Journal of Molecular Structure 565566 (2001) 329334

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Analysis of human cancer prostate tissues using FTIR microspectroscopy and SRIXE techniques
Czesawa Paluszkiewicz a,b,*, Wojciech M. Kwiatek c
a

Regional Laboratory of Physicochemical Analysis and Structural Research, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland b Department of Materials Science and Ceramics, University of Mining and Metallurgy, Al. Mickiewicza 30, 30-059 Krakow, Poland c Henryk Niewodniczanski Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Krakow, Poland Received 31 August 2000; revised 5 February 2001; accepted 5 February 2001

Abstract It is known that Fourier transform infrared (FTIR) spectra of human tissues are specic and can be used to discriminate between various disease states. In this study, cancer and healthy parts of prostate tissues were examined. The human prostate tissues were obtained during surgical operation. Sections of samples were mounted onto Mylar foils and measured by both FTIR microspectroscopy and synchrotron radiation induced X-ray emission (SRIXE) methods. Neighboring sections of tissues analyzed by FTIR and SRIXE were also examined by a histopathologist. Since the SRIXE technique is suitable for trace element analysis the two-dimensional scans on both cancerous and non-cancerous parts of the prostate tissues were done in order to nd elemental distribution of trace elements. The single point analysis on selected areas were also performed. Then the same samples were studied in the mid infrared region on Excalibur spectrometer with infrared microscope UMA-500 equipped with an automatic xy-stage and video camera. Both FTIR spectra and elemental distribution show differences between cancerous and non-cancerous parts of the analyzed tissues. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Prostate; Cancer; FTIR; SRIXE

1. Introduction The prostate cancer disease occurrence seems to have been increasing over recent years [1,2]. Many different analytical techniques have been applied to the study of the cancer genesis. It has been found that there are elemental differences between normal and cancer tissues as well as differences in chemical structural compositions [37]. Among others, the synchrotron radiation induced X-ray emission (SRIXE) technique as well as Fourier transform
* Corresponding author. Tel.: 148-12-172487; fax: 148-12343859. E-mail address: ucpalusz@cyf-kr.edu.pl (C. Paluszkiewicz).

infrared (FTIR) spectroscopy have been complementarily applied to biological samples [8,9]. Standard FTIR analyses of tissue sections are performed on samples placed on transparent materials for IR such as KBr and BaF2 or microscopic glass. In this study we have analyzed tissues placed on Mylar foils using SRIXE and FTIR methods. For the rst time the FTIR spectroscopy was applied to tissues placed on Mylar. Since Mylar does not contain trace elements that could be analyzed by the SRIXE method and it could be used as substrate. The other substrates like KBr, BaF2, and glass would give very intense X-ray uorescent characteristic lines in SRIXE spectra and that is why they cannot be used. The differences between FTIR spectra of healthy

0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(01)00527-0

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and cancer tissues have been discussed elsewhere [1,3,6] with respect to vibration region below 2000 cm 21, which corresponds to characteristic 2 bands of Amid I, Amid II, and PO2 groups. Li and co-workers show the differences between the cancer and healthy tissues of the colon, rectum, and stomach with respect to chemical bonds of CH in the region from 2800 to 3000 cm 21 [10]. Therefore the aim of this study was to investigate the prostate cancer tissue in the above-mentioned IR region in addition to the elemental differences determinations. 2. Materials and methods Samples were obtained during surgical treatments of prostate cancer disease. Malignant parts of the tissue were compared with the healthy parts. The healthy part of the tissue was taken from the same prostate from more than 1 cm away of the malignant part. The fresh tissues from resected human prostate were frizzed in liquid nitrogen for rapid cryosectioning at 2158C. The tissues were sliced into 12 mm thick sections. These sections were put on a 3 mm thick Mylar foils. The neighboring sections were put on a microscopic glass and on KBr windows. Samples were stored at room temperature under dry conditions. The sections mounted on the conventional glass slides were analyzed by an experienced pathologist to conrm cancer or healthy regions. The pathologist also provided the selection of the area to be analyzed by SRIXE and FTIR methods. Fig. 1a presents a histopathological view of one of the analyzed prostate tissues. The investigated sections were also analyzed by FTIR spectroscopy. The SRIXE method was used to determine the concentration of trace elements. For this purpose, the samples placed on Mylar foil were used. The experiments were done at the X26A beam line at the National Synchrotron Light Source in Brookhaven National Laboratory, USA. The beam line was equipped with standard apparatus for SRIXE measurements [11]. The spectra were recorded with a conventional Si(Li) detector with the energy resolution of 140 eV at 5.9 keV energy. The SRIXE measurements were performed with a monochromatic beam of 17 keV. The monochromatic beam of

16 14 mm 2 beam size was applied. The spectra for single point analysis were collected for 300 s live time. For proper calculation, selected standards were measured at the same conditions [12]. The FTIR method was used to determine the differences in CH2 and CH3 groups interactions in cancer and non-cancer prostate tissues. In order to determine these interactions the samples placed on Mylar, KBr and microscopic glass were analyzed. The FTIR spectra were collected at 4 cm 21 resolution in the frequency region from 700 to 4000 cm 21 in transmission mode. Excalibur spectrometer with microscope UMA-500 equipped with an automatic xy-stage, video camera and MCT detector, was used for all the measurements. A video camera enabled optical imaging and recording of the investigated area. The redundant apertures were set to 40250 mm. Since the samples were put on different substrates such as KBr disks, Mylar foils, and standard microscopic glass the spectra of those materials were used for background subtraction. 3. Results and discussion SRIXE analysis of trace element concentrations in cancer and healthy parts of prostate tissues show signicant differences between elemental concentrations [4]. Fig. 1b shows a histogram of averaged elemental concentrations for selected elements such as Ca, Fe, and Zn. As is seen the levels of Ca and Fe concentrations in cancer tissue are much higher than in non-cancer tissues. The opposite relation is observed for Zn. The 2D Zn concentration level distribution in the cancer part of prostate tissue is shown in Fig. 1c. The results obtained in this study conrm the experiments of the others [1,1315]. Fig. 2 presents FTIR spectrum of Mylar foil itself. Although the spectrum is reached in several bands in the region from 730 to 1730 cm 21, it can be used as a background for the spectra of tissues placed on such a foil. The Mylar spectrum is free of bands in the region from 1600 to 1370 cm 21 and above 1730 cm 21, where the characteristic bands belonging to CH2 and CH3 groups appear in biological samples [3,6,9,10,]. The same tissues which had been placed on Mylar foil and previously analyzed with SRIXE were

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331

TE 3
110 17

Zn

490 1370

Fe

non-ca nce r cancer

1870

Ca

2240

500

1000
[g/g]

1500

2000

2500

Fig. 1. (a) Histological view of prostate tissue, (b) histogram of averaged elemental concentration for Ca, Fe, Zn (mg/g) detected in prostate tissue, (c) 2D Zn distribution in the prostate tissue shown on the side, white colors corresponds to the highest Zn concentration while the black one to the lowest concentration.

analyzed with FTIR spectroscopy. For FTIR analysis the same areas of the tissues were chosen. In addition, the tissues placed on KBr and standard microscopic glass were also measured. Fig. 3ac present examples of FTIR spectra of tissues placed on those substrates after background substractions. There are no signicant differences between the spectra of the samples except for the spectrum of the tissue placed on the glass. Such phenomena was obvious since the glass is non-transparent for IR radiation below 1400 cm 21. The existing bands in the spectra presented in Fig. 3 are characteristic for biological samples. Table 1 assigns the major vibrational bands in the FTIR spectra of prostate tissue.

Fig. 4 presents an example of two FTIR prostate tissue spectra in the region from 2800 to 3000 cm 21. Fig. 4a shows the cancer part of tissue spectrum, while Fig.4b the non-cancer part. These spectra are different in terms of band intensities. As is seen, the band at 2930 cm 21 is more intense than the band at 2960 cm 21 for cancer tissue spectrum. The relative band intensity ratio of nas CH2 to nas CH3 (2930 2960 cm 21) changes from 1.05 for healthy parts to 1.5 for cancer parts of the prostate tissue spectra. A similar relation can be observed for the bands due to symmetric stretching vibrations ns CH2 to ns CH3 (28522874 cm 21). The exact evidence of the existing differences is shown in Fig. 5, which presents

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Absorbance (a.u)

3500

3000

2500 2000 Wavenumber (cm-1)

1500

1000

Fig. 2. FTIR spectrum of Mylar foil.

2D map of series spectra in the region from 2800 to 3000 cm 21. Those spectra were taken on prostate cancer and non-cancer tissues. The relative intensity changes of the bands CH2 to CH3 may indicate the disorder of the CH3 groups in the prostate cancer tissue. The similar results were reported by Li and co-workers [10], who analyzed human cancer gastric and colonic tissues and compared them to non-cancer tissues.

4. Conclusions This work shows the possibility of FTIR spectroscopy application to biological tissues placed on Mylar foils. The advantage of such an application is the possible study of the same biological sample by means of different non-destructive techniques such as SRIXE and FTIR spectroscopy. In that case such techniques seem to be fully complementary.

Absorbance (a.u)

c 3500 3000 2500 2000 Wavenumber (cm-1) 1500 1000

Fig. 3. FTIR spectra of prostate cancer tissues placed on: KBr (a), glass (b), Mylar foil (c).

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2960 -

Absorbance (a.u.)

2930 -

- 2876

2980

2960

2940

2920 2900 2880 Wavenumber (cm-1)

2860

- 2852 2840

2820

Fig. 4. An example of FTIR spectra of prostate tissue taken from cancer (a) and non-cancer (b) parts.

The additional advantage is that the samples placed on Mylar foils can be stained after the measurements by SRIXE and FTIR for later histopathological investigations. Our study conrms that the region of CH stretching bands can be used to distinguish healthy and cancer parts of the analyzed tissue.

Acknowledgements The authors would like to thank Dr Marek Galka from Gabriel Narutowicz Hospital in Krakow, Poland for providing prostate tissues. The special thanks are given to Professors Tadeusz Cichocki and Jerzy Stachura both from Collegium Medicum, Jagiellonian

Fig. 5. 2D map of series prostate FTIR spectra taken on cancer and non-cancer parts of the tissue.

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C. Paluszkiewicz, W.M. Kwiatek / Journal of Molecular Structure 565566 (2001) 329334 [2] M. Diem, L. Chiriboga, H. Yee, in: K. Itoh, M. Tasumi (Eds.), Fourier Transform Spectroscopy Twelfth International Conference, Waseda University Press, Tokyo, Japan, 1999, p. 99. [3] C.P. Schultz, H. Mantsch, Cellular Mol. Biol. 44 (1) (1998) 203. [4] M. Jackson, J.R. Manseld, B. Dolenko, R.L. Somorjai, H.H. Mantsch, P.H. Watson, Canc. Det. Prev. 23 (1999) 245. [5] R.A. Goyer, Toxic and essential metal interactions, Ann. Rev. Nutr. 17 (1997) 37. [6] E. Benedetti, E. Bramanti, F. Papineschi, I. Rossi, E. Benedetti, Appl. Spectrosc. 51 (1997) 79. [7] S. Boydston-White, T. Gopen, S. Houser, J. Bargonetti, M. Diem, Biospectroscopy 5 (1999) 219. [8] C. Paluszkiewicz, M. Galka, W. Kwiatek, A. Parczewski, S. Walas, Biospectroscopy 3 (5) (1997) 403. [9] C. Paluszkiewicz, W. Kwiatek, M. Gaka, D. Sobieraj, E. Wentrup-Byrne, Cellular and Molecular Biology 44 (1) (1998) 65. [10] W. Li, Q. Peng, Y. Xu, X. Zhou, S. Weng, J. Wu, in: K. Itoh, M. Tasumi (Eds.), Fourier Transform Spectroscopy twelfth International Conference, Waseda University Press, Tokyo, Japan, 1999, p. 479. [11] W.M. Kwiatek, A.L. Hanson, K.W. Jonson, Nucl. Instr. Meth. Phys. Res. B50 (1990) 347. [12] W.M. Kwiatek, A.L. Hanson, C. Paluszkiewicz, T. Cichocki, M. Gaka, SRIXE and XANES application to iron oxidation state determination in prostate tissue sections, to be published. [13] S. Toyokuni, Free Radic. Biol. Med. 20 (4) (1996) 553. [14] T.V. Vye, S.V. Sviridova, Zaichick, Int. Urol. Nephrol. 29 (5) (1997) 565. [15] K.E. Tvedt, G. Kopstad, O.A. Haugen, J. Halagunset, Cancer Res. Jan 1 47 (1) (1997) 323.

Table 1 Assignment of the major vibrational bands in the FTIR spectra of prostate tissue Frequency (cm 21) 3295 3050 2960 2930 2874 2852 1655 1545 1476 1380 1240 1170 1078 1050 1030 Assignment Amid A (NH stretching) Amid B nas CH3 nas CH2 ns CH3 ns CH2 Amid I (nCyO, dCN, dNH) Amid II (d NH, n CN) d CH2 d CH3 2 nas PO2 nas COOC 2 ns PO2 ns COOC ns CO

University for their help in histopathological investigations of the samples obtained. This work has been partially supported by State Committee for Scientic Research (KBN), Poland, Grants No. IFJ0202, AGH11.11.160.92, and the National Synchrotron Light Source, General User Grant No. 3680. References
[1] L.C. Costello, R.B. Franklin, Prostate 35 (1998) 285.