- Email: [email protected]
X-ray analysis of aerosol samples from a therapeutic cave
Nuclear Instruments and Methods in Physics Research B 174 (2001) 361±366
www.elsevier.nl/locate/nimb
X-ray analysis of aerosol samples from a therapeutic cave B. Alf oldy a
a,*
, Sz. T or ok a, A. Kocsonya b, Z. Sz} okefalvi-Nagy b, Md.I. Balla
c
Health Physics Department, KFKI Atomic Energy Research Institute, P.O. Box 49, H-1525 Budapest 114, Hungary b KFKI Research Institute for Particle and Nuclear Physics, P.O. Box 49, H-1525 Budapest 114, Hungary c Astrazeneca, 2045 Torokb alint, Park u. 3., Hungary Received 1 August 2000; received in revised form 19 October 2000
Abstract Cave therapy is an ecient therapeutic method to cure asthma, the exact healing eect, however, is not clari®ed, yet. This study is motivated by the basic assumption that aerosols do play the key role in the cave therapy. This study is based on measurements of single aerosol particles originating from a therapeutic cave of Budapest, Hungary (Szeml} ohegyi cave). Aerosol particles have been collected in the regions arranged for the therapeutic treatment. Samples were further analysed for chemical and morphological aspects, determining the particle size distribution and classifying them according to elemental composition. Three particle classes have been detected based on major element concentration: alumino-silicate, quartz and calcium carbonate. Calcium ions have well-known physiological in¯uence: anti-spastic, anti-in¯ammation and excretion reducing eects. In¯ammation, accompanying spasm and extreme excretion production cause the smothering stigma, the so-called asthma. Therefore it could be assumed that calcium ions present in high concentration in the cave's atmosphere is the major cause of the healing eect. Ó 2001 Elsevier Science B.V. All rights reserved. Keywords: Cave therapy; Healing aerosols; Elemental composition; Size fraction; EPMA analysis; PIXE analysis; Cluster analysis
1. Introduction X-ray spectroscopy is a traditional method for trace element analysis in environmental sciences. A cave's atmosphere ± notwithstanding it is sheltered ± is periled by the air pollution. It is especially true in case of therapeutic caves, like the Szeml} ohegyi cave in Budapest, where asthma therapy has been
*
Corresponding author. Tel.: +36-1-392-2222/1176. E-mail address: al®@sunserv.kfki.hu (B. Alf oldy).
carried out for two decades. It is not surprising that considerable attention is paid to study the atmosphere of this cave [1]. Despite the fact that the exact curative property is not clari®ed yet, cave therapy is a very popular therapeutic method against asthma in Hungary [2,3]. Several speculations try to explain the healing in¯uence of the cave. Some of them consider the aerosol as the therapeutic agent of the cave's atmosphere, while other ideas regard radon or the special cave climate (temperature 12°C, humidity roughly 100%, clean air, etc.) responsible for the
0168-583X/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 0 ) 0 0 5 8 6 - 3
362
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
healing in¯uence. It is believed that aerosols play the main role in the cave therapy, and this paper intends to provide experimental data to support this hypothesis. Since the aerosol deposition in the lung depends on the particles' morphological characteristics, individual particle analysis was needed in addition to PIXE bulk analysis, which can provide accurate quantitative results, but only for the mean value of all particles.
2. Pulmonological background Asthma bronchiale is a syndrome with clinical manifestation of hampered respiration (wheeze) caused by diuse bronchial obstruction, bronchial mucus hypersecretion and thickening of the epithelial layer [4]. Pathologically, asthma is a destructive in¯ammation with in®ltration of certain cellular elements of the immune system (so-called eosinophyl cells). Its pathological development is characterised by bronchial hyperresponsiveness to dierent exogen agents (allergens, coming from the external environment), or intrinsic, not clearly de®ned factors of the internal environment `milieu interior'. The pathophysiological background of the clinical signs is based on the impaired interaction between bronchial smooth muscle, epithelium, mucus-secreting glands and the cellular elements of the immune system (lymphocytes, macrophages, mast cells, etc.). Certain neural and humoral factors, i.e., interleukins (IL3, IL4 and IL5), immunglobulins (IgE) complement factors (C3a, C5a) are responsible for extracellular, cell-to-cell signalling (that is communication between the cellular elements). Intracellular messengers that is the cAMP/adenilate cyclase and the changes in intracellular Ca2 ion concentration transfer their messages (as ``second messengers'') to produce the ®nal cellular response that is manifested in bronchial smooth muscle contraction, mucus secretion and release of certain humoral factors from cells of the immune system. In addition, Ca2 ions are important in the determination of both the resting and the activated cell membrane potential, so Ca2
ions play key roles in the normal, physiological cell functions. 3. Sampling and analysis Szeml} ohegyi cave is a thermal cave with three warm air sources. The therapy is carried out in the closest chamber to those warm air sources. Sampling sites were selected according to the research objective, namely at representative locations of the cave where the therapy is carried out. Three samples were collected in the therapeutic region, close to the warm air sources. The next three samples came from the vertical entrance, which is far from the warm air sources. Since there is an out¯ow from the cave at this entrance the microclimate of this site is determined by the cave and not by the outdoor conditions. Some patients spend the therapeutic period at this location and the eciency of their healing does not dier from that of those patients staying in the therapeutic chamber. Finally, three samples were collected at a third location called ``clay chamber'' that has a thick clay layer on its ¯oor. Recently, a facility was established to carry out therapy at this site, too. The clay stanches the rifts of the rock thus separating this region from the atmosphere of the rest of the cave. Sample collection was carried out by pumping air through a 25 mm diameter Nuclepore ®lter with 0.4 lm pore size. The ¯ow rate was 10 l/min for 16±24 h. First, the areal densities of the elements in the samples was determined by particle-induced X-ray spectrometry (PIXE) [5]. The proton beam of 2.5 MeV energy was generated by the 5 MV Van de Graa accelerator of KFKI RMKI. The ®nal beam spot was formed by a Ta collimator of 2 mm diameter. To avoid pile-up peaks and high dead time losses beam currents were kept below 10 nA. The samples were positioned at 45° with respect to the beam direction. For heavier element determination a 0.5 mm thick plexiglass absorber was used to reduce the background in the low energy region. When Ca or lighter elements were measured a 25 lm thick polypropylene absorber was used. The X-rays emitted by the target were detected by a
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
vertical Canberra 7333E Si(Li) detector positioned at 90° to the beam. The pulses were processed by a Canberra 2020 spectroscopy ampli®er and a Canberra 35 plus multichannel analyser. Recorded spectra were o-line transferred into a PC. X-ray spectra were evaluated by the AXIL computer code [6]. The quanti®cation was based on the sensitivity curves determined by irradiating a set of thin calibrated Micro Matter standard foils. For elements with no standards the sensitivities were calculated by interpolation. It was checked by direct weighing that the samples were thinner than 1 mg/cm2 . Assuming that the bulk composition of the sample is practically SiO2 , preliminary calculations have shown samples having less than 1 mg/cm2 may be considered as thin ones with respect to the proton energy loss and X-ray attenuation. This simpli®cation has caused errors less than 8% in the concentrations. Therefore the (qdZ area density of the element Z was calculated by the thin target yield formula YZ SZ Q qdZ ; where YZ ; SZ and Q are the X-ray yield, the sensitivity and the collected proton charge, respectively. For individual particle analysis one part of the ®lter was glued to a Cu±Zn sample-holder and coated by 25 nm carbon layer for electron probe micro analysis (EPMA) [7±9]. 350 particles per sample were measured by a PHILIPS 505 scanning electron microscope connected to a computer that was used for controlling the EPMA measurement of individual particles. Morphological parameters such as diameter and shape factor were calculated by the image processing routine of the measuring program. For acceptable image quality sample current of 1.3 nA, excitation energy of 20 keV and sample-detector distance of 31 mm were chosen. The obtained characteristic X-ray spectra of the particles have been evaluated by a least-squares ®tting method (AXIL code [6]). However, the calculation of the elemental concentrations for each individual particle is also possible using particle ZAF correction [10,11], the ®nal result of the classi®cation based on the quantitative concen-
363
trations does not dier signi®cantly from that based on normalised intensities [12]. For classi®cation of the particles, the commonly used method of hierarchical clustering was performed [13]. The net X-ray intensities of N chemical elements form an N-dimensional space, and the M particles to be classi®ed can be considered as M points in this space. The measure of similarity of the points is their Euclidean distance, in most of the cases. The strategy of the clustering is based on the calculation of the distance of two clusters. For environmental applications the socalled Ward's method is the most suitable, where the squares of distances of the objects inside the clusters is minimised [12]. The algorithm can be stopped by obtaining a pre-set number of clusters but more sophisticated stopping rules can also be induced. The data processing program (DPP) [14] is a cluster analysis package, which has three types of clustering methods, namely hierarchical, non-hierarchical and fuzzy clustering. The hierarchical cluster analysis program, which has been applied in this study, uses the Ward's strategy [15].
4. Results The elemental composition of the total aerosol inhaled by the patients was measured by PIXE. The elemental concentration for each inhaled litre of air is presented in Table 1 for one characteristic sample of each area. It is clear from the data that the elemental composition of the sample from the ®rst sampling site and that of the therapeutic region have very similar concentrations. Since the therapy is carried out successfully at those two chambers of the cave, it is reasonable to suppose that the searched healing agent is related to an element that has signi®cant concentration in both samples, like calcium. A secondary electron microscope image of typical cave aerosol particles is shown in Fig. 1. Most of the particles have spherical shape. Results of the hierarchical cluster analysis obtained from the characteristic net X-ray intensities of the sample originating from the therapeutic region are
364
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Table 1 Elemental composition of aerosol samples originating from three dierent sampling sites of the cave Concentration (ng/l)
a
Element
Entrance
Clay chamber
Therapeutic region
K Ca Ti V Cr Mn Fe Ni Cu Zn Br Sr Pb
3.029 0.6 3.058 0.6 0.037 0.004 Nda 0.007 0.0002 0.019 0.004 0.113 0.02 0.006 0.001 0.008 0.001 0.008 0.001 Nd Nd Nd
0.01 0.002 0.307 0.06 0.009 0.002 Nd 0.003 0.0006 0.003 0.0006 0.092 0.02 0.003 0.0006 0.002 0.0003 0.005 0.0006 0.004 0.001 Nd 0.0018 0.0004
0.21 0.04 3.22 0.6 0.103 0.02 0.014 0.003 0.003 0.0006 0.027 0.006 1.064 0.2 0.004 0.0008 0.005 0.0006 0.016 0.002 0.003 0.0006 0.0127 0.003 0.0017 0.0004
Not detected.
displayed in Fig. 2. Particle classes are shown along the X-axis marking their abundance (%) and average diameter (lm). The Y-axis corresponds to the normalised net X-ray intensity of the element marked below the columns. Classi®cation yielded three main groups with similar average diameter: alumino-silicate with calcium, quartz and calcium carbonate classes. It seems that silicates and limestone are the main components of the cave aerosol.
Fig. 1. Electron microscope image of cave aerosol particles.
The size distribution of the same aerosol sample is presented in Fig. 3. Size fractions with their average diameter (lm) are shown on the horizontal axis versus their abundance (%). Calculating the average elemental composition on each selected size fraction a very similar results were found (Fig. 4). Size fractions are shown along the X-axis, the Y-axis corresponds to the normalised net X-ray intensity of the element marked by grey scale. This result indicates that the particles have the same size distribution in every class, which means that the aerodynamic behaviour of the dierent chemical particles is similar (see Fig. 3). As a result we can suppose that the size distribution of calcium rich particles is the same as that of
Fig. 2. Classi®cation of the aerosol particles originating sampling at the therapeutic region.
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Fig. 3. Size distribution of the aerosol particles sampled at the therapeutic region.
the total sample and the average diameter is 2.4 lm (Fig. 2). This value can further be used as input for the lung deposition models [16]. 5. Discussion Three main particle classes were found in the atmosphere of the cave and only one of them could have healing eect. To consider the Ca2 ion physiological in¯uences it could be assumed that the relative high calcium carbonate content of the cave aerosol is the explication for the therapeutic eect of the cave. Since calcium carbonate has not any known physiological in¯uence, only the Ca2 ion has, a chemical reaction is needed to reach calcium ions. The inhaled and deposited calcium carbonate particles in the lung are solved in the mucous membrane in consequence of its carbonic properties. The following well-known processes are going on: CaCO3 H2 CO3 ! Ca OH2 2CO2 Ca OH2 $ Ca2 2OH The deposited calcium is going into solution and can exert their bene®cial eect on the calcium homeostasis of the bronchial cells. As a consequence, the chronic in¯ammation and bronchial smooth muscle contraction, the key factors of asthma bronchiale symptoms decrease and the clinical status of the asthmatic patient improves. In order to quantify this hypothesis the exact deposited calcium amount has to be determined. It
365
Fig. 4. Elemental composition of the size fractions.
could be simulated by several computational lung models [16,17]. Our results shows that in the therapeutic region of the cave 3.2 ng calcium is present in the solid phase of the aerosol of 1 l (roughly one breath). Patients usually spend 2±3 h in the cave at each therapy. Since special physical activity is not pronounced during the therapy, the breathing period could be considered as 4 s and the breathing volume as 1 l. Supposing that the eciency of the deposition is 100% and the eciency of the solution as well, furthermore the deposition is homogeneous, on the average 0.58 lg/m2 Ca2 is deposited on the lung surface according to the Yeh-Schum lung geometry model [18].
6. Conclusions Single particle EPMA combined with PIXE is a useful tool for the characterising inhalation aerosol particles of karstic caves. The amount of inhaled calcium of one breathing was determined. Taking into account, however, that neither the deposition eciency is 100% nor the deposition is homogeneous, a detailed study of the stochastic deposition model is needed.
Acknowledgements This work was supported by the National Committee for Technical Development (OMFB) under UNDP-HUN-95-002-0121 contract and the
366
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Hungarian National Research Foundation (OTKA) under Research Contract No. T22664. References [1] Zs. Kertesz, I. Borbely-Kiss, I. Hunyadi, Nucl. Instr. and Meth. B 150 (1999) 384. [2] T. Szab o, A. V arhegyi, Medicina Thoracalis 51 (1998) 138. [3] T. Horv ath, Karszt es barlang (1989) 89. [4] W. Busse, S.T. Holgate (Eds.), Asthma and Rhinitis, Blackwell, Oxford, 1995. [5] S.A.E. Johansson, J.L. Campbell, PIXE: A Novel Technique for Elemental Analysis, Wiley, Chichester, UK, 1988. [6] P. Van Espen, K. Janssens, J. Nobels, Chemometrics Intell. Labor System 1 (1985) 109. [7] J.I. Goldstein, H. Yakowitz, Practical Scanning Electron Microscopy, Plenum, New York, 1977.
[8] D.B. Holt, M.D. Muir, P.R. Grant, I.M. Borsawa, Quantitative Scanning Electron Microscopy, Academic Press, London, 1974. [9] S.J.B. Reed, Electron Microprobe Analysis, Cambridge University Press, Cambridge, MA, 1975. [10] J.T. Armstrong, Anal. Chem. 47 (1975) 2178. [11] J.T. Armstrong, P.R. Buseck, X-Ray Spectrom. 14 (1985) 172. [12] P.C. Bernard, R.E. Van Grieken, D. Esima, Environ. Sci. Technol. 20 (1986) 467. [13] D.L. Massart, L. Kaufman, The Interpretation of Analytical Chemical Data by the Use of Cluster Analysis, Wiley, New York, 1983. [14] P. Van Espen, Anal. Chem. Acta 165 (1984) 31. [15] G.N. Lance, W.T. Williams, Austral. Comput. J. (1967) 15. [16] L. Koblinger, W. Hofmann, J. Aerosol Sci. 21 (1990) 661. [17] I. Balashazy, W. Hofmann, J. Aerosol Sci. 24 (1993) 745. [18] H.C. Yeh, G.M. Schum, Bull. Math. Biol. 42 (1980) 461.
www.elsevier.nl/locate/nimb
X-ray analysis of aerosol samples from a therapeutic cave B. Alf oldy a
a,*
, Sz. T or ok a, A. Kocsonya b, Z. Sz} okefalvi-Nagy b, Md.I. Balla
c
Health Physics Department, KFKI Atomic Energy Research Institute, P.O. Box 49, H-1525 Budapest 114, Hungary b KFKI Research Institute for Particle and Nuclear Physics, P.O. Box 49, H-1525 Budapest 114, Hungary c Astrazeneca, 2045 Torokb alint, Park u. 3., Hungary Received 1 August 2000; received in revised form 19 October 2000
Abstract Cave therapy is an ecient therapeutic method to cure asthma, the exact healing eect, however, is not clari®ed, yet. This study is motivated by the basic assumption that aerosols do play the key role in the cave therapy. This study is based on measurements of single aerosol particles originating from a therapeutic cave of Budapest, Hungary (Szeml} ohegyi cave). Aerosol particles have been collected in the regions arranged for the therapeutic treatment. Samples were further analysed for chemical and morphological aspects, determining the particle size distribution and classifying them according to elemental composition. Three particle classes have been detected based on major element concentration: alumino-silicate, quartz and calcium carbonate. Calcium ions have well-known physiological in¯uence: anti-spastic, anti-in¯ammation and excretion reducing eects. In¯ammation, accompanying spasm and extreme excretion production cause the smothering stigma, the so-called asthma. Therefore it could be assumed that calcium ions present in high concentration in the cave's atmosphere is the major cause of the healing eect. Ó 2001 Elsevier Science B.V. All rights reserved. Keywords: Cave therapy; Healing aerosols; Elemental composition; Size fraction; EPMA analysis; PIXE analysis; Cluster analysis
1. Introduction X-ray spectroscopy is a traditional method for trace element analysis in environmental sciences. A cave's atmosphere ± notwithstanding it is sheltered ± is periled by the air pollution. It is especially true in case of therapeutic caves, like the Szeml} ohegyi cave in Budapest, where asthma therapy has been
*
Corresponding author. Tel.: +36-1-392-2222/1176. E-mail address: al®@sunserv.kfki.hu (B. Alf oldy).
carried out for two decades. It is not surprising that considerable attention is paid to study the atmosphere of this cave [1]. Despite the fact that the exact curative property is not clari®ed yet, cave therapy is a very popular therapeutic method against asthma in Hungary [2,3]. Several speculations try to explain the healing in¯uence of the cave. Some of them consider the aerosol as the therapeutic agent of the cave's atmosphere, while other ideas regard radon or the special cave climate (temperature 12°C, humidity roughly 100%, clean air, etc.) responsible for the
0168-583X/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 0 ) 0 0 5 8 6 - 3
362
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
healing in¯uence. It is believed that aerosols play the main role in the cave therapy, and this paper intends to provide experimental data to support this hypothesis. Since the aerosol deposition in the lung depends on the particles' morphological characteristics, individual particle analysis was needed in addition to PIXE bulk analysis, which can provide accurate quantitative results, but only for the mean value of all particles.
2. Pulmonological background Asthma bronchiale is a syndrome with clinical manifestation of hampered respiration (wheeze) caused by diuse bronchial obstruction, bronchial mucus hypersecretion and thickening of the epithelial layer [4]. Pathologically, asthma is a destructive in¯ammation with in®ltration of certain cellular elements of the immune system (so-called eosinophyl cells). Its pathological development is characterised by bronchial hyperresponsiveness to dierent exogen agents (allergens, coming from the external environment), or intrinsic, not clearly de®ned factors of the internal environment `milieu interior'. The pathophysiological background of the clinical signs is based on the impaired interaction between bronchial smooth muscle, epithelium, mucus-secreting glands and the cellular elements of the immune system (lymphocytes, macrophages, mast cells, etc.). Certain neural and humoral factors, i.e., interleukins (IL3, IL4 and IL5), immunglobulins (IgE) complement factors (C3a, C5a) are responsible for extracellular, cell-to-cell signalling (that is communication between the cellular elements). Intracellular messengers that is the cAMP/adenilate cyclase and the changes in intracellular Ca2 ion concentration transfer their messages (as ``second messengers'') to produce the ®nal cellular response that is manifested in bronchial smooth muscle contraction, mucus secretion and release of certain humoral factors from cells of the immune system. In addition, Ca2 ions are important in the determination of both the resting and the activated cell membrane potential, so Ca2
ions play key roles in the normal, physiological cell functions. 3. Sampling and analysis Szeml} ohegyi cave is a thermal cave with three warm air sources. The therapy is carried out in the closest chamber to those warm air sources. Sampling sites were selected according to the research objective, namely at representative locations of the cave where the therapy is carried out. Three samples were collected in the therapeutic region, close to the warm air sources. The next three samples came from the vertical entrance, which is far from the warm air sources. Since there is an out¯ow from the cave at this entrance the microclimate of this site is determined by the cave and not by the outdoor conditions. Some patients spend the therapeutic period at this location and the eciency of their healing does not dier from that of those patients staying in the therapeutic chamber. Finally, three samples were collected at a third location called ``clay chamber'' that has a thick clay layer on its ¯oor. Recently, a facility was established to carry out therapy at this site, too. The clay stanches the rifts of the rock thus separating this region from the atmosphere of the rest of the cave. Sample collection was carried out by pumping air through a 25 mm diameter Nuclepore ®lter with 0.4 lm pore size. The ¯ow rate was 10 l/min for 16±24 h. First, the areal densities of the elements in the samples was determined by particle-induced X-ray spectrometry (PIXE) [5]. The proton beam of 2.5 MeV energy was generated by the 5 MV Van de Graa accelerator of KFKI RMKI. The ®nal beam spot was formed by a Ta collimator of 2 mm diameter. To avoid pile-up peaks and high dead time losses beam currents were kept below 10 nA. The samples were positioned at 45° with respect to the beam direction. For heavier element determination a 0.5 mm thick plexiglass absorber was used to reduce the background in the low energy region. When Ca or lighter elements were measured a 25 lm thick polypropylene absorber was used. The X-rays emitted by the target were detected by a
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
vertical Canberra 7333E Si(Li) detector positioned at 90° to the beam. The pulses were processed by a Canberra 2020 spectroscopy ampli®er and a Canberra 35 plus multichannel analyser. Recorded spectra were o-line transferred into a PC. X-ray spectra were evaluated by the AXIL computer code [6]. The quanti®cation was based on the sensitivity curves determined by irradiating a set of thin calibrated Micro Matter standard foils. For elements with no standards the sensitivities were calculated by interpolation. It was checked by direct weighing that the samples were thinner than 1 mg/cm2 . Assuming that the bulk composition of the sample is practically SiO2 , preliminary calculations have shown samples having less than 1 mg/cm2 may be considered as thin ones with respect to the proton energy loss and X-ray attenuation. This simpli®cation has caused errors less than 8% in the concentrations. Therefore the (qdZ area density of the element Z was calculated by the thin target yield formula YZ SZ Q qdZ ; where YZ ; SZ and Q are the X-ray yield, the sensitivity and the collected proton charge, respectively. For individual particle analysis one part of the ®lter was glued to a Cu±Zn sample-holder and coated by 25 nm carbon layer for electron probe micro analysis (EPMA) [7±9]. 350 particles per sample were measured by a PHILIPS 505 scanning electron microscope connected to a computer that was used for controlling the EPMA measurement of individual particles. Morphological parameters such as diameter and shape factor were calculated by the image processing routine of the measuring program. For acceptable image quality sample current of 1.3 nA, excitation energy of 20 keV and sample-detector distance of 31 mm were chosen. The obtained characteristic X-ray spectra of the particles have been evaluated by a least-squares ®tting method (AXIL code [6]). However, the calculation of the elemental concentrations for each individual particle is also possible using particle ZAF correction [10,11], the ®nal result of the classi®cation based on the quantitative concen-
363
trations does not dier signi®cantly from that based on normalised intensities [12]. For classi®cation of the particles, the commonly used method of hierarchical clustering was performed [13]. The net X-ray intensities of N chemical elements form an N-dimensional space, and the M particles to be classi®ed can be considered as M points in this space. The measure of similarity of the points is their Euclidean distance, in most of the cases. The strategy of the clustering is based on the calculation of the distance of two clusters. For environmental applications the socalled Ward's method is the most suitable, where the squares of distances of the objects inside the clusters is minimised [12]. The algorithm can be stopped by obtaining a pre-set number of clusters but more sophisticated stopping rules can also be induced. The data processing program (DPP) [14] is a cluster analysis package, which has three types of clustering methods, namely hierarchical, non-hierarchical and fuzzy clustering. The hierarchical cluster analysis program, which has been applied in this study, uses the Ward's strategy [15].
4. Results The elemental composition of the total aerosol inhaled by the patients was measured by PIXE. The elemental concentration for each inhaled litre of air is presented in Table 1 for one characteristic sample of each area. It is clear from the data that the elemental composition of the sample from the ®rst sampling site and that of the therapeutic region have very similar concentrations. Since the therapy is carried out successfully at those two chambers of the cave, it is reasonable to suppose that the searched healing agent is related to an element that has signi®cant concentration in both samples, like calcium. A secondary electron microscope image of typical cave aerosol particles is shown in Fig. 1. Most of the particles have spherical shape. Results of the hierarchical cluster analysis obtained from the characteristic net X-ray intensities of the sample originating from the therapeutic region are
364
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Table 1 Elemental composition of aerosol samples originating from three dierent sampling sites of the cave Concentration (ng/l)
a
Element
Entrance
Clay chamber
Therapeutic region
K Ca Ti V Cr Mn Fe Ni Cu Zn Br Sr Pb
3.029 0.6 3.058 0.6 0.037 0.004 Nda 0.007 0.0002 0.019 0.004 0.113 0.02 0.006 0.001 0.008 0.001 0.008 0.001 Nd Nd Nd
0.01 0.002 0.307 0.06 0.009 0.002 Nd 0.003 0.0006 0.003 0.0006 0.092 0.02 0.003 0.0006 0.002 0.0003 0.005 0.0006 0.004 0.001 Nd 0.0018 0.0004
0.21 0.04 3.22 0.6 0.103 0.02 0.014 0.003 0.003 0.0006 0.027 0.006 1.064 0.2 0.004 0.0008 0.005 0.0006 0.016 0.002 0.003 0.0006 0.0127 0.003 0.0017 0.0004
Not detected.
displayed in Fig. 2. Particle classes are shown along the X-axis marking their abundance (%) and average diameter (lm). The Y-axis corresponds to the normalised net X-ray intensity of the element marked below the columns. Classi®cation yielded three main groups with similar average diameter: alumino-silicate with calcium, quartz and calcium carbonate classes. It seems that silicates and limestone are the main components of the cave aerosol.
Fig. 1. Electron microscope image of cave aerosol particles.
The size distribution of the same aerosol sample is presented in Fig. 3. Size fractions with their average diameter (lm) are shown on the horizontal axis versus their abundance (%). Calculating the average elemental composition on each selected size fraction a very similar results were found (Fig. 4). Size fractions are shown along the X-axis, the Y-axis corresponds to the normalised net X-ray intensity of the element marked by grey scale. This result indicates that the particles have the same size distribution in every class, which means that the aerodynamic behaviour of the dierent chemical particles is similar (see Fig. 3). As a result we can suppose that the size distribution of calcium rich particles is the same as that of
Fig. 2. Classi®cation of the aerosol particles originating sampling at the therapeutic region.
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Fig. 3. Size distribution of the aerosol particles sampled at the therapeutic region.
the total sample and the average diameter is 2.4 lm (Fig. 2). This value can further be used as input for the lung deposition models [16]. 5. Discussion Three main particle classes were found in the atmosphere of the cave and only one of them could have healing eect. To consider the Ca2 ion physiological in¯uences it could be assumed that the relative high calcium carbonate content of the cave aerosol is the explication for the therapeutic eect of the cave. Since calcium carbonate has not any known physiological in¯uence, only the Ca2 ion has, a chemical reaction is needed to reach calcium ions. The inhaled and deposited calcium carbonate particles in the lung are solved in the mucous membrane in consequence of its carbonic properties. The following well-known processes are going on: CaCO3 H2 CO3 ! Ca OH2 2CO2 Ca OH2 $ Ca2 2OH The deposited calcium is going into solution and can exert their bene®cial eect on the calcium homeostasis of the bronchial cells. As a consequence, the chronic in¯ammation and bronchial smooth muscle contraction, the key factors of asthma bronchiale symptoms decrease and the clinical status of the asthmatic patient improves. In order to quantify this hypothesis the exact deposited calcium amount has to be determined. It
365
Fig. 4. Elemental composition of the size fractions.
could be simulated by several computational lung models [16,17]. Our results shows that in the therapeutic region of the cave 3.2 ng calcium is present in the solid phase of the aerosol of 1 l (roughly one breath). Patients usually spend 2±3 h in the cave at each therapy. Since special physical activity is not pronounced during the therapy, the breathing period could be considered as 4 s and the breathing volume as 1 l. Supposing that the eciency of the deposition is 100% and the eciency of the solution as well, furthermore the deposition is homogeneous, on the average 0.58 lg/m2 Ca2 is deposited on the lung surface according to the Yeh-Schum lung geometry model [18].
6. Conclusions Single particle EPMA combined with PIXE is a useful tool for the characterising inhalation aerosol particles of karstic caves. The amount of inhaled calcium of one breathing was determined. Taking into account, however, that neither the deposition eciency is 100% nor the deposition is homogeneous, a detailed study of the stochastic deposition model is needed.
Acknowledgements This work was supported by the National Committee for Technical Development (OMFB) under UNDP-HUN-95-002-0121 contract and the
366
B. Alfoldy et al. / Nucl. Instr. and Meth. in Phys. Res. B 174 (2001) 361±366
Hungarian National Research Foundation (OTKA) under Research Contract No. T22664. References [1] Zs. Kertesz, I. Borbely-Kiss, I. Hunyadi, Nucl. Instr. and Meth. B 150 (1999) 384. [2] T. Szab o, A. V arhegyi, Medicina Thoracalis 51 (1998) 138. [3] T. Horv ath, Karszt es barlang (1989) 89. [4] W. Busse, S.T. Holgate (Eds.), Asthma and Rhinitis, Blackwell, Oxford, 1995. [5] S.A.E. Johansson, J.L. Campbell, PIXE: A Novel Technique for Elemental Analysis, Wiley, Chichester, UK, 1988. [6] P. Van Espen, K. Janssens, J. Nobels, Chemometrics Intell. Labor System 1 (1985) 109. [7] J.I. Goldstein, H. Yakowitz, Practical Scanning Electron Microscopy, Plenum, New York, 1977.
[8] D.B. Holt, M.D. Muir, P.R. Grant, I.M. Borsawa, Quantitative Scanning Electron Microscopy, Academic Press, London, 1974. [9] S.J.B. Reed, Electron Microprobe Analysis, Cambridge University Press, Cambridge, MA, 1975. [10] J.T. Armstrong, Anal. Chem. 47 (1975) 2178. [11] J.T. Armstrong, P.R. Buseck, X-Ray Spectrom. 14 (1985) 172. [12] P.C. Bernard, R.E. Van Grieken, D. Esima, Environ. Sci. Technol. 20 (1986) 467. [13] D.L. Massart, L. Kaufman, The Interpretation of Analytical Chemical Data by the Use of Cluster Analysis, Wiley, New York, 1983. [14] P. Van Espen, Anal. Chem. Acta 165 (1984) 31. [15] G.N. Lance, W.T. Williams, Austral. Comput. J. (1967) 15. [16] L. Koblinger, W. Hofmann, J. Aerosol Sci. 21 (1990) 661. [17] I. Balashazy, W. Hofmann, J. Aerosol Sci. 24 (1993) 745. [18] H.C. Yeh, G.M. Schum, Bull. Math. Biol. 42 (1980) 461.