Lasers in Analytical Biochemistry
Laser Based Ortho-Molecular Medical Diagnostics
Tech Area / Field
- PHY-OPL/Optics and Lasers/Physics
3 Approved without Funding
VNIIEF, Russia, N. Novgorod reg., Sarov
- Institute of General Physics named after A.M. Prokhorov RAS, Russia, Moscow\nMedical Radiological Scientific Center, Russia, Kaluga reg., Obninsk
- University of Alabama at Birmingham, USA, AL, Birmingham\nImperial College of Science, Technology and Medicine / National Heart and Lung Institute, UK, London
Project summaryImportance of the medical problem
At present, notions of very important role of endogenous nitrogen and carbon monoxides in human physiology are developing intensively. These molecules act as mediators taking part in signal transfer in course of various selfregulation processes in organism [1-10].
Nitrogen oxide was found to play an important role in blood pressure control, in maintaining of blood vessel tonus, in providing blood microcirculation in tissues, in performing of a set of immunity reactions by effector cells and in information transfer between neurons. In organism NO is secreted by a specific NO-syntaze enzymes. A level of constituitive and inducible NO synthesis reflects a state of many important systems in organism and could be used as an indicator of health or disease as well as a measure of applied therapy efficiency.
The last studies of carbon monoxide activity in organism indicate a specific role not only in metabolism regulation of heme-containing protein structures like hemoglobin, mioglobin and citochroms, but also in more complicated biochemical processes like transmission of nerve pulses, maintaining of micro vessel muscle tonus, inducing of chemoreceptor activity, control of some hypothalamus hormone secretion etc.. Besides, CO elimination with breath is sensitive to blood pH variations and to extent of blood saturation with oxygen and thus could characterize gas transportation properties of blood, its acid-base balance and biochemistry.
Intensive development of new treatment approaches and pharmacological preparations for therapy of various diseases caused by different abnormal changes in generation of these endogenous molecule are in progress now on the basis of the last biochemical investigations of CO and NO role in organism. For instance, an inhibition of arteriosclerosis processes discovered recently and caused by elevated constitutional NO synthesis allows development of new technologies to prevent arteriosclerosis and tissue ischemiasation. This could have specific importance taking into account that about 70% of people deaths is caused by diseases of cardio-vascular system.
Importance of the Proposed Technology Innovation
In connection with the key role of these molecules in the numerous biochemicl processes in organism, research and development of a technique for the noninvasive diagnostics of endogenous CO and NO generation, transport and removal by organism became topical. Such technique could provide a possibility for deeper research of processes going on in human and animal organisms, for development of new therapeutic and pharmacology approaches to disease treatment and for prompt diagnostics in course of any therapy.
At present several methods like mass-spectrometry, UF-chemoluminescense, FT-IR spectroscopy and semiconductor chemical sensors are conventionally used in different analytical problems for CO and NO detection. However application of these methods to simultaneous high sensitive analysis of both studied molecules in exhaled air with a sensitivity necessary for biomedical diagnostics is not possible. Mass-spectrometry can not provide necessary speed of analysis, enough sensitivity and selectivity. Besides, in this case it is necessary to manage preliminary removal of water vapor and carbon dioxide from studied gas sample by using gas-chromatography separation. Analytical instruments using UF-chemoluminescent technique can not give sufficient accuracy because they need in preliminary conversion of studied substances into the detectable ones and more they are not sensitive to carbon monoxide. FT-IR spectrometry can not provide accuracy, sensitivity and speed of the detection. Semiconductor chemical sensors suffer from high humidity, has no enough sensitivity, selectivity, speed and shelf time.
It is essential to note here that the most of biochemical studies of CO and NO role in organism are running on animals and with use of specific analytical methods that can not be applied in medical practice because they need in destructurisation of organs or tissues studied.
Careful analysis of the alternative approaches shows that application of diagnostics based on high resolution spectral analysis with use of tunable diode lasers could be the most adequate to the problem. Due to the combination of remarkably high analytical characteristics, such as sensitivity, speed of response, and selectivity, that could be realized with the use of TDL for the detection of light molecule traces in multi-component gas mixtures, it is possible to analyze chemical composition of the exhaled air carefully and in real time [11-13]. The approach proposed in the Project and based on the high sensitive laser analysis of endogenous CO and NO traces in exhaled air is new and unique and has its origins in the last data and experience of the Project authors obtained in their experimental and fundamental studies in laser gas analysis [14-23] as well as in its applications to biomedical diagnostics [24-33]. A development of this scientific approach may considerably widen the existing range of conventional analytical techniques of medical diagnostics or enhance their capabilities.
A highly sensitive and selective chemical analysis of the exhaled air composition may be effectively applied in the research of the condition of the human organism or its system as well as in the diagnostics of various diseases, i.e. in the ortho-molecular diagnostics [34-37]. Actually, the exhaled air composition is rather informative because such air contains several hundreds of endogenous volatile substances, which result from different physiological and biochemical processes. The diseases, which may be diagnosed using the aforementioned technique are rather different and numerous and are defined by a set of molecules available for the analysis. In particular, the diagnostics of the processes occurring in the human organism is mostly efficient, if it allows combining its non-invasive character with the continuos real-time monitoring capability. This may be fully referred to the diagnostic technique based on TDL use [30-33].
The present Project is aimed at the fulfillment of one of the mostly crucial task of the modern medical diagnostics, i.e. the research of the cardio-vascular and respiration systems condition. Under the Project it is planned to develop physical, technological and medico-biological principles of the aforementioned systems diagnostics based on the use of a TDL in a highly sensitive gas analyzer of the exhaled air composition. It will be developed a specialized multi-component TDL-gas-analyzer, as well as new effective non-invasive diagnostic techniques allowing to find out certain diseases and based on the monitoring of the content of the mostly important from the physiology viewpoint endogenous molecules such as CO, NO and CO2 in the exhaled air.
It was shown by the Project authors [24-33] that gas analysis of the exhaled air with the help of a TDL has several serious advantages over other techniques, if microconcentrations serve as the basis of the gas exchange studies. Among these advantages the following are worth mentioning:
· possibility to simultaneously measure in the exhalation the microconcentrations of several gaseous substances which are significant from the diagnostics viewpoint;
· high sensitivity with regard to concentration, i.e. at the level of 0.1-1 g/m3;
· fast recording and analysis of data with the time constant of 5 sec, which allows to diagnose the physiological processes dynamics;
· high selectivity detection of the substances under research in the presence of the dominating components of the exhaled air (N2, O2, CO2, H2O), and the traces of hundreds of other volatile substances;
· on-line detection of the substances under research in the exhalation without a preliminary concentration and/or accumulation of the studied air.
The application of the exhaled air analyzer with a TDL will allow to:
· Improve and simplify traditional diagnostic techniques due to the multi-gas approach, enhanced testing capabilities; fast analysis and reliability of the results obtained.
· Reduce the degree of the invasion into the organism under examination due to the analysis sensitivity increase and reduction of the exogenous gases concentration required for the conduct of physiological respiratory tests.
· Research subtle physiological effects connected with the respiration and blood circulation and previously inaccessible for the observation, as well as the process of the pulses transfer in peripheral and central parts of the nervous system, where CO and NO are produced as mediators.
· Develop new techniques allowing diagnosing the diseases of the respiration and cardio-vascular systems.
Basically, the laser gas-analyzers to be developed under the present Project will be used as the prototypes for serial models, while new diagnostic techniques will be able to find a large-scale application in clinical practice. From the viewpoint of practical medicine, it will be significant to have the possibility of obtaining dynamic characteristics of the organism’s functional condition, as well as the possibility to monitor the effectiveness of the prescribed therapy based on the assessment of the external respiration characteristics, diffusion ability, blood circulation in lungs and blood circulation system. The aforementioned approach is believed to be suitable for the diagnostics of acute and chronic cardio-respiration and congestion (stomach/liver) diseases including professional lungs illnesses (silicosis, enphisema etc.), chronic inflammations, tuberculosis, chronic heart ischemical, syndrome of an insufficient blood circulation in a small loop, infarct of myocardium. It will also allow diagnosing the cases connected with the metabolism malfunctions and blood system diseases. The introduction into medical practice of the gas-analyzers to be developed may play a revolutionary role in the research of the contribution of CO and NO into physiological and pathological processes. In particular, recently it was found out that the increase of the constituitive synthesis of NO might lead to the impedance of the processes of atherosclerosis and tissues ischemisation. This proves the acute character of the proposed Project because about 70% of people’s death rate are caused by the diseases of the cardio-vascular system. In the present Project it is planned to use the equipment to be developed in order to clarify the role of CO and NO in the stimulating effect of the induced electromagnetic fields. Recently, it was found out using the electro-paramagnetic technique that these fields cause the formation of NO in tissues [38-40].
Besides, the proposed technique may be effectively used in the area of physiology connected with the environmental effects. Apart from a considerable statistical data on the organism inpidual reactions to the environmental conditions, the aforementioned technique will allow to work out an inpidual health record and to assess the result of the extreme physical factors effect on the organism (i.e. high temperature, radiation, pressure effects etc.). The proposed technique may be suitable for the diagnostics of other diseases, such as diseases of inner organs, atherosclerosis, metabolism pathologies, degradation and rejection of tissues etc., if to modify the set of the endogenous molecules subject to detection.
The fulfillment of the present Project will allow to engage in the socially significant work the experts formerly involved in the weapons RD, scientists representing the academy of sciences, and medical specialists. The Project is aimed at the development of new techniques and means based on the application of the most advanced technology to non-invasive diagnostics of various diseases. In particular, former weapon scientists will be engaged in the development of an optico-mechanical unit of the gas-analyzer, algorithms of a highly sensitive detection of gaseous molecules, and the software. Besides, they will participate in the issue of the design documentation assembling, adjustment and testing of gas-analyzers, as well as in the development of the functional diagnostics techniques. The labor effort of such specialists will be 228.75 person/months.
If a serial production of the gas-analyzers is to be set up, it is proposed to use the capabilities of several defense production facilities in order to start-up the production of the gas-analyzers and their components (i.e. IR optics, cryogenics, IR photo-detectors, TDLs, electronics etc.).
It is expected to obtain the following results of the Project fulfillment:
1. Development of physical, technological and medico-biological basics for the diagnostics of the cardio-vascular and respiration systems diseases using the technique of highly sensitive and fast analysis of the CO and NO traces and of the CO2 content in the exhaled air with the help of a TDL.
2. Development and manufacturing of the experimental prototype of a specialized multi-component TDL-based gas-analyzer, which is intended for a simultaneous recording of CO and NO traces and of the CO2 content in the exhaled air, while the sensitivity of the aforementioned equipment will be 0.1-1.0 g/m3 in a real time mode.
3. Further development of multi-component models of gas exchange in the human organism as a result of testing the equipment and laboratory research conduct. These models will allow to describe the basic regularities of the behavior of different gaseous substances, the trace concentrations of that are present in the exhaled air, as well as to define/verify the main constants of the gas exchange regarding the microconcentrations of gases.
4. Theoretical and experimental development of low-invasive techniques allowing to measure the parameters of the cardio-vascular and respiration systems. Offer of new effective techniques of several disease diagnostics using the developed gas-analyzer.
5. Obtaining of data on the production of CO and NO in the human organism in case of the organism’s resistance stimulation by means of the modulated electro-magnetic fields.
6. Issue of the recommendation on the use of the ortho-molecular laser diagnostic technique in medical practice.
1. S.Moncada, R.M.J.Palmer, E.A.Higgs, Nitric oxide: physiology, patophysiology and pharmacology, Pharmacol. Rev., Vol. 43, pp. 109-142, 1991.
2. G.S.Marks, Brien J.F., Nakatsu K., McHaughlin B.E., Does carbon monoxide have a physiological functure? Trends in Pharm.Sci., 1991, 12, 185-188.
3. Schmidt H.H.H.W., NO, CO and OH. Endogenous soluble guanylyl cyclase-activating factors, FEBS-Lett., Vol. 307, No. 1, pp.102-107, 1992.
4. Verma A., Hirsch D.J., Glatt C.E., Ronnett G.V., Snyder S.H., Carbon monoxide: A putative neural messenger, Science-Wash.. 1993. vol. 259, no. 5093, pp. 381-384.
5. Marks G.S., Heme oxygenase: The physiological role of one of its metabolites, carbon monoxide and interactions with zinc protoporphyrin, cobalt protoporphyrin and other metalloporphyrins, Cell.-Mol.-Biol., 1994 vol. 40, no. 7, pp. 863-870.
6. Prabhakar N.R., Dinerman J.L., Agani F.H., Snyder S.H., Carbon monoxide: A role in carotid body chemoreception, Proc.-Natl.-Acad.-Sci.-USA, 1995 vol. 92, no. 6.
7. Ingi T., Ronnett G.V., Direct demonstration of a physiological role for carbon monoxide in olfactory receptor neurons. J-Neurosci. 1995 Dec; 15(12): 8214-22.
8. К.М.Маrkov, Nitrogen oxide and carbon oxide – new class of signal molecules, Uspekchi Fiziologicheskikch Nauk, pp. 30-43, 1996 Oct-Dec. (in Russian).
9. Ingi T., Cheng J., Ronnett G.V., Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system., Neuron. 1996 Apr; 16(4): 835-42 /USA.
10. Maulik N., Engelman D.T., Watanabe M., Engelman R.M., Das D.K., Nitric oxide-a retrograde messenger for carbon monoxide signaling in ischemic heart. Mol-Cell-Biochem. 1996 Apr 12-26; 157(1-2), 75-86 /USA.
11. Monitoring of gaseous pollutants by tunable diode laser, Proc. of the Int. symposium, held in Freiburg, FRG, 11-13 November 1986, Edited by R.Grisar, H.Preier, G.Shmidtke, G.Restelli, 176 p, D.Reided Publishing Company, Dordrecht, Holland, (1987).
12. Monitoring of gaseous pollutants by tunable diode laser, Proc. of the Int. symposium, held in Freiburg, FRG, 17-18 October 1988, Edited by R.Grisar, G.Shmidtke, M.Tacke, G.Restelli, 305 p., Kluwer Academic Publishers, Dordrecht, Holland, (1989).
13. Monitoring of gaseous pollutants by tunable diode laser, Proc. of the Int. symposium, held in Freiburg, FRG, 17-18 October 1991, Edited by R.Grisar, H.Bottner, M.Tacke, G.Restelli, 305 p., Kluwer Academic Publishers, Dordrecht, Holland, (1992).
14. A.I.Kuznetzov, A.I.Nadezhdinskii, E.V.Stepanov, Computerized Infrared Fiber-Optic System for Gas Analysis Based on Diode Lasers, Proc. SPIE "Infrared Fiber Optics II", Vol.1228, pp.262-265, 1990.
15. A.M.Bloch, A.I.Kuznetsov, A.I.Nadezchdinskii, E.V.Shvets, Diode-laser-based open-path analyzer of carbon monoxide concentration in atmosphere, Selected papers on TDLS, SPIE Series, Vol.1724, 1992, 251-255.
16. V.G.Artjushenko, A.I.Kouznetsov, Yu.G.Selivanov, E.V.Stepanov, P.V.Zyrianov, Multicomponent Gas Sensor Based on MIR Tunable Diode Lasers and Fiber Optics,. Proc of SPIE, Vol. 2508-27, 1995.
17. E.V.Stepanov, A.I.Kouznetsov, P.V.Zyrianov, V.G.Plotnichenko, Yu.G.Selivanov, V.G.Artjushenko, Multicomponent Fiber-Optical Gas Sensor Based on MIR Tunable Diode Lasers, Proc. of Int. Symp. on TDLAMGP, October 1994, Freiburg, FRG, 1995, Journal of Infrared Physics and Technology, Vol.37, No.1, Feb 1996, p.149-153.
18. A.N.Khusnutdinov, A.I.Kouznetsov Ya.Ya.Ponurovskii, E.V.Stepanov, P.V.Zyrianov, Multicomponent Gas Analyzers Based on Tunable Diode Lasers, Proc. SPIE of the Int. Symp. on Advanced Technologies for Environmental Monitoring and Remediation, 1996, Denver-96.
19. A.I.Nadezhdinskii, G.G.Devjatych, A.I.Kuznetsov, G.A.Maksimov, V.A.Khorshev, S.M.Shapin, E.V.Stepanov, Application of Tunable Diode Lasers in Control of High Pure Material Technologies, Proc. of SPIE, Vol.1418, pp.487-495, 1991.
20. A.I.Nadezhdinskii, E.V.Stepanov, Diode Laser Spectroscopy of Atmospheric Pollutants, Proc. of SPIE, Vol.1433, 10 p., 1991.
21. A.I.Nadezhdinskii, I.I.Zasavitskii, E.V.Stepanov, Spectral Gas Analysis of Polyatomic Molecules by Tunable Diode Lasers, Selected papers on TDLS, SPIE Series, Vol.1724, 1992, pp.238-250.
22. Adamenkov A.A., Bulkin Yu.N., Buzoverya V.V., Kolobyanin Yu.V., Kudryashov E.A., Diagnostics of the active medium of an electric discharge CO2 module by the “laser spectrograph” method / Report at XI International Symposium on gas-flow and chemical lasers, Edinburg, Great Britain, 25-30 August 1996.
23. Adamenkov A A, Bulkin Yu.N., Buzoverya V.V., Kolobyanin Yu.V., Kudryashov E.A., V.Vyskubenko, Study of a chemical gasdynamic laser by the “laser spectrograph” method / Report at the XIX International Conference on Lasers, Portland, USA, 26 December, 1996.
24. A.I.Kuznetzov, A.P.Logachev, E.V.Stepanov, “Computerized Diode Laser System for CO Content Investigation in Human Expiration", Proc.SPIE, vol.1201, pp.487-494, 1990.
25. А.I.Кouznetsov, А.P.Logachev, Е.V.Stepanov, Analysis of human exhaled air by tunable diode laser spectroscopy, Izvestiia AN SSSR, 1990, №10, с.1909-1914 (in Russian).
26. E.V.Stepanov, K.L.Moskalenko, Gas Analysis of Human Exhalation by Tunable Diode Laser Spectroscopy, Optical Engineering, vol.32, No.2, pp.361-367, February, 1993.
27. I.I.Zasavitskii, K.L.Moskalenko, A.I.Nadezhdinskii, E.V.Stepanov, Application of Tunable Diode Lasers for Human Expiration Diagnostics, Proc. of Int.Symp. on Application of Tunable Diode Lasers, Freiburg, Germany, 1991, pp.203-216.
28. I.A.Adamovskaya, K.L.Moskalenko, S.McKenna-Lawlor, A.I.Nadezhdinskii, N.V.Sobolev, E.V.Stepanov, Tunable Diode Lasers Application for Fully Automated Absolute Measurements of CO and CO2 Concentrations in Human Breath, Proc. of SPIE, 1993, Vol. 2205, 440-447.
29. K.L.Moskalenko, A.I.Nadezhdinskii, E.V.Stepanov, Tunable Diode Laser Spectroscopy Application for Ammonia and Methane Content Measurements in Human Breath, Proc. of SPIE, 1993, Vol. 2205, 448-452.
30. Е.V.Stepanov, V.А.Skrupskii, Yu.А.Shulagin, Monitoring of Endogenous CO Elimination with Exhalation in Rats at Hyperoxia”, J. Aviokosmicheskoi I Ekologicheskoi Medicini, 1995, No.1, pp.49-53 (in Russian).
31. A.I.Kouznetsov, E.V.Stepanov, Yu.A.Shulagin, V.A.Skrupskii, Endogenous CO Dynamics Monitoring in Breath by Tunable Diode Laser, Proc. SPIE, “Laser Diodes and Applications II”, Vol.2682, pp.247-256, 1996.
32. A.I.Kouznetsov, E.V.Stepanov, Applications of Tunable Diode Laser Spectroscopy for Detection Exhaled Endogenous Gases: CO, NH3, CH4, N2O, CO2, Proc. SPIE, Biomedical Optics, BiOS’96, 27 Jan-2 Feb., 1996, San Jose, Vol.2676, pp.272-282, 1996.
33. E.V.Stepanov, A.I.Kouznetsov, P.V.Zyrianov, Y.A.Shulagin, A.I.Diachenko, Yu.I.Gurfinkel, Monitoring of rapid blood pH variations by CO detection in breath with tunable diode laser, Proc. SPIE, “Biomedical Sensing, Imaging, and Tracking Technologies II.” Vol.2976., 8 p., 1997.
34. M.Phillips, Breath Tests in Medicine, Scientific American, pp.74-79, July 1992.
35. A.A.Tiunov, V.V.Kustov, Metabolic products at radiation lesion., Moskow: Atomizdat, 1980. – pp. 56-77. (in Russian).
36. U.Lachish, S.Rotter, E.Adler, U.El-Hanany, Tunable Diode Laser Spectroscopic System for Ammonia Detection in Human Respiration, Rev.Sci.Instrum, vol.58, #6, pp.923-927, 1987.
37. P.Shu-Ti Lee, R.F.Maykovski, D.L.Partin, Methods and Apparatus for Measuring Stable Isotopes, US Patent, #4684805, Aug.4, 1987.
38. Konoplyannikov A.G. et.al., Production of nitric oxide in mouse liver after treatment with pulsed extra-low frequency electrical field with certain parameters. In: “EPR-spectroscopy of nitric oxide in biological systems”, Suzdal, December 1996, Int.Conf., pp. 31-32.
39. Kolesnikova A.I., Konoplyannikov A.G., Radiobiological basis of the new methods for stimulation of haematopoietic stem cells proliferation after irradiation of animals and humans. In: “Int. Consortium for research and treatment of radiation-induced injury (Book of abstracts of Int. Conf. on the health effects of low-dose radiation, Houston, Texas, March 30 - April 2, 1993), 1993, pp.31-32.
40. Konoplyannikov A.G., Molecular and Celular Mechanizms of late radiation damages, Rаdiatcionnaia biologia. Radioecologiia, 1997, 37, 4, pp.621-628 (in Russian).
The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.
ISTC facilitates international science projects and assists the global scientific and business community to source and engage with CIS and Georgian institutes that develop or possess an excellence of scientific know-how.