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Biophoton Research in Blood Reveals Its Holistic Properties

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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).
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