I is constant V is directly proportional to R. A shift in the phase angle between the current and voltage denes reactance or a complex impedance measurement including the dielectric nonconducting space attributed to cell membrane capacitance. Electrode placement, measurement frequency and skin impedance are the primary procedural specications that must accompany impedance data. Skin impedance ranges from approximately 300 to one million ohm/cm. To accurately assess body volume electrically, skin impedance must be bypassed using either the two electrode or four electrode techniques. The two electrode technique used by Thomasett has several limitations (24). Results from this technique are often irreproducible due to excessive interference by electrochemical reactions at the subcutaneous needle electrode surface causing additional electrode polarization anomalies. In addition, the small diameter of the electrode needles results in a much greater current density near the electrodes than in the rest of the body. Therefore, the integrity of tissue near the electrodes and electrode size can effect the impedance measurement between electrodes, and confuse desired data (23).

-8Sinusoidal constant current source

Constant current source

Phase sensitive voltmeter

Current source electrode

Detecting electrode

High input impedance

Detecting electrode

Current source electrode

Measured biological resistance and reactance

The four electrode techniques used by RJL Systems largely avoids these difculties. Four surface electrodes are used situated either ipsilaterally or contralaterally on the dorsal surfaces of the right hand and foot at the distal metacarpals and metatarsals, respectively, and the distal prominences of the radius and ulnar and between the medial and lateral malleoli at the ankle. The RJL System delivers 800 uA at 50 KHz which is passed between the outer two electrodes. The voltage drop between the inner two is measured with a high input impedance amplier. The impedance of the skin and the electrode polarization impedance does not effect the measurement of total body impedance with the four surface electrode technique since negligible current is drawn through the skin by the passively coupled input circuits. The four surface electrode technique utilizing a constant deep homogeneous electrical eld in the variable conductor of the human body also minimizes problems with eld distribution and electrode irregularities. The constant current source is regulated to 1% accuracy from 0 to 8,000 ohms. The detecting electrodes have input characteristics that do not require complex electrodes or conductive bands. Safety The safety of bioelectrical instrumentation is assessed by two parameters. One is the aspect of electrical isolation from ground potentials for the subject. The second is the denition of what is a harmless current vs. frequency that can be deliberately introduced into the subject. There are few references that have explicitly established the standards for what is a safe subject current and frequency. Dr. L.A. Geddes and L.E. Baker in Applied Biomedical Instrumentation describe the threshold of electrical perception of alternating currents of varying frequency (4). At frequencies of 50 to 60 Hz (those used in power lines), nerve and muscle cells are stimulated. The sensation threshold is a few uA while pain and involuntary muscle contractions occur with much higher currents (80). Due to the large magnitude of cell membrane capacitance, the sensation and pain thresholds increases an order of magnitude as frequencies increase (4). The RJL Systems impedance analyzers have a precision oscillator tuned to 50 KHz. This oscillator is connected to a network of transformer coupled feedback circuits to generate a constant current at the subject source electrodes. The constant current supply has an accuracy of 1% from 1 to 8,000

-9ohms at 800 microamps RMS maximum. Geddes and Baker determined that the pain threshold at this frequency would be approximately 40 milliamps (4). This is 50 times the nominal current of these two instruments. Therefore the high frequency currents used by RJL BIA products present no hazard to the subject. There have been many applications of electrical impedance plethysmography at frequencies from 10 KHz to 5 MHz that have been introduced to critical human organs. Nyboer and Kornmesser (20) applied an impedance plethysmograph (designed and built by Rudolph J. Liedtke) to the area of the uterus to monitor pregnancy labor movements. There were no reported abnormalities after these observations using this method. The instruments that were used had a frequency of 100 KHz (crystal controlled) at approximately 3 milliamps. Bishop and Nyboer (2) applied the same instrument directly to the eye with an electrode array congured in a contact lens with no ill effects at this frequency and current (100 KHz at 3 milliamps). In addition, Nyboer (21) applied this equipment to many areas of the human body to pioneer the clinical application of electrical impedance plethysmography. In a personal communication to the author "we never have had any ill effects from the application of this equipment." The National Aeronautics and Space Administration (NASA) published an extensive book titled Development and Evaluation of an Impedance Cardiographic System to Measure Cardiac Output and Other Cardiac Parameters by W.G. Kubicek (9). In this publication an electrical impedance plethysmograph is applied directly to the thorax to predict cardiac output. The source electrode frequency is 100 KHz at 4 milliamps. There were no published hazards at this frequency and current. There are several institutions that are able to evaluate medical electronic instruments for electrical isolation standards of safety. Underwriters Laboratories (UL) has had the most experience in specifying electrical safety standards. In addition, most cities or states have their own standards and companies to make these determinations. The RJL Systems instruments has been approved by the ITT Research Institute of Chicago, Illinois for electrical isolation safety. In addition, many international safety institutes have approved all RJL products for safety. The never been a rejection of a IRB approval based on BIA sciences. References
1. 2. 3. 4. 5. 6. 7. 8. 9. Barnett, A.: Electrical method for studying water metabolism and translocation in body segments. Proc. Soc. Exp. Biol. Med. 44: 142, 1940. Bishop, S., Nyboer, J.: Electrical impedance of the anterior eye chamber. Annals of the New York Academy of Science. Vol 170, (2): 793-800, 1970. Durnin, J.U.G.A., and Womersly, J.: Body fat assessed from total body density and its estimation from skinfold thickness: measurement on 481 men and women aged 16-72. British Journal of Nutrition, 32: 77-97, 1974. Geddes, L.A. and Baker, L.E.: Principles of Applied Biomedical Instrumentation, 2nd Edition, John Wiley and Sons, New York, pp. 616, 1975. Geddes, L.A., Baker, L.E.: Applied Biomedical Instrumentation. Second Edition. John Wiley and Sons, New York, pp. 276-300, 1975. Geddes, L.A. and Sadler, C.: The specic resistance of blood at body temperature. Med. Biol. Eng., 11: 336, 1973. Hoffer, E.C., Meador, C.K. and Simpson, D.C.: Correlation of whole body impedance with total body water volume. J. Appl. Physiol., 27: 531, 1969. Hoffer, E.C., Meador, C.K. and Simpson, D.C.: A relationship between whole body impedance and total body water volume. Annals N.Y. Acad. of Sciences, 197: 452-469, 1970. Kubicek, W.G.: Development and Evaluation of an Impedance Cardiographic System to Measure Cardiac Output and Other Cardiac Parameters. National Aeronautics and Space Administration (NASA). July 1, 1968 to June 30, 1969. Contract No. NAS 9-4500.

-1010. 11. 12. Kushner, R.F. and Schoeller, D.A.: Estimation of total body water by bioelectrical impedance analysis. Am. J. Clin Nutr., 44: 417-424, 1986. Kushner, R.F. and Schoeller: Estimation of total body water by bioelectrical Impedance analysis. Am. J. Clin. Nutr., 44: 417-424, 1986. Lukaski, H.C. and Bolonchuk, W.W.: Theory and validation of the tetrapolar bioelectrical impedance method to assess human body composition. Int. Symp. on In Vivo Body Composition Studies, Sept. 28 - Oct. 1, 1986, Brookhaven National Laboratory. Lukaski, H.C., Johnson, P.E., Bolonchuk, W.W., Lykken, G.I.: Assessment of fat free mass using bio-electrical impedance measurements of the human body. Am. J. Clin. Nutr., 41: 810-817, 1985. Lukaski, H.C., Bolonchuk, W.W., Hall, C.B., and Siders, W.: Validation of tetrapolar bioelectrical impedance method to assess human body composition. Journal of Applied Physiology. 60 (4): 1327-1332, 1986. Nyboer, J., Liedtke, R.J., Reid, K.A. and Gessert, W.A.: Nontraumatic electrical detection of total body water and density in man. Proceedings of VIth ICEBI, 381-384, 1983. Nyboer, J., Polasek, P. and Giliard, R.: Bioelectric impedance analyzer. Proc. Second Int. Conf. of Bioelec. Impedance, p. 5 1976. Nyboer, J.: Electrorheometric properties of tissues. Ann. N.Y. Acad. of Sciences, 170 (2): 410-420, 1970. Nyboer, J.: Workable volume and ow concepts of biosegments by electrical impedance plethysmography. T.I.T. Journal of Life Sciences (2): 1-13, 1972. Nyboer, J., Bango, S., Barnett, A. and Halsey, R.H.: Radiocardiograms - the electrical impedance changes of the heart in relation to electrocardiograms and heart sounds. J. Clin. Invest., 19: 963, 1940. Nyboer, J., Kornmesser, J.G.: Electrical impedance of the abdomen during maternal labor. Annals of the New York Academy of Science, Vol. 170, art. 2: 801-803, June 1970. Nyboer, J.: Electrical Impedance Plethysmography. Second Edition. Charles C. Thomas, Springeld, IL, 1970. Segal, K.R., Gutin, B., Presta, E., Wang, J., Van Itallie, T.: Estimation of human body composition by electrical impedance methods: a comparative study. Journal of Applied Physiology, 58 (5): 1565-1571, 1985. Settle, R.G., Foster, K.R., Epstein, B.R. and Mullen, J.L.: Nutritional assessment: whole body impedance and uid compartments. Nutrition and Cancer, 2 (1): 72-80, 1980. Thomasett, A.: Bioelectrical properties of tissue impedance. Lyon Med. 207: 107-118, 1962.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.