INDIAN J. EXP. BIOL., V. 43, MAY 2003, 473-482.
BIOPHOTON RESEARH IN BLOOD REVEALS ITS HOLISTIC
V.L. Voeikov*#, R. Asfaramov,* E.V. Bouravleva*, C.N. Novikov*,
#International Institute of Biophysics, Neuss, FRG, *Faculty of Biology, Moscow State University,
Moscow 119234, Russia. E-mail: firstname.lastname@example.org
Monitoring of spontaneous and luminophore amplified photon emission (PE) from non-diluted
human blood under resting conditions and artificially induced immune reaction revealed that blood is
a continuous source of biophotons indicating that it persists in electronically excited state. This state
is pumped through generation of electron excitation produced in reactive oxygen species (ROS)
reactions. Excited state of blood and of neutrophil suspensions (primary sources of ROS in blood) is
an oscillatory one suggesting of interaction between individual sources of electron excitation.
Excited state of blood is extremely sensitive to the tiniest fluctuations of external photonic fields but
resistant to temperature variations as reflected in hysteresis of PE in response to temperature
variations. These data suggest that blood is a highly cooperative non-equilibrium and non-linear
system, whose components unceasingly interact in time and space. At least in part this property is
provided by the ability of blood to store energy of electron excitation that is produced in course of
its own normal metabolism. From a practical point of view analysis of these qualities of blood may be
a basement of new approach to diagnostic procedures.
Ultra-weak photon emission (PE) in the optical spectral range from many cells is commonly
thought to represent random imperfections accompanying normal physiological processes of oxygen
consumption, and no biological function is usually ascribed to it. However evidence is accumulating
that electron excited species are regular products of biochemical processes1 and that energy of their
relaxation may be used by living systems in different ways, such as provision of excitation energy for
endergonic chemical reactions, photomodulation of enzyme activities, ets. 2. Energy of electron
excitation may be transmitted from the sites of its generation (donors) to the sites of its utilization
(acceptors) both by radiation-less as well as by radiative mechanisms3.
The major sources of electron excitation in living systems are the reactions with the
participation of reactive oxygen species (ROS), in particular, the reactions of superoxide anion
radical (O2• ) recombination, yielding hydrogen peroxide (H2O2) and singlet (electronically excited)
oxygen (*O2), reactions of H2O2 reduction to water and oxygen or of oxidation by it of chlorine ions,
reactions of direct oxidation of carbonyl compounds with oxygen from which reaction products in a
triplet excited state arise, etc. These reactions may go on non-enzymatically or be enzyme catalyzed
and are under strict external regulation besides a probable ability to be self-regulatory4. A substantial
portion of oxygen consumed by aerobic organisms is permanently used for generation ROS, thus,
electronic excitation should also be permanently generated.
Among the mostly intensively studied biological sources of PE are stimulated neutrophils and
other phagocyting cells. They react to multiple stimuli by a respiratory burst (RB) – strong
intensification of ROS production followed with PE5. As intensity of this emission is very low, PE
indicators, such as luminol or lucigenin are introduced into a cellular suspension to increase quantum
yield6. Luminol and lucigenin are known to be indicators of different oxygenation activities.
Lucigenin is regarded as a relatively selective probe for O2• , while luminol is less specific and
reports of a variety of reactive oxygen species (H2O2, ClO, OH•, etc.) production7. Non-diluted
blood is a priori considered to be completely non-transparent for visible light because of a very high
hemoglobin` content, and it is practically never used for PE studies. However, here we show that
significant photon emission can be registered from non-diluted human blood, both in a resting state
in the presence of lucigenin and especially when agents inducing RB of neutrophils are added to it.
Patterns of PE revealed due to continuous monitoring of it allowed to expose peculiar systemic
properties of blood.
Materials and methods
All reagents unless otherwise specified were obtained from Sigma Chemical Co., USA. Stock
solution (10-1 M) of luminol was prepared in analytical grade dimethyl sulfoxide. It was diluted 50-
fold in saline just before use and added to a blood sample to a final concentration of 10 -4 M. Stock
solution (10-2 M) of lucigenin was prepared in saline (0.9% sodium chloride solution). It was added
to a blood sample to a final concentration of 10-4 M. Zymosan was opsonized with human blood
serum by a routine procedure and was added to blood to a final concentration of 0,1 mg/ml.
Preparation and treatment of blood samples
Blood from healthy volunteers was obtained by venous puncture between 9 and 11 hours a.m.
and was stabilized by heparin or sodium citrate. Blood was kept in 5 or 10 ml plastic disposable
syringes without air bubbles at 20 oC or at 4 oC if it was stored for more than 6 hours. In these cases
blood was kept at room temperature for 1 hour before the measurements. For experiments described
in this paper blood of healthy donors (males, 20 - 51 years old) was used. Individual differences in
PE kinetic curves progression, PE maximal intensity were noted, though they were reproducible in
experiments with blood of each particular donor. General trends of PE from blood exemplified at
figures presented in the paper were typical for blood of all the donors.
The ability of neutrophils to reduce nitro-blue tetrasolium (NBT), expressed as percent
reducing neutrophils was evaluated by a common method 8. In brief an aliquot of blood (50 mkl) was
taken from a blood sample, mixed with 50 mkl of 0,1% NBT solution in 0,15 M Na-P buffer (pH
7,2), the mixture was incubated at 37 oC for 20 min and at 20 oC for 20 min. Blood was smeared on
a slide, dried out, fixed with methanol for 10 min. Slides were dyed with methylene green. 100
neutrophils were counted at each slide. NBT test was considered positive when dark blue diformasan
granules (the product of NBT reduction) occupied from 5 to 100% of neutrophils cytoplasm. All the
cells were grouped in 6 ranks according to their activity: range 1 -- 0%, range 2 -- 5-7%, range 3--
up to 30%, range 4 -- 30-50%, range 5 -- 50-90%, range 6 -- 100% of cytoplasm is filled with
reduced diformasan granules.
Detection of photon emission
PE from blood was registered either in a liquid scintillation counter Mark-II (Nuclear-Chicago,
USA), equipped with photomultipliers EMI 9750QB/1 or on a single photon counter equipped with
PMT type EMI 9558 QA, cooled to –200С +- 0,2 0С9. Mark II counter was used in the mode of
single photon counting (out-of-coincidences mode) in a tritium window. The measurements were
performed at room temperature (19-21 oC). PE was recorded as counts per 0.2 or 0.1 min. 1 ml
Eppendorf polyethylene test tubes were used as blood containers. Test tubes were fixed in empty
standard borosylicate glass vials for liquid scintillation counter in one and the same position. Vials
and test tubes having short decay time of own luminescence after insertion into the counting chamber
were selected. Dark counts with an empty test-tube in a counting chamber varied in the range of 40-
50 counts/sec. All the operations were performed at dim ambient illumination. Sequence of addition
of blood, luminol, lucigenin and zymosan are described in figure legends. Other experimental details
are described in the section “Results". Single photon counter7 was used in experiments where
temperature dependence of PE from blood was measured. Blood was placed in standard disposable
transparent plastic cuvettes for spectrophotometers, and a cuvette was fixed in a copper container
with one transparent wall and equipped with the Peltier element for its heating and cooling. A
thermistor coupled to the Peltier element was inserted to blood and using a special software
temperature in blood could be changed and PE and blood temperature were simultaneously
Photon emission from blood and its dependence on oxygen.
Addition of either lucigenin or luminol to blood as soon as 5 min after it had been taken out
by a finger puncture or elbow vein drainage is followed with an increase of PE in the absence of RB
stimulants (Figure 1). Fresh blood response to lucigenin was much more pronounced than that to
luminol. During the first hours of blood storage after its withdrawal luminol-dependent PE (LM-PE)
level was decreasing, but addition of luminol to 1 day old blood resulted in rapid and strong
elevation of LM-PE (Fig 2a). Unlike complex pattern of LM-PE changes during blood storage,
lucigenin-dependent PE (LC-PE) was not decreasing, but was rather gradually increasing during
blood storage. Similar to LM-PE strong enhancement of LC-PE was observed in 1-day old blood
(Fig 2b). Blood even in a resting state (without addition of inducers of RB) to which lucigenin was
added continued to emit photons for many hours indicating that the process of ROS production and
generation of electron excited species persistently proceeds in it.
The very fact that pronounced PE may be registered from non-diluted blood -- a highly
opaque liquid due to very high concentration of hemoglobin -- indicates that hemoglobin packed in
erythrocytes does not quench efficiently PE. However, if free hemoglobin is added to blood at a
concentration of only 0,5% of already present in it, LC-PE practically disappears (Figure 2). Taking
into account that concentration of hemoglobin in erythrocytes may reach as high value as 35-40%
(hemoglobin can not reach such high concentration in a free solution) one may suggest that
hemoglobin in erythrocytes is present in a liquid crystalline state. In such a form it may provide
transfer of excitation energy over long distances without its dissipation, unlike hemoglobin in a
solution that absorbs and dissipates energy of electron excitation.
Figure 1. Photon emission changes in non-diluted blood (0.2 ml) supplemented with luminol (a) or lucigenin
(b) in relation to time of blood storage. Aliquots (0.2 ml) for measurements were taken from this sample at
time moments marked by inscriptions at each curve. Note, that the curves for 5 min., 1 hr, and 3 hours in (a)
apply to the left ordinate and the curve for 24 hours -- to the right ordinate.
Time, 0,1 min
0 300 600 900
Figure 2. Lucigenin-dependent PE from blood diluted 1:1 with physiological saline (curve 1) or physiological
saline to which human hemoglobin was added at a concentration of 2 mg/ml (curve 2).
High intensity of LC-PE in fresh blood indicated of a production of O2• in it, and a weak
response to luminol suggests that generation and/or accumulation of more reactive oxygen species
does not occur. However, after zymosan addition to fresh blood strong LM-PE developed (Figure 3,
curve 2). Figure 3 shows also that even in the absence of luminol PE response to induction of RB in
the same blood is pronounced (curve 1). It should be noted that these two samples of blood were
monitored simultaneously on two identical counters under same conditions differing only by the
presence or absence of luminol in blood. Elevation of PE intensity in the absence of luminol indicates
that blood contains own fluorophores that are able to transfer energy of electron excitation and that
part of it is released as photon emission. Luminol as a very efficient fruorophore amplifies PE from
blood nearly 100 x fold. However, it may be seen that besides amplification maximal intensity of PE
in the presence of luminol is reached 30 min earlier than in blood without luminol and that it decays
faster. This may suggest that excessive PE from blood devastates its energy resources.
Counts/6 sec (curve 1)
Counts/6 sec (curve 2)
0 1200 2400 3600 4800 6000 7200 8400 9600 10800
2 4 6 8 10 12 14 16 18
Figure 3. Photon emission from non-diluted blood (0.1 ml) without luminol (curve 1, left Y-scale) or with it
(curve 2, right Y-scale) after induction of respiratory burst by addition of zymosan to blood (without zymosan
and luminol PE from blood sample was 250-300 counts/6 sec).
5 10 15 20
Figure 4. Effect of blood dilution with physiological saline upon photon emission from blood to which either
lucigenin or luminol+zymosan were previously added. Initial blood volume – 0,1 ml. Dilution did not change
cell contents in blood.
Comparison of PE from non-diluted blood and from suspensions of neutrophils isolated from
the same blood and adjusted in cell concentration to that of concentration of neutrophils in whole
blood revealed differences as well as similarities in PE patterns from these two experimental systems.
We suggested that these differences could be at least in part be explained by different conditions of
oxygen supply to neutrophils in blood and in neutrophil suspension. Concentration of dissolved
oxygen due to its high affinity to hemoglobin is very low in blood plasma, and white blood cells, in
particular neutrophils may get it for respiration only from erythrocytes. On the contrary, in
neutrophil suspensions cells consume oxygen dissolved in cell medium. Thus it is possible that high
level of LC-PE in non-diluted blood may be related to interactions of neutrophils and erythrocytes in
blood. In fact, when blood to which luminol+zymosan or only lucigenin had been added was diluted
with saline (supplemented with either luminol or lucigenin) only LC-PE, but not LM-PE intensity
was progressively declining with the increase of the degree of dilution (Figure 4). This result agrees
with the suggestion that persistent LC-PE from blood is provided by a close contact of neutrophils
and oxygen-donating erythrocytes in it.
Oscillatory behavior of PE from neutrophil suspensions and from blood.
Under certain conditions PE from suspensions of isolated neutrophils may gain oscillatory
patterns with high amplitude of oscillations reaching up to 25% of the mean PE intensity. These
conditions include lack of agitation of a suspension, an optimal buffered medium containing nutrients
and access to air. As it can be seen in Figure 5 prominent oscillations under these conditions develop
about 1 hour after RB initiation, and last for many hours. In the absence of air access oscillatory
behavior can also be seen, however, PE fades much earlier and amplitudes of oscillations are smaller.
0 2 4 6 8 10 12 14
0,0 0,5 1,0 1,5
Figure 5. Sustained oscillations of LM-PE in neutrophil suspensions (20000 cells in 0.1 ml of M-PRM
medium (ICN) after RB initiated with zymosan. Curve 1 – aerobic conditions, Curve 2 -- suspension is
isolated fron air. Insert – the initial period of RB.
Oscillations of LC-PE can be also registered from non-diluted blood (figure 6). Significant
oscillations could be seen at the shoulder of the major peak of PE, and then they appeared again at a
new smaller peak long time later. It is interesting to note that some similarity may be seen between
oscillatory behavior of neutrophils in suspensions and blood: in both cases there could be
distinguished two macroscopic waves of PE, and the second wave was modulated by oscillations of
rather large amplitude.
Time, 0,1 min
10 100 1000 10000
8600 9200 9800 10400 11000
Figure 6. Oscillatory behavior of LC-PE from non-diluted blood (0,1 ml). Insert – oscillations at the
second peak of PE arising about 16 hours after addition of lucigenin to blood and start of the experiment.
Thermal hysteresis of PE from blood
Dependence of LM-PE intensity during the development of zymosan-induced RB upon slow
(period about 20 min) and regular temperature changes in the range of physiological temperatures is
illustrated in Figure 6. During the lag-period preceding RB development and until PE reaches
maximal intensity it practically does not depend upon temperature. It can be seen that at this stage
PE elevates even when temperature declines that formally accounts for the “negative energy of
activation” (Figure 7, A&B). Only after maximal intensity of PE is reached and starts to decline the
major tendency of curves in Ahrrenius coordinates starts to approach a classical one (Fig. 7C),
however, even at this stage a strong temperature hysteresis is still observed.
Violation of periodicity of temperature changes or its elevation to extreme values were
followed with the paradoxical blood response (Figure 8). It can be see that blood reacts to changes
in the gradient of temperature decrease (time points 1800, 3000 and 4200 sec) by elevation of PE
intensity. On the other hand when temperature reaches 39,5 оС PE intensity drops abruptly, in spite
of further temperature rise, as if blood is denaturized. However, this is not the case because as soon
as temperature after reaching its maximum begins to decline PE from blood starts to elevate. Thus
decrease of PE from blood is not caused by irreversible changes in it.
Effect of back reflected photons upon the development of RB in blood.
If PE from blood reflects functionally significant processes of electron excited states generation
one can expect that irradiation of blood with low level intensity photon flux with the same spectral
properties as emitted by it may modulate the character of the processes related to EES generation. In
fact previously we10, 11, and others12 have demonstrated that two populations of neutrophils or blood
being in an optical (but not chemical) contact with each other can influence intensity of oxidative
processes in each other if in one of them RB is induced. It was interesting to see if back reflection of
photons emitted by blood can influence the processes, that lead to photon emission, in particular,
because this experimental approach could demonstrate effect of ultra-weak irradiation of blood more
convincingly than others.
0 600 1200 1800 2400 3000 3600 4200
3,205 3,215 3,225 3,235 3,245 3,255
3,205 3,215 3,225 3,235 3,245 3,255
Figure 7. Dependence of LM-PE from blood in which RB is induced with zymosan upon temperature
waves. (A) Original data. (B and C) Ahrrenius plots for the initial stage of RB (ca. up to 1700 sec) and later
stage of RB, respectively.
To achieve high degree of reflection of photons emitted by blood back to it Eppendorf test
tubes were wrapped in aluminum foil screens. As it is demonstrated in Figure 9 (A and B), kinetics
of LM-PE development during RB induced by zymosan was different in test tubes with blood
covered with aluminum foil and in control test tubes. The “sign” of the effect: enhancement or
attenuation of PE intensity in experimental test tubes after the foil was moved away from the test
tube depended upon the rate of PE development in blood. If PE was accelerating slowly (count rate
in the control samples did not exceed 100 000 counts/0.2 min at the moment of foil removal) PE
intensity in the initially screened sample just after a screen removal usually exceeded that of a control
one 1.5-5 fold and continued to increase faster that in control samples (Fig. 9A). On the contrary, at
high rates of PE development in control blood samples back reflection of photons either did not
modify the process or even attenuated PE in wrapped samples in comparison to the control ones
0 600 1200 1800 2400 3000 3600 4200
Figure 8. Effect of interruption of periodicity of temperature changes or its elevation to extreme values upon
the patterns of LM-PE from blood in which RB was induced 2,5 hour before the time point 0 at this panel.
Figure 9. Effect of aluminum foil screen over the test tube with blood upon LM-PE development in zymosan
treated preparations of blood with slow (A) and fast (B) PE development. Curve 1 – control test tube, curve 2
-- test tube was wrapped with foil at the moment indicated with the arrow, curve 3 -- test tube was wrapped
with foil before luminol and zymosan addition. In each experiment a preparation of blood was distributed in 3
equal portions counted in the mode of rotation (in turns).
It can be seen from Figure 10 that effects of photons reflection from aluminum foil on LC-PE
development are generally the same as those observed with LM-PE development after RB inducing
in blood. In the sample with slow development of LC-PE screening accelerates photon emission (Fig.
10, A), and in a blood preparation with fast development of LC-PE its inhibition is observed (Fig.
NBT test revealed the effect of foil screens upon activity of neutrophils in blood where RB
was induced with zymosan and in which LC-PE was registered. In this experiment LC-PE was
prominently higher after foil removal from the experimental sample than in the control sample (data
not shown). It can be seen in the Figure 11 that after zymosan addition to blood the number of active
neutrophils significantly increased in both samples. However, the total number of active cells in the
sample screened with foil is larger than in the control sample (39% vs. 30%). It is notable that the
number of cells belonging to high ranks (more than 50% of cytoplasm is filled with diformasan
granules) is twice as high in the former sample in comparison to the control one (compare the bars
##5 and 6 in 1c and 1f). Thus, the NBT test gives an independent confirmation that sample screening
with aluminium foil at the initial stage of RB development enhances reactive oxygen forms
generation by neutrophils in whole non-diluted blood.
Figure 10. Effect of aluminum foil screen on LC-PE development in two preparations of blood with slow (A)
and fast (B) PE development. Curve 1 – control test tube, curves 2-4 -- test tubes were wrapped with foil
before lucigenin addition. Foil was removed from respective test tubes at moments indicated by arrows. In
each experiment a preparation of blood was distributed in equal portions among 4 samples, which were
counted in the mode of rotation. Note the difference in ordinate scale values in A and B.
Figure 11. Effect of foil screening on neutrophil activity in whole blood evaluated by NBT test. 0c and 0f –
distribution of neutrophils according to the ranges of their activity in a control and a foil screened samples just
before the addition of zymosan and lucigenin to them.1c and 1f – same 6 minutes after addition of zymosan
and lucigenin to the control and foil screened blood samples.
We used also another experimental set-up to reveal effects of back reflected photons upon
RB patterns in blood. Eppendorf test tubes were fixed in glass vials as it is shown in the upper right
corner of Figure 12. Part of light emitted from blood samples can be reflected from the glass wall of
a vial back to a sample. We compared the level of radiation registered by PMT from test tubes with
blood inserted in empty vials and in vials filled with water. These measurements has shown that PE
intensity levels measured from the same sample of blood were 16 ± 4% (mean ± s. d.) higher when a
vial was filled with water than when a test tube was inserted in an empty (containing air) vial.
Increased light emission from test tubes with blood positioned in water filled vials is due to lack of
photon reflection from the inner wall of the vial back to its source due to the immersion effect of
water (reflection coefficient of water and glass are similar unlike those for air and glass). In these
experiments it was noted that even this small part of light that was reflected back and irradiated a
test tube with blood had a measurable effect upon the kinetic parameters of PE response in blood
during RB. As it can be seen in figure 12, when the sample was transferred to a water filled vial at
the initial stage of PE development its intensity initially leaped by 16% due to the immersion effect
and then its acceleration temporarily retarded (Fig. 12, A). Sharp leaps of PE intensity were
observed when the sample was transferred from an empty vial to a water filled one and back at the
quasi-stationary stage of LM-PE (Fig. 12, B). However, it can also be noted, that when after a few
minutes of the sample stay in a water-filled vial it was returned back to a empty one the stationary
level of LM-PE in it increased to a much higher level that could be expected if blood continued to
stay in an empty vial. On the other hand, when sample transfers from an empty to a water-filled vial
and back were made at the stage of PE decay (Fig. 12, C), PE intensity in water-filled vial was
rapidly declining. After the sample was transferred back to an empty vial it appeared initially at a
much lower value than previously, but began to rise up. Thus, the effect of irradiation of blood
samples by back reflected photons may be opposite at different stages of RB which in turn differ in
the absolute levels of PE and the direction of its change. Lack of self-irradiation depresses photon
emission from blood at relatively low levels of PE (at the stage of PE development and even more
prominently at the stage of its decay). On the other hand, at high levels of photon emission
(stationary stage) back reflected photons seem to depress PE (Fig. 12, B).
Blood provides vital functions for an organism as a whole. It carries gases, nutrients and
evacuates by-products of metabolism, participates in humoral regulation of physiological functions of
all other tissues and systems, supports water, salt and acid-base homeostasis, executes
immunological defense, etc. A lot of knowledge about particular functions of blood components has
been gained due to the relative ease of their isolation from whole blood. However, this approach
does not allow to get information about interactions of blood components in whole blood, though
their interactions are undoubtedly very significant for efficient blood functioning. In a frame of the
dominating biochemical paradigm interactions of blood components are provided by chemical
signaling through diffusion of bio-regulatory molecules such as cytokines and hormones. However,
there may exist an alternative, or, to be more precise, a complementary route of regulation of blood
functions – through a non-stationary biophotonic field, or, in other words, through a field of
electronically excited states of molecular components of this tissue. It has been long ago stated that
in complex and structured systems energy of electron excitation does not immediately dissipate into
heat but rather may migrate along the common energy levels and perform chemical, mechanical and
other forms of useful work13, 14, 15, 16. Such energy migration and storage may be provided by
structural organization of a biological systems. However, most of these conclusions were made
basing on data obtained from experiments with external irradiation of a system under study and
analysis of fluorescence or delayed photon emission data (e. g. 14-16). It was only in studies of
mitogenetic radiation by A.G. Gurwitsch and his school where internal oxidative metabolism was
suggested to be the source of energy that pumps biophotonic fields of living organisms (see e. g. 17).
It is shown here that addition of lucigenin – the probe for superoxide anion radical (O2• ) to
non-diluted blood is followed with PE of rather high intensity even in a resting state, indicating that
ROS are permanently produced in blood (Fig. 1B). Due to high activity of superoxide dismutase in
blood O2• is rapidly converted into hydrogen peroxide, and the latter is immediately decomposed
with catalase present in human blood. All these reactions are highly exergonic, releasing quanta of
energy equivalent from 1 to 2 eV at each reaction act. Thus electron excited states are continuously
generated in blood. When the immune reaction – respiratory burst of neutrophils – is induced in
blood, intensity of PE dramatically increases (Fig. 3). It is known that under these conditions other
highly reactive oxygen species appear, in particular, hypochlorite (ClO), the product of oxidation of
Cl with hydrogen peroxide, in a reaction catalyzed with myeloperoxidase. Luminol amplifies PE
under these conditions nearly 100-fold. However, PE elevation is registered even in the absence of
luminol and sustains at high level for many hours. This again argues for the generation of electron
excited state in native blood.
The very opportunity to register photon emission from such an opaque liquid is possible only
because energy of electron excitation can migrate in blood at least partially without dissipation. We
suggest that hemoglobin – the major candidate for dissipation of energy of electron excitation does
no do it in intact blood because it is present in erythrocytes in a liquid crystalline state. In fact,
hemoglobin dissolved in blood even in a very low concentration readily quenches photon emission
It has recently been shown that individual neutrophils produce ROS in a strictly oscillatory
manner with a period of oscillations in the range of tens of seconds18. Spontaneous arousal of multi-
period oscillatory regime including very low frequency oscillations of PE from neutrophil
suspensions and whole blood indicates that production of ROS by individual cells manifested as
generation of electronic excitation is to a certain degree correlated in systems containing tremendous
quantities of these cells (Fig. 5&6). Such correlated behavior may be seen even in such a complex
system as non-diluted blood in which only a tiny fraction of cells (granulocytes do not exceed
together 0,1% of erythrocytes in quantity) is responsible for ROS generation. Such a correlated,
cooperative behavior can be in principle provided by diffusible regulatory molecules that activate or
inhibit granulocyte activity. However this explanation was ruled out in the experiments in which PE
from blood was registered under the conditions of oscillatory temperature changes.
For the first time thermal hysteresis of PE from a living system (etiolated barley) was
observed by Slawinsky and Popp19. Their data argued for the presence in living matter of a
delocalized coherent electromagnetic field far away from thermal equilibrium. Here we confirmed
and extended their observations. In our case PE from blood originated definitely due to a set of
biochemical reactions induced in neutrophils and resulting in generation of electron excited species.
Very prominent temperature hysteresis of PE especially at a stage of the development of RB
suggests that either the kinetics of these biochemical reactions and/or the kinetics of the processes
resulting in photon emission do not obey simple laws of chemical kinetics, in other words these
processes are at least partially not determined by the rate of diffusion of reagents which should
strongly depend upon temperature. As soon as RB starts to fade, temperature dependence of PE
more and more resembles of a “classical” one, though even many hours after RB was initiated
pronounced temperature hysteresis is still observed . Of a special interest is the behavior of blood
when the periodicity of temperature changes was violated or temperature was raised to extreme
values. Paradoxical changes in PE -- elevation of its intensity when temperature continued to decline
though at different rate than before, or abrupt drop in PE intensity when it continued to raise –
indicate that blood has some peculiar mechanism sensing temporal variations of temperature. The
nature of this mechanism is currently obscure.
Modulation of activity of neutrophils in whole blood by back reflected photons and changes
of patterns of PE from it in response to this very low intensity irradiation (Fig. 10-12) also argues in
favor of the functional role of the biophotonic field in this tissue. As it has been pointed out above,
non-diluted blood is a system with a very high optical density and the ratio of neutrophils to
erythrocytes in it is approximately 1:1000, so the question arises as how these very low intensity
photon fluxes may have any measurable effect upon neutrophil activity it. It may be suggested that at
least in part these effects may be related to specific ways by which neutrophils obtain oxygen for
their respiration in blood. It was demonstrated here that LC-PE in blood is strongly dependent upon
the interaction of neutrophils and erythrocytes, and that LC-PE depends on oxygen supplied by
erythrocytes (Fig. 4). Taking into consideration that λmax for luminol emission is 427 nm and for
lucigenin emission it is 470 nm, and that several hemoglobin absorption maximums are not far from
this spectral region, hemoglobin may be excited by back reflected photons. It is interesting to
speculate that due to liquid crystalline state of hemoglobin in erythrocytes absorption of photons by
few hemoglobin molecules may provide enough energy of excitation for dissociation of oxygen from
multiple oxyhemoglobin molecules belonging to this crystal thus providing the mechanism for
cascade amplification of a signal and additional substrate supply for generation of ROS by
neutrophils and enhancement of PE intensity in blood.
However, these considerations cannot readily explain attenuation of PE intensity by back-
reflected photons in blood preparations with initially high rate of PE intensity growth. Previously we
observed that the high rate of LM-PE without artificial induction of RB in it is characteristic of blood
of patients with cardiovascular diseases20. Besides it has been shown here that both LM-PE and LC-
PE intensity is dramatically high after long storage of blood even of healthy donors’. After being
stored in certainly unnatural conditions blood in a certain sense becomes “sick”. It can be supposed,
that in such blood preparations hemoglobin is already highly excited for the reasons discussed below,
and that auxiliary irradiation of blood with back reflected photons does not provide an additional
stimulus for oxygen release. On the other hand, additional irradiation of blood with weak photon
fluxes originated from blood itself and reflected back to it, may play an organizing role so that less
energy is lost from it, and the photon flux from such blood registered by a photomultiplier is lower
after the reflective screen removal than the control sample.
The hypothesis that reactive oxygen species generation by white blood cells is enhanced due
to an increase in oxygen availability, ensuing the enhancement of photon emission from “normal”
blood, is supported by the results of our studies of the effect of carbon monoxide upon whole blood
and isolated neutrophils21, 22. We found that while CO sharply intensified LC-PE in non-diluted
blood; it had much weaker effect in saline-diluted blood, and inhibited LC-PE in a neutrophil
suspension. CO is known to bind to heme 100-fold more tightly than O2. In whole blood its major
target is deoxy-Hb. Sharp elevation of LC-PE after CO addition to blood is a strong indication of
highly co-operative processes proceeding in blood. Presumably, high energy released when CO binds
to heme of hemoglobin molecules packed in erythrocytes induces accelerated oxygen release from
many HbO2 molecules resulting in the similar effect upon PE from whole blood as that produced by
blood self-irradiation. Inhibitory effect of CO upon LC-PE in neutrophil suspensions is most
probably explained by its ability to bind to heme enzymes of neutrophils responsible for oxygen
Thus, blood displays many features of an active physical medium, constituents of which are
permanently present in an electronically excited state. Electronic excitation of blood constituents is
provided by the reactions of ROS generation permanently proceeding in it and providing energy for
pumping internal “biophotonic” field of blood. Efficient migration and storage of energy in blood is
provided by its peculiar structure. It is reasonable to suggest that this energy field plays a very
important role in efficient performance of all blood functions.
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