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PIXE analysis of elements in gastric cancer and adjacent mucosa
Nuclear Instruments North-Holland
and Methods
PIXE ANALYSIS LIU
Qixin.
Institute
XU
in Physics
OF ELEMENTS
ZHONG
Ming,
ZHANG
Research
231
B49 (1990) 231-233
IN GASTRIC CANCER AND ADJACENT Xiaofeng
and YAN
MUCOSA
Lingnuo
of High Energv Physics. Academia Sinica, Beying, PR Chinu
Yongling
Thrrd Subsidiqv
and YE Simao Hospital, Beijing Uniuersity
of Medicine. Be&g,
PR China
The elemental regional distributions in 20 resected human stomach tissues were obtained using PIXE analysis. The samples were pathologically divided into four types: normal. adjacent mucosa A, adjacent mucosa B and cancer. The targets for PIXE analysis were prepared by wet digestion with a pressure bomb system. P, K. Fe, Cu. Zn and Se were measured and statistically analysed. We found significantly higher concentrations of P, K. Cu, Zn and a higher ratio of Cu compared to Zn in cancer tissue as compared with normal tissue, but statistically no significant difference between adjacent mucosa and cancer tissue was found,
1. Introduction Recently, a large amount of epidemiological data and the results of animal experiments showed an anomaly in trace element concentrations of Cr. Mn. Ni. Cu, Zn, As and Se, which was correlated to cancer [l-4]. However, the mechanism of the action is not clear. Gastric cancer is the most common one of all cancers. The distribution of trace elements in the human stomach has been studied by several investigators using PIXE [5]. but still very little is known about the distribution of trace elements in human gastric tumours. It is very important to collect all possible information about the characteristics of the tumour, tumour front and the surrounding normal stomach tissue. Our main object was to determine the trace elements in human gastric cancer tissue, adjacent mucosa and normal mucosa by the PIXE technique and to analyse the information so obtained by statistical methods in order to establish the probability that trace elements act as a chemical mark by which the gastric cancer can be diagnosed.
2. Materials and methods The specimens of resected stomach were stored at -40 o C. Part of each sample was submitted to histopathological investigation with subsequent classification into four groups: normal, adjacent mucosa A, adjacent mucosa B and cancer. The normal mucosa tissue or chronic superficial gastritis (CSG) tissue which are at a 0168-583X/90/$03.50 (North-Holland)
‘B Elsevier Science Publishers
B.V.
long distance from the cancer centre belong to normal group, adjacent A is tissue of CSG or of CSG with intestinal metaplasia, adjacent B is tissue of chronic atrophic gastritis with intestinal metaplasia or a typical plasia. According to ref. [6], bromine and selenium are lost when using low-temperature ashing. Wet-ashing, suggested by Maenhaut et al. [6] for the preparation of biological samples is the most suitable technique. From each sample approximately 50 mg was taken and then freeze-dried for 24 h. The dry matter was wet-ashed by adding 100 ~1 ultrapure nitric acid per 10 mg dry matter to the sample container (pressure bomb system). The container was then placed in an oven at 150°C until the tissue was completely dissolved by the acid. As internal standard, yttrium was added. Finally 10 1.11of the solution was put on Mylar foils and allowed to evaporate. In order to check the analytical procedure. standard reference material Horse Kidney (IAEA H-8) was prepared in a similar way. The proton beam from a 2.5 MV Van de Graaff accelerator at the Institute of High Energy Physics was used for the sample irradiations. The energy of the proton beam was 2.4 MeV. The area of the beam spot was 0.9 cm’ and the beam current intensity was about 30 nA. A 80 mm’ Si(Li) detector connected with a standard electronic chain with an overall resolution of 180 eV at 5.9 keV was used for the measurement of the X-ray spectra. The detector was mounted at 90” to the incident beam. An absorbing layer of 400 pm thickness of Mylar with a 1 mm hole was placed in front of the detector to reduce the background and the intense IV. BIOLOGICAL APPLICATIONS
Lm Qixtn et al. / PIXE ana(wis of elements WIgastric cancer
232 Table 1 The mean values and the relative
standard
deviations
of the measured
element
levels in IAEA H-8 Horse
Kidney
Element
P
K
Fe
Cu
Zn
Se
Measured value (ppm) Certified value (ppm)
11300~700 112OOzk604
15700+500 117OOF749
275 + 48 265i15
35.855.7 31.3k1.8
200 + 21 193+ 6
4.8 k 0.9 4.7 * 0.3
Table 2 The mean values and the relative standard deviation tissues. adjacent mucosa A and adjacent mucosa B
of the measured
Element
Cancer tissues (n =15)
P
1.16k 0.24 0.88i 0.14 118 *51 24.9 + 10.0 104 *18 4.2 + 2.2 0.23+ 0.10
< < > < i > <
@pm)
P(B)
Normal tissues (n=lO)
K (%) Fe cu Zn Se Cu/Zn
0.66 0.43 104 5.9 67.3 1.5 0.084
low-energy
radiation.
X-ray
analysis
program
the
spectral
f 0.07 f 0.02 *16 + 1.8 k 3.9 * 0.9 + 0.027
The results were analysed
3. Results
spectra
AXIL statistically
were
0.02 0.02 0.05 0.01 0.01 0.05 0.02
analysed
by
on a PDP 11/23. by a T-test.
and discussion
In the samples evaporated on Mylar foils elements heavier than aluminum were determined. The results obtained by PIXE analysis of standard reference material Horse Kidney are presented in table 1. Acceptable agreement between values quoted by the manufacturer and measured values was obtained. The element concentrations of P, K, Fe, Cu, Zn. and Se in four groups (normal. cancer, adjacent mucosa A and B) are shown in figs. 1 and 2 respectively, and are also listed in table 2.
Fig. 1. The concentrations
of P and K in the four groups.
element
concentrations
Adjacent mucosa A (n =19)
P
1.18+ 0.19 0.89i 0.04 195 *35 27.4 +10.3 115 +22 2.6 + 1.2 0.23+ 0.10
i i z > c: >
0.01 0.01 0.05 0.05 0.005 0.05
in normal
stomach
tissues.
Adjacent mucosa B (n =lO)
P
0.90+ 0.11 0.9Ok 0.08 131 f 19 19.6 +18.7 100 +13 2.6 f 2.1 0.20f 0.20
> i > i < >
cancer
0.05 0.01 0.05 0.05 0.05 0.05
As is evident from table 2, the concentrations of P, K, Cu and Zn in the cancer group are significantly highter than in the normal group, while the concentrations of Fe and Se show no significant difference between both of them. The elevation of K concentration in cancer tissue shows good agreement with the reports of other investigators [7]. However, there is no information on elevation of P concentration in cancer. Elevation of Zn concentration in gastric, breast, lung and colon cancer has been reported [7-121. Our result is in agreement with these studies. In addition, the ratio of Cu to Zn shows a difference ( p < 0.02) between cancer tissues and normal tissues. No significant difference was found among element concentrations in the cancer tissue, adjacent mucosa A and B. On the other hand, we found a large difference for the element concentration between normal and ad-
Fig. 2. The concentrations
of Fe. Cu and Zn in the four groups.
Liu Qixin et al. / PIXE analysis of elements m gastric cancer
jacent A and B groups. According to pathological classification, adjacent A and normal tissues belong to the same classification of CSG, only the distances from the cancer are different. The adjacent A is closer to the cancer than normal. When cancer cells have just invaded the adjacent mucosa A, the differences in tissue cannot be found by histo-pathological investigation. However, the variation of some element concentrations (like as K. Cu. and Zn), which is quite evident, can be detected early by the PIXE method. It indicates that abnormality of some element concentration in stomach tissue might give a possibility for cancer diagnosis.
References [l] U.A.S. Tapper, K.G. Malmqvist, A. Brun and L.C. ford, Nucl. Instr. and Meth. B22 (1987) 176.
Sal-
233
[2] E. Johansson.
[3] [4] [5] [6] [7]
U. Lindh. H. Johasson and C. Sundstrom, ibid., p. 179. M. Uda. K. Maeda, Y. Sasa, H. Kusuyama and Y. Yokode, ibid., p. 184. K. Maeda. Y. Yokode, Y. Sasa, H. Kusuyama and M. Uda, ibid., p. 188. Xu Li Qiang. Liu Zheng Xian, Zhou Pei Fang and Shi Kui Xiong, Trace Elements (China) No. 2 (1986) 19. W. Maenhaut, L. DeReu and J. Vandenhaute, Nucl. Instr. and Meth. B3 (1984) 135. G.C. Gregoriadis, N.S. Apostolidis, A.N. Romanos and T.P. Paradellis, Cancer 52 (1983) 508.
[8] E.J. Margalioth,
J.G. Schenker and M. Chevion. Cancer 52 (1983) 868. [9] A.S. Frank, M.K. Schauble and I.L. Kreiss. Am. J. Pathol. 122 (1986) 421. [lo] F.A. Hoffman, Cancer 55 (1983) 295. [ll] S.L. Risk and H.H. Sky-Peck, Cancer Res. 44 (1984) 5390. [12] I.L. Mulay, R. Roy, B.E. Knox, N.H. Suhr and W.E. Delaney, J. Nat. Cancer Inst. 47 (1971) 1.
IV. BIOLOGICAL
APPLICATIONS
and Methods
PIXE ANALYSIS LIU
Qixin.
Institute
XU
in Physics
OF ELEMENTS
ZHONG
Ming,
ZHANG
Research
231
B49 (1990) 231-233
IN GASTRIC CANCER AND ADJACENT Xiaofeng
and YAN
MUCOSA
Lingnuo
of High Energv Physics. Academia Sinica, Beying, PR Chinu
Yongling
Thrrd Subsidiqv
and YE Simao Hospital, Beijing Uniuersity
of Medicine. Be&g,
PR China
The elemental regional distributions in 20 resected human stomach tissues were obtained using PIXE analysis. The samples were pathologically divided into four types: normal. adjacent mucosa A, adjacent mucosa B and cancer. The targets for PIXE analysis were prepared by wet digestion with a pressure bomb system. P, K. Fe, Cu. Zn and Se were measured and statistically analysed. We found significantly higher concentrations of P, K. Cu, Zn and a higher ratio of Cu compared to Zn in cancer tissue as compared with normal tissue, but statistically no significant difference between adjacent mucosa and cancer tissue was found,
1. Introduction Recently, a large amount of epidemiological data and the results of animal experiments showed an anomaly in trace element concentrations of Cr. Mn. Ni. Cu, Zn, As and Se, which was correlated to cancer [l-4]. However, the mechanism of the action is not clear. Gastric cancer is the most common one of all cancers. The distribution of trace elements in the human stomach has been studied by several investigators using PIXE [5]. but still very little is known about the distribution of trace elements in human gastric tumours. It is very important to collect all possible information about the characteristics of the tumour, tumour front and the surrounding normal stomach tissue. Our main object was to determine the trace elements in human gastric cancer tissue, adjacent mucosa and normal mucosa by the PIXE technique and to analyse the information so obtained by statistical methods in order to establish the probability that trace elements act as a chemical mark by which the gastric cancer can be diagnosed.
2. Materials and methods The specimens of resected stomach were stored at -40 o C. Part of each sample was submitted to histopathological investigation with subsequent classification into four groups: normal, adjacent mucosa A, adjacent mucosa B and cancer. The normal mucosa tissue or chronic superficial gastritis (CSG) tissue which are at a 0168-583X/90/$03.50 (North-Holland)
‘B Elsevier Science Publishers
B.V.
long distance from the cancer centre belong to normal group, adjacent A is tissue of CSG or of CSG with intestinal metaplasia, adjacent B is tissue of chronic atrophic gastritis with intestinal metaplasia or a typical plasia. According to ref. [6], bromine and selenium are lost when using low-temperature ashing. Wet-ashing, suggested by Maenhaut et al. [6] for the preparation of biological samples is the most suitable technique. From each sample approximately 50 mg was taken and then freeze-dried for 24 h. The dry matter was wet-ashed by adding 100 ~1 ultrapure nitric acid per 10 mg dry matter to the sample container (pressure bomb system). The container was then placed in an oven at 150°C until the tissue was completely dissolved by the acid. As internal standard, yttrium was added. Finally 10 1.11of the solution was put on Mylar foils and allowed to evaporate. In order to check the analytical procedure. standard reference material Horse Kidney (IAEA H-8) was prepared in a similar way. The proton beam from a 2.5 MV Van de Graaff accelerator at the Institute of High Energy Physics was used for the sample irradiations. The energy of the proton beam was 2.4 MeV. The area of the beam spot was 0.9 cm’ and the beam current intensity was about 30 nA. A 80 mm’ Si(Li) detector connected with a standard electronic chain with an overall resolution of 180 eV at 5.9 keV was used for the measurement of the X-ray spectra. The detector was mounted at 90” to the incident beam. An absorbing layer of 400 pm thickness of Mylar with a 1 mm hole was placed in front of the detector to reduce the background and the intense IV. BIOLOGICAL APPLICATIONS
Lm Qixtn et al. / PIXE ana(wis of elements WIgastric cancer
232 Table 1 The mean values and the relative
standard
deviations
of the measured
element
levels in IAEA H-8 Horse
Kidney
Element
P
K
Fe
Cu
Zn
Se
Measured value (ppm) Certified value (ppm)
11300~700 112OOzk604
15700+500 117OOF749
275 + 48 265i15
35.855.7 31.3k1.8
200 + 21 193+ 6
4.8 k 0.9 4.7 * 0.3
Table 2 The mean values and the relative standard deviation tissues. adjacent mucosa A and adjacent mucosa B
of the measured
Element
Cancer tissues (n =15)
P
1.16k 0.24 0.88i 0.14 118 *51 24.9 + 10.0 104 *18 4.2 + 2.2 0.23+ 0.10
< < > < i > <
@pm)
P(B)
Normal tissues (n=lO)
K (%) Fe cu Zn Se Cu/Zn
0.66 0.43 104 5.9 67.3 1.5 0.084
low-energy
radiation.
X-ray
analysis
program
the
spectral
f 0.07 f 0.02 *16 + 1.8 k 3.9 * 0.9 + 0.027
The results were analysed
3. Results
spectra
AXIL statistically
were
0.02 0.02 0.05 0.01 0.01 0.05 0.02
analysed
by
on a PDP 11/23. by a T-test.
and discussion
In the samples evaporated on Mylar foils elements heavier than aluminum were determined. The results obtained by PIXE analysis of standard reference material Horse Kidney are presented in table 1. Acceptable agreement between values quoted by the manufacturer and measured values was obtained. The element concentrations of P, K, Fe, Cu, Zn. and Se in four groups (normal. cancer, adjacent mucosa A and B) are shown in figs. 1 and 2 respectively, and are also listed in table 2.
Fig. 1. The concentrations
of P and K in the four groups.
element
concentrations
Adjacent mucosa A (n =19)
P
1.18+ 0.19 0.89i 0.04 195 *35 27.4 +10.3 115 +22 2.6 + 1.2 0.23+ 0.10
i i z > c: >
0.01 0.01 0.05 0.05 0.005 0.05
in normal
stomach
tissues.
Adjacent mucosa B (n =lO)
P
0.90+ 0.11 0.9Ok 0.08 131 f 19 19.6 +18.7 100 +13 2.6 f 2.1 0.20f 0.20
> i > i < >
cancer
0.05 0.01 0.05 0.05 0.05 0.05
As is evident from table 2, the concentrations of P, K, Cu and Zn in the cancer group are significantly highter than in the normal group, while the concentrations of Fe and Se show no significant difference between both of them. The elevation of K concentration in cancer tissue shows good agreement with the reports of other investigators [7]. However, there is no information on elevation of P concentration in cancer. Elevation of Zn concentration in gastric, breast, lung and colon cancer has been reported [7-121. Our result is in agreement with these studies. In addition, the ratio of Cu to Zn shows a difference ( p < 0.02) between cancer tissues and normal tissues. No significant difference was found among element concentrations in the cancer tissue, adjacent mucosa A and B. On the other hand, we found a large difference for the element concentration between normal and ad-
Fig. 2. The concentrations
of Fe. Cu and Zn in the four groups.
Liu Qixin et al. / PIXE analysis of elements m gastric cancer
jacent A and B groups. According to pathological classification, adjacent A and normal tissues belong to the same classification of CSG, only the distances from the cancer are different. The adjacent A is closer to the cancer than normal. When cancer cells have just invaded the adjacent mucosa A, the differences in tissue cannot be found by histo-pathological investigation. However, the variation of some element concentrations (like as K. Cu. and Zn), which is quite evident, can be detected early by the PIXE method. It indicates that abnormality of some element concentration in stomach tissue might give a possibility for cancer diagnosis.
References [l] U.A.S. Tapper, K.G. Malmqvist, A. Brun and L.C. ford, Nucl. Instr. and Meth. B22 (1987) 176.
Sal-
233
[2] E. Johansson.
[3] [4] [5] [6] [7]
U. Lindh. H. Johasson and C. Sundstrom, ibid., p. 179. M. Uda. K. Maeda, Y. Sasa, H. Kusuyama and Y. Yokode, ibid., p. 184. K. Maeda. Y. Yokode, Y. Sasa, H. Kusuyama and M. Uda, ibid., p. 188. Xu Li Qiang. Liu Zheng Xian, Zhou Pei Fang and Shi Kui Xiong, Trace Elements (China) No. 2 (1986) 19. W. Maenhaut, L. DeReu and J. Vandenhaute, Nucl. Instr. and Meth. B3 (1984) 135. G.C. Gregoriadis, N.S. Apostolidis, A.N. Romanos and T.P. Paradellis, Cancer 52 (1983) 508.
[8] E.J. Margalioth,
J.G. Schenker and M. Chevion. Cancer 52 (1983) 868. [9] A.S. Frank, M.K. Schauble and I.L. Kreiss. Am. J. Pathol. 122 (1986) 421. [lo] F.A. Hoffman, Cancer 55 (1983) 295. [ll] S.L. Risk and H.H. Sky-Peck, Cancer Res. 44 (1984) 5390. [12] I.L. Mulay, R. Roy, B.E. Knox, N.H. Suhr and W.E. Delaney, J. Nat. Cancer Inst. 47 (1971) 1.
IV. BIOLOGICAL
APPLICATIONS