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A NONLINEAR MICROWAVE RADIATION EFFECT ON THE PASSIVE EFFLUX OF SODIUM AND RUBIDIUM FROM RABBIT ERYTHROCYTES

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Olcerst, Robert Benjamin
A NONLINEAR MICROWAVE RADIATION EFFECT ON THE PASSIVE
EFFLUX OF SODIUM AND RUBIDIUM FROM RABBIT ERYTHROCYTES
PH.D. 1982
New York University
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University
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A NONLINEAR MICROWAVE RADIATION EFFECT
ON THE PASSIVE EFFLUX OF SODIUM AND RUBIDIUM
FROM RABBIT ERYTHROCYTES
by
ROBERT B. OLCERST
QctoLiWi, T981-
A dissertation in the Program in Environmental Health Sciences
submitted to the faculty of the Graduate School of Arts and Science
in partial fulfillm ent of the requirements fo r the degree of Doctor
of Philosophy at New York U niversity.
Approved
Merril Eisenbud, Advisor
Professor of Environmental Medicine
Sidney Belflifin, Co-Ad visor
Professor(qY Environmental Medicine
ABSTRACT
The passive efflux rates of sodium-22 and rubidium-86 from the
red cells of male New Zealand white rabbits were measured Jr» vitro
a fte r one hour irradiation at 2.45 GHz.
The temperature of the
samples and the power absorbed were controlled.
A rrhenius plots
of measurements made in the absence of radiation exposure revealed
th a t both the sodium and rubidium efflux have fo ur regions with
transitions at 8-13, 2 0 .0 -2 2 .5 and 36°C .
The efflu x rates with
microwave exposure were identical to the control rates, except at the
critical tem peratures, where irradiation increased the efflu x of both
cations.
This response was examined at the 22.5°C phase transition
at three specific absorption rates (100, 190, 390 m W /g).
A t all three
levels the cation efflux was statistically greater than one would
predict from a s tric tly thermal response.
The response does not
increase monotonically as a function of absorbed power.
The data
suggest th at the existence of two phases within the membrane is
necessary for the observation of the increased e fflu x .
interpretations of these observations are discussed.
Mechanistic
T A B L E OF C O N TE N TS
Page
Acknowledgements
I
List of Tables
fv
List of Figures
v
CHAPTER
I. Introduction
1
I I . The
Physics and Biophysics of Microwave Interactions
8
A.
Physical Descritpion
8
B.
Microwave Heating Mechanism
8
C.
Effects of Microwaves on Membrane Permeability
10
1. Red Cells and Sodium and Potassium
11
2. Active T ran sp o rt— Na-K ATPase A c tiv ity
15
3. Microwave Heating and T ran sp o rt in
O ther Membrane Systems
D.
S tru ctu re and Composition of E rythrocite Membranes
15
20
1. Membrane Component Characteristics
22
2 . The Effect of Tem perature on Red Cells
28
3. The A rrhenius Plot and Phase Changes
30
4. Erythrocyte Passive Tran sp o rt
33
I I I . The Irradiation System
A . Calorimeter Irradiation
B. The Waveguide
45
System
47
49
C.
The Tem perature Jacketed SampleCompartment
51
D.
Calorim eter Calibration andTem perature Distribution
53
E.
Discussion
55
IV . Passive Ion T ran sp o rt
73
A.
Materials and Methods
73
B.
Results
82
C.
Sodium Efflux
82
D.
Rubidium Efflux
83
E.
Discussion
84
F.
Conclusion
94
V . Summary
106
V I.
110
B ibliography
V II. Appendices C hapter I I I .
Appendix 111-A
Derivation
124
of Equation IV -1
125
Describing the Therm istor Tem perature
D ifferential
Appendix lll- B
Derivation of the Case 1
Solution
130
Appendix lll- C
Derivation
Solution
134
of the Case 2
iv .
L IS T OF TA B LE S
C hapter II
11-1
Page
Percentage D istribution of Phospholipids in
Human Red Cells
37
11-2
Lipid Phase Tran sition Tem peratures
38
11-3
Human and Rabbit E rythrocyte Parameters
39
C hapter IV .
IV -1
Medium Composition
96
IV -2
Isotope Radiological Properties
97
IV -3
Sodium Efflux Tem perature Control Data
98
IV -4
Rubidium Efflux Tem perature Control Data
99
IV -5
Sodium E fflux Irrad iatio n Data
100
IV -6
Rubidium Efflux Irradiation Data
101
IV -7
Effect of Microwaves on Efflux
102
V.
L IS T OF FIGURES
C h a p te r I I .
Page
11-1
T h e E lectro m ag n etic S p ectru m
40
11-2
Red C ell M em brane S t r u c t u r e
41
11-3
L ip id A m p h ip a tic S t r u c t u r e
42
11-4
Phase T ra n s itio n s o f DPPC V esicles
43
II-5
M ixed L ip id B ro ad en ed Phase T r a n s itio n s
44
C h a p te r I I I .
I II-1
W aveg uid e Irr a d ia tio n System o f A . S .
Presman
I I I -2
W aveg uid e Irr a d ia tio n System o f B e lk h o d e , Johnson
57
and Muc
58
III-3
S chem atic o f M icro w ave C a lo rim e te r S ystem
59
III-4
W aveg u id e C o o rid in a te System
60
ill-5
T h e rm is to r M o u n tin g
61
111-6
W heatstone B rid g e C ir c u it
62
111-7
E lectro m ag n e tic W ave C o o rd in a te A x is and
B o u n d a ry C o n d itio n s
111-8
63
A ssem bly D ra w in g o f th e T e m p e ra tu re Jacketed
Sample C o m p artm ent
I ) I -9
T e m p e ra tu re Jacketed Sample C o m p artm en t
64
-
O u te r S ection
111-10
T e m p e ra tu re Jac keted Sample C o m p artm en t
In n e r S ection
111-11
In te rc h a n g e a b le T e flo n Sample Cell
*
65
66
67
v i.
111-12
B ridge Zero Power C haracteristic
68
111-13
In
69
111-14
In R ^ /R f Versus Power at 31.24°C
70
111-15
Rotation of Z -X Plane Field Profile
71
III- 1 6
B ridge Recorder Trace
72
Versus Power at 2 1 .8 4 °C ‘
C hapter IV .
IV -1
Semi-Logarithmic Plot of the Rb-86 Efflux
A c tiv ity Versus Time at 37°C
103
IV -2
A rrhenius Plot of Sodium Efflux
104
IV -3
A rrhenius Plot of Rubidium Efflux
105
Appendix l l l - C .
III- C - 1
Waveguide Coorinate Transform ation
140
CHAPTER I:
INTRODUCTION
The technological use of microwave radiation dates to a time fifty
years ago, b u t the microwave devices which are presently used are fa r
more complex than were th e ir early forerunners and are being used in
a v a rie ty of diverse applications (Mum ford, 1976).
For example, the
water content of geological samples is being measured by microwave
radiation (B irc h a k , 1974;
H ipp, 1974;
Castle and Roberts, 1974).
The food industry relies heavily on microwave heating, thawing and
sterilization (Bengtsson, 1974;
Goldblith, 1966).
Telephone, telegraph
and satellite microwave links are appearing in ever increasing numbers
(Jansky, 1972;
Jones, 1977;
Glasser, 1972a,b;
Brown, 1974).
Navigational, collision avoidance and radar devices are appearing for
increasing numbers of small c ra ft and vehicles.
1974; T e ll, 1977;
Peak et a L , 1975).
(T e ll and Nelson,
In in d u s try, microwave heating,
soldering and welding are gaining acceptance.
A survey of industrial
applications conducted by the Bureau of Radiological Health predicted
th a t by 1974, there would be 6000-8000 workers using microwave
devices which at th a t time were being introduced at a rate of sixty new
units per year (B R H /D E P 7010).
It was also estimated in 1970 th a t by
1976 there would be 1.8 million consumer microwave ovens in this
country and that this would represent a q u arter of all ovens purchased
yearly (E den, 1970;
H arris, 1970).
Since the time of these predictions
the use of microwave devices has increased at a rate that is greater
than was originally estimated.
Medical and research applications of microwave radiation are the
most interesting areas of endeavor.
The Federal Communications
Commission has allocated seven frequencies specifically fo r industrial,
scientific and medical use.
Prominent among these is 2.45 GHz, a
frequency th a t is also used in the majority of consumer microwave
ovens and the frequency at which this study was conducted.
Biochemical researchers rely on microwave radiation to quickly inactivate
enzymes and th e re b y , preserve substrate levels in biological materials
(Schmidt et a L , 1972;
Brown et a l. , 1978).
Microwave diatherm y
has been used to reduce swelling and inflammation in joints and
muscles and has even been used to promote healing of detached
retinas (G u y , 1974;
Merriam, 1974).
Microwaves may soon provide
a technique fo r thawing frozen organs and blood (R ajotte, 1978;
et a l., 1973).
Voss
Microwaves have proven useful in deep layer
thermography diagnostic techniques used in the detection of breast
cancer and tumors (B a rre t et a [ ., 1977).
Microwave instrumentation to
measure respiratory rate and lung edema noninvasively are under
development (Pedersen et a L , 1978).
The resurgent interest in
hyperthermia as a therapeutic modality has generated interest in
microwave applicator design, cancer cell biological interaction and
the dynamics of tissue heating (Rossi-Fanelli et a l., 1977;
et a l., 1976;
Forster et a l. , 1978;
K anter, 1977;
LeVeen
Stamm et a [ .,
1974).
The steadily increasing levels of environmental microwave radiation
have sparked interest in studies concerning the interaction of microwave
radiation with biological systems.
Significant efforts toward elucidating
mechanisms of interaction date back over th irty years (C le a ry , 1970).
During this tim e, biological effects such as hypertherm ia and protein
denaturation were observed and understood in terms of a classical
thermal mechanism (Berm an, 1965;
Schwan, 1957).
Thermal
characteristics of biochemical reactions are often described by A rrhenius
plots.
For example, Linden, W right, McConnell and Fox (1973),
constructed A rrhenius plots of various fS-glucose and p-gaiactose
tran sp o rt processes in E. coli.
They found the transition temperature
to be dependent on the fa tty acid composition of the membrane.
have observed two critical transition tem peratures:
They
an upper critical
tem perature which corresponds to the fir s t appearance of more ordered
domains in the membrane;
and a lower transition tem perature which
th ey in te rp re t as the point at which th e transition is complete.
A t the
upper transition tem perature, they measured a striking increase in the
lateral compressibility of membrane lipids and a twofold increase in
sugar tran sp o rt as the tem perature was decreased to 0 .7 °C .
They
a ttrib u te this change to the increased penetration of protein carriers
into the bilayer facilitated by the increased flu id ity th at they observed
in the remaining unordered lipids.
In general, when tran spo rt is
studied at a tem perature other than at critical transition tem peratures,
passive ionic efflux increases as heat is added to the system.
A number of recent experiments report microwave induced biological
effects th at are not supportive of a thermal mechanism.
Among these
are the developmental effects th a t Carpenter has observed on microwave
irradiated larvae (C arp en ter and Livstone, 1971);
changes in the
mitotic and blastic transformation rate of lymphocytes observed by
Huang and associates and others (Huang et a [ . , 1977;
Baranska, 1967);
Stodolnki-
the microwave induced changes in the bursting
potentials and frequency of Aplysia neural cells (Wachtel et a L , 1974
and Seaman, 1975);
low intensity electromagnetic effects on cell
,
membrane calcium binding (Bawin et a [ ., 1974); and changes in
choliriesterase enzyme activity (B aran sky, 1972).
An increased number of experiments have demonstrated microwave
influences on membrane tran spo rt processes in rabb it lenses (W eiter et
a L , 1975), blood b a rriers (Oscar and Hawkins, 1977;
A lb e rt, 1977),
synovial membranes (Latsenko, 1967) and brain tissue (Bawin et a L ,
1975;
Bawin and A dey, 1976, 1977a,b ).
Th ere is as y e t no underlying
body of knowledge th at can serve to explain the observed effects in
terms of recognized biophysical mechanisms.
Recently, several general
theories have been proposed which suggest new modes of interaction
between microwaves and biological systems ( I (linger, 1970;
1970;
G rodsky, 1975;
and Hu, 1977;
Kaczmarek, 1976;
Vogelhut,
Rabinowitz, 1973;
Barnes
Spiegel and Joines, 1975).
The effect of microwave radiation on erythrocyte cation tran sp o rt
has been studied in several laboratories (Gordon et a l., 1974;
Stodolnik-B aranska, 1971;
Stem ler, 1968, 1972, 1973).
Baranski has
irradiated rabbit blood with 3 GHz microwaves at 1, 5 and 10 mW/cm2
(B aranski et a L , 1974), and suggested th at microwave radiation
interacts directly with the Na-K pump.
Recently, Hamrick and Zinkl
(1975) attempted to replicate Baranski's original study but were unable
to observe an effect on potassium tran sp o rt at power densities from
1-25 mW/cm2 at 2.45 or 3 GHz.
A similar study was conducted by
Ismailov (1971), using human erythrocytes.
He found th at a 1 /2 h r
exposure at 1 GHz resulted in a decrease in active cation tran spo rt
and an increase in passive diffusion at 37°C.
The experiments described in this dissertation and which constitute
the main body of my research deal with the effects of 2.45 GHz
5.
microwave radiation on the passive efflux of ions from rab b it erythrocytes
maintained at a constant tem perature.
In preparation fo r this, study, a microwave exposure system was
designed th at is suitable fo r the exposure of cell suspensions and
solutions to 2.45 GHz microwave radiation under conditions in which
the tem perature and absorbed power are controlled (R abinow itz, Olcerst
and Mumford, 1977).
Experiments were then conducted to ascertain
the effect of microwave radiation on the passive efflu x of sodium-22 and
rubidium -86 from erythrocytes of male New Zealand white rabbits after
a one hour irradiation .
Rubidium-86 served as an analog fo r potassium
and was used because of its long half life compared to available potassium
isotopes.
A rrhenius plots of the efflux were obtained and the irradiated
samples, whose average tem perature resulted from combinations of
microwave and conventional heating, were compared with control cells
maintained in a constant tem perature water bath.
The paradoxical decrease in tran sp o rt reported by Linden and
associates (1973) which occurred at a membrane phase transition
tem perature provides a system capable of d ifferen tiating effects of
continuous wave 2.45 GHz microwave radiation from a classically thermal
response.
If microwave radiation affects ionic tran sp o rt by a thermal
mechanism, irradiation of erythrocytes at a transition temperature would
also result in a decrease in passive ionic efflux similar to the control
response of cells maintained in a water bath.
Both Baranski et aL
(1974) and Ismailov (1971) rep o rt increases in passive efflu x in response
to microwave irrad iatio n .
Confirmation of these increases at a transition
temperature would demonstrate the presence of another mechanism by
which microwaves could be absorbed in addition to the classical thermal
mode.
The following results are reported:
1.
A rrhenius plots (e fflu x versus inverse tem perature) exhibit four
regions fo r both cations with transitions at 8-13, 20 to 22.5°C and 36°C.
2.
The rubidium efflux data indicate no difference
between irradiated samples and controls except at the transition
temperatures of 36°C , 2 2 .5 °C , and 13°C.
A t these temperatures
there is a statistically significant increase in e fflu x .
3.
The sodium efflux data demonstrate differences between
control and irradiated samples th a t are statistically significant at 3 5 .5 °C ,
36°C , 37°C , 22.5°C and 13°C.
Irradiated sample efflux rates at
temperatures other than critical temperatures do not d iffe r from
control efflu x rates.
4.
Both cations exhibit passive effluxes at the 22.5°C critical
tem perature th at exceed control values but th a t do not increase
monotonically with specific absorption rate.
The following conclusions can be drawn from this work:
1.
Microwave radiation increases passive tran spo rt near critical
temperatures when the membrane undergoes phase transitions,
and not at intermediate temperatures between critical points.
2.
The data suggest that the simultaneous existence of two
phases within the membrane is necessary for the observation
of critical e fflu x .
3.
The efflux rates at critical temperatures do not increase
monotonically with increased absorbed power.
4.
The absorption of microwave energy under isothermal
conditions persisting during a critical phase transition result
in increases in passive ionic tran spo rt th a t do not rely on a
macroscopic thermal mechanism.
The increased ionic efflux
triggered by small microwave-induced perturbations may
result from cooperative interactions within the membrane
during phase transitions which result in charge separation
and transient dipole formation.
A lte rn ativ ely , the increase in
lateral compressibility of the membrane lipids which occurs
at phase transitions may facilitate extracellular or intracellular
dipole penetration.
In both cases, microwave energy could
couple into the membrane under these conditions by polarizing
the charge distribution, affecting dipole orientation and
biological function d ire c tly .
CHA PTER II
THE PHYSICS AND BIOPHYSICS OF MICROWAVE INTERACTIONS
A . PHYSICAL DESCRIPTION
A schematic diagram of the electromagnetic spectrum is given in
Figure 11-1.
The spectrum is continuous and so divisions into various
radiation bands is a matter of a rb itra ry convention.
The region
associated with microwave radiation is generally between 300 and
300,000 MHz.
Frequencies in this region, propagating through free
space, correspond to wavelengths between 1 mm and 1 m.
The
frequency and wavelength are related to the speed of light according
to the following relation:
\u = c
Eq. 11-1
where
\ = the wavelength (m)
u =. the frequency (H z )
c = the speed of light (m /s e c ).
For the case, of an electromagnetic wave propagating through a simple
homogenous isotropic medium, the product of th e wavelength and the
frequency yields a velocity th a t is less than the speed of lig ht.
\u = v
v < c
•
Eq. 11-2
This decrease occurs because the electromagnetic wave must now
propagate through a medium whose electric and magnetic properties
o ffe r a larger impedance to its passage.
B. MICROWAVE HEATING MECHANISM
Electric fields interact with free charges and dipoles found in
biological materials.
When a dipolar molecule is subjected to an electric
fie ld , a torque develops, which tends to align the dipole with the fie ld .
The torque (x ) is given by the following equation:
x = p x E
Eq. II-3
where p is the dipole moment.
The dipole moment is a vector equal
to the charges of the molecule multiplied by the distance by which they
are separated.
When a field is impressed, the dipoles tend to align
with the electric fie ld , reducing th e ir potential energy.
In biological
media the viscous drag causes a delay in the time th a t it takes fo r
molecules to respond to the field .
The frictional viscous drag results
in energy losses in the form of heat.
principle of microwave cooking.
This is the essence of the
The frictional force will depend on
the size and shape of the dipole, its charge d istribu tion , and the
types of interactions it makes during its alignment.
Sim ilarly, when
the field is ab ru p tly removed, the aligned and ordered dipoles will
slowly wander back to a totally random orientation by means of
tem perature-dependent brownian interactions.
The time it takes the
number of oriented dipolar molecules to be reduced by a factor of 1/e
is defined as the relaxation time of th at molecule.
The complex dielectric constant e* depends on the freq uen cy, the
stru ctu re of the polarizable elements, the viscosity of the medium and
tem perature.
The frequency dependence of the complex dielectric
constant has been described by Debye (1929) and can be given as
follows:
qj - £'
os
Eq. 11-4
1 + (u it)2
(qj - e')u>t
Eq. II-5
e" =
1 + (tu t) 2
10.
°o ) ( w t) 2
T v -f f t j a —
■
a = a° +
Eq- " - 6
where the subscript » indicates in fin itely high frequencies and the
subscript 0 indicates low frequencies approaching DC.
The variable,
t , represents the relaxation time.
As the frequency of an electromagnetic field increases, the
molecule will follow the orientational influence of the fie ld .
As the
frequency is fu rth e r increased past the relaxation tim e, the frictional
damping will no longer allow the molecule to rotate with the fie ld .
Thus, at high frequencies dipole orientation will not occur.
Water has
relaxation times up to 24 picoseconds, which corresponds to a frequency
of 30 GHz and a wavelength of 1 cm.
The relaxation times fo r both
free and bound w ater are both within the microwave range and they
dominate the absorption characteristics of biological materials (Clegg
and Drost-Hansen, 1977).
Orientational effects on long chain molecules
resulting in non-thermal interactions have been discussed in a recent
theoretical description by Barnes and Hu (1977).
C. EFFECTS OF MICROWAVES ON MEMBRANE PERMEABILITY
Th ere exists a large body of literatu re concerning microwave
effects on neural immune, endocrine and lenticular systems.
mutagenic and teratogenic effects have also been investigated.
Behavioral,
There
are several excellent comprehensive reviews th at can be consulted for
additional information dealing with the biological effect of microwave
radiation (C le a ry , 1970, 1973, 1977;
1970;
Glazer, 1971;
McLees and Finch, 1973;
Pressman,
and Baranski and C zerski, 1978).
A generalized overview of the literature concerning the biological
effects of microwave radiation is beyond the scope of this chapter, which
will be confined to a more specific discussion of effects on membranes
and neural systems.
V ery little mechanistic information exists on the interactions
between microwaves and biologically active molecules and how these
interactions might influence biological function.
Microwave effects on
the diverse biological processes cited above appear to be compatible
with changes in the ac tivity and perm eability of cell membranes.
1.
RED CELLS AND SODIUM AND POTASSIUM
Baranski et aL (1974) irradiated rab b it blood with 10 cm
electromagnetic waves at 1, 5, and 10 mW/cm2 power densities and
reported increases in potassium and hemoglobin in the external cell
media as well as an increase in osmotic fra g ility .
He reported that
exposures of 1 mW/cm2 fo r 15 minutes will result in significant
increases in extracellular potassium concentrations th a t can be
attributed to microwave effects on the sodium-potassium pump.
His
A
experiments were conducted in a potassium-free saline medium with
washed cells.
Th ere is no mention as to whether the medium contained
b u ffe rs , and the osmolarity and pH are not given.
Under these
conditions there will be a natural loss of potassium from the cells by
diffusion alone.
Control suspensions were maintained at room
tem perature under the assumption that field densities of 10 mW/cm2
could lead to no appreciable macroscopic term perature rise.
Th ere
was no attempt to control the irradiated or control sample tem peratures.
Tem perature was monitored during the whole time of irradiation
but by an undisclosed method.
Objection to the introduction of
therm istor or thermocouple transducers has often been expressed
because these elements would pertu rb the field and might induce
differen tial absorptions of energy and hot spots (Tyazhelov et a h ,
1977).
Potassium was assayed by a titration method which was less
sensitive than other analytical techniques.
Additional experiments were reported with rabb it granulocytes
(B aran ski, 1975;
Szmigielski, 1975).
used as an indicator of cytotoxicity.
The uptake of nigrosin stain was
A fte r one hour irradiations at
5 mW/cm2 , 80% of the granulocytes were extensively stained.
In
addition, the liberation of 85% of the acid phosphatase and lysozyme
content of the granulocyte and 45% of the alkaline phosphatase indicated
irreversible in ju ry to the cell membrane.
Recently, Hamrick and Zinkl (1975) have attempted to replicate
Baranski's findings.
They used both buffered and unbuffered suspending
media, frequencies of 2450 and 3000 MHz, and assayed potassium
levels by flame photometry.
Tem perature was monitored by placing a
therm istor in a dummy suspension located in the exposure fie ld .
They
also attempted to bracket the temperature of the irradiated sample
by providing controls one degreee above and below its tem perature.
In one series of irradiations at 10 mW/cm2, a 1.5°C temperature rise
above ambient was found and the temperature control's water baths
were adjusted to reflect this change.
Th ey were unable to reproduce
Baranski's experimental conditions and results of potassium loss,
osmotic fra g ility or hemolysis.
More recently, Liu, Nickless and Cleary
(1979) and Petersen (Petersen et a L , 1978) have published research
th a t is contrary to the findings of Baranski.
As study on microwave irradiation of human erythrocyte sodium
and potassium has been presented by Ismailov (1971).
Exposures for
30 minutes at 45 mW/cm2SHF radiation of 1 GHz were conducted.
The
sample tem perature was maintained at 32°C with a tem perature thermostat
while an additional 5°C microwave heating brought the final temperature
to 37°C .
He reports an increase in total potassium released from the
irradiated cells, whereas the control cells showed an uptake of
potassium from the medium.
The final concentration of potassium from
irradiated samples increased 0.365 mM as compared to controls, which
decreased 0.164 mM.
This represents a change of approximately 10%
in the cells' potassium levels which was in itially 5.1 mM.
C oncurrently,
he measured the total sodium concentrations fo r irradiated and control
samples.
The tem perature control samples released 0.237 mM to the
medium while the irradiated samples exhibited an uptake of 0.771 mM.
A second series of irradiations was then conducted with the
addition of monoiodoacetic acid to the medium.
Monoiodoacetic acid
is an inhibitor of Na-K ATPase, and as such allowed Ismailov to
differen tiate microwave effects on both active and passive tran sp o rt.
His data revealed th a t both the irradiated and control sample released
85% more potassium during a th irty minute incubation.
Sim ilarly, sodium
was found to passively enter both control and irradiated cells b u t,
once again, the irradiated cell intake was 79% greater than th at of the
control cell in flu x .
Ismailov concludes th at microwaves can in hibit active tran spo rt
substantially and increase passive diffusion of sodium and potassium
in human e ry trh ro cytes.
The la tte r, he suggests, could be due
to a microwave-induced change in the effective area of pores within
the membrane, the formation of new pores, or tran spo rt channels, or
to changes in the ion's hydration.
He associates these changes in
sodium and potassium permeabilities with changes he has observed in
the membrane electroconductivity of unicellular Opalina ranarum
(Ism ailov, 1966).
U nfortunately, nowhere in his communication does he disclose
an exact description of his experimental apparatus or the chemical
composition of his media.
Because his work was performed at a
hematocrit of 54.4%, it is likely th at there were problems assuring
adequate glucose concentrations necessary to maintain healthy active
tran sp o rt.
If unbuffered media were used, the production of lactic
acid from glycolysis might lead to decreasing pH as irradiations and
incubations proceeded.
Y et, Ismailov's findings are corroborative
of those of the Russian investigator, Stolodnik-Baranska (1971), who
also used human erythrocytes to examine microwave effects on tran spo rt
of sodium and potassium.
A t frequencies of 3000 MHz and power
densities of 1-45 mW/cm2 fo r periods up to 3 hours, she found that
both active and passive tran spo rt could be affected.
The magnitude
of these effects increased both with the time of exposure and power
density, and was measurable after one hour.
A study cited by Gordon at the Warsaw Symposium investigated the
effects of 900 and 2340 MHz radiation at power densities of 1 to 100 mW/cm2
on human erythrocytes (Gordon, 1975).
This w ork, conducted by
Stemler, resulted in a dose response curve describing a frequency
independent threshold of 1 mW/cm2 fo r 2 hours.
A mathematical model
was developed th at describes the microwave effects on membrane sodium
and. potassium perm eability as the sum of two processes:
a linear
decline in the rate of potassium e n try with increasing microwave field
strength, and a "change of the rate of transport under conditions of
diminished adaptation possibility" (Stem ler, 1968, 1972, 1973).
2.
A C T IV E TRANSPORT - Na-K ATPase A C T IV IT Y
A definitive answer as to the existence of microwave effects on
Na-K ATPase is still lacking.
In studies aimed a t elucidating the
biochemical pathways influenced by microwave radiation during the
development of cataracts, the formation of opacities occurred at the
same time as did changes in the lens' sodium, potassium and water
levels (Kinohita et a L , 1966).
In a study of the ATPase a c tiv ity on
19 rabbits, an average decrease of 40% ATPase activity was measured.
Despite this decrease, no correlation seemed to exist between the
severity of the cataract produced and the changes in en 2 yme a c tiv ity .
It was concluded th at microwave damage had occurred to the lens
capsule membrane or its components causing them to become "leaky".
Straub and C arver (1975) examined the ac tivity of Na-K ATPase
prepared from guinea pig brain following irradiation with 1.6 -1 2 GHz
microwave radiation at power densities of up to 2 mW/cm2 .
were conducted in the field at 22°C fo r fifteen minutes.
Incubations
The authors
were unable to detect any microwave induced differences between
irradiated and control samples.
3.
MICROWAVE HEATING AND TRANSPORT
IN OTHER MEMBRANE SYSTEMS
Microwave induced heating has caused changes in the tran sp o rt
and permeability in synovial membranes.
Latsenko (1967) has found
a synergistic effect when diatherm y and combinations of corticotropin
or cortisone are used to tre a t joint inflammation.
The rate of absorption
of radiophosphorus in the synovial fluid of rabbit knee joints
demonstrated that the combined effect of microwaves with either
agent caused substantially greater uptake than did either microwave
radiation, cortisone or corticotropin alone.
The mechanism underlying
this observation was not discussed nor was it determined whether
microwave heating was the sole cause of these results.
Other heating effects have been observed with X band radiation on
skin slices from guinea pigs (C a rn e y , 1968).
She measured decreases
in the uptake of phosphorus, sulfates and proline which were radioactively labeled.
The results of this study are d iffic u lt to evaluate
because short exposure times of 1, 2 and 4 seconds, high power
densities of 1-3 W/cm2 and large tem perature rises in the sample cell
as compared to the control were parameters included in her experimental
design.
An interesting study of microwave effects on cellulose acetate
membranes used in desalinization procedures was conducted by Wasilewski
(1 966 ).
He measured the microwave absorption spectra of these
membranes at 82°C over a frequency range of 1.8 to 10 GHz.
Absorption
peaks at 2 .2 5 , 3 .4 3 , 5 .8 5 , 8 .6 , 8 .9 and 9 .2 GHz were calculated
assuming rotation of side branches and functional groups.
He has
identified the 3.43 GHz absorption peak as arising from the principal
quantum state of a rotating acetate group.
Such rotations are implicated
in the formation of new intermolecuriar bonds within the membrane that
reduce the effective pore size and change the membrane perm eability.
Wasilewski also observed th at a critical tem perature of 65°C must be
exceeded before these events can be observed in cellulose acetate
membranes.
Recently, Sheridan and co-workers (1977) have demonstrated in
artificial lipid membranes th a t microwave radiation can induce lipid
disorder.
Membranes composed of bovine sphingomyelin in single
and binary mixtures with other naturally occurring membrane lipids
were exposed to 10 mW/cm2, 2 GHz CW and pulsed microwave fields.
N on-perturbative examination of the lipid membranes using laser Raman
spectroscopy revealed lipid chain disorientation which was especially
prominent in the tem perature range of 32-37°C .
Th ey were able to
observe microwave induced melting of lipids by these small power
densities at temperatures lower than those at which the lipids would
normally change state.
A drop in lens ascorbic acid levels was among the fir s t biochemical
lesions observed in the formation of a microwave induced cataract
(Kinoshita et a L , 1966).
This observation has prompted several
investigations and some controversy.
Weiter and Finch (1975) have also
demonstrated a decrease in the total ascorbic acid content of rabbit lenses
irradiated in v itro by exposure to 2.45 GHz microwave radiation.
This
decreased ascorbate level becomes more pronounced as the absorbed
power is increased.
Control samples exposed to the same temperature
histories as irradiated lenses demonstrated similar ascorbic acid levels.
Weiter interprets these results as a therm ally induced loss of membrane
in te g rity .
His claim th a t the membrane becomes leaky has been questioned
by Rabinowitz and Olcerst (1976), who were unable to observe a
decrease in lens ascorbate transport post irrad iatio n .
Recently, Ferri
(1977), although also unable to v e rify the earlier studies of Kinoshita
and C arpenter, has found decreases in the concentrations of ascorbic
acid within the aqueous humor.
Her results indicate that the prim ary
damage occurs at the blood-ciliary b a rrier resulting in reduced
permeability and lower aqueous humor concentrations of ascorbic acid
which are ultimately reflected in the lower lens levels.
Support for the ciliary-blood b a rrie r hypothesis can be found in
the research of A lbert (1977).
He observed th at 2.45 GHz microwave
radiation at a power density of 10mW/cm2 altered the perm eability of
blood brain barriers of Chinese hamsters.
Thermal effects were not
considered as likely mechanisms at the low power density employed in
this study.
He used horse radish peroxidase as a marker fo r large
proteins and examined sections of the hamster brains under an electron
microscope.
He observed an increase in the number of pinocytic
vesicles loaded with the peroxidase in the irradiated sections.
He did
not perceive any significant tran spo rt within the tig h t junctions that
connect adjacent cells within the endothelial lining of the microcapillaries.
He believes th at microwaves might be able to initiate or enhance
permeability of large molecules, predominantly by vesicular or other
transcellular paths.
Similar work concerning microwave influences on the permeability
of the blood brain b a rrie r of rats has been conducted by Oscar and
Hawkins (1977).
They examined continuous wave power densities of
up to 1 mW/cm2 and pulsed densities of 0.1 mW/cm2 .
The increase in
permeability that they found was not apparent at higher power
densities (see Bawin, 1977a,b).
Changes in the binding and release of radioactive calcium from
neural tissue of cats and chickens has also been found to exh ib it such
a "power window" (B aw in, 1975, 1976, 1977a,b).
Working with
extremely low frequency fields and also when modulating 147 MHz VHF
fields with brain wave frequencies of 4-35 Hz, Bawin and associates
have observed th at weak electrical signals may act as trigg ers fo r
more dynamic cellular events.
They have found th a t ELF signals
resulted in an increased calcium binding that was dependent on both
the frequency and amplitude of the incident fie ld .
Significant binding
was found at 6 and 16 Hz with 56 V/m field strengths bu t not at 1,
32 and 75 Hz.
No discernible effects were seen at field strengths of
10 and 100 V/m (B aw in, 1976).
In another study, Bawin (1977b)
modulated 147 MHz, 0 .8 mW/cm2 fields at 16 Hz and found an 18%
increase in neural membrane calcium release.
No effect was found with
the 147 MHz electromagnetic wave in the absence of the lower frequency
modulation.
Similar increases with a 16 Hz modulation were observed
with a 450 MHz ca rrier at power densities of 1, 5 and 15 mW/cm2.
A 10% change was observed a fte r a tw enty minutes exposure to a power
density of 1 mW/cm2 .
Although these experiments were reported as
being prelim inary, no effect was seen at field strengths higher than
2 mW/cm2 .
D.
STRUCTURE AND COMPOSITION OF ERYTHROCYTE MEMBRANES
The cu rre n t understanding of the stru ctu re of the erythrocyte
membrane-can be conceptualized by the illustration in Figure 11-2
(C apaldi, 1974).
This model is still consistent with the concept of
a lipid bimolecular leaflet as originally proposed over fifty years ago
by Gorter and Grendel (1925) and later emphasized by Davson and
Danielli (1956).
Evidence firm ly establishing this basic structural
framework has been gained through X -r a y diffraction studies, freeze
frac tu re electron microscopy and from the characteristics and properties
of artificial membranes.
Individual lipid molecules are amphipathic, having a hydrophilic
head group and two extended chains of fa tty acids forming hydrophobic
tails.
The lipids' arrangement within the membrane bilayer accomodates
th e ir amphipathic s tru c tu re , the head groups forming the top and
bottom surfaces and the hydrophobic tails projecting inward toward
the center of the membrane (see Figure 11-3, Capaldi, 1974).
Lipids
comprise approximately half the weight of the cell membrane.
Up to
ten percent of the membrane mass may be carbohydrate with the
remainder being protein (B retsch er, 1973).
of three broad classes of lipids:
The bilayer is composed
phospholipids (65% by w eig h t),
neutral lipids (25%), and glycoiipids (10%) (B retsch er and R aff, 1975).
The percentage phospholipid compositions are listed in Table 11-1
(Ways and Hanahan, 1964).
T h e predominant neutral lipid is cholesterol,
which accounts fo r 23% of the membrane weight (Reed et a[, 1960).
The s tru c tu re , placement and function of cholesterol in the membrane
will be examined below.
As seen in Figure 11-2 , various proteins.
21 .
are associated with the erythrocyte membrane.
Proteins th at enter or
traverse the lipid bilayer are referred to as intrinsic proteins, whereas
those adjacent to the bilayer are extrin sic.
Examples of intrinsic
proteins are mitochondrial cytochrome oxidase and rhodopsin.
Mitochondrial
ATP-ase and red cell spectrin, protein fractions having molecular
weights of 255,000 and 220,000 are both extrinsic (C apaldi, 1974).
Spectrin represents roughly a th ird of all the protein in the red
cell (C apaldi, 1974).
For some time it was thought th a t this protein
frac tio n , which is found to cover the cytoplasmic surface of the red
cell, formed myosin-like contractile fib ers (B rets ch e r, 1973).
This
suggestes a possible functional role of spectrin in controlling red
cell deform ability. The myosin-like nature of this protein fraction
has been challenged on the basis th a t the solubility of spectrin is the
exact opposite of myosin.
Spectrin has been shown to be soluble in
80% alcohol, a property th at infers a hydrophobic nature (B rets ch e r,
1973).
Thus, p art of the spectrin fraction may be intercalated with the
inner surface of the red cell membrane.
Th ere are two major glycoproteins exposed on the outer red cell
surface, comprising another th ird of the red cell protein complement.
Since it has been found th a t there are no proteins associated only
with the external cell surface, both of these span the b ilaye r.
larger is globular and has a molecular weight of 100,000.
The
It has
little carbohydrate and may be present in the membrane as a dimer
(B re ts c h e r, 1975).
There is also exellent reason to believe that this
protein is associated with the sugar tran sp o rt site (Lin et aL_, 1974).
The other glycoprotein is the most completely characterized of all
mammalian membrane proteins.
It has a molecular weight of 30,000
22 .
and is composed of 13 amino acids.
The amino-terminal hydrophilic
end carries about 100 sugar residues including more than 90% of the
charged sialic acid residues found on the cell surface.
Functionally
this protein carries blood group alloantigens, and acts as a receptor
fo r influenza virus and some plant lectins (B rets ch e r, 1975).
Freeze fra c tu re microscopy has revealed th at the proteins are
distributed asymmetrically within the cell membrane favoring the
internal surface.
A similar lipid asymmetry has been found,
characterized by more phosphatidyl serine and amino phospholipids on
the inner surface.
These types of phospholipids contain a greater
number of polyunsaturated fa tty acids than do choline phospholipids,
sphingomyelins and the glycolipids.
T h e ir unsaturated components
result in a less ordered state th at can b etter accomodate the higher
distribution of proteins on the inner surface (B rets ch e r, 1973, 1975).
1.
MEMBRANE COMPONENT CHARACTERISTICS
The mobility of membrane lipids has been examined with NMR and
ESR techniques.
These studies have revealed th a t lipid lateral diffusion
within each monolayer of the leaflet is rap id , with molecules exchanging
with neighbors at a rate of a million per second.
In contrast, there
is an extremely low probability that a lipid from one monolayer may
exchange or flip between layers.
Consequently, such in terlayer
exchanges might occur less than once in two weeks (B rets ch e r, 1973).
Th us, the lipid monolayers may be thought of as a two-dimensional
liquid.
The flu id ity of this liquid has often been found to closely
resemble the term perature characteristics of its components.
For
example, in artificial membranes composed of dipalmitoyl phosphatidylcholine
a sharp melting point is found at 4 1 .5 °C , the same tem perature at which
this pure lipid material is known to undergo a transition from a fluid
state to a rigid crystalline gel phase.
C oncurrently, the permeability
of these membranes to Na-22 changes about this phase change (see
Figure 11-4 ) (Jacobson and Papahadjopoulos, 1975).
Th us, the
structural status of the membrane is directly related to its functional
characteristics.
By altering the head groups, chain lengths and degree of
saturation, the transition temperatures of lipids are known to change.
The degree of hydration can also influence these transition temperatures
(Chapman, 1973).
Increasing the w ater content results in a higher
hydrocarbon chain mobility and decreases the phase transition tem perature.
For sim ilarly hydrated lipids, a higher melting point is found fo r lipids
with longer hydrocarbon chains.
T h e higher the degree of
unsaturation, the lower the transition tem perature.
This results from
the fact th a t the unsaturated hydrocarbon molecules are more mobile,
have a larg er interneighbor distance and are less ordered than more
unsaturated lipids.
Sim ilarly, cis bonds are harder to pack than
trans bonds, a fact consistent with th e ir lower transition temperature
(Chapman, 1973).
Alteration of the head group chemically th at results in an increased
ordering of the lipid molecules w ill, by the same rationale, increase the
transition tem perature.
In a similar manner, m etal-lipid interactions
and pH changes have also been found to influence phase transition
temperatures (Jacobson and Papahadjopoulos, 1975).
In other studies,
mixtures of lipids have been found to exhibit broader transition
tem perature ranges indicating th at a "phase separation" may occur
24 .
where clusters or gel domains exist within the liquid monolayer (see
Figure 11-5).
Mixtures of lipids and proteins have also been examined
and found to exhibit a v e ry complex polymorphism.
Rand (see Chapman,
1973) using lecithin-cardiolipin mixtures with bovine serum albumin,
found th at there was an electrostatic interaction which brought the
components close enough together so th at shorter range polar and
hydrophobic interactions occurred.
changes in the protein.
He noted gross conformational
Spectrin has been added to Jipid vesicles
of phosphatidylserine resulting in increased sodium diffusion rates
(Chapman, 1973).
The reciprocal nature of the lipid-protein interaction
is illustrated by the viscotropic effect certain lipids have on specific
enzymes.
Kimelberg and Papahadjopoulos (1972) found th at (N a-K )A TP ase
incorporated into lipid vesicles can be activated by additions of
phosphatidylserine or phosphatidylglycerol.
Furtherm ore, the activation
is inhibited below the transition tem perature of the lipid or in the
presence of cholesterol.
"This specificity fo r the (Na-K)AT.Pase
reactivation correlated with the observation th a t only phosphatidylserine
and phosphatidylglycerol vesicles show substantial discrimination for
K+ over Na+ in terms of perm eability."
1972).
(Kimelberg and Papahadjopoulos,
Th ey theorize th at the ion tran sp o rt site may involve such an
electrostatic activation of the ATPas by phosphatidylserine in the
membrane.
Electrostatic alterations in the rhodopsin protein on retinal
discs afte r exposure to light have been shown to change the degree
of insertion of this protein into the membrane (C apaldi, 1974).
Thus
it would appear that proteins are mobile within the membrane .
They
are also able to diffuse laterally, a fact demonstrated by the ability of
antigens to aggregate proteins within the membranes.
Although it
25 .
was thought th at spectrin formed a filamentous network on the cytoplasmic
surface of the red cell that anchored red cell proteins, making this,
type of aggregation impossible, (C apaldi, 1974) recent studies have
shown th a t this is not the case (L arsen , 1975;
1975).
Gordon and M arquardt,
Another concept that has been recently called into question
concerns the nature of cholesterol within the membrane.
X -r a y
diffraction data suggest th at the plate-like steroid rings interact with
and hinder the mobility of parts of the chain closest to the polar head
groups, leaving the remaining chain flexib le.
This interaction prevents
the chains from crystallizing while inhibiting the overall fle x ib ility of
the lipids and thus keeping the membrane lipids in an intermediate
fluid state (S hipley, 1973).
It had been thought that temperature
induced phase transitions within membranes of high cholesterol content
would not be found (S hipley, 1973;
Chapman, 1968, 1973).
This
hypothesis gained fu rth e r support from the finding th at mixtures of
cholesterol and phosphatidylcholine did not exhibit phase transitions.
Mixtures of cholesterol and trans-phosphatidyiethanolam ine, however,
demonstrate clearly observable phase transitions (Chapman, 1967).
Erythrocyte ghosts do not show a phase transition when examined by
means of differential scanning calorimetry (D S C ), although a transition
can easily by seen when cholesterol depleted ghosts are examined.
When intact ghosts are examined by laser Raman spectroscopy, the
transition unobservable by DSC can be demonstrated but is found
over a broader temperature range.
Lippert and Peticolas (1971)
(see also Chapman, 1973) in te rp re t this transition as being noncooperative.
Dehydration of an erythrocyte membrane results in a
crystallization of membrane cholesterol.
Under dehydrated conditions,
a phase transition again becomes observable by standard DSC
techniques (Chapman, 1973).
Functional evidence of the occurrence
of a phase transition within a high cholesterol content membrane has
been given by Elford and Solomon (1 974 ).
Th ey observed phase
transitions in dog erythrocyte sodium and potassium influx experim ents.
Th ey cite the constancy of the activation energy fo r water transport
in these cells over a tem perature range of 7-37°C as evidence th at
these ions and water each enter the erythrocyte at d iffe re n t membrane
sites and furtherm ore, th at sodium and potassium ions do not enter
through w ater-filled pores.
The phase transition of cholesterol occurs
in physiological range of 35-40°C (Ladbrooke and Chapman, 1969;
Chapman, 1967).
This transition is characterized by a low transition
enthalpy of only 0 .7 kcal/mole (Van Putte et all., 1968).
Cholesterol
transitions in myelin are found at 35°C and are due to polymorphic
transitions to a crystalline form (Chapman, 1967).
Phase transitions
of various lipids, mixtures and artificial membrane systems th at are
found within physiological temperatures are listed in Table II- 2 .
Recently, potential-dependent transition tem perature of ionic
channels have been induced in locust muscle by the addition of glutamate.
This work by Anderson and co-workers (1977) presents A rrhenius
plots of the membrane channel closing rate and single channel conductance.
Th ree degree shifts in the break points of these plots were observed
when the transmembrane potentials were changed by as little as 30 mV.
Th ey describe the mean lifetime of an open gate ( t ) according to the
folowing relationship:
27 .
I = to
Thus th e energy
[E_ + H ( T ) ] /R T
+ e
Eq. 11-7
b a rrie r model ismodified by coupling the activation
energy in the system to a tem perature-dependent dipole (p ).
Th ey
believe th a t th e ir results are due to a potential-dependent phase
transition in the membrane around
the channel th at affects the gating
molecule.
Similar studies with electric field pulses at intensities of kV/cm
and durations of microseconds have demonstrated th at the incorporation
of impermeant molecules in red cells is possible.
Kinoshita and Tsong
(1977) have found th a t by varying the electric field strength they
were able to control the size of the pores within the erythrocyte
membrane to allow the passage of large molecules of varying size.
They were also able to obtain larger pore sizes by increasing the
pulse duration and reducing the ionic strength of the suspending
medium.
O ffn er and Kim (1976a, 1976b) have considered the activation
energies fo r the diffusion of hydrated ions through the membrane
pores.
Th ey have introduced the term "electroformation" to describe
the effect of electric fields upon the membrane.
The model they
consider features a pore lined with long chain hydrocarbons with
phosphate head groups.
These phosphate head groups strongly
influence the electric field at the mouth of the pores.
Small deflections
of these groups by electric fields or mechanical collisions by impinging
ions, are capable of resulting in large changes in activation energy.
They estimate the magnitudes of these energies for sodium and
potassium with various degrees of hydration.
Support fo r th e ir general
28 .
hypothesis is found in the w o rk.o f May and associates (1 970 ).
These
researchers demonstrated electric field effects on brain cephalins and
lecithin films , via in frared spectroscopy.
They observed spectral
changes in intensity at frequencies th at correspond to changes in
conformation of the CH2 groups as the electric field is applied.
They
also noted th at the strongly dipolar phosphate groups tend to align
themselves with the electric field at low applied voltages.
Parry-Jones
and Gregson (1972) have presented data on pure egg yolk lecithin
membranes th at indicate an additive effect of pulsed fields in aligning
these membrane lipids.
Certain photochromic systems in non-polar
solvents have also been shown to be aligned to form quasi-crystalline
structures by absorbances in the 60 MHz microwave frequency.
The
flu id ity of this membrane region has been assessed using NMR
resonance absorptions in this region (C erbo n, 1970).
2.
THE EFFECT OF TEMPERATURE ON RED CELLS
The effect of high temperatures on erythrocytes has been well
studied.
A t temperatures between 52-65°C , erythrocytes exposed fo r
one hour become fragm ented.
There is a great amount of cell damage;
cell debris and hemolysis are found.
Intact cells are of varied size
and irre g u lar shape and spheroidal cells are common.
This drastic
thermal damage is characteristic of erythrocytes from human, dog, cat
and ra b b it.
burns.
It has often been'seen ]n v itro and jn vivo following
Thermal changes in morphology and osmotic fra g ility in the
range of 45-50°C depend on the tim e-tem perature history of the sample.
Slow progressive changes start with the formation of budlike protrusions
on the cell surface.
These are.e vid en t both by microscopic examination
29 .
and by an increasing hematocrit as the buds make the cells more
d iffic u lt to pack by centrifugation.
Increased exposure to the effects
of tem perature results in multiple budding followed by buds breaking
free and separating into closed hemoglobin containing fragments th at
may be spherical or elliptical in appearance.
Ham and associates indicate th at there is a critical temperature
range where a change of 1 - 1 .6°C will result in a rapid increase in
osmotic fra g ility .
is 4 7 -4 8 .6 °C .
For experiments of one hour duration, this range
In experiments where the rate of heating was increased
the critical region shifted to 5 0 -5 1 .6°C .
They heated samples of human
and canine erythrocytes fo r periods of one hour to 46°C without effect
(Ham, 1948).
Maintaining human erythrocytes fo r fifteen minutes at
49°C caused no change in osmotic fra g ility , although the few buds
th at developed altered the cell viscosity enough to p revent filtration
through a filte r with an 8 micron pore size (Ham et a [ . , 1968).
In
comparison, dog erythrocytes exposed fo r fifteen minutes to 49°C
showed no budding, and no increase in osmotic fra g ility .
They did
demonstrate an increased viscosity and a decreased survival when
chromium-51 labeled cells were injected into live animals (Carlson and
Ham, 1968).
These experimenters also observed a change a fte r one
hour in the acetylcholinesterase ac tivity which decreased th irty percent
in dog erythrocytes but was unaffected in human red cells. Sodium and
potassium cell concentrations remained unchanged in either type of
cell under these conditions.
They also measured a hemoglobin decrease
from 4 m g/g found in control cells to 3 .2 -3 .5 mg/g in temperature
treated cells.
The loss in cholesterol, although not large in magnitude,
infers a membrane structural change th at has previously been associated
with increasing erythrocyte osmotic fra g ility (M u rp h y , 1962,a ,b ) .
V ery few studies have been conducted on the effects of lower
temperatures on erythrocytes.
One exceptional paper by Karle
described studies of erythrocyte changes occurring at temperatures
corresponding to those found during fevers (K a rle , 1969).
Osmotic
fra g ility in rabb it erythrocytes was examined m vitro a t 37, 41 and
44°C fo r incubations of six and twelve hours.
The results are
consistent with the picture seen at higher temperatures where the
osmotic fra g ility generally increases with tem perature.
Karle also
examined hemolysis at 38 and 4 1 .5 °C , fo r both human and rabbit
erythrocytes.
The time periods of significance in this study are 20
to 40 hours after heating.
seen
From these experiments rabb it cells are
to be more temperature sensitive than are human erythrocytes.
In another stu d y, Karle (1970) has observed a "remarkable p relytic
loss of potassium during incubations at 4 2 °C ".
By a p relytic loss,
it is meant th at the loss of potassium from the cell was statistically
greater than could be accounted fo r by the loss of hemoglobin.
This
occurred with no loss of glycolytic a c tiv ity , no morphological changes
and no loss in cell volume control.
Karle also found a progressive
cell rig id ity as the time and tem perature increased as measured by
cell filtratio n times (K a rle , 1970).
3.
THE ARRHENIUS PLOT AND PHASE CHANGES
A rrhenius plots of tran sp o rt versus inverse absolute tem perature
are used as a technique for determining phase transitions resulting
during the course of the experiments to be described.
This technique
has often been used in the study of membrane tran sp o rt systems.
31 .
Arrhenius plots of the logarithm .of the process velocity versus
inverse absolute tem perature do not always yield linear graphs.
Discontinuities of slope are sometimes found which indicate changes in
activation energy occurring at the critical transition tem perature.
Dixon and Webb (1958) o ffe r several explanations of such
discontinuities th a t may occur in enzyme systems or transport models
of permeation which follow• Michaelis-Menton kinetics.
Th ey propose
th a t discontinuities may result from the following:
1)
Phase transitions.
2)
Parallel reactions with d ifferen t active centers each having
a unique tem perature coefficient.
3)
Processes occurring through two serial steps each with its
own tem perature coefficient.
4)
Enzymes or carriers which exist in several stereospecific
conformations having d ifferin g activation energies.
5)
Reversible inactivation of enzymes or carriers.
The work of Elford and Solomon (1974) dealing with the influx of
labeled sodium and potassium into dog erythrocytes is an example of
the use of this technique.
They generated A rrhenius plots th at were
characterized by a well defined minimum at 12°C fo r potassium influx
and a maximum at 22.4°C fo r sodium ion in flu x .
Since dog erythrocyte
tran sp o rt occurs predominantly by a passive mechanism, these findings
indicate th at tran sp o rt of sodium and potassium ions occurs by two
independent processes.
They proceed to discuss lipid membrane phase
changes such as those reported in human erythrocytes at 22°C, as
being responsible fo r th e ir observations.
32 .
The A rrhenius plot technique has been used by Dockter and
Magnuson (1975) to demonstrate th at the active tran sp o rt of chlortetracycline by Staphylococcus aureus is dependent on the o rder-diso rd er
phase transition tem peratures.
They were able to alter the transition
tem perature by changing the fa tty acid composition of the membrane.
Sim ilarly, Linden and associates (1973) have discussed a lateral phase
separation in E. coli membrane which was also found to be dependent
on th e fa tty acid composition of the membrane.
distinct temperatures of transition:
They describe two
the higher tem perature corresponds
to the fir s t appearance of solid domains in the membrane, whereas the
lower of the two critical temperatures corresponds to the temperature
at which the formation of the solid phase has been completed.
A t the
upper transition temperature they found a strikin g increase in the
lateral compressibility th a t resulted in a twofold increase in facilitated
sugar tran spo rt as the tem perature was decreased less than 1°C .
Such lipid characteristic phase phenomena can ex ert considerable
influence on membrane enzyme activities as well.
Kimelberg and
Papahadjopoulos (1 972 ), working with Na-K ATPase in delipidized
preparations noted th at a reactivation of this enzyme be dipalmitoyl
phosphatidylglycerol was dependent on the lipid liq u id -to-crystallin e
transition tem perature.
Cholinesterase activities have also been shown
to be dependent on the lipid composition of th e ir surroundings (Morero
et a L , 1972).
Kimelberg and Papahadjopoulos (1972) have introduced
the term "viscotropic" to describe the effect of membrane flu id ity on
enzyme a c tiv ity .
33 .
4.
ERYTHROCYTE PASSIVE TRANSPORT
Rabbit erythrocytes are similar to human erythrocytes with respect
to th e ir w ater, sodium and potassium content (see Table 11-3;
et a L , 1969).
Rettori
Both types of cells maintain low internal sodium
concentration and high external potassium concentrations.
The use of
rab b it erythrocytes is preferred to human erythrocytes as an experimental
system because in the rabb it the sodium e fflu x is three times slower,
and there is also a larger component th a t is actively tran spo rted.
Passive tran sp o rt from erythrocytes has been described
mathematically by J .F . Hoffman (1 962 ).
Since the experiments to be
described involve the passive efflux of isotopically labeled sodium-22
and rubidium -86, the mathematical description developed by Hoffman
can be adapted to sodium and rubidium passive tran spo rt as follows:
Let:
l . ( t ) = the concentration of
i
22
Na within the cell
22
loCt) = ^ e concentration of
Na in the extracellular medium
(cpm /m l)
K. = the rate constant fo r sodium influx (h x)
K0 = the rate constant for sodium outflux (h x)
Tm
= total monodirectional flu x (mmoles/l of R B C 's /h r)
l»l •
i
where
S ubscript i = in flu x ;
o = outflux
S uperscript T = to tal; meaning the sum of both passive (P ) and active (A )
The rate of change of the internal Na concentration is equal to the
sum of
th e influx of sodium from the external medium andthe outflux of
sodium
from the cell to th e medium.
simple differential equation:
This can be expressed by the
34 .
d l .( t )
1
= K.l 0( t ) - KoI»( t )
Eq. 11-8
rearranging
d>i( t )
dt
+
K0l . ( t ) = K .le( t )
1
1
K t
m ultiplying both sides of the equation by e °
e Kot +
K0l. ( t ) e Kot =
[ l . ( t ) e Kot] = K .I0( t ) e Kot
now assuming l 0( t ) and both rate constants to be constant, the above
expression can be integrated to yield:
.K0t _
l.( t ) e ° =
l
^K0t
K .l0( t ) e
n\'° y
rz---------r\0
+ constant
Eq. 11-9
Now consider the situation where at t=0, all the isotope is in the e x tra ­
cellular solution and thus l.(t= 0 )= 0 .
Under this initial condition, we can
evaluate the constant of integration as follows from Equation 11-9:
K .l0( t )
constant = -
K,
Substituting this constant back into equation 11-9 yields:
K t
K .l0( t ) e K°t
K ,l„ (t)
K„
K„
"
m ultiplying both sides by K,
K0l . ( t ) e Kot = K .l0( t ) ( e Kot - 1)
dividing both sides by e
K0t
K0l , ( t ) = K .l0( t ) ( 1 - e‘ Kot)
solving fo r K.
Ij ( t ) K 0
K'
l 0(t)< 1
Tm
- e " K o t)
=
I
If a sodium in flux experiment were conducted with ouabain it will
provide the passive unidirectional influx
. A concurrent study
i
conducted in the absence of ouabain would provide a measure of the
total unidirectional sodium influx T .. .
M.
The active sodium unidirectional
i
influx would be the difference of these two quantities:
AM.
i
=
T M.
i
p ivi
.
i
with ouabain
T M . = PM .
i
i
The outflux constant K0 will be determined after the fashon of
J .F . Hoffman (1962), by preloading erythrocytes with isotope and
conducting the outflux into what is initially unlabelied media.
Hoffman has demonstrated by working with blood cell hematocrits
of less than 0.3% that the term in Equation II- 8 fo r the backflux of
isotope from the medium into the erythrocyte can be considered
equal to zero.
Th us, the outflux is a fir s t order occurrence described
by the following equation:
d l .( t )
3t
= " K<»l i^t ^
The solution of this relation fo r l . ( t ) yields:
36 .
1,(1) = l.(o )e ‘ K ot.
If , at t=0 we have internal to the cells an activity 1 .(0 ),
then at a
later time t= t the activity lost from the cells would be
1.(0) - l.(0 )e ~ Kot
The a c tiv ity lost from the cell is
1.(0) - l . ( t ) = l.(0 )(1 - e ' Kot)
and
1 - l . ( t ) / l . ( 0 ) = (1 - e‘ Kot)
Taking the natural logarithm of both sides of the above equation yields:
loge [ l . ( t ) / l . ( 0 ) ] = K„t
solving fo r K0
Ke = 1 /t loge [ l . ( t ) / l . ( 0 ) ]
Eq. 11-10
T h u s , a plot of time » t versus the logg term will yield a straig h t
line whose slope is the outflux constant.
Hoffman's derivation resulted
in this equation:
1
[ I j( t ) / l . ( 0 ) ]
K° ” T" lo9e (1 - fractional hemolysis)
II 11
Note th a t when there is no hemolysis during the execution of this
procedure, then the equation reduces to Equation 11-10.
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5
LIPID PHASE TR A N S ITIO N TEMPERATURES*
TABLE 11-2
Lipid or
Membrane
T ransition
Tem perature
(°C )
Egg yolk phosphatidyl
ethanolamine
15
55
Egg yolk lecithin
20
Cholesterol
1,2 dipalm itoyl-Lphosphatidyl choline
Dimyristoyl phosphatidyl
ethanolamine
Sphingomyelin
Liquid crystal -*• gel
Lamellar •* hexagonal II
35-45
25,50
48
40
Acholeplasma Laidowii B.
35-40
E. Coli
15-45
Decholesterolized red cells
37
Decholesterolized myelin
37
Dehydrated myelin
37
♦(Chapman, 1973)
Description
Liquid crystal -*• gel
TABLE 11-3
ERYTHROCYTE PARAMETERS*
Na meq/kg wet wt
K meq/kg wet wt
H20 m l/kg wet wt
Rabbit
10.3 ± 0 .4
96.4 ± 1.1
661 ± 5
Human
11.1 ± 0 .5
89.2 ± 3 .4
650 ± 3
* (R etto ri et a L , 1969)
FIGURE 11-1
THE ELECTROMAGNETIC SPECTRUM
Cosmic
Gamma
X
U ltra v io le t
V isible
In fra re d
Radio
Microwaves
Electric
1Q23
1022
1021
102°
1019
1018
1017
1Q16
1015
1014
1013
1012
1011
1010
109
10®
107
10®
10®
104
10®
Wave Length
3 x 10-S A
3 x 1 0 -4 A
3 x 10—3 A
3 x 1 0 -2 A
0 .3 A
3 A
30 A
300 A
3 x 103 A
3 x 104 A
30 m
300 |j
3 mm
3 cm
30 cm
3 m
30 m
300 m
3 km
30 km
300 km
_________________ Energy________
ev
ergs
4
4
4
4
4
4
4
4
4
0
4
4
4
4
4
4
4
4
4
4
4
1 x
1 x
1 x
1 x
1 X
1 X
1 X
1 X
1
41
1 x
1 x
1 X
1 X
1 X
1 X
1 X
1 X
1 X
1 X
1 X
10®
107
10®
10s
104
10®
102
10
1 0 -2
1 0 -3
1 0 -4
1 0 -5
10-®
1 0 -7
10-®
1 0 -9
10—10
1 0 -11
1 0 -12
6
6
X
6
6
X
1 0 -4
1 0 -5
10-®
10—7
10-®
1 0 -9
10—10
1 0 -11
1 0 -12
1 0 -13
1 0 -14
1 0 -15
10-1®
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
O
1
M
Frequency
(cycles/sec)
6
6
X
6
6
X
1 0 - 1®
10-20
6
6
X
6
6
X
6
6
X
6
6
X
6
6
X
1 0 -19
10-21
10-22
1 0 -23
1 0 -24
FIGURE 11-2
RED CELL MEMBRANE STRUCTURE
*(C a p a ld i, 1974)
SUPRAMOLECULAR AGGREGATES in red-cell m embrane
completely penetrate the membrane. One is a glycoprotein
w ith a molecular weight of 87,000 (b). T he third protein in
spectrin (c). Evidently the three proteins are so linked that if
include ihe two proteins that
(a ); the other is a protein
the hypothetical aggregate is
one moves, the others follow.
FIGURE 11-3
LIPID AMPHIPATHIC STRUCTURE*
*(C a p a ld i, 1974)
H— C
H-------
A M PHIPATHIC STRUCTURE of a lipid
m olecule, with a hydrophilic head and twin
hydrophobic tails, is exemplified by this
typical phospholipid, specifically a molecule
of phosphatidylcholine. Various lipid mole­
cules comprise about half of the mass of
m am m alian membrane, forming the mem­
b ran e’s structural framework. T h eir fattyacid tails may be saturated (left), with a hy­
drogen atom linked to every carbon bond,
o r unsaturated (rig ht), with carbons free.
FIGURE 11-4
PHASE TR A N S ITIO N OF DPPC VESICLES*
. *(Jacobson and Papahadjopoulos, 1975)
25
*
30
35
40
45'
'
50
55
T e m p e ra tu re ( " t )
I IOURI- 9 Phase transition of D P P C vesicles. (A ) Intensity o f perslenc
fluorescence (arb itrary units) at various temperatures in D P P C disper­
sion IP e r /D P P C < I | D P P C ' l = 0.8 p m o l/n tl). ( I)) Pulariration
o f perylene embedded in D PP C dispersions as in ( A ): brosen line rep­
resents first derivative o f p -T scan, evaluated as described in text. (C )
Differential scanning calorimeter thermogram for D P P C dispersion;
heating rate. 1.2J#/'inin. ( D ) : ; N a : * self-dilfusion rate through unsonicated D PP C vesicle membrane at various temperatures (efflu x ex­
pressed as per cent o f captured ions per h : Papahadjopouls'S c l c l..
| 9 “ Jti). A ll dispersions prepared in IUO nvst N a C I-4 inM llis -T F .S -0 .1
ntM E D I A at pi I 7.4. without sonication.
FIGURE 11-5
MIXED LIPID BROADENED PHASE TR A N S IT IO N S *
♦(Chapman, 1973)
263
323
Tempers lure (*K)
Fig. .*3 Differential scanning calorimetry curves of dimyristoyl lecithin D M L dimvristovl phosphatidyiethanolamine D M PE. (a) D M L 100 mol %: (b) D M L
95 mol % -fDM PF. 5 mol %; (c) DML. 70 mol % -f-DMPE 30 mol %; (d) D M L
50 mol U
/„H D M PE 50 mol %: (c) D M I’E 100 mol % all in excess water
45.
CHAPTER III
THE IR R A D IA TIO N SYSTEM
Much of the previous work dealing with the biological effects of
microwave radiation was performed by irradiating whole animals with
electromagnetic waves directed by an antenna.
These open field
irradiations were compared by measuring the exposure as registered
by a field probe placed where the animal would be positioned during
illumination.
Because of variations in anatomical s tru c tu re , weight,
shape and positioning between animals, these types of experimental
procedures could provide little knowledge of the energy absorbed
(Leich er-P reka and Ho, 1977).
Q uantitative results were approximations
at best within experimental groups and interlaboratory replications of
experimental results were often d iffic u lt.
Microwave radiation has
long been known to be able of inducing rotational states in water
molecules.
As a consequence of the rapid spinning of water molecules
caused by microwave radiation, numerous intermolecular collisions
occur, leading to increases in the kinetic energy and temperature of
the absorbing substance.
The thermal absorption mechanism is the
criterion upon which the present United States exposure limits are
predicated (Mum ford, 1961;
T e ll, 1978).
The possibility of the existence
of other modes of microwave absorption th at may be nonthermal in nature
must be investigated under conditions of controlled temperature and
absorbed power.
These experimental conditions can best be achieved
in a waveguide irradiation system.
Presman (1961) has described an apparatus fo r the irradiation of
aqueous samples with microwave radiation.
It is illustrated in Figure l l l - l
46.
where it can be seen to be simply a test tube stuck through a waveguide.
There is no provisiBq^for measuring or controlling the tem perature of
the system.
More recently, Belkhode, Johnson and Muc (1974) have
described a microwave waveguide system th a t attempts to accomplish
similar objectives as the device presented in this chapter.
They have
incorporated a circuitous sample tube into the waveguide (see
Figure I I I - 2 ) .
They have insulated this tube with an amorphous
material and circulated water through a concentric tube as a heat
exchange flu id .
U nfortunately, the high absorption characteristics
of th e ir heat exchange fluid have probably caused as much energy
to be deposited in this material as was absorbed in the sample its elf.
In addition, the convolutions of tubing in the waveguide expose the
sample to differin g electric and magnetic field strengths.
Guy (1977) has developed a coaxial waveguide irradiation system
with a frequency range of 0-100 MHz.
His design features a teflon
sample compartment of toroidal geometry.
Th is system controls the
sample tem perature and measures the absorbed power and will doubtlessly
find important research applications.
B ut, as with the other irradiation
systems described, it does not lend itself to studies involving the use
of radioactive isotopes because of the d iffic u lty th at would be
encountered in decontaminating the apparatus between experiments.
The design of the microwave calorimeter described in this chapter is
an improvement over previous systems and offers several unique
features that will aid research with cell culture systems.
47.
A.
THE CALORIMETER IR R A D IA TIO N SYSTEM
The irradiation system used in this research has been previously
described (Rabinow itz, Olcerst and Mumford, 1977).
illustrated in Figure 11-3.
It is schematically
By locating the sample cell as shown in
Figure 11-4, the sample was exposed to a region of maximum electric
field strength.
Microwaves at a frequency of 2.45 GHz ± 50 MHz (a
wavelength of 12.4 cm) were generated by a FGMX-10 Raytheon diathermy
u n it.
The generator output was fed to a directional coupler that
sampled a portion of the generated energy which then went to a fixed
value precision attenuating device (P A D ).
Two Narda PAD's were
employed to reduce the power propagating through the exposure
system by 3 and 6 db ± 0 .2 db.
In this manner the samples could
be exposed to three levels of microwave power ( i.e . 0 db, -3d b ,
-6db attenuation at any given generator power output se ttin g ).
The
generator output power was controlled by a precision potentiometer
th at enabled the power to be varied from 0 to 85 Watts.
Microwave
energy th at was not directed into the waveguide portion of the
apparatus was dissipated into an anechoic chamber.
The waveguide
portion of the system consists of a coaxial to waveguide adapter, a
bidirectional coupler, a tu n er section, sample cell and sliding short.
The bidirectional coupler provides a means of measuring both the
incident and reflected microwave power by means of Hewlett Packard
432 and 435A meters.
The tu n e r section consists of two movable
stainless steel blocks th a t allow matching of the inductive and capacitive
discontinuities th at occur in the system as well as those introduced
by insertion of the samples.
By adjusting the sliding short and the
tu n e r, it was possible to match a sample into the system so that almost
48.
all the incident energy is absorbed within the sample and ve ry little
energy is reflected back toward the bidirectional coupler or dissipated
within th e walls of the waveguide.
A power reflection coefficient
(reflected pow er/incident power) of 0.001 has been measured when
blood samples were inserted into the sample chamber.
In this system the total power absorbed by the sample is measured
calorim etrically.
This is accompliched by maintaining a temperature
controlled flow of a heat exchange liquid through a compartment
surrounding the sample cell.
The coolant tem perature is controlled by a
Haake FKP circulating bath which maintained the tem perature over a
range of 0-100°C ±0.02°C .
Into this flow circu it were placed two
therm istors (Fenwall Electronics Co. model UUT51J1) having resistance
characteristics matched to 2%.
The two thermistors were connected as
legs of a Wheatstone bridge circu it (see Figure I II - 5 and I I I - 6 ) .
A
therm istor mounted in the inflow port monitors the incoming temperature
of the dodecane and the other therm istor at the outflow of the sample
cell measures the final tem perature.
When a sample absorbs microwave
en ergy, its increased tem perature heats the circulating flu id and changes
the resistance of the outflow therm istor.
By changing the decade
resistance (General Radio seven decade resistor) sufficiently to null
the Hewlett Packard model 419 D C -null meter, the absorbed power can
be determined by the following equation, the derivation of which is
given in Appendix 111—A .
Pa * =-''-4T = F T p ( n(^ r)- - f - - K)
Eq- ' " - 1
49.
where c is the specific heat of the dodecant (c a l/g °K )
v is the flow rate (g /m in )
C is cv (c a l/°K min)
T is absolute tem perature ( ° K )
A{3 is the difference in the exponential constants fo r the two
thermistors ( ° K )
P is the average exponential constant fo r the two thermistors (° K )
R^ is the measured decade resistance in the bridge circu it (Ohms)
Rj. is the value of the fixed resistance in the bridge circu it (Ohms)
K is A p /T e, a constant of the system (u nitless)
P
B.
is the absorbed power (cal/m in )
THE WAVEGUIDE
The waveguide used in the construction of this microwave calorimeter
had a WR-430 El A designation.
It was fabricated from aluminum by
Maury Microwave Inc. and had a width (a ) of 10.922 cm and a height (b )
of 5.461 cm.
The frequency of propagation in the transverse electrical
(T E 10) mode ranged from a cutoff frequency of 1.737 to 2 .6 GHz.
For
electromagnetic waves propagating in the fundamental T E 10 mode, a
solution to Maxwell's equations fo r a coordinate axis as shown in
Figure 111-7, yields electric and magnetic fields of the following form
(Belkhode et a [ ., 1974;
Reich et a L , 1953):
Eq. III - 2
j(u)t - y + <f>)
Ez = E0 sin (7xx/a)e‘
Eq. III - 3
50.
sin(7tx/a) e}E0\
Hy =
Where
j(u)t - y + $)
C 03(,lx/a) e'
Eq. 111-4
Eq.- 111-5
E0 Is the amplitude of th e electric field
7
0 is the impedance of fre e space
X is the free space wavelength
Xg is the wavelength of the electromagnetic wave within the
waveguide
<j> is the phase angle of the electromagnetic wave
Since the perm eability of biological material is approximately
equal to th at of free space, variations of the magnetic field within the
guide are of little concern, and interest in E2 , the electric fie ld , will
predominate.
As examination of the equation given above fo r E^ will
show that the electric field varies from a value of zero at each side
wall of the waveguide to a maximum value, E0 / at the center.
The
total power transm itted along the waveguide in the +X direction is
given by the following relationship:
2
E0Xab
Eq. 111-6
Solving equation 11-6 fo r the amplitude of the electric field at the
center of a transverse section of the waveguide (E 0) results in the
following (Reich et a L , 1953;
Corson and Lorraine, 1962):
Eq. 111-7
51.
C.
THE TEMPERATURE JACKETED SAMPLE COMPARTMENT
The key innovation of the irradiation assembly is the sample cell
and temperature jacketed holder illustrated in Figure 111 -8 .
They have
been designed to meet specific electrical and mechanical parameters.
E lectrically, they must act as a cylindrical dielectric absorber.
If the
radius of a sample cell and its dielectric constant are of the rig h t
proportions, the sample cell will act as a cylindrical waveguide
propagating electromagnetic energy and passing it ve rtically out of
the waveguide.
Since the objective of the sample cell was to supply
a known amount of microwave energy to a sample, it would not suffice
to allow the sample cell to act as a cylindrical waveguide.
The critical
relationship describing whether the sample cell would conduct or absorb
microwave energy has been described by Reich et aL (1953):
r =
where
f
c
3.412 s1* f
----------------------------------------------c
Eq.
is the critical frequency 2.45 GHz
c is the speed of light (m /sec)
r is the radius of the cylindrical sample holder (m )
s is the dielectric constant of the cylinder
The dielectric cylindrical sample holder considered above is in
reality fo u r concentric cylinders of differin g dielectric properties.
Consequently, an exact solution to this situation would be d iffic u lt to
obtain;
however, certain simplifying assumptions and approximations
have allowed a cell design th at assures th at total energy
occurs
within the sample cell.
absorption
Dodecane, a liquid alkane with
e' = 1.89 and e" = <10~4 was used fo r a heat exchange coolant.
The
11
52.
choice of dodecane as a coolant was based on the sim ilarity of its
dielectric properties with those of teflon (e 1 = 2.1 and e" < 10”3)
and consideration of viscosity, vapor pressure and heat capacity
(B h a rtia , 1975).
The resulting composite cylinder and the central
cylindrical sample chamber were designed to be less than th e critical
value of the radius ( r ) calculated from equation I I I - 8 .
From this dual
concentric cylindrical model, the maximum physical dimensions were
obtained that would allow the largest sample size bu t still not exceed
the equivalent critical radius where the sample holder would start to
act as a propagating cylindrical waveguide.
A set of machine shop
drawings of the various components of the sample cell are given in
Figures 111-9 through 111-11.
The sample tube had a volume of 1.5 ml contained within the
waveguide but was designed to accomodate an additional 4 .5 ml of
solution.
This feature allowed the termination of isotopic tran spo rt
experiments to be accomplished by the addition of large volumes of cold
stopping solutions containing inhibitors of tran sp o rt processes.
The
additional length of the sample tube external to the waveguide vortex
mixing of the sample without introducing material into the waveguide
and perturbin g the fie ld .
The teflon surfaces of the sample tube and
the inner section of the temperature jacketed holder act as a relatively
low friction bearing surface fo r the vortex rotation.
The frictional
heat generated by the mixing process was measured calorimetrically
in the absence of microwave radiation.
The teflon of the sample tube
offered an in e rt surface that was extremely easy to clean and free
of radioactive material.
This allowed successive irradiations to be
conducted with a minimum of inconvenience and no c a rry -o v e r contamination.
The tem perature jacketed holder Is constructed of two threaded
sections.
The inner section provides a flow directing baffle th at
channels the dodecane from the input port around the sample cell to
the outflow p o rt.
The large cylindrical block of teflon located on the
outer section provides some degree of thermal isolation from ambient
temperature changes while providing a firm vertical alignment of the
sample tube within the waveguide.
D.
CALORIMETER CALIBRATIO N AND TEMPERATURE D IS TR IB U TIO N
The rate of energy absorbed within the sample cell is proportional
to the tem perature difference between the two thermistors (A T ).
An
expression fo r AT has been derived in Appendix 111-A (see Eq. 3 ).
The constant parameters of this equation can best be evaluated by
calibrating the calorimeter, dissipating known amounts of power within
the sample cell.
A conventional resistive heating element was placed
in the sample cell and connected to a power supply, digital voltmeter
and ammeter.
From Equation 111-1, the power dissipated was proportional
to the specific heat of the dodecane multiplied by its flow rate.
Unknown parameters of Equation 2 of Appendix 111-A were found
by measuring ln (R d/R f ) as a funciton of coolant temperature with no
absorbed energy ( i. e . AT=0) as shown in Figure 111-12.
The variable
AP was found to be much less tem perature dependent than p.
assumed to be a constant.
It was
Figures 111-13 and 111-14 plot power
as a function of ln (R d/R f ) at two constant tem peratures.
The
absorbed power measured by means of the therm istor calorimeter
agreed with the net power measured with the waveguide bidirectional
coupler to within 0.1 db.
54.
The temperature distribution within the sample cell can be
expressed in a form known as Poisson's equation:
V2T(r,<|>,z) - -1/Kq(r,<J>,z)
Eq. III- 9
where K is the thermal conductivity of the sample and q is the heat
generated within the sample.
The rate of energy absorption can be
found by integrating over the whole sample tube:
Eq. 111-10
P = Jq(r,<j>,z)2nr*dr*d<|»*dz
Methods fo r calculating the electric field inside concentric cylinders
in a rectangular waveguide have been published (B h a rtia , 1976;
Neilsen, 1969;
Ho, 1975;
Vee, 1965 and Richmond, 1965).
In th e o ry ,
Equation 111-9 can be solved using Green's function fo r a cylinder
with the fixed tem perature (T = T 0) on the surface as a boundary
condiction.
Rather than solve Equation 111-9 numerically, it is more
illu strative to consider two extreme cases:
Case 1, where q, the heat
generated at any point within the sample cell, is a constant and
Case 2, where the heat generated at a given point within the sample
cell is proportional to the electric field strength at th at point and is
represented by a cosine term .
These two cases give an upper and
lower bound to the difference between the maximum temperature at
the axis of the sample cell and the tem perature at the periphery of
the cell cooled by the dodecane.
The firs t case assumes th at the
rate of energy absorption is uniform over the sample cell.
In the
second case, the field profile in the Z -V plane is rotated 180° to
give a three-dimensional field (see Figure 111-15).
4pVK
For Case 1:
Eq. 111-11
55.
and fo r Case 2:
-377cct\ P
f nr 2
a
fn r A
* T = - ^ r ^ T ^ r - a? sin2 ( , - r j j
Eq. m * i 2
These equations firs t presented by Rabinowitz, O lcerst and Mumford
(1977) are derived in Appendices l ll- B and lll- C .
For an absorption rate of 620 mW (an SAR o f 413 mW/g) the
maximum tem perature difference within the sample is 1.65°C fo r Case 1
and 1.16°C fo r Case 2.
Measurements of th e term perature differential
occurring at an SAR of 100 mW/g revealed a 0 .5 °C difference between
the central axis of the sample cell and the edge.
This value differed
by only 2.25% from the theoretical value calculated under the Case 1
assumption.
Belkhode, Johnson and Muc (1974) have arrived at an
equation identical to Equation 111-11 and have found th a t it adequately
describes the tem perature differen tial for a small radius sample cell.
E.
D IS C U S S IO N
There are several improvements and suggested modifications
th a t should be discussed to aid any fu tu re extension of the concepts
of the irradiation system th at has been described in this chapter.
Examination of a typical recorder trace presented as Figure 111-16,
demonstrates th at there is a constant fluctuation in the bridge balance.
Although this fluctuation is smalt and wihtout functional significance,
th ere are several contributing sources whose influence might be
reduced.
F irs t, the two thermistors have characteristics matched to 2%.
Replacement with custom fabricated thermistors whose characteristics
are better matched would fu rth e r reduce these random fluctuations.
Secondly, the flow system is maintained by means of a Fluid Metering
56.
pump from FMI In c ., which was selected because of its stable long term
pumping rate.
The outflow from this pump is, however, pulsate and as
such may contribute to the random fluctuations observable on the
recorder tra c e .
Maintaining flow be a g ra v ity syphon system was
attempted and did provide a more stable trace bu t proved unworkable
over long times when air bubbles freq u en tly became trapped around
the thermistors th e rb y changing th e ir thermal impedance.
It should
be possible to obtain a constant flow by resorting to a compressed
air system.
This might consist of a large sealed reservoir suspended
in a constant tem perature bath equipped with inflow and outflow tubes
and a pressure valve to connect it to a source of compressed air.
The time constant of 2 minutes could be improved by positioning the
thermistors closer to the sample cell (b u t still outside the microwave
fie ld ) and molding the sample tube's teflon walls th in n er than the
1/32 inch thickness th a t was machinable.
57.
FIGURE IN -1
WAVEGUIDE IR R A DIA TIO N SYSTEM OF A .S . PRESMAN (1961)
Z
58.
FIGURE 111-2
WAVEGUIDE IR R A DIA TIO N SYSTEM OF BELKHODE, JOHNSON AND MUC (1974)
K aat transfer
COil
atr
II
!
Sliding
short
ATT
LU LU
LU GC
microwave
I
calorimeter system
FIGURE 111-3
Q_ UJ
Schematic of
REVERSE
POWER
METER
59.
X
M
Waveguide Coordinate
System
60.
Epoxy Coating Covered with
Conducting Epoxy Coat
E
§
a>
00
22X
"O
o
CD
a
CD
UJ
LO
I
o
LU
o
OC
3
g
uu
Thermistor mounting
61.
O
co “o
<C
i
c
o
CO
CD
FIGURE III-©
Wheatstone bridge circuit
Null
Meter
Source
j y
When V )i4=0
^Decade
R t,
R-
FIGURE 111-7
63.
Removable Sample Tube
Dodecane
Coolant
Flow Directing
Baffle
Sample
FIGURE III—8
Assembly drawing of the temperature
jacketed sample compartment
65.
1
2>
.§
os
N
H < *>
<3
05
/
s
8
O )
I
■
§
t e
LLi
DC
ID
O
L i.
a
^
1
fe
fe
§
fc
S
5
.-O
££
66.
o
Ss
Qs
_ [_
—
LU
p )
CC
^
£
k} §
Qr
S
Q
=> ,-~ j
ki
th
£
r
5
S
- j
s
!
&
fcy
I
**^|oa
I
Z>
CD
LL
67.
E
o
O
o
Q
o
<3*
O
o
OR
OJ
<D
O
cri
o
ro
FIGURE 111-11
LO
ro
Q .
E
CG
CO
c
o
CD
■*->»
o
o
o
JQ
o
CG
O )
C=
CG
JC
o'
O
o
k-
CD
o
in
ro
co
c
o
0
1
cvj
*
MATERIAL - l.27cm TEFLON ROD
c
o
~
6_2
T In RD/R F=-1.40X T -0.261T+78.036
R*=0.998
FIGURE 111-12
Bridge Zero Power
Characteristics
305
300
295
290
J
-3 :0
L
-2 .0
J
L
I-
-1.0
0
-I—
I
+ 1.0
J
L
+ 2.0
FIGURE 111-13
\6 9 .
Ln Rd /R f VERSUS POWER A T 2 1 .84°C.
In R d/R fx |0
fO
TBath=21.84°C.
y = 6.894 x10 6x + 3.013x |0"3
R2= 0.999
■ 1500
Power (mw)
FIGURE 111-14 ;Ln R ./R
VERSUS POWER A T 31.24°C .
In Rd/R f x |0
+ 2.0
TBotll=31.240C.
y=5.741 x|0"6x -4.435X 10
R2= 0.997
-5 .0
0
1000
500
Power (mw)
1500
FIGURE 111-16
Bridge recorder trace
73..
CHAPTER IV
PASSIVE ION TRANSPORT
The experim ents described in this chapter constitute the main
body of my research.
T h ey were designed to investigate the effects
of continuous wave 2.45 GHz microwave radiation on the passive
e fflu x of ions from rab b it erythrocytes maintained at a constant
tem perature.
MATERIALS AND METHODS
Blood was drawn from the medial ear a rte ry of male New Zealand
white rabbits between 9:15 and 9:30 on the morning of experimental
runs.
As rabbits m ature, th ere is an increasing internal eryth ro cyte
sodium concentration and a decreasing internal eryth ro cyte potassium
concentration.
Smith et aL (1975) have found these parameters
change little a fte r rabbits reach 9 months of age.
between 5 and 9 lbs were used.
standard laboratory chow ad |ib .
T h erefo re , rabbits
Th ey were allowed water and
Approxim ately 16 ml of blood was
drawn from the medial a rte ry of the ear and collected in 4 ml yellow
capped Vacutainers (Becton-Dickinson C o .) containing one ml of
a c id -c itrate -d e xtro s e formula A anticoagulant. T h e blood was split into
two aliquots and then tran sfe rred to 50 ml conical bottom Nalgene
centrifuge tubes.
To each tu b e, 20 ml of room tem perature medium
was added and th e cells were centrifuged fo r 10 minutes at 3000g in a
Lourdes model VRA u n it.
The medium and top layer of white cells
were removed by aspiration.
Fresh medium was then introduced and
the washing procedure performed twice more to insure th a t all the
white
cells were removed and th a t the .erythrocytes had equilibrated with
the medium.
The composition of the medium is given in Table IV -1 .
Ouabain in a concentration of 0.1 mmoles/l inhibited active ionic
tra n s p o rt.
Double glass distilled deionized w ater was used
exclusively and the medium had a to nicity of 305 mOsmols.
The
medium was sterilized by passage through a 0.22 micron Millipore
Corporation Swinex filte r .
Loading Tracers into E rythrocytes
Sodium-22 and rubidium -86 were obtained from New England
Nuclear Co. The specific a c tiv ity of the rubidium -86 was 9 .5 m Ci/m g.
The sodium-22 isotope was c a rrie r fre e .
Rubidium-86 provided a
suitable analog fo r potassium while offering a longer half life than any
available potassium isotope (Kim elberg and Mayhew, 1974).
Radiological properties of these isotopes are listed in Table IV -2 .
These labeled chlorides were each evaporated to dryness and then
dissolved in 0 .5 ml of sterile medium and then mixed to form 1ml of
combined isotopic stock fo r use in a dual labeled experim ent.
N ext,
0.1 ml of the combined isotopes ( i . e . 20 pCi of sodium-22 and 403.2
pCi rubidium -86) was mixed in a screw cap 16 x 150 mm Kimax test
tube with Chaney adapters to insure precision.
These radioactive
aliquots were stored in a cold room at 4°C until they were used.
A fte r washing the erythrocytes with medium, the two aliquots
were recombined and packed du ring a ten minute centrifugation at
3000 g in a 75 x 100 mm p yrex te st tu b e.
One ml of packed
erythrocytes was withdrawn from the bottom of each tu b e .
This 1 ml
of erythrocytes was incubated with 0.1 ml of combined radioactive
sodium and rubidium stock. The specific activities present during
incubation were 18.2 pCi/ml in of sodium-22 and 366.5 pCi/ml of
rubidium -86 .
This incubation was accomplished in the screw cap test
tubes which were placed fo r two hours in a Forma Scientific bath
shaken 120 times per minute through a 1 inch path.
Rettori et a|;
(1969) determined th at two hours was a suitable incubation time to
introduce su fficien t a c tiv ity in tra c e llu la rly , so th a t the subsequent
e fflu x could be measured.
This incubation resulted in internal
e ryth ro cy te a c tiv ity concentrations th a t were 11% of the maximum
equilibrium value. Aliquots of these incubated packed erythrocytes
were used fo r both tem perature control and microwave irradiation
experim ents.
A fte r incubation, the erythrocytes were washed three
times with 0°C medium and packed by cen trifu g atio n .
The
supernatant was aspirated and the loaded packed cells were placed on
ice until th ey could be used.
Periodic samples of these loaded cells
were measured to determine cell efflu x and lysis.
were used within 14 hours after incubation.
All erythrocytes
Hematocrits were taken
with a Drummond heparinized m icrocapillary tube and clinical
ce n trifu g e.
Five to seven capillaries were filled fo r each
determ ination, a fte r the packed cells had been thoroughly mixed fo r
five minutes with a Clay Adams aliquot blood tube m ixer in the cold
room.
Irradiation Experiments
Irradiations were conducted in a microwave calorim eter system
th a t has been previously described (see C hapter I I I ) .
The
calibration and theoretical analysis of the tem perature distribution
within th e sample cell are presented in Chapter III and its
appendices.
The field strengths induced by the microwave radiation
can be calculated from the following expression (B aranski and
C zersk i, 1977):
E (V /m ) = (2 P /0 )15
Eq. IV -1
where
P is the absorbed power density
a is the conductivity in mhos. T h e field strengths under the
conditions encountered in these experiments are on the o rder of
several kV /m .
P rio r to an irra d ia tio n , 1 .5 ml of non-radioactive medium was
tra n s fe rre d to th e teflon sample tubes which were subsequently
capped with parafilm .
These filled tubes were then b rie fly
centrifuged in an I EC clinical centrifu ge to remove air bubbles
trapped on the sides or bottom of the tu be.
Tubes were stored in
the cold room until needed. Fifteen minutes before irra d ia tio n , the
tubes were inserted into the waveguide holder and allowed to reach
th e ir final equilibrium tem perature.
This tem perature was the result
of the tem perature of the dodecane circulated from a Haake FKP bath
and the heating due to microwave energy absorbed within the sample.
The Haake bath was equipped with a platinum resistor sensor and
proved rem arkably stable while maintaining the tem perature of the
coolant over a range of 0 to 100°C ± 0 .0 2 °C .
The tem perature of the
dodecane in the Haake circulator was monitored with a standard bomb
calorim eter thermometer with a range of 19-35°C and which was
graduated in hundredths of a degree C entigrade.
The average
tem perature of the medium was taken by tu rn in g o ff the Raytheon
PGMX-10 generator and qu ickly inserting a Yellow Springs Instrum ent
Company 18 gauge therm istor probe th a t had been preheated to
77.
approxim ately th e same tem perature as the bath.
Measurements
performed with this probe revealed a 0 .5 °C tem perature d ifferen tial
from the sample cell axis to its p e rip h e ry at specific absorption rates
of 100 mW/g.
This value d iffe rs by only 2.3% from the theoretical
value calculated under the assumption th a t microwave energy is
absorbed uniform ly over th e en tire sample cell (Case 1 assumption,
see C hapter I I I ) .
Irradiations were of one hour duration and were
eith e r timed with a GRA model 171 laboratory tim er connected in
series with the generator power or by means of a T 'mex ^SQ digital
watch which was readable in seconds.
To commence an irradiation experim ent, 0.005 ml of erythrocytes
containing labeled ions were mixed with the preheated medium in the
sample compartment.
This tra n s fe r was accomplished using a
calibrated Schwartz-Mann capillary tu b e.
A fte r one ho ur, the
irradiations were term inated and the blood cells and medium separated
e ith er by filtra tio n or refrig erated ce n trifu g atio n .
Hemolysis was
measured according to the standard cyanomethemoglobin technique of
the Hycel Corporation (H yc el, 1973).
Tem perature Control Experiments
Tem perature profiles of the passive e fflu x rates of sodium-22 and
rubidium -86 were measured in a Gilson respirom eter shaker bath
o u tfitted with a Cqoltemp immersion re frig e ra tio n unit th a t maintained
the tem perature to ± 0 .0 2 °C .
The shaking u n it had a 1 inch travel
and moved at one cycle per second to provide adequate m ixing.
Ten
ml of medium were tra n s fe rre d into 20 ml glass liquid scintillation
vials which served as the experimental container.
These vials were
allowed to equilib rate with the bath tem perature fo r fifteen minutes
before the s ta rt of an efflu x experim ent.
To begin a control experim ent, 0.03 ml of incubated erythrocytes
was drawn by suction into a Schwartz-Mann calibrated capillary tube
and mixed with 10 mis of the preheated washout medium in the liquid
scintillation v ia l.
Tem perature control e fflu x experiments were
determined at 0 .5 , 1, 1 .5 and 2 .5 hours.
A t a tem perature, five
replicate samples were taken and duplicate aliquots were counted from
each replicate.
The control tem perature experiments ranged from 2 .5
to 37°C , but p a rticu lar attention was given to th e region between 20
to 25°C , where the intervals were no more than one degree ap art.
Term ination of Efflux Experiments
Two series of experiments were perform ed, one in December 1976
(Series 1) and a second (Series 2) in August of 1977.
The only
difference in procedure between th e two series was the method of
term ination of th e efflu xes.
For Series 1, the method of Dalmark and
Weith (1972) was employed.
According to this method, Millipore filte r
cassettes were fitte d with 0.22 micron acetate filte rs and glass
p re filte rs .
The filte r cassettes were wrapped with parafilm to
p rev en t leaks. Erythrocytes were tran sfe rred out of the sample tube
or liquid scintillation vial with a Pasteur pipette and deposited in a 3
ml polyethylene Becton-Dickinson syringe to which the cassettes were
attached.
T h e plungers were inserted and th e erythrocytes quickly
separated from the medium.
Separations were accomplished in 15 to
20 seconds a fte r the termination of irra d ia tio n .
Dalmark and Weith
claim th a t such filtra tio n s could be accomplished with hemolysis under
1%. A fte r all th e medium passed through the filte r , continued
depression of th e plunger forced air into the syringe through the
79.
filte r ru p tu rin g eryth ro cytes.
coloration of the filtra te .
Hemolysis was evident by the
Consequently, only the fir s t 12-15 drops of
filtra te were collected.
Experiments comparing the filtra tio n method with the refrig e ra ted
centrifugation used to term inate Series 2 experim ents resulted in no
statistically significant differen ce between the two methods.
Filtration
was used in Series 1 to insure quick separation of erythrocytes from
the washout medium.
The results of the Series 1 ionic efflu x
experiments indicated th at in th e tem perature range of concern, such
quick filtra tio n rates were not c ritic a l.
During Series 2
experim entation, a Lourdes model VRA refrig e ra ted centrifuge was
used which removed any question of hemolysis during the separation
procedure.
Termination of a Series 2 efflu x was accomplished by
tra n s fe rrin g the eryth ro cy te suspension by Pasteur pipette to
p re-chilled 10 x 75 mm test tubes resting in an ice bath.
A fte r
centrifugation for 2 minutes at 3000 g at 0 °C , the tubes were
replaced in the ice bath until they could be sampled.
A
Schwartz-M ann pipettor was used to sample 0 .2 ml of the supernatant
washout medium.
These samples were collected in 12 x 100 mm Falcon
Company polyethylene vials.
A c tiv ity Determination
A Picker N a l(T I) well crystal gamma ray spectrophotometer was
used to count the a c tiv ity of Series 1 filtra te s .
Sample filtra te s with
volumes of 0 .2 ml were added to 2 ml of distilled water in a 16 dram
snap cap polycarbonate v ia l.
The samples were counted fo r fo rty
minutes during which time between 3000 and 5000 counts were
ty p ica lly accumulated in the photopeak window regions.
Rubidium
a c tiv ity was found by correcting the net count in a window from 940
80 .
to 1170 Kev fo r scattered sodium a c tiv ity .
Sodium ac tivity was found
by summing the net count in the region from 1170 to 1340 Kev.
Ten
standards of various activities were prepared fo r each isotope and
these provided detector efficiencies and contribution facto rs.
Minimum detectable tru e activities (M D T A ) were calculated at a 95%
confidence level fo r both ty p e I and ty p e II erro rs (A ltsch u ler and
Pasternak, 1963).
MDTA's of 0.88 and 2.5 7 pCi were found fo r
sodium and rubidium , respectively.
Series 2 supernatant activities were determined on a Beckman
th re e channel auto-gamma counter.
The gamma spectrum above 1170
Kev served as th e sodium window w heras, the region below this
energy was used to assess the. rubidium a c tiv ity a fte r correction fo r
scattered sodium counts.
Ten standards of each isotope were
prepared at various concentrations to provide detector efficiencies and
contribution facto rs.
MDTA's of 0.37 and 1.12 pCi were found fo r
the sodium and rubidium activities respectively.
Data Analysis and Statistics
The average a c tivity of the control efflu x at any of the sampled
time intervals was graphed as a semi-logarithm ic plot with time as the
abscissa.
(F o r example see Figure IV - 1 ) .
T h e data were fitted by
least square linear regression to the single exponential model of zero
tran s efflu x (see Hoare, 1973 fo r a comparison of the types of
membrane tra n s p o rt processes) as described by Hoffman (1962) and
R ettori and colleagues (1 9 6 9 ).
T h e ir model is based on the
assumption th a t backflux into the erythrocytes is negligible when the
hematocrit is <0.3%.
less than 0.25%.
The hematocrit in these experiments was always
Correlation coefficients close to 1 .0 were typically
found and indicated the strength' of this model in in terp retin g the
data.
The data from tem perature control experiments were used to
obtain the best estimate of the efflu x a fte r one hour. These hour
e fflu x values were used to form A rrheniu s plots which were fitte d by
least square linear regression.
The one hour tem perature control
efflu x values were compared to the one hour irradiation e fflu x values.
Because both Baranski et a[. (1974) and Ismailov (1971) reported
increase in passive tran sp o rt under conditions of microwave
irra d ia tio n , a one-sided hypothesis th a t microwave radiation increases
efflu x relative to a control experim ent is an appropriate hypothesis to
te s t. The variances of the control and irradiated experiments were
found to d iffe r through the use of the "F" test (H oel, 1966).
T h e re fo re , statistical significance was determined by use of a t"
statistic re fe rre d to a standard normal d istrib u tio n , according to
Brown and Hollander (1 9 7 7 ).
RESULTS
A rrhenius plots (in v e rs e tem perature vs . the logio of the
a c tiv ity existing from one ml of erythrocytes a fte r one h o u r) are
given in Figures IV -2 and IV -3 for sodium and rubidium respectively.
Between 2 .5 and 37°C these plots exh ib it two linear regions with a
transition occurring at 2 0 -2 2 .5°C (see Tables IV -3 and IV - 4 ) .
T h ere
are an insignificant number of data points to conclusively characterize
th e upper and lower regions as being lin ear, however, the data do
not disagree with a linear approximation.
The slope of each linear
region represents the activation energy of the tra n s p o rt process.
T h e activation energies determined fo r the linear regions are positive,
whereas within th e transition region a negative activation energy was
found.
SODIUM EFFLUX
The sodium ion irradiation data are shown in Figure IV -2 and
Table IV -5 .
A t 36°C , th e highest tem perature of the uppermost
critical region, an increased passive efflux statistically d iffe re n t from
th e tem perature control values was observed (p < 0 .0 0 0 1 ).
Additional
statistically significant increases were also seen at tem peratures of
0 .5 °C above and below this critical region.
Irradiation at th ree specific absoption rates of 100, 190 and 390
mW/g near the end of the middle transition (2 2 .5 ° C ) resulted in
statistically significant increases in passive efflux (p < 0 .0 0 0 1 ).
The
83.
e fflu x increased as th e specific absorption rate was increased from
100 mW/g to 190 mW/g but the e fflu x at an SAR of 390 mW/g was
sig nifican tly less than even at the smallest specific absorption ra te .
The factors by which these exposures exceed control efflu x
experiments ranged from 1.38 to 10.57 (see Table IV - 7 ) .
Irradiations
at other tem peratures along th e linear regions between transitions are
indistinguishable from points predicted from the tem perature control
p ro file.
An exception to this is the singular irradiation with a
specific absorption rate of 190 mW/g at 13°C .
This sample exceeded
the control value by a factor g rea ter than 10.
R UBIDIUM EFFLUX
The rubidium irradiation data (see Figure IV -3 and Table IV -6 )
indicate no difference between irrad iated samples and controls except
at the transition tem peratures of 36°C , 22.5°C and 13°C.
A t these
tem peratures, exposure to‘ microwave radiation, with specific
absorption rates g rea ter than of equal to 100 mW/g resulted in
statistically significant increases in efflu x as compared to control
exp erim en ts'at confidence levels g rea ter than 0.002.
The efflu x of
exposed samples at this critical tem perature exceed th a t of control
samples by factors ranging from 1.64 to 7.66 (see Table IV - 7 ) .
Irradiations were conducted at th e 22.5°C tem perature transition at
th re e specific absorption rates of 100, 190 and 390 mW/g.
The
rubidium passive efflu x does not increase monotonically when the
specific absorption rate is doubled.
In fa c t, th e highest exposure (a t
an SAR of 390 mW/g) resulted in an increase in rubidium e fflu x ,
which was not statistically d iffe re n t from the lowest exposure rate at
an SAR of 100 mW/g.
84.
DISCUSSION
I have found temperatures at which the A rrhenius plots fo r the
efflu x of sodium and rubidium ions from rabbit erythrocytes change
slope.
At these transition tem peratures, the combination of
conventional heating and the absorption of microwave energy result in
substantial increases in passive e fflu x , exceeding control values by a
factor of as much as an order of magnitude.
A temperature increase
of more than 7 .5 °C would be required to obtain a rate of sodium
efflu x similar to th at found at 22.5°C with an SAR of 190 mW/g.
A
13.0°C temperature rise would be needed to cause the rubidium efflux
observed under the same conditions.
The sample cell design is such
as to preclude a temperature increase of more than 2°C above the
tem perature of the dodecane coolant.
The control data indicate th a t, as the tem perature increases
0 .5 °C through a transition from 22.0°C to 2 2 .5 °C , sodium efflux
decreases be a factor of 1.62.
A similar reduction in rubidium
e fflu x , by a factor of 1.54, is seen when the temperature is increased
by 1.5 °C from 21.0°C to 22.5°C through the same transition.
Changes of similar magnitude were found at both the 36°C and
1 1 .0 -1 3 .0 °C transitions for each ion.
Linden and associates (1973) investigated the transport of
p-glucose and 0-galactose in E. coli.
They demonstrated two tra n ­
sition temperatures th at are dependent on the fa tty acid composition
of the cell membrane.
At the higher of the two transition
tem peratures, they measured a striking increase in lateral
compressibility of the membrane lipids and a doubling of the
rate of sugar tran sp o rt as the tem perature decreased 0 .7 °C .
Similar increases in the diffusion rates of sodium and sucrose
85.
have been reported at the 41 °C transition of artificial
dipalm itoyl-phosphatidyl choline liposomes by Jacobson and
Papahadjopoulos (1975).
Elford and Solomon (1975) have described Arrhenius plots for
sodium and rubidium in flux in dog erythrocytes th at are consistent
with our observations of membrane transition temperatures in rabbit
erythrocytes.
They found two linear regions with a transition at
22.4°C for sodium ions, and two linear regions with a transition at
1 1 .0 -1 3 .0 °C fo r potassium in flu x .
T h e ir results indicate th at ion
influx in dog erythrocytes occurs at two d ifferen t regions of the
membrane.
In the experiments presented in this dissertation a
transition was observed at 20 .0 -2 2 .5 °C fo r both sodium and rubidium
ions.
This' suggests th at in the rabbit eryth ro cyte, transport of
sodium and rubidium may take place at the same locus or loci.
The
observation of a 22.5°C transition in A rrhenius plots of facilitated
tran sp o rt of 3-O-m ethylglucopyranoside in rabbit erythrocytes
(unpublished observations) supports a more general interpretation
th a t at 22.5°C there occurs a phase change which results in
reordering of the erythrocyte membrane.
The sodium ion efflu x at 13.0°C fo r erythrocytes irradiated at
an SAR of 390 mW/g exceeds the 13.0°C temperature control efflux
by a factor of 10.57.
This microwave induced effect occurs near
the 8 .0 -1 1 .0 °C transition tem perature depicted in the Arrhenius
plot of Figure IV -2 , page 104.
Sodium efflux data at 11.6°C
obtained during irradiation at an SAR of 95 mW/g and also data
measured at 13.8°C during irradiation at an SAR of 375 mW/g
both fall on the linear region of. the Arrhenius plot between
transitions. The irradiated 13.0°C sodium efflux data therefore
86.
does not support the hypothesis suggested by observations of
microwave effects at higher transition tem peratures, i.e . that
s
erythrocyte membranes are sensitive to microwave radiation near
phase transition temperatures.
However, the Arrhenius plot of
passive rubidium ion e fflu x , shown in Figure IV -3 , page 105, exhibits
an upper transition tem perature of 13 .0°C .
Perhaps, at this lower
tem perature, passive ionic sodium and rubidium transport occurs at
d iffe re n t membrane sites as described by Solomon and Elford (1975) in
canine erythrocytes. The data suggest th at passively transported
sodium ions maybe exiting the erythrocytes through regions,
succeptible to perturbation by microwave radiation, where rubidium
ions are predominantly transported.
Additional experimentation with
simple lipid systems might cla rify the nature of this observation.
Rettori and associates (1969) have investigated ouabian sensitive
sodium efflux in rabbit erythrocytes.
They observed th at concen­
trations of O.ImM ouabain in the media inhibit the active component of
ionic tran sp o rt mediated by Na-K ATPase.
The energy dependent process of active transport removes in tra ­
cellular sodium ions against a concentration gradient to maintain a
relatively low internal sodium concentration and a high potassium
concentration.
Rabbit erythrocytes have similar intracellular sodium,
potassium and water content when compared to human erythrocytes as
seen in Table 11-3, page 39.
Plasma in both species contain approxi­
mately 5 meq/l of potassium and 137-142 meq/l of sodium.
The rate of
sodium efflux in rabbit erythrocytes is three times faster than that found
in human erythrocytes.
Approximately fifty percent of the sodium is
87.
transported by passive exchange diffusion.
Ouabain inhibition of
Na-K ATPase provides a direct method fo r investigating the passive
transport of ions through erythrocyte membranes (Hoffman, 1962, See
pg. 33).
Rettori (1969) equilibrated 1ml of rabbit erythrocytes with 20mls
of labeled media during a 2 hour incubation to introduce radioactively
labeled tracers in tracellu larly.
He reports that when such incubations
are performed in the presence a ouabain that a 50% increase in
intracellular sodium can be measured.
Subsequent passive sodium
ionic efflux remain unchanged when ouabain is present in the ex tra ­
cellular media.
As described in the material and methods section of Chapter IV,
the incubation procedure used in these experiments differed from the
classical procedure used by Rettori.
One m illiliter of packed erythro­
cytes was incubated for two hours with 0.1ml of media containing both
sodium-22 and rubidium-86 isotopes.
The volume ratio of
erythrocytes to incubation media d iffe r from that used in classical
transport experiments and consequently, equilibrium was not obtained
during the two hour incubation.
Aliquots of erythrocytes used in
both microwave irradiation and temperature control passive efflux
studies were prepared following and identical incubation protocol.
The author is unaware of other studies of passive transport of
sodium and rubidium in rabbit erythrocytes at temperatures below
37°C. Rettori and co-workers (1969) have reported an efflux rate for
sodium at 37°C of 9 .3 m e q /K g /h r th a t exceeds the value of 0.43
m e q /K g /h r reported in this present study by an order of magnitude.
This difference resulted p rim arily from the incubation procedure used
which only attained internal isotope levels th a t were approxim ately 11%
of the maximal equilibrium values.
R ettori's study and this
experim ent cannot be d irec tly compared because of other differences
in media and possible differences in experimental animal age (see
Smith et a L , 1975). Nevertheless, the experimental work reported in
this dissertation was designed to d iffe re n tia te between tem perature
control efflu x and microwave efflu x measured on aliquots of
erythrocytes incubated in the same manner.
Rubidium-86 was used as an analog fo r potassium efflux in these
experiments because of its suitably long radiological half life.
Ru-
bidium -86 has been used to measure passive ion efflu x and in flux in
plant celis (M a lle ry , 1979; Gronewald, 1979).
Kimelberg and Mayhew
(1974) employed rubidium -86 as a potassium analog to investigate
ouabain sensitive A TP -ase a c tiv ity in transform ed mammalian cell
lines.
Delamere (1979) found rubidium -86 useful in studies of
membrane depolarization of ra b b it lenses both in the presence and
absence of ouabain.
Pang and co-w orkers (1979) investigated changes in rubidium -86
e fflu x and membrane flu id ity in egg phospholipid vesicles exposed to
anesthetic compounds.
Th ey re p o rt a correlation between cation
perm eability and lipid bilayer perturbations measured by a 1 ,6 -d i­
phenyl 1 ,3 ,5 hexatriene fluorescent probe.
Th ey rep o rt th a t 2-5%
changes in anesthetic concentration increase the flu id ity of th e inner
hydrophobic regions of the membrane bilayer inducing functional
increases in perm eability of from' 20-50%.
The perm eability increases
reported were an o rder of magnitude greater than the associated
stru ctu ral changes.
Passive tra n s p o rt of cationic ionophores, th e re ­
fo re , provides a sensitive system with which to investigate
tem perature dependent membrane processes.
Ismailov (1971) and Baranski (1974) have both reported
increased potassium e fflu x from microwave irradiated erythrocytes as
described in more detail in Chapter II (p g 1 1 -1 4 ).
The efflux
experiments described in this dissertation using rubidium -86 as an
anolog fo r potassium ions were designed specifically to investigate
these reported microwave induces effects as well as the existence of a
“leaky membrane" hypothesized by Weiter and Finch (1975, see pg.
17 ).
The microwave field has a significant effect on the efflux of
sodium and rubidium at transition temperatures (w ith the possible
-0
exception of the anomalous. irrad iated sodium efflu x at T 3 1 C ). The
discontinuities in the A rrheniu s plot are likely to be the result of
stru ctu ral changes at the e fflu x site.
Because I have not observed
increases in efflu x fo r passive ionic efflu x or the e fflu x of
3-O -m ethylglucopyranoside along the linear regions of the A rrhenius
plot, the hypothesis of a leaky membrane can be discounted.
The eryth ro cyte membrane and the sodium and rubidium
tran sp o rt site are chemically complex systems and it is not possible to
id en tify a specific stru ctu ral change associated with each tran sitio n .
Such changes may occur in the membrane lipids, proteins, surface
w ater (see Clegg and D rost-H ansen, 1978), or in combinations of
these elements.
Furtherm ore, transitions may involve various
stru ctu ral changes of a d iffe re n t n a tu re .
While I cannot provide a
d e fin itive mechanism fo r an increase in absorber concentrations near
th e efflu x site, two altern ative mechanisms should be discussed:
1.
During the stru ctu ra l tran sitio n s, new absorbers are
introduced into th e e fflu x site or its v ic in ity .
Th ey may be created
by th e stru ctu ral change at the site o r introduced from the
extracellular spaces;
2.
The dynamics of the stru ctu ra l transition makes some p a rt
of th e efflux system p a rtic u la rly sensitive to microwave fields even
though no additional absorption occurs.
The major absorber of microwave radiation in a biological system
is w ater.
In comparison to the aqueous fractio n , the cell membrane is
a poor absorber and most of the energy tra n s fe rre d to it from the
*
microwave field is tra n s fe rre d by collision.
Free and bound water in
the pores and channels of the membrane or portions of large molecules
th a t have rotational freedom (R abinow itz, 1973) are excellent
absorbers. If the number of these absorbers is increased locally,
microscopic tem perature gradients can be maintained in the membrane
(see Sheridan et a [ . , 1979).
Clegg and Drost-Hansen (1978) have considered the importance
of interfacial and intracellular water in detrmining electromagnetic
effects. They believe th a t collections of w ater moldecules are capable
of energy interaction over long distances and will exh ib it cooperative
effects of the ty p e described by Frolich (1 9 4 8 ), 11linger (1 9 7 8 ),
Monod, Changeaux and Jacob (1 963 ).
Th ey suggest th a t the large
electrical potentials observed at the interface between freezing ice
crystals and unfrozen solution by Workman and Reynolds (1950) are
caused by such cooperative behavior.
Potentials of as much as 230
volts have been observed at the interface between phases fo r some
solute-w ater combinations.
Such potentials over distances of 100 A
would yield potential gradients approaching 105 kV /cm .
Drost-Hansen
(1969) has proposed a mechanism fo r the generation of these freezing
potentials based on th e selective absorption of anions by vicinal
crystalline w ater.
Such an absorption is necessary to relieve the
strain energy of ice crystals as th ey assume a more nearly tetraedral
shape in th e ir frozen state.
He theorizes th e existence of sheets of
oriented dipoles capable of accepting anions and repelling cations
during the freezing process.
Such sheets sould act cooperatively to
bring ions across th e energy b a rrie r.
The resu ltan t freezing
potentials exist only during changes of state and once the transition
is complete, they are no longer observable.
Following a similar
conceptualization as D rost-H ansen, cooperative effects th a t lead to
freezing potentials might be found at critical tem peratures where two
phases exist.
Such potentials would lower the b a rrie r potential and
resu lt in increased pore size and the observed changes in lateral
com pressibility.
Microwave radiation could couple into the membrane
under these conditions by polarizing the transition charge distribution
or effecting tran sitio n dipole orientation.
If the s tru ctu ra l change involves the reordering of lipids at a
transition tem p eratu re, two phases exist simultaneously and the lateral
compressibility of th is region is g re a tly increased (Linden et a L ,
1973).
This increased compressibility will allow dipolar molecules to
penetrate the membrane at the interface between phases and provide
local absorbers in th a t region.
This increased lateral compressibility
also allows more freedom fo r th e constrained rotors already in the
membrane and will increase absorption of microwave radiation at the
interface between the phases.
If these new tran sien t absorbers are
functional parts of the tra n s p o rt system, the effect on passive sodium
and rubidium efflu x could be d ire c t.
T h e nonlinearity of the response of passive e fflu x to microwave
radiation could be a resu lt of a change in th e three-dim ensional
configuration of a protein involved with e fflu x .
In th a t case, at the
transition tem perature two protein structu res could exist within the
transition region during a phase tran sitio n .
If at least one
interm ediate s tru c tu re is a b e tte r absorber than th e initial and final
configurations, this would provide a suitable mechanism.
Furtherm ore, if orientation by the field would enhance the transition
from one intermediate state to another, this transitional state would
provide an irre v e rs ib le path fo r th e absorption of energy from a
microwave field th a t is not available at either higher or lower
tem peratures.
It is not necessary to postulate increased total absorption by the
e ryth ro cy te membrane in o rder to explain the observed results.
If
two local phases exist simultaneously, the constituents would be
expected to segregate between the phases.
If one of the phases
contained both the absorbers and the efflux sites, this would provide
a mechanism fo r the interaction between microwave fields and the
tra n s p o rt system.
T h e increases in efflu x we observed at 22.5°C are not monotonic
functions of an increasing specific absorption rate .
In fa c t, fo r
sodium ions, at the greatest SAR of 390 mW/g th e effect was th e
•
93.
smallest.
T h e duration of th e applied field and the efflu x
measurement were the same fo r each absorption rate .
If the
simultaneous existence of two lipid phases or an interm ediate protein
configuration could cause an increased tra n s p o rt, then one might
expect this re s u lt.
As the absorption rate increased at fir s t the
increased energy would populate this interm ediate configuration from
the initial configuration, but as absorption continued, the population
of the interm ediate configuration would be depleted.
For higher
absroption rates the amount of time each system was in the
interm ediate state would decrease and the integral R ( x , t ) (Rabinowitz
et a h , 1977) over the en tire cell cu ltu re tu be fo r the duration of the
exposure will be less than th a t at lower SAR's.
Changes in the binding and the release of radioactive calcium
from neural tissue of cats and chickens has also been found to exh ib it
such a “power window1' (B aw in, 1975, 1976, 1 9 77a ,b ).
Working with
extrem ely low freq uen cy fields and also when modulating 147 MHz VHF
fields with brain wave frequencies of 4-35 H z, Bawin and associates
have observed th a t weak electric signals may act as trig g e rs fo r more
dynamic cellular events.
Th ey have found th a t ELF signals resulted
in an increased calcium binding th a t was dependent on both the
frequency and amplitude of the incident fie ld .
Significant binding
was found at 6 and 16 Hz with 56 V/m field strength but not at 1, 32
and 75 Hz. No discernible effects were seen at field strengths of 10
and 100 V/m (B aw in, 1976).
In another s tu d y , Bawin (1977b)
modulated 147 MHz, 0 .8 mW/cm2 fields at 16 Hz and found an 18%
increase in neural membrane calcium release.
No effect was found
with th e 147 MHz electromagnetic wave in the absence of the lower
frequency modulation. Similar increases with a 16 Hz modulation were
observed with a 450 MHz carrier at power densities of 1, 5 and 15
mW/cm2 .
A 10% change was observed a fte r a tw enty minutes exposure
to a power density of 1 mW/cm2 .
Although these experiments were
reported as being prelim in ary, no effect was seen at field strengths
higher than 2 mW/cm2 . Recently, these experiments were repeated by
Blackman et aL (1 9 8 0 ), who were also able to demonstrate a "power
window".
T h e w ork presented here at SAR's of 290 mW/g may be an
example of a similar power dependent e fflu x .
CONCLUSION
VI have demonstrated th a t microwave fields can affect the passive
e fflu x of both sodium and rubidium ions from rab b it erythrocytes at a
tem perature where the A rrhenius plot fo r sodium and rubidium change
slope.
I have discussed possible mechanisms fo r this effect but
cannot provide a defin itive mechanism fo r this phenomenon.
In
general, such mechanisms depend on interm ediate configurations th at
exist only a t a critical transition tem perature and is th erefo re only
tran sien t.
These results could serve to reconcile discrepancies in the
lite ra tu re concerning microwave effects on ionic tra n s p o rt (see
S todolnik-B aranska, 1971; Stem ler, 1968, 1972, 1973;
a L , 1974;
Baranski et
Hamrick and Z in k l, 1975; and Ismailov, 1971).
The
existence of intermediate configurations th a t are postulated to explain
the experimental results presented in this chapter may be influenced
not only by tem perature, bu t by cation concentration (see Kimelberg
and Papahadjopoulos, 1972), trace components in the membrane and
pH (see T rau ble and Eibl, 1974).
F u rth er understanding of the
underlying mechanisms of microwave interaction would result from
experim ents with simple lipid combinations.
T A B L E IV-1
BUFFERED MEDIUM COMPOSITION
mmoles/l
NaCI
150.00
KCI
5.00
Na2H P 04
5.20
NaH2P 0 4 -H 20
0.80
CaCI2 -2H20
1.00
MgCI2
0.25
'
Ouabain
0.10
Rabbit Albumin fraction V
2.00 g /l
TABLE IV -2
ISOTOPE PROPERTIES*
Sodium-22
Half Life
2.6 2 years
Betas
1.820 max (0.05%) Mev
0.545 max
Gammas
Rubidium-86
*(L e d e re r et a L , 1968)
Half Life
<
Mev
0.511
(180%) Mev
1.275
(100%) Mev
18.66 days
Betas
1.780 max
Mev
Gammas
1.078
Mev
98.
TABLE IV-3
SODIUM EFFLUX TEMPERATURE CONTROL DATA
TEMPERATURE
# SAMPLES
(°C )
Na-22 NET EFFLUX ± S .E .
(n C i/m l R B C /h r)
0.13 + 0.26^
8
8.0
41
0.69 + 0.01
10.0
10
0.33 + 0 .0 2 *
10.0
41
0.36 + 0.01
13.0
23
0.35 +
15.0
23
0.51 + 0 .2 5 *
20.0
12
1.48 + 0 .2 5 *
20.0
36
1.67 + 0.01
21.0
43
2.02 + 0.02
22.0
30
2.20 + 0.01
22.5
37
1.36 + 0 .0 2
23.0
30
1.43 ± 0.01
24.0
37
1.61 ± 0.01
25.0
32
2.34 ± 0 .0 3 *
25.0
39
2.31 ± 0.02
30.0
15
4.47 ± 0 .0 6 *
30.0
40
3.67 + 0.03
35.5
28
8.84 + 0.05
36.0
39
5.14 + 0.05
37.0
37
5.82 + 0.07
* Series 1 Data
*
CVJ
o
o
2.5
99.
TABLE IV-4
RUBIDIUM EFFLUX TEMPERATURE CONTROL DATA
TEMPERATURE
# SAMPLES
(n C i/m l R B C /h r)
00
o
(°C )
Rb-86 NET EFFLUX ± S .E .
27
3.31 ± 0.17
10.0
40
4.53 ± 0.09
13.0
16
3.29 ± 0 .5 4 *
13.0
39
3.25 ± 0.48
20.0
39
9.10 ± 0.12
21.0.
42
8.63 ± 0.12
22.0
30
5.86 ± 0.11
22.5
39
5.59 ± 0.12
23.0
13
5.97 ± 0 .6 0 *
24.0
38
5.82 ± 0.10
25.0
39
7.79 ± 0.09
30.0
27
8.60 ± 0 .4 8 *
35.5
29
15.35 ± 0.26
36.0
39
11.28 ± 0.17
37.0
39
14.68 ± 0.21
* Series 1 Data
100.
TABLE IV -5
SODIUM EFFLUX IR R A D IA TIO N DATA
TEMP.
°C
S .A .R
mW/g
Na-22 NET E X IT +S .E .
ttSAMPLES
STAT.
T
S IG N IF .
n C i/m l/R B C /h r
CONFIDENCE
LEVEL
95
0.28 ± 0.03
3
No
13.8
375
0.47 ± 0.11
4
No
16.5
100
0.69
4
No
17.4
210
1.09 ± 0.15
3
No
18.7
410
1.17 ± 0.49
1
No
22.5
100
2.62 ± 0.19
2
Yes
23.5
190
1.51 i 0.3 6
2
No
25.0
400
2.1 2 ± 0.3 6
2
No
25.4
100
2.17 ± 0 .9 4
3
No
26.0
170
1.99 ± 0.2 7
3
No
26.9
380
2.8 7 ± 0.28
3
No
i+
o
•
11.6
00
SERIES 1
6.57
P<0.0001
SERIES 2
13.0
390
3.70 ± 0.2 7
7
Yes
12.37
P<0.0001
22.5
100
3.16 ± 0 .1 7
10 .
Yes
10.52
P<0.0001
22.5
190
4.47 ± 0 .1 7
6
Yes
18.17
P<0.0001
22.5
390
1.88 ± 0 .1 4
9
Yes
3.68
P<0.0001
35 .5
190
15.75 ± 0.43
10
Yes
15.96
P<Q.0001
36.0
190
10.36 ± 0.39
10
Yes
13.28
PC0.0001
37.0
190
14.11 ± 0.26
7
Yes
30.79
' P<0.0001
101.
TABLE IV-6
RUBIDIUM EFFLUX IR R A D IA TIO N DATA
'
TEMP.
°C
S .A .R
mW/g
Rb-86 NET E X IT +S .E .
#SAMPLES
n C i/m l/R B C /h r
STAT.
T
CONFIDENCE
LEVEL
S IG N IF .
SERIES 1
11.6
95
4.00 ± 0.47
1
No
13.8
375
3.26 ± 1.96
4
No
16.5
100
5.20 ± 5.93
2
No
17 .4
210
6.29 ± 3.34
1
No
18 .7
410
6 .5 4 ± 9.20
1
No
22 .5
100
7.6 9 ± 0.42
2
Yes
23 .5
190
6.42 ± 1.91
3
No
25 .0
400
7.15 ± 0.74
2
No
2 5 .4
100
6.82 ± 2.92
4
No
2 6 .0
170
7.93 ± 0.77
3
No
26 .9
380
8 .3 4 ± 3.82
4
No
13.0
390
24.90 ± 4.71.
8
22.5
100
9.15 ± 1.65
22 .5
190
22 .5
4.83
PC0.0001
Yes
4.57
P<0.0001
9
Yes
2.16
P<0.002
21.26 ± 1.93
8
Yes
8.10
P<0.0001
390
9.66 ± 1.34
10
Yes
3.03
P<0.001
35.5
190
22.27 ± 1.71
10
Yes
4.00
P<0.0001
3 6 .0
190
27.28 ± 1.93
10
Yes
8.13
P<0.0001
37.0
190
17.29 ± 2.05 *
7
No
SERIES 2
TABLE IV -7
EFFECT OF MICROWAVES ON EFFLUX
Average Temp.
(° C )
Specific Absorption Rate
E fflux Radiation/Control
mW/g
I. Rubidium-86
13.0 ( 8 )a
390
7.66 ± 8.71
22.5 ( 9)
100
1.64 ± 0.91
22.5 ( 8)
190
3.8 0 ± 0.47
22.5 (1 0)
390
1.73 ± 0.7 9
35.5 (1 0 )
190
1.45 ± 0.38
36.0 (1 0)
190
2.42 ± 0.59
13.0 ( 7)
390
10.57 ± 3.5 4
22.5 (1 0)
100
2.32 ± 0.45
22.5 ( 6)
190
3.29 ± 0.42
22.5 ( 9)
390
1.38 ± 0.33
36.6 (1 0 )
190
2.02 ± 0.27
37.0 ( 7)
190
2.42 ± 0.21
lodium-22
( ) a = number of irradiated samples
103.
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Figure IV -2
A rrhenius plot of sodium e fflu x .
U nirradiated
tem perature controls are represented by open
symbols ( 0 ) .
Irra d ia ted samples are represented
as darkened symbols ( ♦ ) .
Specific absorption rates
are in units of mW/g.
( • = 100,
Temperature (°C)
36
225 15 8
20
10
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3.4
3.5
0/TIK)X10J)
see Table IV -5 series 1
36
■ = 190,
A = 39 0).
Figure IV -3
A rrheniu s plot of rubidium e fflu x .
U nirradiated
tem perature controls are represented by open
symbols ( O ) .
Irradiated samples are represented
$
darkened symbols ( ♦ ) . Specific absorption rates
(S A R ) are in units of mW/g. ( •
▲ = 39 0).
Temperature (°C)
O
CD
K 10
OS
see Table IV -6 series 1
= 100,
■ = 190,
V . SUMMARY
According to th e classical thermal mechanism of microwave
absorption, dipoles are induced to reo rien t under the influence of an
oscillating electromagnetic fie ld .
A tem perature increase occurs
through the collisional tra n s fe r of kinetic energy of the dipole with
neighboring molecules.
Biological effects resulting from microwave
absorption according to a classical thermal mechanism resu lt from
general heating and increase in d ire c t proportion to the total absorbed
power unless there occurred an irre v e rs ib le change such as thermal
denaturation.
Athermal absorption mechanisms have been theorized
to occur when electromagnetic energy is absorbed d irec tly by a
ta rg e t molecule.
Biological functions associated with this ta rg et
molecule might then be affected by microwave radiation in the absence
of macroscopic tem perature increases and w ithout the tran sfe r of
kinetic en erg y.
T h e existence of athermal absorption mechanisms has
proved d iffic u lt to demonstrate in biological systems because of the
dominant thermal absorption resulting from the ubiquitous presence of
water molecules.
This thesis describes th e design, construction and calibration
of a microwave irradiation system suitable fo r exposing cell suspensions
or solutions to 2.45 GHz microwave radiation under conditions of controlled
tem perature and absorbed power.
This irradiation system was used to
examine the influence of microwave radiation on the passive tran sp o rt
of sodium-22 and rubidium -86 in th e erythrocytes of male New Zealand
white rabb its.
The e fflu x of radioactively labeled cations from the
erythrocytes to the extracellular medium was measured after irradiations
107.
of one hour duration.
Arrhenius plots of the efflux of irradiated cell
suspensions were compared with plots of control cells maintained in a
conventional water bath.
The control efflux experiments revealed th at rabbit erythrocytes
exhib it a decrease in tran sp o rt as the tem perature is increased
through a critical phase transition.
This phenomenon provides an
experimental system th at can be used to d ifferen tiate effects of
microwave radiation from a classically thermal absorption mechanism.
Both Ismailov (1971) and Baranski et aL (1974) have reported th at
microwave radiation of erythrocytes results in increases in passive
ionic tran sp o rt.
The confirmation of such microwave induced
increases in erythrocyte suspensions maintained at a critical
tem perature where the control cell thermal response would result in a
decrease in e fflu x , demonstrates th at microwave energy can be
absorbed by mechanisms other than those of a classical thermal mode.
The following results are reported:
1.
Arrhenius plots (e fflu x versus inverse tem perature) exhibit
two linear regions for both cations with transitions at 11.0°C to
13 .0 °C , 20.0°C to 22.5°C and 35.5°C to 36 .0°C .
2.
The rubidium efflux data indicate no difference between
irradiated samples and controls except at transition temperatures of
3 6 .0 °C , 22.5°C and 1 3 .0 °C .
A t these temperatures there are
statistically significant increases in e fflu x .
3.
The sodium efflu x data demonstrate differences between
control and irradiated samples th a t are statistically significant at
3 5 .5 °C , 3 6 .0 °C , 3 7 .0 °C , 22.5°C and 1 3 .0 °C .
Irradiated sample efflux
rates at temperatures other than these critical temperatures do not
d iffe r from control efflux rates.
4.
Both cations ex h ib it passive effluxes at the 22 .5°C critical
tem perature th a t exceed control values but th a t do not increase
monotonically with specific absorption rate.
The following conclusions are presented in this thesis:
1.
Microwave radiation increases passive tran sp o rt at certain
critical tem peratures when the membrane undergoes phase
tran sitio n s.
2.
T h e data suggest th a t th e simultaneous existence of two
phases within th e membrane is necessary fo r the observation
of increased e fflu x .
3.
T h e e fflu x rate at these critical tem peratures does not
increase monotonically with increased absorbed power.
4.
The absroption of microwave energy under isothermal
conditions persisting during a critical phase transition resu lt
in increases in passive ionic tran sp o rt th a t do not rely on a
macroscopic thermal mechanism.
The increased ionic efflu x
trig g e re d by small microwave-induced perturbation may result
from cooperative interactions w ithin the membrane during
phase transitions which result in charge separation and
tran sien t dipole form ation.
A lte rn a tiv e ly , the increase in
lateral compressibility of the membrane lipids, which occurs
at phase transitions may fa cilitate extracellular dipole
penetration.
In eith er case, microwave energy could couple
into the membrane under these conditions by polarizing the
charge distribu tion or affecting dipole orien tation .
Cone (1974) has emphasized the crucial role of in tracellular sodium
and potassium in regulating mitogenic a c tiv ity , DNA synthesis, RNA
IU3.
tran scrip tio n and chromosome p u ff form ation.
He associates conditions
of sustained malignant cell proliferation with membrane depolarization
and elevated in tracellular sodium levels.
Microwave-induced changes
in membrane perm eability to ions o r chemotherapeutic materials at
phase transitions may o ffe r an interesting approach to cancer th erap y
based on th e differences in lipid composition of malignant and normal
cells (Wood, 1970;
S hu ster, 1955;
Sanioto and Scheier, 1975).
The experim ental observations presented in this thesis also have
relevance to the recently proposed chemotherapeutic technique of
encapsulating antimetabolic agents in lipid vesicles fo r a microwaveenhanced d elivery d ire c tly to tumors (Y a tv in et a L , 1978 and
Weinstein et a L , 1979).
Processes of immune response and cell
recognition might also ex h ib it altered responses to microwave radiation
at or near phase tran sitio n s .
J_n v itro experiments of such processes
could be effectively studied with th e cell irradiation system presented
in this thesis.
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A P P E N D IX l l l - A *
DERI VAT I ON OF EQUATION 111-1
DESCRI BI NG THE THERM ISTO R
TEMPERATURE DI FFERENTI AL
^(R abinow itz, Olcerst and Mumford, 1977)
Equation 111-1 states th a t
Eq.lll-1
(page 48)
I t can be derived as follows:
When the bridge is nulled:
where
R x is the resistance of the inflow therm istor
R 2 is the resistance of the outflow therm istor
and
Rd and Rf are defined in chapter 111, pg 49
The resistance of a therm istor can be described as a function of
tem perature by means of the following relationship (M uller and
Stolten, 1953):
R, = R „
where
Rxo is the resistance at 298.16°K (2 5 °C )
T 0 is 298.16°K (2 5 °C )
T. is the tem perature in degrees Kelvin at which the
therm istor has a resistance Rx
0 is the therm istor constant ( ° K ) .
for i a
1 ,2
Therm istor relationships:
P i d / T i - 1 / T 0)
J32 ( V T
2
- 1 / T 0)
Taking th e natural logarithm of both sides of this equation leads to:
let
K' = In
R 10
—=— ,
K2 0
which is ju st a constant
S ubstituting
Pi
° _ 1/1
ln R 7 = K + T x
let
0
*
0
P2
(P i “ P2)
T2
T
i
A0 = 0 2 - 0x
T = Tx
AT = T 2 - T x
Then
In
When
AT=0 ,
= k1 +
■
K '
■
= K1 +
T
—
y 'C £ t ~
+
Eq‘
#1
i . e . , when th ere is no absorbed microwave energy
L - -A£. + -A L .
T
T
T0
AS(+ + Tr}
Thus in the absence of absorbed microwave power K' and A0 can
be found by va ry in g the bath tem perature T and nulling the b rid ge.
Id eally , this should have been a straightforw ard procedure, but the
data obtained demonstrated th a t the therm istors were not well matched
and a tem perature dependent A0 characteristic was by necessity
incorporated into this model.
The V icto ry U U T5IJI therm istors were
obtained with therm istor coefficients
0(t)
matched only to
2 %.
IC l
.
It is assumed th a t A0 varies lin early with tem perature
A0 = aT + c
Then,
In
Expanding,
= K' + (a T + c) (
In —
)
= K1 + -4j£- +
a - -y -
M ultiplying both sides by T
Rl
Tin - k — = K 'T +
K
5
AT2
-— r -
+
J o
cT
*
-a T - c
0
Ri
a
c
Tin - k — = ( - 4 - )T 2 + ( -4 - + K' - a ) T - c
l \ 2
*
0
' o
Rj
Rx
vd _ _____
R^
R2
Since
Tin
r\^
= ( - 4 “ )T 2 + ( - L . + K1 - a ) T - c
1o
'O
Eq. 2
When AT=0, i . e . , no microwave energy absorbed in sample cell.
The decade resistance R^ was measured at tw enty d ifferin g values of
bath tem peratures.
A plot of T InCR^/R^) vs. T was made (see Fig.
and fitte d to a quadratic equation.
The following coefficients were
obtained:
4 “ = - 1 . 4 x 10"s
•0
c + K, _ a = -o.26
■O
c = 78.04
11 - 1 2 )
The correlation coefficient of the" data fitte d to a quadratic form was
found to be
r 2 = 0.9978
at
T 0 = 298.16°K (2 5 °C )
Resolving fo r a and K
a = 298.16 ( - 1 . 4 x 1 0 '6) = -4 .1 7 x 10‘
K = 4.17 x 10 ~ 4 - 0.26 + ■?
!
4
= 2.16 x 10 - 3
A0 = -4 .1 7 x 1 0 '4T + 78.04
R eturning to equation 1:
and defining the variable K as follows:
at
K
=
K'
+
4L
10
Substituting K into equation 1
In ( R d/ R f ) - K = p /T - (0 + A p )/(T + AT)
This equation can be solved fo r AT
T2
f - AP
AT =
^ln
Rd
Rf '
A*>
T
* K)
Eq’
3
The total absorbed power in the sample cell (P ) can be expressed as
a
Pa = cvAT
a
where
P
= the total absorbed power (c a l/m in )
c
= the specific heat of dodecane (c a l/g °K )
v
= the flow rate of the dodecane (g /m in )
3
S u b s titu tin g th e above equation fo r AT yields:
Pa = c - v - A T =
In
- 46-
where C=cv.
This is the desired resu lt Equation 111-1 pg. 48
APPENDIX l l l - B
D ERI VATI ON OF CASE 1 SOLUTION
The solution presented fo r Case'1 is based on the geometry of a rig h t
circu lar cylind er having a radius ( r ) and a height ( I ) .
It is assumed
th a t the microwave energy is uniform ly absorbed within the sample
compartment.
The cylind er's o u ter surface is fixed at a tem perature
T 0 and a tem perature maximum will exist at the axis ( T ) .
a
It is also
assumed th a t the radius r is much smaller than the crosssectiona! width
of th e waveguide and thus th ere is no significant decrease in the
electric or magnetic field strength over this distance. Heat loss
through the top surface of the cell is assumed to be negligible.
A ccordingly:
P = pSV
where
P = absorbed power (mW)
S = Specific absorption rate (m W /g)
p = density (g /m l)
V = Volume = 7t r 2l (cm3)
R = radius of sample cell
I = height of sample cell
q = cP = cpVS
Eq. 1
where
q= heating rate (calories/sec)
c = conversion facto r (2 .3 9 x 10
4 )cal/sec*mW
From elementary thermodynamics the radial heat tra n s fe r from
T
3
to T 0 is expressed as: (B orow itz and B eiser, 1966)
where
K = thermal conductivity (cal/sec-cm ° C )
A = area = 2m*l
Equating Equations 1 and 2 when p = I,
Snr2lc = -2Knrl 4 1
Separating variables:
In teg ratin g :
R
/o r d r = - 3#C
/
To
-rdT
x
Ta
Solving fo r AT:
A-r. _ ScR 2
AT -
5
Eq. 3
where
AT = T a - T 0
Note:
Beikhode, Johnson and Muc (1974) have considered a sample
cell of similar cylindrical geometry in th e ir microwave enzyme
experim ents and have presented an identical equation.
T h ey consider
th at the assumption of uniform power absorption over the sample cell
to be a good estimate of the actural tem perature profile of th e ir sample.
From Eq. 1:
S u b stitu tin g into Eq. 3 yields:
AT =
Case 1 Solution (Eq. 4)
relating the temperature difference between the axis and periphery
of the sample cell to microwave power in the waveguide.
APPENDIX l l l - C
DERIVATION OF CASE 2 SOLUTION
The temperature distribution within a sample held in a rig h t
cylindrical geometry can be expressed in the form
VZTCr^z) =
Eq. 1
where
T = temperature ( ° C )
K = thermal conductivity (cal/sec*cm °C )
q
= the heat generated by microwave absorption per unit volume
per second.
The variable parameters (r,<(>,z) represent the coordinates depicted
in Figure I I I - 4 .
The tem perature at any point in
a conducting medium
will be proportional to the absorbed power density which is related to
the square of the electric field by the following relationship:
P = -S ii
K
2
where
P = absorbed power density (Watts/cm3)
a = conductivity (mhos/cm)
E = electric field (volts/cm )
P can be expressed in dimensions of calories/sec as follows:
q = cP =
c = 2.39 x 10~l (calories/sec‘ W)
Eq. 2
136.
I = height of sample cell (cm)
r
= radius of sample cell (cm)
Solving fo r the heating rate q in equations 1 and 2 and then equating
the results yields:
q = - K W 2 T cal/sec = ( £§ ^ p )
q = -KVV2T =
or equivalently:
(cm3) ( - ^ )
cE2o
K7ir 2 V2TI = cE2 a /2
&
Solving fo r V2 T :
V2T = . "-9 PE2( r i 2.) _
v 1
2Knr2sl
Eq. 3
q
The WR-430 waveguide is propagating in a transverse electrical mode
( T E j o) a* 2.45 GHz.
Under these conditions Maxwell's equations
/
have the following forms:
Ey * Ex = Hz =
H
y
0
= A 1 cos(~~”)
°
]\A
..
_
2b
a /1
y
" v
(A'A C
c )' 2
x ----------------T 7 --------
V
E
=
2
-Vp7 e
H
V1 - ( V A J 2
E , = a/mT T
X
A * sin ( - 2 J - )
, nxN
(_ r)
137 .
E2 = A x sin (- 2
|0
where:
A x is an amplitude
constant and the coordinate origin is asdepicted
in Figure l l l - C - 1 .
The boundary conditions are also illustrated in
Figure l l l - C - 1 .
The electric field E2 is zero at the side walls and
reaches a maximum value A x at the center (x = a /2 ) .
At the sample cell center axis:
Ez (x = a /2 ) = AiCOs (0 ) = A x
Eq. 4
The power propagated down the waveguide in the +y direction
can be given by the following equation (R eich, 1953, also Corson and
Lorraine, 1962):
= E (X=a/2)?A _ab_
y
4r|cAg
where
k
9
E
= ---------%
[1 - ( \ / 2 a )2]
5
is the waveguide wavelength
Ho = the impedance of free space (377 Ohms),
This power (P ) is measured in the waveguide system as the difference
between the incident and reflected power as sampled in the tu rr e t
bidirectional coupler located at the center of the waveguide crosssection .
Substituting Equation 4 into. Equation 5 and solving fo r
A i? =
=
1
4x377\_P ..
-
\a b
g 'y
1508 \„ P
= '
"g~ v
kab
If the origin is transform ed to the center of the waveguide as illustrated
in Figure l l l - C - 1 , then the electric field can be expressed as a cosine
function:
(1 5 0 8 \ P ) %
Ez =
\a b
Substituting Equation
V2T
v
1
6
"
„
ms(-— >
Ec>-
6
into Equation 3 yields:
-ca(1508)X P
„
= _______ —----2_X_ Cos2f~5L')
2 Knrs\a b l
cos ^ a J
Ea 7
tq * '
or if the constant A is equated to all the constant factors preceding
the squared cosine term , then:
V2T = A cos2 ( - ^ )
In Case 2, it is assumed th a t a cosine function of the electric
field will yield the maximum variation of the electric field across the
sample cell.
Furtherm ore, if this typ e of profile were rotated 180°
then it would provide the worst case description of the temperature
differential existing in the sample cell.
Since the tem perature distribution will not va ry with <)) or z , then:
V2 T = -5 7 ? = A
Integrating twice according to standard tables (D w ig h t, 1975 page 101
^Equation 440.20) yields:
T = - I r t - l r + -k
s ir , 2( J r » + C1
Eq- 8
The constant C can be evaluated by fixing the coolant bath tem perature
and measuring, in the absence of microwave radiation, the tem perature
difference between the axis and the edge of the sample cell as follows:
AT - T axis - T edge = T <r=0> '
nr 2
nr
Substituting for the constant (A ) from equation 7,
-c o (3 7 7 )\ P
AT =
nr
nr
2
Kn^r5? b\
+ ~2 n sm2( “
)
1
The solution fo r Case 2.
The Case 2 tem perature differential can also be found in terms
of the specific absorption ra te , if the absorbed power is defined as
in Equation 1 of Appendix 111- B (see page 120).
Py = PTtr^lS
-377ca\ pS
AT =
KbnX
nr
2
a
nr
h>a“ + "2n sin 2 ^ lT ^ ^
Waveguide
transformation
lll-C -1
coordinate
FIGURE
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