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The determination of trace metals in human fluids and tissues
Analytico Chimica Acta, 157 (1984) 183-186 Elsevier Science Publishers B.V., Amsterdam -Printed
in The Netherlands
Short Communication
THE DETERMINATION OF TRACE METALS IN HUMAN FLUIDS AND TISSUES Part 2. The Homogeneity of Liver Tissue for Sampling
V. HUDNIK Boris KidriE Institute of Chemistry, Ljubljana (Yugoslavia) M. MAROLT-GOMISCEK Faculty of Medicine, Edvard Kardelj University, Ljubljana (Yugoslavia) S. GOMISCEK* Faculty of Natural Sciences and Technology, (Yugoslavia)
Edvard Kardelj University, Ljubljana
(Received 26th July 1983)
Summary. The homogeneity of liver tissue of healthy persons is examined with regard to the distribution of copper, zinc, cadmium and manganese. The relative standard deviations of results for particular samples of the same human liver were 6.1-9.9, 4.0-6.0, 6.2-8.7 and 6.2-10.3% for copper, zinc, cadmium and manganese, respectively. Comparison of these values with those obtained for the standard reference material NBS Bovine Liver 1577 (2.8, 3.5, 3.4 and 1.8%, respectively) indicates that small samples of liver tissue can be accepted, with some limitations, as representative of the whole organ for the metals investigated.
The first condition for the reliable investigation of any material is the production of a representative sample. The representativeness of biological materials is often critical, and this is an important problem in clinical work, especially in the determination of trace metals. The representativeness of liver tissue samples has often been discussed and must be accepted as a decisive factor in the applicability of trace element results in medical investigations. KostiE et al. [l] examined the iron, zinc and cobalt contents in tissue samples taken from different parts of healthy and cirrhotic livers; they found that a representative sample of healthy liver tissue was obtained, irrespective of the site. In cirrhotic liver tissue, however, heterogeneous pathological structures were involved, and the distribution of these trace elements was not the same in the tissue samples selected as in the whole liver. Lievens et al. [2] determined the concentrations of 25 trace elements in five normal human livers, taking eight samples from each; the relative standard deviations found for iron and zinc in the same liver were less than 1076, but those for cobalt were higher. Schicha et al. [ 31 observed a heterogeneous but parallel distribu0003-2670/84/$03.00
o 1984 Elsevier Science Publishers B.V.
184
tion of cobalt, iron, phosphorus, selenium and zinc, whereas van Eijk et al. [4] concluded from their results that there were no differences in the iron contents between the left, right and central parts of human liver. Koenig et al. [5] determined 18 elements in homogenized samples taken from three different areas of a single human liver, and concluded that the results obtained for small liver samples were, except for lead, fairly representative of the whole organ. The aim of this study was to obtain additional data on the homogeneity of liver tissue of healthy persons, regarding the distribution of copper, zinc, cadmium and manganese. Experimental Tissue specimens were obtained at autopsy from victims of accidental death. In the initial tests (Table l), only one specimen was taken from each liver; in further tests, 4-6 specimens were taken from different areas of one particular liver by surgical techniques. Each of these specimens was divided into four samples. These samples were lyophilized and stored in polystyrene vessels at -20°C until required for the determination of copper, zinc, cadmium and manganese. The procedures applied have been described [ 61. Results and discussion Homogeneity can be critical in the sampling of liver tissue. In hospitals, such samples are obtained by biopsy, which can be done in diagnosing hepatic disease; only the residual sample is then available for the determination of metals. In addition to contamination during sampling, the representativeness of the small sample (0.5-5 mg) is decisive for reliable results. In order to elucidate this problem, the series of tests outlined above were done. In all cases, the specimens were taken at autopsy with surgical instruments. Afterwards, the upper layers were removed with quartz scalpels in the laboratory before lyophilization in order to avoid the risk of contamination from the surgical instruments. Only samples without any pathological changes TABLE 1 The homogeneity
Human liver N = 120 k = 30 Bovine liverb NBS 1577 N=lO
of liver sample@
2 @g g’) ‘kg I”-’ ) : i (P”gg-’ 1 ) : r‘;;g? 0
Cd
CU
Mn
Zn
7.07 0.76 10.7 0.29 0.01 3.4
34.5 2.7 8.5 186 5.2 2.8
5.18 0.43 8.3 10.6 0.19 1.8
183 12 6.5 130 4.5 3.5
*N is the number of determinations and k the number of different livers examined. bCertified content. Cd 0.27, Cu 193, Mn 10.3, Zn 130 pg-‘.
185
were accepted for examination. The results obtained were statistically evaluated; the deviations calculated by the equation for pooled data [7] were taken as a measure of the homogeneous distribution of the metals in the liver tissues. The initial tests were designed to obtain an overall view of the homogeneity of normal liver tissue. The results are shown in Table 1. The standard deviations and relative standard deviations obtained for the human liver samples are higher than those obtained for the NBS Bovine Liver, but not excessively so. Considering all the efforts put into the preparation of standard reference materials with the highest possible homogeneity, it appears that the liver tissues studied were reasonably homogeneous. In further experiments, the homogeneity of the distribution of copper, zinc, cadmium and manganese in different parts of the same liver was examined. The results are shown in Table 2, where the spread is the difference between the lowest and the highest mean values for each specimen and standard and relative standard deviations are calculated from pooled data of all specimens. From the results obtained, it is obvious that the variations in the contents of copper, zinc, cadmium and manganese in the investigated specimens TABLE 2 Homogeneity of liver tissue with regard to copper, zinc, cadmium and manganese Liver
N
Copper 1 2 3 4
23 22 16 19
Zinc 1 2 3 4
24 22 15 16
Cadmium 1 2 3 4
23 24 16 19
6 6 4 5
6.0-7.6 3.6-5.4 4.2-6.6 3.1-4.3
6.6 4.6 5.5 3.9
0.44 0.30 0.34 0.34
6.6 6.5 6.2 8.7
Manganese 1 2 3 4
23 24 16 19
6 6 4 5
4.5-5.4 4.6-5.6 5.2-5.5 4.9-6.7
5.0 5.1 5.3 5.8
0.32 0.50 0.55 0.36
6.4 9.8 10.3 6.2
Walculated
k
Spread kg g-’ )” 14.1-16.6 32.6-36.9 21.9-27.5 10.0-13.7 139-147 158-177 234-294 121-149
for dry tissue.
x bg g-’ 1 14.8 34.9 24.4 11.9 142 166 265 137
s kg g’) 1.47 3.17 1.48 0.79 8.6 9.9 11.7 5.5
sr (%I
9.9 9.0
6.1 6.6 6.0 6.0 4.4 4.0
186
of the same liver are small. Therefore it can be concluded that small samples of liver tissue can be accepted, at least for the metals investigated, as representative of the whole organ. These conclusions can be related to samples obtained by biopsy if they are not contaminated during sampling. The differences between particular samples of the same liver are negligible, particularly if one recalls that the concentrations of the trace elements under investigation in liver tissues from different people vary significantly [ 61 . The authors are grateful to the Research Community of Slovenia for financial support. We also thank Dr. Jacques Versieck for his critical review of this manuscript. REFERENCES 1 K. KostZ, R. Ristanovie, V. Obradovi&, M. DjordjeviC and R. Drdkovic’, in Trace Element Analytical Chemistry in Medicine and Biology, Walter de Gruyter, Berlin, 1980, p. 601. 2 P. Lievens, J. Versieck, R. Cornelis and J. Hoste, in Proc. 1976 Int. Conf. Modern Trends in Activation Analysis, Vol. 1, 1976, p. 311. 3 H. Schicha, L. E. Feinendegen, K. Kasperek, H. J. Klein and V. Siller, Beitr. Pathol., 141 (1970) 227. 4 H. G. van Eijk, W. F. Wilting, G. Bos and J. P. Goossens, Clin. Chim. Acta, 50 (1974) 275. 5 W. Koenig, F. W. Richter, B. Meinel and J. Ch. Bode, J. Clin. Chem. Clin. Biochem., 17 (1979) 23. 6 V. Hudnik, M. Marolt-GomHEek, Anal. Chim. Acta, 157 (1984) 143. 7 b. A. Skoog and D. M. West, in Fundamentals of Analytical Chemistry, Holt, Rinehart & Winston, New York, 1971, p. 39.
in The Netherlands
Short Communication
THE DETERMINATION OF TRACE METALS IN HUMAN FLUIDS AND TISSUES Part 2. The Homogeneity of Liver Tissue for Sampling
V. HUDNIK Boris KidriE Institute of Chemistry, Ljubljana (Yugoslavia) M. MAROLT-GOMISCEK Faculty of Medicine, Edvard Kardelj University, Ljubljana (Yugoslavia) S. GOMISCEK* Faculty of Natural Sciences and Technology, (Yugoslavia)
Edvard Kardelj University, Ljubljana
(Received 26th July 1983)
Summary. The homogeneity of liver tissue of healthy persons is examined with regard to the distribution of copper, zinc, cadmium and manganese. The relative standard deviations of results for particular samples of the same human liver were 6.1-9.9, 4.0-6.0, 6.2-8.7 and 6.2-10.3% for copper, zinc, cadmium and manganese, respectively. Comparison of these values with those obtained for the standard reference material NBS Bovine Liver 1577 (2.8, 3.5, 3.4 and 1.8%, respectively) indicates that small samples of liver tissue can be accepted, with some limitations, as representative of the whole organ for the metals investigated.
The first condition for the reliable investigation of any material is the production of a representative sample. The representativeness of biological materials is often critical, and this is an important problem in clinical work, especially in the determination of trace metals. The representativeness of liver tissue samples has often been discussed and must be accepted as a decisive factor in the applicability of trace element results in medical investigations. KostiE et al. [l] examined the iron, zinc and cobalt contents in tissue samples taken from different parts of healthy and cirrhotic livers; they found that a representative sample of healthy liver tissue was obtained, irrespective of the site. In cirrhotic liver tissue, however, heterogeneous pathological structures were involved, and the distribution of these trace elements was not the same in the tissue samples selected as in the whole liver. Lievens et al. [2] determined the concentrations of 25 trace elements in five normal human livers, taking eight samples from each; the relative standard deviations found for iron and zinc in the same liver were less than 1076, but those for cobalt were higher. Schicha et al. [ 31 observed a heterogeneous but parallel distribu0003-2670/84/$03.00
o 1984 Elsevier Science Publishers B.V.
184
tion of cobalt, iron, phosphorus, selenium and zinc, whereas van Eijk et al. [4] concluded from their results that there were no differences in the iron contents between the left, right and central parts of human liver. Koenig et al. [5] determined 18 elements in homogenized samples taken from three different areas of a single human liver, and concluded that the results obtained for small liver samples were, except for lead, fairly representative of the whole organ. The aim of this study was to obtain additional data on the homogeneity of liver tissue of healthy persons, regarding the distribution of copper, zinc, cadmium and manganese. Experimental Tissue specimens were obtained at autopsy from victims of accidental death. In the initial tests (Table l), only one specimen was taken from each liver; in further tests, 4-6 specimens were taken from different areas of one particular liver by surgical techniques. Each of these specimens was divided into four samples. These samples were lyophilized and stored in polystyrene vessels at -20°C until required for the determination of copper, zinc, cadmium and manganese. The procedures applied have been described [ 61. Results and discussion Homogeneity can be critical in the sampling of liver tissue. In hospitals, such samples are obtained by biopsy, which can be done in diagnosing hepatic disease; only the residual sample is then available for the determination of metals. In addition to contamination during sampling, the representativeness of the small sample (0.5-5 mg) is decisive for reliable results. In order to elucidate this problem, the series of tests outlined above were done. In all cases, the specimens were taken at autopsy with surgical instruments. Afterwards, the upper layers were removed with quartz scalpels in the laboratory before lyophilization in order to avoid the risk of contamination from the surgical instruments. Only samples without any pathological changes TABLE 1 The homogeneity
Human liver N = 120 k = 30 Bovine liverb NBS 1577 N=lO
of liver sample@
2 @g g’) ‘kg I”-’ ) : i (P”gg-’ 1 ) : r‘;;g? 0
Cd
CU
Mn
Zn
7.07 0.76 10.7 0.29 0.01 3.4
34.5 2.7 8.5 186 5.2 2.8
5.18 0.43 8.3 10.6 0.19 1.8
183 12 6.5 130 4.5 3.5
*N is the number of determinations and k the number of different livers examined. bCertified content. Cd 0.27, Cu 193, Mn 10.3, Zn 130 pg-‘.
185
were accepted for examination. The results obtained were statistically evaluated; the deviations calculated by the equation for pooled data [7] were taken as a measure of the homogeneous distribution of the metals in the liver tissues. The initial tests were designed to obtain an overall view of the homogeneity of normal liver tissue. The results are shown in Table 1. The standard deviations and relative standard deviations obtained for the human liver samples are higher than those obtained for the NBS Bovine Liver, but not excessively so. Considering all the efforts put into the preparation of standard reference materials with the highest possible homogeneity, it appears that the liver tissues studied were reasonably homogeneous. In further experiments, the homogeneity of the distribution of copper, zinc, cadmium and manganese in different parts of the same liver was examined. The results are shown in Table 2, where the spread is the difference between the lowest and the highest mean values for each specimen and standard and relative standard deviations are calculated from pooled data of all specimens. From the results obtained, it is obvious that the variations in the contents of copper, zinc, cadmium and manganese in the investigated specimens TABLE 2 Homogeneity of liver tissue with regard to copper, zinc, cadmium and manganese Liver
N
Copper 1 2 3 4
23 22 16 19
Zinc 1 2 3 4
24 22 15 16
Cadmium 1 2 3 4
23 24 16 19
6 6 4 5
6.0-7.6 3.6-5.4 4.2-6.6 3.1-4.3
6.6 4.6 5.5 3.9
0.44 0.30 0.34 0.34
6.6 6.5 6.2 8.7
Manganese 1 2 3 4
23 24 16 19
6 6 4 5
4.5-5.4 4.6-5.6 5.2-5.5 4.9-6.7
5.0 5.1 5.3 5.8
0.32 0.50 0.55 0.36
6.4 9.8 10.3 6.2
Walculated
k
Spread kg g-’ )” 14.1-16.6 32.6-36.9 21.9-27.5 10.0-13.7 139-147 158-177 234-294 121-149
for dry tissue.
x bg g-’ 1 14.8 34.9 24.4 11.9 142 166 265 137
s kg g’) 1.47 3.17 1.48 0.79 8.6 9.9 11.7 5.5
sr (%I
9.9 9.0
6.1 6.6 6.0 6.0 4.4 4.0
186
of the same liver are small. Therefore it can be concluded that small samples of liver tissue can be accepted, at least for the metals investigated, as representative of the whole organ. These conclusions can be related to samples obtained by biopsy if they are not contaminated during sampling. The differences between particular samples of the same liver are negligible, particularly if one recalls that the concentrations of the trace elements under investigation in liver tissues from different people vary significantly [ 61 . The authors are grateful to the Research Community of Slovenia for financial support. We also thank Dr. Jacques Versieck for his critical review of this manuscript. REFERENCES 1 K. KostZ, R. Ristanovie, V. Obradovi&, M. DjordjeviC and R. Drdkovic’, in Trace Element Analytical Chemistry in Medicine and Biology, Walter de Gruyter, Berlin, 1980, p. 601. 2 P. Lievens, J. Versieck, R. Cornelis and J. Hoste, in Proc. 1976 Int. Conf. Modern Trends in Activation Analysis, Vol. 1, 1976, p. 311. 3 H. Schicha, L. E. Feinendegen, K. Kasperek, H. J. Klein and V. Siller, Beitr. Pathol., 141 (1970) 227. 4 H. G. van Eijk, W. F. Wilting, G. Bos and J. P. Goossens, Clin. Chim. Acta, 50 (1974) 275. 5 W. Koenig, F. W. Richter, B. Meinel and J. Ch. Bode, J. Clin. Chem. Clin. Biochem., 17 (1979) 23. 6 V. Hudnik, M. Marolt-GomHEek, Anal. Chim. Acta, 157 (1984) 143. 7 b. A. Skoog and D. M. West, in Fundamentals of Analytical Chemistry, Holt, Rinehart & Winston, New York, 1971, p. 39.