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Determination of trace element distribution in cancerous and normal human tissues by total reflection X-ray fluorescence analysis1
SAB 1625
Spectrochimica Acta Part B 52 (1997) 1047–1052
Determination of trace element distribution in cancerous and normal human tissues by total reflection X-ray fluorescence analysis1 D. von Czarnowski, E. Denkhaus*, K. Lemke Gerhard-Mercator-University of Duisburg, Department of Instrumental Analytical Chemistry, Lotharstr.1, D-47057 Duisburg, Germany Received 1 August 1996; accepted 21 October 1996
Abstract The intention of this study was to establish a method for cancer diagnosis. For this purpose, different trace element distributions in carcinomas of the digestive tract and in normal tissues of human stomach, colon and rectum in correlation to the type of cancer were determined by total reflection X-ray fluorescence analysis (TXRF). The tissue samples were frozen and cut by a microtome into 10 mm sections, and a modified sample excision technique was introduced according to the aim of this research. After drying and spiking of the tissue sections, more than 20 elements, especially biologically relevant ones, were determined. The repeatabilities of measurements of element concentrations in malignant and normal tissues were calculated to be 10– 30% (RSD) depending on the specific element. The concentration of Ca was found to be virtually constant (0.250 6 0.025 mg per 0.1 mm 3) in normal tissue and in carcinoma of the digestive organs. A significant diminution of Cr, Fe and Ni in carcinoma of the stomach, of Cr and Co in carcinoma of the colon and a significant accumulation of K in cancerous tissue of the colon and of Fe and K in neoplastic tissue of the rectum were discovered for a very limited population of patients. q 1997 Elsevier Science B.V. Keywords: Carcinoma ; Colon; Carcinoma; Rectum; Carcinoma; Stomach; Microtome section; Total reflection X-ray fluorescence analysis; Trace element distribution
1. Introduction The role of metals in the development and inhibition of cancer has a complex character and raises many questions. Drake and Sky-Peck demonstrated
* Corresponding author. 1 This paper was presented at the 6th Conference on ‘‘Total Reflection X-Ray Fluorescence Analysis and Related Methods’’ (TXRF ’96) held in two parts in Eindhoven (The Netherlands) and Dortmund (Germany) in June 1996, and is published in the Special Issue of Spectrochimica Acta, Part B, dedicated to that Conference.
that the concentration ratios of elements in malignant and normal tissues from the same individual differ according to the pattern of the element distribution of normal and cancerous tissues, thus providing an effective methodology for the recognition and classification of cancers—significant elements are established as being organ-specific [1]. The following questions arise: is the type of cancer a function of living conditions (nutrition, working conditions, environmental factors, alcohol consumption, smoking habits etc.); what is the pattern of element distribution in tissues of a similar type, e.g. the digestive tract? This is the first step of the current study. The
0584-8547/97/$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 58 4- 8 54 7 (9 6 )0 1 62 5 -4
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D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
concentrations of metal ions must lie in a fixed range within the cells. The consequence of a low concentration of an essential element is a negative influence on processes for which the metal ion is necessary. Above a certain maximum concentration of an essential element, the metal ion shows a toxic effect. The second step is to discover the influence of metal levels on tumor development or inhibition. Thus, the concentrations of certain metals can be determined in cancerous tissues and in normal tissues, but the elements relevant to bioinorganic chemistry and toxic elements from environmental exposure have to be differentiated. The biological function of essential metal ions is known in part for a few elements, including Na, K, Mg, Ca, V, Mo, W, Mn, Fe, Co, Ni, Cu and Zn [2]. Normally, metals are incorporated as cofactors of proteins in biological processes, known as metalloproteins. Metallo-proteins with a catalytic function are known as metallo-enzymes. The biological function of the selected metal ions in combination with an investigation of element distribution patterns in malignant and in normal human tissues of cancer patients can give some indication of the effect of metal ions on carcinogenesis. The next problem is to select an analytical method that is appropriate to the low concentrations of metal ions in biomaterials and the small available masses of the biological specimens. These basic requirements necessitate a direct elemental analysis of solid samples and simultaneous detection of the elements. Instrumental neutron activation analysis (INAA) and proton induced X-ray emission (PIXE) [3] are largely suitable for this application but have some drawbacks, e.g. they are time-consuming and labor intensive and involve considerable instrumental expenditure. Klockenka¨mper et al. have demonstrated the suitability of total reflection X-ray fluorescence (TXRF) spectrometry for the direct analysis of thin films of biomaterials [4–7]. The samples were prepared as thin sections by a freezing microtome. The TXRF spectrum gives a ‘‘fingerprint’’ of the biomaterial and corresponds to qualitative classification of the sample for screening and monitoring. Furthermore, a simple procedure for quantification with internal calibration was described. Validation of the analytical results was achieved by using certified reference materials (CRM) [7].
Fig. 1. Ground quartz glass tube for excision of tissue samples of definite volume.
2. Experimental 2.1. Sampling and sample preparation The sampling of normal and neoplastic tissues from the same individual took place at the time of surgery. Four carcinomas of the stomach (N = 4), eight carcinomas of the colon (N = 8) and four carcinomas of the rectum (N = 4) were taken from cancer patients of different sex, age, and living conditions. The catchment area of the cancer patients was the city and district of Viersen (Germany), where a high level of mortality of individuals with cancer of the digestive organs has been recorded in the last ten years [8]. The samples were transported and carried out in iced flexible polyethylene bags, and samples were deepfrozen and stored at −158C for no longer than seven days before analysis. The tissue samples were cut into small pieces (1 cm long, 1 cm wide) with a quartz knife and excised into cylindrical pieces of diameter 3.5 mm by a ground quartz tube after cooling with liquid nitrogen, as shown in Fig. 1.
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
1049
radiation of the sample was detected by a Si(Li) detector equipped with a multichannel analyzer and software from Link Systems Ltd., London, UK (QX 2000, System 860/500) with a spectral resolution of 152 eV for the Fe Ka line. 2.3. Analytical procedure
Fig. 2. Flow chart of sample preparation procedure for determination of element concentrations in human tissue samples.
The excised tissue was then fixed onto a microtome support by an embedding medium (SLEE, Mainz) and freeze-cut at − 268C to tissue sections 10 mm thick with a microtome (SLEE, Mainz, model MHR). The thin sections were positioned centrally on a cleaned siliconized quartz glass carrier and dried for 1 h at 808C on a ceramic hot-plate (Schott, Germany). After this procedure they were ‘‘spiked’’ with 10 ml of internal standard solution (10 mg l −1 yttrium and 10 mg l −1 tellurium) and dried for 30 min at 808C. Fig. 2 shows a flow-chart of the sample preparation procedure for determination of the masses of elements in human tissue samples of definite volume. 2.2. Instrumentation To determine the concentrations of the elements P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, Zr, Mo, Cd, and Te in the tissue samples, an EXTRA II TXRF spectrometer (Richard Seifert and Co., Ahrensburg, Germany) was used. For excitation, an X-ray tube with a molybdenum anode (maximum power 2 kW) was operated at 48 kV. The current was adjusted at a dead time ratio of 45% corresponding to a count rate of 8.000 cps. Mo foils 50–150 mm thick were used as filters. An absolute counting time of 300 s was selected. The fluorescence
Because of the inhomogeneous distribution of the trace elements in the tissue samples and the irregularities in density and structure of the tissue layers, the quartz glass support was turned three times through an angle of 1208 (resulting in three measuring cycles) in order to minimize variations in sensitivity. To determine the deviation of the measured concentrations, three sections of each tissue sample were measured. The results of analysis are given as average values of the concentrations of the detected elements. The repeatability of the measurements is presented as the relative standard deviation of the absolute concentrations of three microtome sections and the three rotations of the quartz glass target.
3. Results and discussion In order to demonstrate the applicability of the described sample preparation and analytical procedure, the repeatability of measurements of 16 absolute element concentrations in eight normal and cancerous tissues has been determined. For the elements P, Cl, V, and Mn, the repeatability was , 10% for normal or malignant tissues. The RSD for As, Se and Br was , 15% for both tissue types. The concentrations of Ca, Ti, Cr, Co, Ni, Cu, and Zn in malignant tissues show relative standard deviations , 10% but RSD in normal tissues are . 20%, and in some cases up to 50% for the elements Ti and Cr. Two reasons can be suggested. (i) The preparation of tissue sections by microtome is simpler for cancerous tissue owing to its firmer consistency compared with normal tissue. Therefore the surfaces of cancerous tissue layers are more nearly planar and in consequence the spectral background resulting from Rayleigh scattering of the primary beam is reduced. (ii) The cells of malignant tissue samples show less differentiation and the distribution of the trace elements is more homogeneous than in normal tissues. The influence of the
1050
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
consistency and therefore the reduction of Rayleigh scattering is not so important, because the RSD of the concentrations of K and Fe is up to 30% in neoplastic tissue and 30–50% in normal tissue.
To simplify the evaluation of the analytical results, the measured element concentrations in normal and in cancerous tissues were classified into four groups: (i) elements having constant concentrations;
Fig. 3. Ratio of element concentrations in normal and malignant tissues according to the type of cancer.
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
1051
(ii) elements with variable concentrations; (iii) elements with significant concentrations; (iv) elements for which the occurrence rate of concentrations shows a trend depending on the type of cancer. The first evaluation step was to eleminate those elements present at concentrations below or near the detection limit: S, V, Zr, Sr, Rb, Mo and Cd. These elements were excluded from subsequent evaluations. (i) Elements that show steady concentrations are: in the stomach, P, Cl, Ca, Cu, As, Se and Br; in the colon, P, Cl, Ca, Ni, Cu and As; and in the rectum, Ca, Ni, As, Se and Br. The interpatient concentrations of Ca amount to 0.250 6 0.025 mg per 0.1 mm 3. This result is an indication that the preparation of microtome sections of tissues with a definite volume is reproducible. This result supports the indication that it is possible to determine the sample mass from the total intensity I t within the spectrum when the same type of sample is analysed. This fact results from the linear dependence of the intensity I t on the mass m t of the section. The relative standard deviation is reported as < 25% [7]. Consequently, the use of Ca as an internal standard is possible. (ii) Elements of variable concentration are those elements which show no significant accumulation in normal or cancerous tissues. The ratio of malignant to normal element concentration varied in both directions: in the stomach, K, Ti, and Co; in the colon, Ti, Mn, Zn, Se and Br (Br level below the detection limit); in the rectum, Ti, Co, Zn, P and Cl. (iii), (iv) To identify the elements having significant concentrations, the first step was to calculate the ratio of the malignant to the normal element concentration. The second step was to compare the ratio of element concentrations for each sample. If the ratio of malignant/normal element concentration is , 0.5
(the element is accumulated in normal tissue) or . 2 (the element is accumulated in malignant tissue) for each sample of a specific type of cancer, the element is declared to be significant. An element is considered to show a tendency in concentration if the ratio of element concentration for each sample of a specific type of cancer shows a non-significant accumulation in malignant or normal tissue (the ratio of element concentrations is . 0.5 or , 2 respectively). Fig. 3 shows the ratios of element concentrations in malignant versus normal tissues according to the type of cancer, demonstrated by three selected examples. The results are summarized in Table 1, where the significant and the tendentious elements are listed according to the type of cancer. The positive sign is used to illustrate the accumulation of the element in neoplastic tissue, and the minus sign is used to indicate the depletion of the element in cancerous tissue. The first result is a pattern of element distributions in cancerous versus normal tissues. The malignant tissues of the stomach show a significant reduction in the levels of Cr, Fe and Ni and a questionable decrease of Mn and Zn concentrations. In carcinoma of the colon and the rectum K is accumulated significantly, but Cr and Co decrease in cancerous tissue of the colon. Levels of Ni and Fe show a tendency towards reduction in malignant tissue of the colon, whereas Fe is significantly enriched in neoplastic tissue of the rectum. P, Mn, and Cr are diminished and Cu is concentrated in rectal carcinoma. Therefore the patterns depend on the type of cancer, and consequently neoplastic classification of similar cancer types (digestive tract) is possible, but needs first to be verified by analysing and comparing a larger number of tissue samples.
Table 1 List of significant and tendentious elements in carcinoma of the digestive organs
4. Conclusion
Status
Stomach
Colon
Rectum
Significant
Cr (−) Fe (−) Ni (−) Mn (−) Zn (−)
K (+) Cr (−)
Fe (+) K (+)
Ti (−) Fe (−) Ni (−)
P (−) Mn (−) Cu (+) Cr (−)
Tendentious
TXRF in combination with simplified sample preparation by microtome is a method well suited to the direct instrumental microanalysis of human tissues. It can be demonstrated that a recognition of element distribution patterns in cancerous tissues versus normal tissues is possible. Moreover, it can be shown that the patterns determined depend on the type of cancer. Therefore a good method has been
1052
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
found for the classification of similar types of cancer. If a classification of the described cancer types can be verified by means of a large number of samples, an application in clinical chemistry is conceivable, with the aim of replacing large scale pathological investigations. Therefore a correlation of analytical data and pathological information needs to be corroborated by the application of various statistical methods. The established patterns of distribution of trace elements in malignant versus normal tissues can be used as a basis for further biochemical studies. The significance of the determined elements has to be proved according to their biological relevance. Chromium, as a significant element in carcinoma of the stomach and the colon, shows promise for further research. This can be substantiated, as chromium is a relevant bioinorganic element and its biological function is to date not known [2]. The role of the significant elements in carcinogenesis or the means of inhibition of cancer cannot be readily determined. Further investigations such as in vitro experiments on normal and malignant cells by incubation with different inorganic and organic trace element compounds need to be conducted. For this purpose also, TXRF is a suitible analytical method.
Acknowledgements This work was supported by the Ministerium fu¨r
Wissenschaft und Forschung des Landes NordrheinWestfalen (MWF) and by the Institut fu¨r Umwelttechnologie und Umweltanalytik e.V. (IUTA). We are also indebted to Dr H. Kraemer (medical superintendent of St. Cornelius Hospital Viersen–Du¨lken) for interdisciplinary collaboration. We also thank M. Mu¨ller and A. von Bohlen in connection with many aspects of this investigation.
References [1] E.N. Drake II and H.H. Sky-Peck, Cancer Res., 49 (1989) 4210. [2] S.J. Lippard and J.M. Berg, Bioanorganische Chemie, Spektrum Akademischer Verlag GmbH, Heidelberg, Berlin, Oxford, 1995. [3] R. Pepelnik, A. Prange and R. Niedergesa¨ß, J. Anal. At. Spectrom., 9 (1994) 1071. [4] A. von Bohlen, R. Klockenka¨mper, H. Otto, G. To¨lg and B. Wiecken, Int. Arch. Occup. Environ. Health, 59 (1987) 403. [5] A. von Bohlen, R. Klockenka¨mper, G. To¨lg and B. Wiecken, Fresenius’ Z. Anal. Chem., 331 (1988) 454. [6] A. Niemann, A. von Bohlen, R. Klockenka¨mper and E. Keck, Biochem. Biophys. Res. Commun., 170 (1990) 1216. [7] R. Klockenka¨mper, A. von Bohlen and B. Wiecken, Spectrochim. Acta Part B, 44 (1989) 511. [8] B. Pesch, U. Halekoh, U. Ranft, M. Richter and F. Pott, in Ministerium fu¨r Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen (Ed.), Atlas zur Krebssterblichkeit in Nordrhein-Westfalen, Satz und Druck, Du¨sseldorf 1994.
Spectrochimica Acta Part B 52 (1997) 1047–1052
Determination of trace element distribution in cancerous and normal human tissues by total reflection X-ray fluorescence analysis1 D. von Czarnowski, E. Denkhaus*, K. Lemke Gerhard-Mercator-University of Duisburg, Department of Instrumental Analytical Chemistry, Lotharstr.1, D-47057 Duisburg, Germany Received 1 August 1996; accepted 21 October 1996
Abstract The intention of this study was to establish a method for cancer diagnosis. For this purpose, different trace element distributions in carcinomas of the digestive tract and in normal tissues of human stomach, colon and rectum in correlation to the type of cancer were determined by total reflection X-ray fluorescence analysis (TXRF). The tissue samples were frozen and cut by a microtome into 10 mm sections, and a modified sample excision technique was introduced according to the aim of this research. After drying and spiking of the tissue sections, more than 20 elements, especially biologically relevant ones, were determined. The repeatabilities of measurements of element concentrations in malignant and normal tissues were calculated to be 10– 30% (RSD) depending on the specific element. The concentration of Ca was found to be virtually constant (0.250 6 0.025 mg per 0.1 mm 3) in normal tissue and in carcinoma of the digestive organs. A significant diminution of Cr, Fe and Ni in carcinoma of the stomach, of Cr and Co in carcinoma of the colon and a significant accumulation of K in cancerous tissue of the colon and of Fe and K in neoplastic tissue of the rectum were discovered for a very limited population of patients. q 1997 Elsevier Science B.V. Keywords: Carcinoma ; Colon; Carcinoma; Rectum; Carcinoma; Stomach; Microtome section; Total reflection X-ray fluorescence analysis; Trace element distribution
1. Introduction The role of metals in the development and inhibition of cancer has a complex character and raises many questions. Drake and Sky-Peck demonstrated
* Corresponding author. 1 This paper was presented at the 6th Conference on ‘‘Total Reflection X-Ray Fluorescence Analysis and Related Methods’’ (TXRF ’96) held in two parts in Eindhoven (The Netherlands) and Dortmund (Germany) in June 1996, and is published in the Special Issue of Spectrochimica Acta, Part B, dedicated to that Conference.
that the concentration ratios of elements in malignant and normal tissues from the same individual differ according to the pattern of the element distribution of normal and cancerous tissues, thus providing an effective methodology for the recognition and classification of cancers—significant elements are established as being organ-specific [1]. The following questions arise: is the type of cancer a function of living conditions (nutrition, working conditions, environmental factors, alcohol consumption, smoking habits etc.); what is the pattern of element distribution in tissues of a similar type, e.g. the digestive tract? This is the first step of the current study. The
0584-8547/97/$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 58 4- 8 54 7 (9 6 )0 1 62 5 -4
1048
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
concentrations of metal ions must lie in a fixed range within the cells. The consequence of a low concentration of an essential element is a negative influence on processes for which the metal ion is necessary. Above a certain maximum concentration of an essential element, the metal ion shows a toxic effect. The second step is to discover the influence of metal levels on tumor development or inhibition. Thus, the concentrations of certain metals can be determined in cancerous tissues and in normal tissues, but the elements relevant to bioinorganic chemistry and toxic elements from environmental exposure have to be differentiated. The biological function of essential metal ions is known in part for a few elements, including Na, K, Mg, Ca, V, Mo, W, Mn, Fe, Co, Ni, Cu and Zn [2]. Normally, metals are incorporated as cofactors of proteins in biological processes, known as metalloproteins. Metallo-proteins with a catalytic function are known as metallo-enzymes. The biological function of the selected metal ions in combination with an investigation of element distribution patterns in malignant and in normal human tissues of cancer patients can give some indication of the effect of metal ions on carcinogenesis. The next problem is to select an analytical method that is appropriate to the low concentrations of metal ions in biomaterials and the small available masses of the biological specimens. These basic requirements necessitate a direct elemental analysis of solid samples and simultaneous detection of the elements. Instrumental neutron activation analysis (INAA) and proton induced X-ray emission (PIXE) [3] are largely suitable for this application but have some drawbacks, e.g. they are time-consuming and labor intensive and involve considerable instrumental expenditure. Klockenka¨mper et al. have demonstrated the suitability of total reflection X-ray fluorescence (TXRF) spectrometry for the direct analysis of thin films of biomaterials [4–7]. The samples were prepared as thin sections by a freezing microtome. The TXRF spectrum gives a ‘‘fingerprint’’ of the biomaterial and corresponds to qualitative classification of the sample for screening and monitoring. Furthermore, a simple procedure for quantification with internal calibration was described. Validation of the analytical results was achieved by using certified reference materials (CRM) [7].
Fig. 1. Ground quartz glass tube for excision of tissue samples of definite volume.
2. Experimental 2.1. Sampling and sample preparation The sampling of normal and neoplastic tissues from the same individual took place at the time of surgery. Four carcinomas of the stomach (N = 4), eight carcinomas of the colon (N = 8) and four carcinomas of the rectum (N = 4) were taken from cancer patients of different sex, age, and living conditions. The catchment area of the cancer patients was the city and district of Viersen (Germany), where a high level of mortality of individuals with cancer of the digestive organs has been recorded in the last ten years [8]. The samples were transported and carried out in iced flexible polyethylene bags, and samples were deepfrozen and stored at −158C for no longer than seven days before analysis. The tissue samples were cut into small pieces (1 cm long, 1 cm wide) with a quartz knife and excised into cylindrical pieces of diameter 3.5 mm by a ground quartz tube after cooling with liquid nitrogen, as shown in Fig. 1.
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
1049
radiation of the sample was detected by a Si(Li) detector equipped with a multichannel analyzer and software from Link Systems Ltd., London, UK (QX 2000, System 860/500) with a spectral resolution of 152 eV for the Fe Ka line. 2.3. Analytical procedure
Fig. 2. Flow chart of sample preparation procedure for determination of element concentrations in human tissue samples.
The excised tissue was then fixed onto a microtome support by an embedding medium (SLEE, Mainz) and freeze-cut at − 268C to tissue sections 10 mm thick with a microtome (SLEE, Mainz, model MHR). The thin sections were positioned centrally on a cleaned siliconized quartz glass carrier and dried for 1 h at 808C on a ceramic hot-plate (Schott, Germany). After this procedure they were ‘‘spiked’’ with 10 ml of internal standard solution (10 mg l −1 yttrium and 10 mg l −1 tellurium) and dried for 30 min at 808C. Fig. 2 shows a flow-chart of the sample preparation procedure for determination of the masses of elements in human tissue samples of definite volume. 2.2. Instrumentation To determine the concentrations of the elements P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, Zr, Mo, Cd, and Te in the tissue samples, an EXTRA II TXRF spectrometer (Richard Seifert and Co., Ahrensburg, Germany) was used. For excitation, an X-ray tube with a molybdenum anode (maximum power 2 kW) was operated at 48 kV. The current was adjusted at a dead time ratio of 45% corresponding to a count rate of 8.000 cps. Mo foils 50–150 mm thick were used as filters. An absolute counting time of 300 s was selected. The fluorescence
Because of the inhomogeneous distribution of the trace elements in the tissue samples and the irregularities in density and structure of the tissue layers, the quartz glass support was turned three times through an angle of 1208 (resulting in three measuring cycles) in order to minimize variations in sensitivity. To determine the deviation of the measured concentrations, three sections of each tissue sample were measured. The results of analysis are given as average values of the concentrations of the detected elements. The repeatability of the measurements is presented as the relative standard deviation of the absolute concentrations of three microtome sections and the three rotations of the quartz glass target.
3. Results and discussion In order to demonstrate the applicability of the described sample preparation and analytical procedure, the repeatability of measurements of 16 absolute element concentrations in eight normal and cancerous tissues has been determined. For the elements P, Cl, V, and Mn, the repeatability was , 10% for normal or malignant tissues. The RSD for As, Se and Br was , 15% for both tissue types. The concentrations of Ca, Ti, Cr, Co, Ni, Cu, and Zn in malignant tissues show relative standard deviations , 10% but RSD in normal tissues are . 20%, and in some cases up to 50% for the elements Ti and Cr. Two reasons can be suggested. (i) The preparation of tissue sections by microtome is simpler for cancerous tissue owing to its firmer consistency compared with normal tissue. Therefore the surfaces of cancerous tissue layers are more nearly planar and in consequence the spectral background resulting from Rayleigh scattering of the primary beam is reduced. (ii) The cells of malignant tissue samples show less differentiation and the distribution of the trace elements is more homogeneous than in normal tissues. The influence of the
1050
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
consistency and therefore the reduction of Rayleigh scattering is not so important, because the RSD of the concentrations of K and Fe is up to 30% in neoplastic tissue and 30–50% in normal tissue.
To simplify the evaluation of the analytical results, the measured element concentrations in normal and in cancerous tissues were classified into four groups: (i) elements having constant concentrations;
Fig. 3. Ratio of element concentrations in normal and malignant tissues according to the type of cancer.
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
1051
(ii) elements with variable concentrations; (iii) elements with significant concentrations; (iv) elements for which the occurrence rate of concentrations shows a trend depending on the type of cancer. The first evaluation step was to eleminate those elements present at concentrations below or near the detection limit: S, V, Zr, Sr, Rb, Mo and Cd. These elements were excluded from subsequent evaluations. (i) Elements that show steady concentrations are: in the stomach, P, Cl, Ca, Cu, As, Se and Br; in the colon, P, Cl, Ca, Ni, Cu and As; and in the rectum, Ca, Ni, As, Se and Br. The interpatient concentrations of Ca amount to 0.250 6 0.025 mg per 0.1 mm 3. This result is an indication that the preparation of microtome sections of tissues with a definite volume is reproducible. This result supports the indication that it is possible to determine the sample mass from the total intensity I t within the spectrum when the same type of sample is analysed. This fact results from the linear dependence of the intensity I t on the mass m t of the section. The relative standard deviation is reported as < 25% [7]. Consequently, the use of Ca as an internal standard is possible. (ii) Elements of variable concentration are those elements which show no significant accumulation in normal or cancerous tissues. The ratio of malignant to normal element concentration varied in both directions: in the stomach, K, Ti, and Co; in the colon, Ti, Mn, Zn, Se and Br (Br level below the detection limit); in the rectum, Ti, Co, Zn, P and Cl. (iii), (iv) To identify the elements having significant concentrations, the first step was to calculate the ratio of the malignant to the normal element concentration. The second step was to compare the ratio of element concentrations for each sample. If the ratio of malignant/normal element concentration is , 0.5
(the element is accumulated in normal tissue) or . 2 (the element is accumulated in malignant tissue) for each sample of a specific type of cancer, the element is declared to be significant. An element is considered to show a tendency in concentration if the ratio of element concentration for each sample of a specific type of cancer shows a non-significant accumulation in malignant or normal tissue (the ratio of element concentrations is . 0.5 or , 2 respectively). Fig. 3 shows the ratios of element concentrations in malignant versus normal tissues according to the type of cancer, demonstrated by three selected examples. The results are summarized in Table 1, where the significant and the tendentious elements are listed according to the type of cancer. The positive sign is used to illustrate the accumulation of the element in neoplastic tissue, and the minus sign is used to indicate the depletion of the element in cancerous tissue. The first result is a pattern of element distributions in cancerous versus normal tissues. The malignant tissues of the stomach show a significant reduction in the levels of Cr, Fe and Ni and a questionable decrease of Mn and Zn concentrations. In carcinoma of the colon and the rectum K is accumulated significantly, but Cr and Co decrease in cancerous tissue of the colon. Levels of Ni and Fe show a tendency towards reduction in malignant tissue of the colon, whereas Fe is significantly enriched in neoplastic tissue of the rectum. P, Mn, and Cr are diminished and Cu is concentrated in rectal carcinoma. Therefore the patterns depend on the type of cancer, and consequently neoplastic classification of similar cancer types (digestive tract) is possible, but needs first to be verified by analysing and comparing a larger number of tissue samples.
Table 1 List of significant and tendentious elements in carcinoma of the digestive organs
4. Conclusion
Status
Stomach
Colon
Rectum
Significant
Cr (−) Fe (−) Ni (−) Mn (−) Zn (−)
K (+) Cr (−)
Fe (+) K (+)
Ti (−) Fe (−) Ni (−)
P (−) Mn (−) Cu (+) Cr (−)
Tendentious
TXRF in combination with simplified sample preparation by microtome is a method well suited to the direct instrumental microanalysis of human tissues. It can be demonstrated that a recognition of element distribution patterns in cancerous tissues versus normal tissues is possible. Moreover, it can be shown that the patterns determined depend on the type of cancer. Therefore a good method has been
1052
D. von Czarnowski et al./Spectrochimica Acta Part B 52 (1997) 1047–1052
found for the classification of similar types of cancer. If a classification of the described cancer types can be verified by means of a large number of samples, an application in clinical chemistry is conceivable, with the aim of replacing large scale pathological investigations. Therefore a correlation of analytical data and pathological information needs to be corroborated by the application of various statistical methods. The established patterns of distribution of trace elements in malignant versus normal tissues can be used as a basis for further biochemical studies. The significance of the determined elements has to be proved according to their biological relevance. Chromium, as a significant element in carcinoma of the stomach and the colon, shows promise for further research. This can be substantiated, as chromium is a relevant bioinorganic element and its biological function is to date not known [2]. The role of the significant elements in carcinogenesis or the means of inhibition of cancer cannot be readily determined. Further investigations such as in vitro experiments on normal and malignant cells by incubation with different inorganic and organic trace element compounds need to be conducted. For this purpose also, TXRF is a suitible analytical method.
Acknowledgements This work was supported by the Ministerium fu¨r
Wissenschaft und Forschung des Landes NordrheinWestfalen (MWF) and by the Institut fu¨r Umwelttechnologie und Umweltanalytik e.V. (IUTA). We are also indebted to Dr H. Kraemer (medical superintendent of St. Cornelius Hospital Viersen–Du¨lken) for interdisciplinary collaboration. We also thank M. Mu¨ller and A. von Bohlen in connection with many aspects of this investigation.
References [1] E.N. Drake II and H.H. Sky-Peck, Cancer Res., 49 (1989) 4210. [2] S.J. Lippard and J.M. Berg, Bioanorganische Chemie, Spektrum Akademischer Verlag GmbH, Heidelberg, Berlin, Oxford, 1995. [3] R. Pepelnik, A. Prange and R. Niedergesa¨ß, J. Anal. At. Spectrom., 9 (1994) 1071. [4] A. von Bohlen, R. Klockenka¨mper, H. Otto, G. To¨lg and B. Wiecken, Int. Arch. Occup. Environ. Health, 59 (1987) 403. [5] A. von Bohlen, R. Klockenka¨mper, G. To¨lg and B. Wiecken, Fresenius’ Z. Anal. Chem., 331 (1988) 454. [6] A. Niemann, A. von Bohlen, R. Klockenka¨mper and E. Keck, Biochem. Biophys. Res. Commun., 170 (1990) 1216. [7] R. Klockenka¨mper, A. von Bohlen and B. Wiecken, Spectrochim. Acta Part B, 44 (1989) 511. [8] B. Pesch, U. Halekoh, U. Ranft, M. Richter and F. Pott, in Ministerium fu¨r Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen (Ed.), Atlas zur Krebssterblichkeit in Nordrhein-Westfalen, Satz und Druck, Du¨sseldorf 1994.