Biophoton Research in Blood Reveals Its Holistic Properties

Indian Journal of Experimental Biology Vol. 41, May 2003, pp. 473-482

Biophoton research in blood reveals its holistic properties

V L Voeikov*#, R. Asfaramov*, E V Bourav leva*, C N Novikov* & N D Vilenskaya*

# International In stitute of Bi ophysics, Neuss, FRG, *Faculty of Biology, Moscow State University, Moscow 119234, Ru ssia

Monitoring of spontaneous and luminophore amplified photon emi ssion (PE) from non-di luted human blood under resting conditions and artificiall y induced immune reacti on revealed th at blood is a continuous source of biophotons indicating th at it persists in electroni cally excited state. This state is pumped through generati on of electron excitation produced in reactive oxygen species (ROS) reactions. Excited state of bl ood 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 extre mely sensitive to the tiniest nuctuations of external photonic fie lds but resistant to temperature variations as renected in hysteresis of PE in response to temperature variations. These data suggest that blood is a hi ghl y cooperative non-equilibrium and non-linear syste m, whose compo nents unceasing ly interact in time and space. At least in part thi s property is provided by th e ability of blood to store energy of electron excitati on that is produced in course of it s own normal metabol ism. From a practi cal point of view analysis of these qualities of blood may be a basement of new approach to diagnostic procedures.

Keywords : Biophoton, Diagnostic appli cati ons, Photon emission from bl ood, Reacti ve oxygen species, Thermal hysteresis

Ultra-weak photon emission (PE) in the optical be enzyme catalyzed and are under strict external spectral range from many cells is commonly thought regulation besides a probable ability to be self to represent random imperfections accompanying regulator/ . A substantial portion of oxygen normal physiological processes of oxygen consumed by aerobic organisms is permanently used consumption, and no biological function is usually for the generation of ROS, thus. electronic excitation ascribed to it. However evidence is accumulating that should also be permanently generated. electron excited species are reg ul ar products of Among the mostly intensively studied biological biochemical processes I and that energy of their sources of PE are stimulated neutrophils and other relaxation may be used by living systems in different phagocyting cells. They react to mUltiple stimuli by a ways, such as provision of excitation energy for respiratory burst (RB) - strong intensification of ROS endergonic chemical reactions, photomodulation of production followed with PES. As intensity of thi s 2 enzyme activities, etc. . Energy of electron excitation emission is very low, PE indicators, such as luminol may be transmitted from the sites of its generation or lucigenin are introduced into a cellul ar suspension 6 (donors) to the sites of its utilization (acceptors) both to increase quantum yield . Luminol and lucigenin are 3 by radiation-less and radiative mechanisms . known to be indicators of different oxygenation activities. Lucigenin is regarded as a relati vely The major sources of electron excitation in living systems are the reactions with the participation of selective probe for O2- - , while luminol is less specific and reports of a variety of reactive ox ygen reactive oxygen species (ROS), in particular, the species (H 20 2, ClO- , OH-, etc.) production? Non reactions of superoxide ani on radical (0 - ) 2 diluted blood is a priori considered to be completely recombination, yielding hydrogen peroxide (H 0 ) 2 2 non-transparent for visible light because of a very and singlet (electronically excited) oxygen (*0 ), 2 high hemoglobin content, and it is practically never reactions of H 0 reduction to water and oxygen or of 2 2 used for PE studies. However, here we show that oxidation by it of chlorine ions, reactions of direct oxidation of carbonyl compounds with oxygen from significant photon emission can be registered from non-diluted human blood, both in a resting state in which reaction products in a triplet excited state arise, presence of lu cigenin and especially when agents etc. These reactions may go on non-enzymatically or inducing RB of neutrophils are added to it. Patterns of #* For correspondence: E-mail: vvl @soil.msu.ru PE revealed in the continuous monitoring bring OLit Fax : 007-095-93C)-4309 some peculiar systemic properties of blood. 474 INDIAN J EXP SIOL, MA Y 2003

Materials and Methods -20°C±0.2°C (ref. 9). Mark II counter was used in Reagents- All reagents unl ess otherwise specifi ed the mode of single photon counting (out-of were obtained from Sigma Chemical Co., USA. Stock coincidences mode) in a tritium window. The meas solut ion (l0- 1 M) of luminol was prepared in analyti urements were performed at room temperature (19- cal grade dimethyl sulfoxide. It was diluted SO-fold in 2 1°C). PE was recorded as counts pe r 0.2 or 0. 1 min . sa line just before use and added to a blood sample to a Eppendorf pol yeth ylen e test tubes ( J ml ) were used as final concentration of 10-4 M . Stock solution ( 10-2 M) blood containers. Test tu bes were fixed in empty of luci gen in was prepared in saline (0.9% sodium sta nd ard borosilicate glass vials for liquid sc intill at ion ch loride sol uti on). It was added to a blood sa mple to a counter in one and the same position. Vials and test final concentration of 10-4 M. Zy mosan was tubes having short decay time of own luminescence opsoni zed with human blood serum by a routine pro after inserti on into the counting chamber were se cedure and was added to blood to a fi nal co ncentra lected . Dark counts with an empty test-tube in a ti on of 0.1 mg/ml. count in g chamber va ri ed in the range of 40-50 Preparation and treatll/en! of blood salllples counts/sec. All the operations were performed at di m Blood fro m healthy volunteers was obtained by ve ambient illumination. Sequence of additi on of blood, nous puncture between 0900-1100 hrs and was stabi lum inol , luci genin and zymosan are described in fig 7 lized by heparin or sodi um citrate. Blood was ke pt in ure legends. Single photon counter was used in ex 5 or 10 ml plastic disposable syrin ges without ai r perimc nts wherc temperature dependence of PE from bubbles at 20°C or at 4°C if it was stored for more blood was measured. Blood was placed in stand ard th an 6 hrs. In these cases blood "vas kept at room di sposable transparent plastic cuvettes for spectropho temperature for 1 hr before the measurements. For tometers, and a cuvette was fixed in a copper con experiments blood of healthy donors (mal es, 20-51 tainer with one transparent wa ll and equipped with th e years old) was used. Individual differences in PE ki Peltier element for its heating and cool in g. A thermis netic curves progression, PE maxi mal intensity were tor coupl ed to the Pelti er element was in serted to nored, th ough they were reproducible in experiments blood and using a special software temperature in with blood of each particul ar donor. General trends of blood cou ld be changed and PE and bl ood tempera PE from blood exempli fi ed at fig ures presented were ture were simultaneollsly recorded. typ ical for blood of all the donors. The ab ility of neutrophils to reduce nitro-blue tet Results rasoliu m (NBT), expressed as percent-reducing neu Photon emission ji-om blood and its dependence 011 trophils was eval uated by a common methods. Briefly, oxygen - Addition of ei th er lu cigenin or luminol to an al iqu ot of blood (SO ml) was taken from a blood blood as soon as 5 min after it had been taken out by a samp le, mixed with 50 ml of 0.1 % NBT solution in finger puncture or elbow vein drainage was followed 0. 15 M Na-P buffer (PH 7.2), the mixture was incu wi th an increase of PE in absence of RB stimul ants bated at 37°C for 20 min and at 20°C for 20 min . (Fig. 1). Fresh bl ood response to lu cigen in was much Bl ood was smeared on a slide, dried out, fixed with 8000 methanol for 10 min. Slides were dyed with methyl ene green. Neutrophils 100 were counted at each 6000 slide. NBT test was considered positive when dark c blue diformasan granules (the product of NBT red uc E tion ) occupied from 5 to 100% of neu trophils cyto ~ 4000 plasm. All the cells were grouped in 6 rank s accord C "o ing to their ac ti vity: range 1 = 0%, range 2 = 5-7%, (,) 2000 range 3 = up to 30%, range 4 = 30-50%, range 5 = 50- 90% an d range 6 = J 00% of cytoplasm is filled with reduced diformasan granules. °0~------3-00------6-0-0------~900

Detection of photon emission - PE from blood was Time, 0,1 min J. registered either in a liquid scintillation counter Mark JJ (N ucl ear-Chicago, USA), equipped with photomul Fi g. 1-- Lucigenin-dependent PE from bl ood di luted I: I with physiological saline (curve I) or physiolog ical sali ne to which tipliers EMI 9750QBIl or on a single photon counter human hemoglobin was added at a concentratio n of 2 mg/ml eq uipped with PMT type EMI 9558 QA, cooled to (curve 2). VOE IKOV er 01.: BIOPHOTON RESEARCH IN BLOOD REVEALS ITS HO LI STIC PROPERTIES 475 more pronounced th an th at to luminol. During th e first (Fig. 2) . Taking into account that concentra ti on of hours of bl ood storage after its withdrawal luminol hemoglobin in erythrocy tes may reach as hi gh va lue dependent PE (LM-PE) level decreased, but addition as 35-40% (hemoglobin cannot reach such hi gh con of lumin ol to 1-day-old blood res ulted in rapid and centrati on in a free solution) one may suggest that stro ng elevation of LM- PE (Fig. 2a). Un li ke co mplex hemoglobin in erythrocytes is present in a liq ui d crys pattern of LM -PE changes durin g blood storage, ta ll ine state. In sllch a form it may provide transfer of lucigen in -dependent PE (LC-PE) was not decreased, excitati on energy over long distances without its dis but rather graduall y increased during blood storage. sipation, un like hemoglobin in a solution that absorbs Similar to LM -PE, stro ng enh ancement of LC-PE was and dissipates energy of electron excitati on. observed in I-day-o ld bl ood (Fig. 2b). Blood even in Hi gh intensity of LC-PE in fres h blood indicated of a resting state (w ith out additi on of inducers of RB ) to a production of O2- - in it, and a weak res ponse to which lucigen in was added continued to emit photons lu minol suggests that generati on and/or accu mu lation for many hours ind icating th at the process of ROS of more reactive oxygen species does not occur. prod uction and generati on of electron exc ited species However, after zymosan addition to fresh blood persisten tl y proceeds in it. strong LM-PE developed (Fig. 3, curve 2). Figure 3 The very fact th at pronounced PE rnay be regis also shows th at even in absence of luminol PE re tered from non-diluted blood - a high ly opaq ue li q sponse to induction of RB in the same blood is pro uid du e to very high concentration of hemoglobin nounced (curve 1). It may be noted that these two indicates that hemoglobin packed in erythrocytes does sa mples of blood were mo nitored simu ltaneously on not quench efficiently PE. However, if free hemoglo two identical counters under same conditions di ffe r bin is added to blood at a concentration of onl y 0.5 % ing onl y by the presence or absence of luminol in of al ready present in it, LC-PE practicall y disappears blood. Elevation of PE in tensity in absence of luminol 2500 l e S ind icates th at blood contains own fluorophores that are able to transfer energy of electron excitation and 80000 th at part of it is re leased as photon emi ssion. Luminol ...2 2000 '" ...2 as a very efficient fruorophore ampli fies PE fro m c .. 'E 60000 ~ blood nearl y 100 x-fold . However, besides ampli fica c ~ 1500 'E tion, maximal intensi ty of PE in presence of lu mi nol 'E N ..N 40000 ~.. reached 30 mi n earl ier th an in bl ood wi th out lu mi nol .. '++. 'E and th at it decays faster. This may suggest that exces 1: 1009 ~ "o 0 20000 u u" sive PE from blood devastates its energy resources. Compari son of PE from non-diluted blood and 500 4 from suspensions of neutrophils isolated from the

2,Se 5 same blood and adjusted in cell concentrati on to that ~ 1.Se5 24 hrs 2e S c 'E 1200 1.20 S N l ,SeS ;:- N .. 3hr .. t! ., > '"~ 1: ~ 90000 ~ le5 ~ 900 "0 u u u CJ OJ Vl Vl ~ 60000 5:2 50000 Vl ~ C !: 600 0" "o U . 30000 U 4 Time, min 100 Fig. 2 - Ph oton emi ssion changes in non-diluted bl ood (0.2 ml ) 10 12 I~ Ie la supp lemented with luminol (a) or lucigcnin (b) in relati on to time Time. hrs of blood storage. Ali quots (0.2 ml ) for measure ments we re taken Fig. 3 - Photon emi ssion fro m non-di luted blood (0. 1 ml ) without from th is sample at time moment s marked by insc riptions at each luminol (c urve I, left Y-sca le) or with it (c urve 2, ri ght Y-sca le) curve. Note, that the curves for 5 min ., I hr, and 3 hours in (a) after induction of respiratory burst by addition of zy mosan to appl y to the left ordinate and the curve for 24 hours -- to the right blood (without zy mosan and luminol PE from blood sampl e was ordinate. 250-300 counts/6 sec). 476 INDIAN J EXP BIO L. MA Y 2003 of concentration of neutrophils in whole blood re Oscillations of LC-PE can be also registered from vea led diffe rences as well as similarities in PE pat non-diluted bl ood (Fi g. 6). Si gnificant oscillati ons terns from these two ex perimental systems. It may be could be seen at the shoulder of the major peak of PE, suggested th at these differences could be at least in and then th ey appeared again at a new small er peak part be ex plained by different conditions of oxygen long time later. It is interesting to note that some simi suppl y to neutrophils in bl ood and in neutrophil sus larity may be seen between oscillatory behavi or of pension. Concentrati on of di ssolved oxygen due to its neutrophils in suspensions and bl ood ; in both cases high affinity to hemoglobin is very low in bl ood there could be di stingui shed two macroscopi c waves pl as ma, and white bl ood cell s, in parti cul ar neutro of PE, and the second wave was modul ated by oscill a phils may get it for respirati on onl y from erythrocytes . ti ons of rather large amplitude. On the contrary, in neutro phil suspensions cell s con Th ermal hysteresis of PE from blood sume oxygen di ssolved in cell medium. Thus it is Dependence of LM-PE intensity during the develop possibl e th at hi gh level of LC-PE in non-diluted bl ood ment of zymosan-induced RB up on slow (peri od may be related to interac ti ons of neutrophils and about 20 min ) and regul ar temperature changes in th e erythrocytes in bl ood. In fac t, when bl ood to whi ch range of ph ys iological temperatures is il lustrated in lu min ol+zy mosan or onl y lucigenin had been added Fi g. 6. During the lag-peri od precedi ng RB develop was diluted with saline (supplemented with eith er lu ment and until PE reaches max imal intensity it pract i mino l or lu cigenin ) onl y LC-PE, but not LM -PE in call y does not depend up on temperature. It can be tensity was progressively declined with th e increase 10000 of the degree of dilution (Fig. 4). This res ult agrees with the suggesti on th at pers istent LC-PE from bl ood !SIXIQ 8000 f \ is provided by a close contact of neut ro phil s and oxy J- .. ~ c gen-donatin g eryth rocytes in it. 6000 1 ,.. ,., '.' ,.,2 'E """ 100 .... '.' OscillatofY behavior of PEfrom neutrophil suspen 0" i! sions and ji"om blood- Under certain conditions PE 1: :;J 4000 0 ~ from suspensions of iso lated neutrophil s may ga in u oscillatory patterns with hi gh amplitude of osc ill a 2000 ti ons reaching up to 25 % of the mean PE intensi ty. These co nditi ons inclu de lac k of ag itati on of a sus 0 pension, an optimal buffe red medium containing nu 0 2 6 8 10 12 14 tri ents and access to air. Prominent oscill ati ons under TIme, hours th ese conditi ons develop about I hI' after RB initi a Fi g. 5 - Sustai ncd oscill ati o ns or LM-PE in neutrophil suspcn ti on, and last fo r many hours (Fig. 5). In abse nce of sions (20000 cell s in 0. 1 ml of M-PRM medium (ICN) after RB initiated with zymosan. Curve I-aerobic conditions, Curve 2 - air access osc ill atory behav ior can also be seen, how suspension is isolated fro n ai r. In sert - the initi al pe ri od of RB. ever, PE fades mu ch earli er and amplitudes of oscill a tions are small er. 8000 ~-----~---_=-=-==_~. ~_ ~_======~

7000 "": I

6000 ",·,1~ ""l l ' \ ~LI~) i\l/ I, i1.1 '\,'\ I J I• E I: - I .I ~I Vi • ~\I 'J ,~ U~\~I'\ ,: ' ;.,<= 'E 5000 ~. 80000 1'~ '''' Vi._ _ I ~ i ~j 00'1 ~ 1: 4000 ~ .!: '0 :;J C o :;J E.2 o j .... o () 40000 ...J 0 :: -~ I -kiilllllllllllllllllllllllllllill __ o (~~~::::==~:1I:;1I:1I1:11I1:1I1:11I1:1 1I~1~~~~2~ 1000. LI------~------.J. 5 10 15 20 10 100 1000 10000 Time, min Time. 0.1 min

Fig. 4 - Effect o r blood dilution with physiological saline upon Fig. 6 - 0scill atory behavior of LC-PE from nun-di lu ted blood photon e mi ssion frolll blood to whi ch either lucigenin or lumi nol (0. 1 ml ). In sert-oscill ati ons at the second peak of PE arising + zy mosan were previously added. Initial blood volume - 0, I m!. about 16 hours after addition of lu cigenin to blood and start of the Dilution d id not change cell contents in blood. expe rime nt. VOEIKOV el al.: BIOPHOTON RESEARCH IN BLOOD REVEALS ITS HOLISTIC PROPERTIES 477 seen that at this stage PE elevated even when with low level intensity photon flux with the same temperature declined, that formally accounts for the spectral properties as emitted by it may modulate the "negative energy of activation" (Fig. 7 A&B). Only character of the processes related to EES generation. IO 12 after maximal intensity of PE is reached and starts to ]n fact it has been c1emonstrated - that two popula decline the major tendency of curves in Arrhenius tions of neutrophils or blood being in an optical (but coordinates starts to approach a classical one (Fig. 7C), not chemical) contact with each other can influence however, even at this stage a strong temperature hys intensity of oxidative processes in each other if in one teresis is still observed. of them RB is induced. It was interesting to see if Violation of periodicity of temperature changes or back reflection of photons emitted by blood can influ- its elevation to extreme values were followed with the 5 paradoxical blood response (Fig. 8). ]t can be seen Photon t · 43 that blood reacts to changes in the gradient of tem ! perature decrease (time points 1800, 3000 and 4200 141 sec) by elevation of PE intensity. On the other hand ; 0° 1+-+---+I1-+-- \ 39 ! when temperature reaches 39.S oC PE intensity drops abruptly, in spite of further temperature rise, as if 137 8. E blood is denaturized. However, this is not the case i 35 ~ because as soon as temperature after reaching its 33 maximum begins to decline PE from blood starts to elevate. Thus decrease of PE from blood is not caused 1000 1L------__~ o 600 1200 1800 2400 3000 3600 4200 by irreversible changes in it. Time, sec Effect of back reflected photons upon the develop Fig. 8 - Effect of interruption of periodicity of temperature ment of RB in blood - If PE from blood reflects func changes or its elevation to extreme values upon the patterns of tionally significant processes of electron excited states LM-PE from blood in which RB was induced 2,5 hour before the generation, one can expect that in·adiation of blood time point 0 at this panel.

A 6500 ,------·...., 39

5500 38

4500 U lil 37 o~ ~ 3500 .a C ~ ~ o 36 8. U 2500 E ~ 35 1500 Temperature emission 500 34 o 600 1200 1800 2400 3000 :l600 4200 Time, sec B 35500 C 28000 38°C 29500 36°C 26000 23500 ~~ ,,', ~ ~ 17500 I 24(,00 '14 C ~ ~ 22000 8 11500 0 ()" 20000 1 I ----"' START ~ E~ 5500 1 18000 3,205 3,215 3,225 3,23S 3,245 3,255 3,WS 3,215 l,225 3,235 3,245 3,255 1000fT 1000fT

Fig. 7 - Dependence of LM-PE from blood in which RB is induced with zymosan upon temperature waves. (A) Original data. (8 and C) Arrhenius plots for the initial stage of RB (ca. up to 1700 sec) and later stage of RB , respectively. 478 INDIAN J EXP BIOL, MAY2003 ence the processes, that lead to photon em iSSion, in wrapped sampl es in compari son to the control ones particul ar, because this experimental approach could (Fig. 9B). [t can be seen from Fig. 10 that effects of demonstrate effect of ultra-weak irrad iat ion of bl ood ph otons reflection from al uminum foil on LC-PE de • more convin ci ngly th an others. velopment are generall y the same as those observed To ac hi eve hi gh degree of reflecti on of photons with LM-PE development after RB inducing in blood. emitted by bl ood back to it Eppendorf tes t tubes were In th e sampl e with slow developmen t of LC-PE wrapped in aluminum fo il screens. Kinetics of LM-PE screening accelerates photon emission (Fig. lO A), and development during RB induced by zymosan was dif in a blood preparation with fast development of LC fe rent in test tubes with blood covered with aluminum PE its inhibition is observed (Fig. lOB ). foil and in co ntrol test tubes (Fig. 9). The "sign" of NBT test revealed th e effect of foi l screens upon the effect: enhancement or attenu ation of PE intensity activity of neutrophils in bl ood where RB was in in experimental test tubes after the foil was moved duced with zymosan and in which LC-PE was regis away from the test tu be depended on the rate of PE tered. In thi s experiment LC-PE was prominently development in bl ood. If PE was accelerating slow ly hi gher after foil removal from th e experimental sam (cou nt rate in the co ntrol sampl es did not exceed ple th an in th e control sampl e (data not shown ). After 100,000 counts/0.2 min at the moment of foil re zy mosan addition to blood the number of active neu moval) PE intensity in the initi all y sc reened sa mpl e trophil s significantly increased in both sa mpl es just after a screen removal usually exceeded that of a (Fig. 11 ). However, the total number of active cells in co ntrol one by I.S-S-fold and continued to increase the sa mpl e screened with foil is larger than in th e co n faster th at in control samples (Fig. 9A). On the con trol sampl e (39 vs. 30%). It is notabl e that th e number trary , at hi gh rates of PE development in control of cells belongi ng to high ranks (more than 50% of blood sampl es back reflection of photons eith er did cytoplasm is filled with diformasa n granul es) is twi ce not modify the process or even attenu ated PE in as hi gh in th e former sa mpl e in comparison to th e co nt rol one (compare the bars ##5 and 6 in I c and I f). 8e5 A 8e4 -0- 1 Foil is 6e5 ...... 2 A C -0- 3 t removed 6e4 -0- 1 'E C + c: Sample 2 ...... 2 N 'E c: 0 4e5 g ~ is wrapped ..... 3 .- 0 N 4e4 'c 0 :; 2e4 ...J u 0 1 U 0 0

6 12 18 24 6 12 18 24 8e5 3e5 B r B

6e5 -<,)- 1 -0- 1 t c:~:~ c c: ...... 2 ...... 2 2e5 'E - 4- 3 Sample 2 Foil is 'E ..... 3 N 4e5 is wrapped removed N ...... 4 0 + c: 0

Fi g. 9 - Effect of aluminum fo il scree n over the test lUbe with Fi g. 10- Erfe ct of alumi:1um foil screen on LC-PE development blood upon LM-PE developmelll in zymosan treated preparations in two pre parations of blood with slow (A) and fast (8 ) PE devel of blood with slow (A) and fast (8) PE de velopment. Curve 1- opment. Curve I - contro l test tube, curves 2-4 - test tubes were control test tube, eurve 2 - test tube was wrapped with fo il at the wrapped with foil before lueigenin addition. Fo il was removed moment indicated with th e arrow, curve 3 - test tube was from respective test tubes at moments indicated by arrows. In wrapped with foil before luminol and zymosan addition. In each each experiment a pre paration of blood was distri but ed in equal ex periment a preparati on of blood was distributed in 3 equal por portions among 4 samples, which we re coullted in the mode of ti ons counted in th e mode of rotation (in turns). rotation. Note the difference in ordinate scale va lu es in A and B VOEIKOV el al.: BIOPHOTON RESEARCH IN BLOOD REV EALS ITS HOLISTIC PROPERTI ES 479

Thus, the NBT test gives an independent confirmation (Fig. 12). Part of li ght emitted from blood sa mpl es that sample screening with aluminium foi l at the ini can be reflected from the glass wa ll of a vial back to a tial stage of RB development enhances reactive oxy sample. The level of radiation registered by PMT gen forms generation by neutrophils in whole non from test tubes with blood inserted in empty vials and diluted blood. in vials filled with water was compared . These meas urements showed that PE intensity levels measured Another experiment was set-up to reveal effects of back reflected photons up on RB patterns in blood. from the same sample of blood were 16±4% hi gher Eppendorf test tubes were fixed in glass vials when a vial was filled with water than wh en a test tube was inserted in an empty (containing air) vial. Increased light emi ssion from test tubes with bl ood 100 positioned in water fill ed vials is due to lack of ph o 90 ton reflection from the inner wall of the vi al bac k to its source due to the immersion effect of water (refl ec 80 tion coefficient of water and glass are similar unlike ~ 0.- 70 those fo r air and glass). In these experiments it was I- Z 1f noted that even this small part of li ght th at was re 60 Oc Of 1c I I I ~ flected back and irradiated a test tube with blood, hacl 10 a measurable effect up on the kinetic parameters of OU _ _ b PE response in blood during RB. As it can be seen in 1 234 5 6 1 234 5 6 1 2 3 4 5 6 1 2 345 6 Fig. 12, when the sample was transferred to a water No of rank filled vial at the initial stage of PE development, its Fi g. II - Effec t of foil sc reening on neutrophil ac ti vity in whole intensity initially leaped by 16% due to the immersion bl ood evaluated by NBT tes t. Oc and Of - di stribution of neutro effect and then its acceleration temporarily retarded phi ls according to the ran ges of th eir acti vit y in a cont rol and a (Fig. 12A). Sharp leaps of PE intensity were observed foil sc ree ned sa mp les just before th e addition of zy mosan and when th e sample was transfen-ed from an empty vial lucigenin to th em. lc and I f- same 6 minutes aft er addition of zy mosan and lucigenin to the control ano foil sc reened bl ood to a water-filled one and back at the quasi-stationary sa mpl es. stage of LM-PE (Fig. 12B ). However, it can al so be

20000 A Sample with bl od c: 15000 ·E -g; :l .. "0c: ~ 10000 :l .. o C iu ! 6, :l o 5000 \ ~ t .l< u .." \...... \._--- CD D Vial with 20 40 60 80 air or water

...------,2,6e5

4,2e5 c 2,4e5 c: c: ·E 3,8e5 2,2e5 ·E N N ~ ~ ~:l 3,4e5 _ In 2e5 ~ L.. :l o C( ::l o o U 3e5 .r. 1,8e5 u o 2,6e5 '------' N o 10 20 30 40 50 60 70 o 50 100 Time, 0.2 min Time, 0.2 min

Fig. 12 - Effec t of parti al li ght renecti on from th e inner surface of th e glass vial upon LM -PE at different stages of RB . Uppe r ri ght cor ner: ex pe rimen tal set-up. An Eppendorf test tube wi th bl ood is inse rt ed by turns into either empty or water fill ed glass vial s. A·- th e ini tial stage of RB ; B - the stati onary stage of RB ; C - th e stage of RB decay. Upper ri ght corner- experi menta l setup. 480 INDIAN J EXP BIOL, MA Y 2003

noted, that when after a few minutes of the sample tion of biological systems. However, most of th ese stay in a water-filled vial it was returned back to a conclusions were made on the basis of data obtain ed empty one, the stati onary level of LM-PE in it in from experiments with external irradiation of a system creased to a much hi g her level that could be expected under study and analysis of fluorescence or delayed I4 16 if blood continued to stay in an empty vial. On the photon emission data - . It was onl y in studies of oth er hand, when sample transfers from an empty to a mitogenetic radiation by Gurwitsch l7 where internal water-filled vial and back were made at the stage of oxidati ve metaboli sm was suggested to be th e source PE decay (Fig. 12, C), PE intensity in water-fi ll ed vial of energy that pumps biophotonic fields of living or was rapidly declining. After th e sample was trans gani sms. fen'ed back to an empty vial , it appeared initially at a Addition of lu c igenin - the probe for superox ide

much lower value than previously, but began to ri se ani o n radical (02- -) to non-diluted blood is followed up. Thu s, the effect of irradiati o n of bl ood samples by with PE of rather high intensity even in a resting state, back reflected photons may be opposite at different indicating that ROS are permanently produced in stages of RB w hi ch in turn differ in the absolute lev blood (Fig. I B). Due to high activity of superoxid e els of PE and the directi o n o f its change. Lack of self dislllutase in blood O2- - is rapidly converted into irradiatio n depresses photon emission from blood at hydrogen peroxide, and the latter is immediately de relati vel y low levels of PE (at the stage of PE devel composed with catalase present in human blood. All opment and even more prominently at the stage of its these reactions are hi ghl y exergonic. releasing quanta decay). O n the other hand, at hi gh levels of photon of energy equi valent from I to 2 eV at each reacti on emission (stati onary stage) back reflected photons act. Thus electronic excited states are continuously seem to depress PE (Fig. 12B). generated in blood. When the immune reaction respiratory burst of neutrophils- is induced in blood, Discussion intensity of PE dramatically increases (Fig. 3). It is Bl ood provides vital functions for an organi sm as a known th at under these conditions oth er hi g hl y reac whole. It carries gases, nutrients and evacuates by tive oxygen species appear, in particular, hypochlorite products of metabolism, participates in humoral regu (CIO) , the product of oxidation of cr- with hydro lation of physiological functi o ns of a ll other tissues gen peroxide, in a reaction catalyzed with myeloper and systems, supports water, salt and acid-base ho oxid ase. Luminol amplifies PE under these conditions meostasis, executes immunological defense, etc. nearly 100-fo ld. However, PE e levation is registered Knowledge about particular functions of blood com even in absence of luminol and sustains at hi gh level ponents has been gained due to the re lative ease of .tor many hours. This again argues for the generation their isolation from whole blood. However, thi s ap of electron-excited state in native blood. proach does not allow to get in formati on about inter The very opportunity to register photon e mi ssio n acti o ns of blood components in whole blood, tho ug h from such an opaque liquid is possibl e only because their interactions are undoubtedly very signifi cant for energy of e lectron excitati o n can migrate in blood at efficient blood functioning. In a frame of the dominat least parti all y without dissipation. We suggest that ing biochemical paradigm interactions of blood com hemoglobin -lhe major candidate for dissipation of ponents are pro vided by chemical signaling through energy of e lectron excitation does no do it in intact diffu sion of bio-reg ulatory molecules such as cyto blood because it is present in erythrocytes in a li quid Kines and hormones. However, there may exist an crystalline state. In fact, hemoglobin dissolved in blood even in a very low concentration readily al te rn ati ve, or, to be more precise, (l complementary route of regulation of blood functions - through a quenches photo n emi ssion (Fig. 2). non-stationary bi ophotonic fie ld , or, in other words, lndividual neutrophils produce ROS in a stri ctly through a field of electronically excited states of mo oscillatory manner wi th a period of oscill ations in th e lecular components of this ti ssue. It has been stated range of tens of seconds 18. Spontaneous arousal of that in complex and tructured systems energy of multi-period osci ll atory regime including very low electron excitati on does not immediatel y dissipate frequency osci ll ati o ns of PE from neutroph il suspen into heat but rather may mi grate al ong the commol1 sions and whole blood indicates th at producti o n of energy levels and perform chemical, mechanical and ReS by individual ce lls manifested as generati on of at her f orms a f LIse f·u I wor k 11-16. . Suc h energy mi.grati.o n electro ni c excitation is to a certain degree correlated and storage may be provided by structura l organ iza- in systems co ntaining tremendous quantities of these VO EIKOV ('/ al.: BIOPHOTON RESE ARCH IN BLOOD REVEALS ITS HOLISTIC PROPERTI ES 481

cell s (Fi gs 5 and 6). Such correlated behavior may be measurabl e effect upon neutro phil activity. It may be see n even in such a compl ex system as non-diluted su ggested th at at least in part these effects may be bl ood in whi ch onl y a tin y fraction of cells (granulo related to specific ways by whi ch neutrophils obtain cy tes do not exceed together 0.1% of erythrocytes in oxygen fo r th eir respiration in blood. It was demon qu antity) is responsible for ROS generation . Such a strated here that LC-PE in bl ood is strongly dependent co rrelated, cooperati ve behavior can be in principl e upon the interaction of neutrophils and erythrocytes, prov ided by di ffu sibl e regulatory mol ecul es that acti and th at LC-PE depends on ox ygen supplied by eryth vate or inhibit granul ocyte ac tivity. However thi s ex rocy tes (Fi g. 4). Taking into considerati on th at .A.,nax pl anation was rul ed out in the experiments in whi ch for luminol emi ssion is 427 nm and for luci genin PE from bl ood was registered under th e conditions of emi ssion it is 470 nm , and th at several hemoglobin oscillatory temperature ch anges. absorpti on maximums are not far from thi s spectral For th e first time thermal hysteres is of PE from a region, hemoglobin may be exc ited by bac k refl ected li ving system (etiolated barl ey) was observed by photons. It is interesting to specul ate that due to liquid Slaw in sky an d POpp1 9. Their data argued for the pre~ crystalline state of hemoglobin in eryth rocy tes ab ence in livin g matter of a delocali zed coherent elec sorption of photons by fe w hemoglobin molecul es tro mag neti c field far away from th ermal equilibriu m. may provide enough energy of excitation fo r di ssocia Present results confirmed and ex tend th ese observa ti on of oxygen from multiple oxyhemoglobin mo le ti ons. In the present study PE from bl ood ori gin ated cul es belonging to thi s crystal thu s prov iding th e definitely due to a set of bi ochemi cal reacti ons in mechani sm for cascade amplification of a signal and duced in neu trophil s and res ulting in generati on of additi onal substrate supply fo r generation of ROS by elec tro n exci ted spec ies. Very prominent temperature neutrophils and enh ancement of PE intensity in bl ood. hysteres is of PE especiall y at a stage of th e develop However, these considerati ons cann ot readil y ex ment of RB suggests that either the kin eti cs of these pl ain attenuati on of PE intensity by bac k reflected bi oc hemi cal reactions and/or the kineti cs of th e proc ph otons in bl ood preparati ons with initiall y hi gh rate esses res ulting in ph oton emi ss ion do not obey simpl e of PE intensity growth . Prev iously it was observed laws of chemical kin eti cs, in other words these proc th at th e hi gh rate of LM-PE without artificial induc esses are at least parti all y not determined by the rate ti on of RB in it is characteri sti c of bl ood of patients 2o ." of di ffu sion of reagents whi ch shoul d strongly depend with cardi ovascul ar di seases . Besides, it has been upon temperature. As soon as RB starts to fade, tem shown here th at both LM-PE and LC-PE in te nsity is perature dependence of PE more and more resembles dramati ca ll y hi gh after long storage of blood even of of a "classical" one, though even many hours after RB health y donors. After being stored in certain ly unnatu was initi ated pronoun ced temperature hysteresis is ral conditio ns blood in a certain sense becomes still obse rved. Of a spec ial in terest is the behavi or of "sick". It can be supposed, th at in such bl ood prepara blood wh en the peri od icity of temperature changes ti ons hemoglobin is already hi ghl y excited for the rea was violated or temperature was raised to ex treme sons discussed below, and that auxili ary irrad iation of val ues. Paradoxical changes in PE--elevati on of its bl ood with back refl ected photons does not provide an in tensity when temperatu re continu ed to deciine additi onal stimulu s fo r oxygen release. On the other though at di ffe rent rate than before, or abrupt drop in han el , additional irradi ati on of bl ood with weak ph o PE intensity when it continued to raise -- indicate that ton flu xes ori gin ated from bl ood itsel f and reflected blood has some pec ul iar mec hani sm sensing tempo ral back to it, may pl ay an organi zin g ro le so th at less variations of te mperature. The nature of thi s mecha energy is lost fro m it, and the ph oton flu x fro m such nis m is currentl y obsc ure. bl ood reg istered by a photomul tipli er is lower after Modul ati on of acti vity of neutrophil s in whole the refl ective screen re moval th an the control sample. blood by back refl ected photons and changes of pat The hypothesis th at reacti ve oxygen species gen tern s of PE fro m it in response to thi s very low inten erati on by white bl ood cell s is enh anced due to an sity irradi ati on (Fi gs 10- 12) also argues in favour of increase in oxygen availability, ensuing th e enhance the functional ro le of the biophotonic field in thi s tis ment of photon emi ss ion fro m "normal" blood, is sue. As it has been poi nted out above, non-d iluted sup ported by the res ults of studies of the effect of CO 2 blood is a system with a very high optical density and on who le blood and isolated neutrophils 1.22. It was the ratio of neutrophils to erythrocytes in it is ap observed th at whil e CO sharpl y intensified LC-PE in prox imately I: 1000, so the question arises as how non-d iluted blood; it had mu ch weaker effect in sa these very low intensity photon fl uxes may have any lin e-diluted blood, and in hibited LC-PE in a neutro- 482 INDIAN J EXP BIOL, MA Y 2003 phi I suspension . CO is known to bind to heme 100- 9 Mieg C, Mei W P. & Popp F A, Technical Notes to Bi ophoton Emiss ion. in: Recent advances in Biophoton research and its fold more tightly than O2. In whole blood its major target is deoxy-Hb. Sharp elevation of LC-PE after applicatiollS, edited by F A Popp, K H Li , & Q Gu Q (World Scientific Publi shin g Co. Pte.Ltd., Singapore) 1992, 197. CO addition to blood is a strong indication of hi gh ly 10 Voeikov V L & Novikov C N, Interac ti on of two opticall y co-operative processes proceeding in blood. Presuma coupled who le blood samples in the course of respi ratory burst bly, high energy rel eased when CO binds to heme of in one of them, in Advances in fluorescence sensing technol hemoglobin molecules packed in erythrocytes induces ogy in clinical diagnostics III, edi ted by. J A Lakowicz, R D accelerated oxygen release from many Hb0 mole Thompso n (SPIE Proe.) 2980 (1997) 470. 2 II Novikov C N, Voeikov V L & Popp F-A, Analys is of li ght cu les res ulting in the similar effect upon PE from emi ss ion by neut rop hil s in the process of respi ratory bu rst sug who le blood as that produced by blood self gests that phys ical field s are involved in intercellular co mmu irrad iation. Inhibitory effect of CO upon LC-PE in nications," Biophotonics. Non-equilibriulII alld Coherelll Sys· neutrophil suspensions is most probably ex plained by tel/IS in Biology, Biophysics and Biotechnology. Proe. Int. 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