Path. Res. Pract. 186, 197-201 (1990)
Trace Metal Analysis of Chromium and Nickel in Lung Tissue Fixed and Stored in Formalin 1 J. Seemann, P. Wittig and H. Kollmeier Bundesanstalt fur Arbeitsschutz, Dortmund, FRG
K.-M. Muller and V. Schejbal Institut fUr Pathologie der Berufsgenossenschaftlichen Krankenanstalten "Bergmannsheil Bochum'; Universitatsklinik, Bochum, FRG
SUMMARY
In an attempt to answer the question whether the determination of chromium and nickel concentration in lung tissue, fixed and stored in formalin is still tolerable despite the related sources oferror, the entire system (tissue, tissue abrasion, formalin, plastic container) was analysed by means of f/ameless atomic absorption spectrometry. It appears that such a procedure can be justified for practical purposes. In order to reduce possible errors, well defined rules are to be observed during specimen collection and processing and specialities have to be taken into account.
Introduction Tissue residues of toxic/carcinogenic material in organs (in which uptake and/or - maximal - deposition take place) serve as an indicator for a preceding total load and are used as important additional information for the determination of causality between exposition and disease. Since trace analysis is almost exclusively performed in order to obtain these results, all sources of error have serious consequences. This is true for exogenous contaminations in particular, especially through material which is more or less ubiquitous. We examined whether under the circumstances mentioned above trace element measurements (in this case of chromium (Cr) and nickel (Ni)) can still be carried out on lung tissue specimens being fixed and stored in formalin. For that purpose the mass distribution of these metals in the entire system must be determined: tissue specimen, tissue abrasion, formalin, plastic contain-
er. 1 Dedicated to Prof. Dr. Dr. h.c. Franz Biichner on the occasion of his 95th birthday.
© 1990 by Gustav Fischer Verlag, Stuttgart
Material and Methods The Cr and Ni contents of 8 postmortem lung specimens were determined, which belonged to 4 men who had died in 1988/89. The autopsies were carried out at the Institute of Pathology (Berufsgenossenschaftliche Krankenanstalten "Bergmannsheil Bochum" - Universitiitsklinik). Table 3 presents the data of the cases. 1. Place of residence (pIRe): Hattingen, 40 cigarettes per day for many years, cigars only about 5 years before his death, coal miner between 1936 and 1975, metastasising small cell bronchial carcinoma, condition after irradiation, very high pulmonary total dust content and silicosis II. 2. Oer-Erkenschwick and Datteln (pIRe), non-smoker, coal miner for 32 years, metastasising squamous cell carcinoma of the right lower lobe bronchus, very high pulmonary total dust content and fibrous anthraco-silicosis III. 3. Bochum (pIRe), 40 cigarettes per day since 1948, chronic alcoholic, plumber (soldering and welding) from 1944-1985, metastasising small cell bronchial carcinoma. 4. Liinen (pIRe), presumably heavy smoker, metastasising squamous cell carcinoma of the 2nd right bronchus segment. The postmortem specimens were taken in the usual manner with common metal alloy instruments and stored in plastic containers, filled with 5% formalin. 0344-0338/90/0186-0197$3.50/0
198 .
J. Seemann et al.
The Cr and Ni concentrations were determined by means of flameless atomic absorption spectrometry (AAS) after Iyophilisation, wet ashing under pressure, chelating, and extraction. The analytical method used has been described elsewhere 3,8. Four specimens of 100 mg were analysed from each tissue, applying standard additions before ashing. Measured or mean values that vary considerably were rejected. The following exclusion criteria were employed: coefficient of variation (CV) ;:: 3% with 4 measured values, as well as r::; 0.997 or CV of the slope and axial section of the linear correlation;:: 10%.
Table 1. Recovery in percent of metal ions in 5% formalin 50 ng/ml
5 hours
24 hours
CrH
96% 96% 107%
66% 89% 105%
CrO~
NiH
Table 2. Specimens fixed and stored in formalin and Cr and Ni concentration of the conservation solution No.
tissue g
Quantityl tissuesolution abrasion ml g
Difference to primary solution2 chromium nickel ng/ml ng/ml
1.
5.7
0.10
40
-0.3
+ 13.7
2a. 2b.
29.3 14.9
0.35 0.01
16 4
-0.1 +0.5
+ 0.4 + 1.1
3a. 3b.
44.1 26.9
0.04 0.15
12 31
-0.1 -0.5
3.6 4.8
4a. 4b. 4c.
8.9 15.3 18.3
0.05 0.01 0.05
15 16 16
-1.0
3.1 2.2 + 1.0
-1.1
-1.0
1 Before Iyophilisation. - 2 In the primary formalin-solution: 0.6 ng Crimi and 6.3 ng Ni/ml.
The analytical characteristics were: - detection limit (threefold variation from five blind values): Cr 8 ng/g, Ni 5 ng/g dry weight; - precision: Cr 1.9%, Ni 11.7%; - accuracy (checked with NBS SRM 1577 bovine liver) Cr 106 ng/g dry weight (certification value: 88 ± 12 ng/g) (the National Bureau of Standards (NBS), Washington D.C., does not provide Standard Reference Material (SRM) for Ni determination). Metal ion concentrations in tissue abrasions were determined in the following way: after removal of the tissue the solution containing tissue abrasions was centrifuged, the jutting-out material pipetted, the residue lyophilised. After weighing the dried abrasion material, it was analysed in the same way as the tissue specimens. To prove the validity of the method the question arises, how much of minor concentrations of metal ions in formalin can be recovered with time. To determine the amount of metal ions in the conservation solutions, they were centrifuged and measured directly. They were calibrated in the graphite tube applying standard additions before ashing. Metal ions at the inner surface of the plastic container were removed by exposure to 6% nitric acid for half an hour and then measuring them directly. Mass balance analyses were carried out in cases 4a-c (Tables 2-4).
Results The movement and the related recovery of small amounts of Cr3+-, CrO~- and NiH-ions in 5% formalin are fairly limited (Table 1). The Cr and Ni concentration of the primary formalin solution2 is more or less identical with that of the conservation solutions of the individual specimens (Table 2); minor deviations do only exist in relation to the Ni concentration, and this more evidently in only one case (Table 2: 1). 2 By Fa. "Sealand Recycling" (Braunschweig, FRG) 5% in a 10 Liter plastic container.
Table 3. Cr and Ni concentration of 8 lung tissue specimens from 4 autopsies No.
Age
Specimen from
1.
72
1 L(ower} L(obe}
4.39
2a. 2b.
81
rt lung I lung
7.51 2.77
3a. 3b.
58
1 U(pper}L 1 LL
4a. 4b. 4c.
76
rt UL UL UL
rt rt
dev. 2 %
Cf3
dry I-tg/g
Nickel form. l I-tg/g
dev. 2 %
0.529
33
8.3
0.547
0.066
-9
1.25 0.318
82 -33
6.0 8.7
0.846 0.628
0.141 0.072
6 -21
13.4 12.8
2.39 2.03
480 450
5.6 6.3
2.98 3.94
0.523 0.625
670 910
1.58 2.18 2.71
0.255 0.427 0.437
-57 -40 -26
6.2 5.1 6.2
0.297 0.264 0.414
0.048 0.052 0.067
-57 -61 -39
dry I-tg/g
Chromium form. l I-tg/g
1 Formalin-fixed tissue before lyophilisation. - 2 Dev.: % deviation from "expected value". weight after and before lyophilisation: x = 6.8, CV = 19%.
3
CF: conversion factor: ratio of
Chromium and Nickel in Lung Tissue . 199 Table 4. Mass balance of the specimens 4a-e Tissue, dry 4a: weight
(g)
Abrasion dry
1.43
Formalin solution)
0.0088
14.6
Container adhesion
Sum
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
1.58 2.26 99.6
2.41 0.02 0.9
-0.001 -0.02 -0.9
0.01 0.4
2.27 100.0
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.297 0.425 43.1
1.67 0.015 1.5
- 0.003 - 0.044 -4.5
0.590 59.8
0.986 100.0
0.0006
16.3 -0.001 -0.02 -0.3
0.01 0.02
6.50 100.0
0.013 1.7
0.784 100.0
4b: weight
(g)
2.98
16.0 0.01 0.2
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
2.18 6.50 100.0
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.264 0.787 100.4
28.2 0.017 2.2
-0.002 -0.033 -4.2
3.00
0.0078
16.3
7.91 0.06 0.7
-0.001 -0.02 -0.2
0.01 0.01
8.18 100.0
10.8 0.084 6.2
+0.001 +0.016 +1.2
0.012 0.9
1.352 100.0
4e: weight
(g)
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
2.71 8.13 99.4
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.414 1.240 91.7
) In relation to the primary solution.
Table 5. Measured values (x + s) of previous test groups3,4 n
Chromium dry x±s f-tg/g 0.001
wet) x±s f-tg/g 0.0002
Nickel dry x ± s f-tg/g 0.002
wet) x±s f-tg/g 0.0003
A.
Extreme min. stillbirth
1
B.
Extreme max. Bronehial-Ca
1
1210.0
219.0
20.6
3.75
C.
Bronehial-Ca without B. (only men)
5
6.37 ± 3.25
1.14 ± 0.56
1.38 ± 0.93
0.26 ± 0.18
D.
All remaining Ca.
5
3.57 ± 1.56
0.58 ± 0.31
1.03 ± 0.50
0.17 ± 0.10
E.
Whole group (BOIDO) without extreme min. and max. a) men b) women
85
3.17 ± 3.09
0.57 ± 0.56
0.67 ± 0.94
0.12 ± 0.17
53 32
4.04 ± 3.47 1.74 ± 1.50
0.73 ± 0.62 0.31 ± 0.32
0.87 ± 1.13 0.35 ± 0.31
0.16 ± 0.21 0.06 ± 0.06
E.a without Bronehial-Ca
48
3.80 ± 3.44
0.69 ± 0.61
0.81 ± 1.15
0.15 ± 0.21
F.
) "Individual" conversion factor.
200 .
J. Seemann et al.
Table 3 shows the Cr and Ni concentrations of the examined lung tissue specimens. The percentage deviation of an age-, sex- and regional-adjusted "expected value" is also given in order to provide information about the proportions. The former were determined in previous measurement series (Table 5)4. The ratio of the specimen weight in a formalin-fixed condition to that of dry weight amounts to a mean-value x = 6.8 and the deviation is characterized by a CV of 19% (Table 3). The metal content in the tissue abrasion is higher than in the tissue itself. Due to the abrasion quantity, that characteristic is insignificant for the mass balance. The measured Cr and Ni concentrations of the conservation solutions are mostly lower than those of the primary solution (therefore marked with a minus sign). The adsorption at the inner surface of the container is considerable in one case, otherwise relatively small (Table 4). Discussion The increase in pulmonary Cr and Ni content is caused by certain working places (local) but also by a steady low level exposure to air pollution (regional) and by smoking habits with its uptake of Cr and Ni 3 • Probably a greater portion of the chromium added to the atmosphere by man is initially present in the hexavalent oxidation state; some of this will be reduced by organic matter in the air and some will be deposited on vegetation, soils and in the water2. With regard to the oxidation state and chemical binding form during uptake by inhalation, little is known as to where and to which amount pulmonary accumulated Cr and Ni originate from. Pitfalls of trace metal analyses are numerous. This particularly holds true for Cr and Ni measurements of human lung tissue 3,8. Thus, the value of those measurements is not better than their validity and reliability. However, control of accuracy is difficult due to the lack of appropriate standard reference material. Once more efforts have recently been made to provide this material, including that for nickel l . The prevention and control of extrinsic contamination by chromium and nickel during specimen collection and processing are discussed in full detail elsewhere3, 8. The very complex problems and possibilities of errors of the actual chemical analysis can not be discussed here. The metal concentrations in the primary and conservation solutions are very low. Usually certifical aqueous standard solutions are relatively unstable over a long period of time, because ions then move out of the solution to the container wall and therefore can not be measured any longer. This effect arises particularly with aqueous solutions near a neutral ph-valueS. The formalin in use has a ph-value of 4-4.5 and is therefore within that range. The measured effect of ion movement can be tolerated but has to be taken into account (Table 1). Concerning the validity of the measurements it is important to be able to estimate the shifts of the metal contents of different sections of the total system and to ascertain which contamination effects are possible.
Tissue: Under these circumstances it can not be determined exactly, whether and how much the tissue specimens are contaminated (through common metal alloy instruments, dirty container and contaminated solution, handling etc.). However, the comparison with a previous test group from the same residential area, the Ruhr District, shows that the pulmonary Cr and Ni concentrations are within the same range (Tables 3 and 5). Previous measurements revealed that age-, sex- and regionally adjusted "expected values" can be calculated for chromium and nickel (which in part are obviously accumulated in the lung tissue with a long biological half-time4 • In this respect, the difference between the observed and expected values is interesting, too (Table 3: % dev.). Load dependent interindividual differences of course still occur. It must also be taken into account that there is a considerable intraindividual variability of Cr and Ni concentrations in lung tissue3, 6; in our measurement series it was sectoral less than 10% for chromium and less than 15% for nickel at the lateral base of the right upper lobe 3 • It was evidently higher between remote bronchopulmonary segments, for instance between the apex and the base of the lung6• Therefore, a standardised procedure for specimen collection is a prerequisite for measured values to be comparable and interpretable. Furthermore, lyophilisation as well as the concentration reference to dry weight are necessary, because the ratio of formalin-fixed to dry lung tissue before and after lyophilisation interindividually resulted in a considerable deviation (Table 3: UF), depending upon (patho-)physiological conditions like hypostases etc.; this was similar in wet specimens (n = 258): x = 6.65, CV = 16%3. Tissue abrasion: The Cr/Ni concentration of the abrasion material is relatively high due to its surface area (Table 4). This is a consequence of metal adsorption on the one hand, on the other hand, it can be assumed that the abrasion material has mainly come off the contaminated surfaces of cut. The Cr/Ni content of the abrasion material was of little importance for the mass balance (Table 4). Thus, it may be neglected in practice. Besides, the determination of its metal content requires a lot of skill and time. Formalin: For the conservation of the examined specimens the 5% formalin filled in 10 I plastic containers from Fa. "Sealand Recycling" (Braunschweig/FRG) was used. It contained 0.6 ng Cr and 6.3 ng Ni/ml and with this (accidentially) a Cr and Ni content similar to the drinking water in Bochum (Gelsenwasser, water purification area Bochum-Stiepel, in late 1988): 1 ng Cr and 6 ng Ni/ml. The maximal total concentration of heavy metals in 37% formalin of Merck (stabilised with 10% methanol) is 2 ppm (the catalogue does not provide any information about formalin for histology). In connection with production-, dilution-, filling- and storage-procedures and container material remarkably higher Cr and Ni concentrations of the primary solution are possible. The maximum possible concentrations of CrH , CrO~- and NiH in water are higher by many orders of magnitude. In particular, it depends upon the presence of precipitant, e.g. with Cr H depending upon N~N02, or
Chromium and Nickel in Lung Tissue . 201
other substances disturbing the hydrolysis-equilibrium. For example, in the primary formalin solution in use 1 gil of the ions mentioned above was still well soluble. Therefore if there is any doubt, Cr and Ni concentrations of the formalin charges used for these measurements must be known or determined. The fact that there are almost equal Cr and Ni concentrations in the primary formalin solution and each conservation solution cannot be interpreted to the effect that formalin does not become contaminated in the process. It is rather more probable that all possible effects of Cr/Ni contamination (elution out of the specimens, wash off from the contaminated surface of cut, through common metal alloy instruments, further impurities through handling) are masked through adsorption on all existing surfaces in formalin (specimens, "natural" abrasion, plastic container) with the effect that low Cr and Ni concentrations similar to the primary solution remain (Table 2). Furthermore, the content of these metal traces is subject to their specific solubility-time pattern (Table 1). Plastic container: Container adhesions are of little consequence for the mass balance. This holds true with one exception, namely when it makes up more than one half the Ni content of a specimen of 1.43 g dry weight (Table 4: 4a). The distribution of Cr and Ni masses in all parts of the total system, the Cr/Ni concentration in formalin before and after tissue conservation and the seemingly realistic Cr/Ni concentrations in the individual postmortem specimens of lung provide good reasons to state that measurements of trace elements in formalin-fixed tissue can be justified even in normal cases. Singular relevant exogenous contaminations, as they occurred with Ni adhesions on the container surface of specimen 4a, can only be detected when the whole system is analysed, which requires a great deal of asservation and measurement procedures. The trace metal analysis of Cr and Ni in specimens fixed and stored in formalin can be justified for practical purposes, e.g. for questions concerning causality in the field of occupational diseases and workers' compensation 7,9, 10, since those usually are based on a relatively wide range of threshold values, as far as the Cr and Ni content of lung tissue serves as an indicator for preceding expositions to the causal factor. This procedure does not suffice for scientific measurement. Certain rules (••) must be followed in order to attain comparability of measured values and to reduce possible sources of error (.) to an acceptable degree: • Exogenous contaminations of specimen through specimen-collection, -transport, and -storage. •• Avoidance of contact with contaminated common metal alloy instruments (through autopsy instruments made of quartz-glass, titan etc.), with hands (by means
of contamination-free gloves), with surfaces (through contamination-free plates, packing material, etc.), with fluids like transudate and exsudate and conservation solutions as well as pollution (dust)3, 8. • Intrapulmonary variability of metal concentrations. •• Always collecting the specimen from the same area (at least exact description of the area of specimen-collection), measurement of many specimens and quantification of the pulmonary variability3, 8. • Different water content of (lung-)tissue. •• Standardised specimen preparation: freezing, lyophilisation and concentration reference to dry weight.3, 4, 8. • Dependence of tissue metal concentration on age, sex, occupation, place of residence and smoking habits (total inhalative dust load)3,4. •• Adequate personal data recording. References 1 Belliardo 11, Wagstaffe PJ (1988) BCR reference materials for food and agricultural analysis: an overview. Fresenius Z Anal Chern 332: 533-538 2 Cary EE (1982) Chromium in air, soil and natural waters. In: Langards S (Ed) Biological and Environmental Aspects of Chromium, pp 49-64. Elsevier Biomedical Press, Amsterdam 3 Kollmeier H, Muller KM, Seemann J, Rothe G, Wittig P, Schejbal V, Hummelsheim G (1988) Untersuchungen wr Chromund Nickel-Belastung der Lunge. Wirtschaftsverlag NW, Bremerhaven (BAU-Forschungsbericht Nr 548, pp 1-222) 4 Kollmeier H, Seemann J, Rothe G, Muller KM, Wittig P (1989) Age-, sex-, and regional-adjusted concentrations of chromium and nickel in lung tissue. Br J Int Med (submitted) 5 Nackowski SB, Putnam RD, Robbins DA, Varner MO, White LD, Nelson KW (1977) Trace metal contamination of evacuated blood collection tubes. Am Ind Hyg Assoc J 38: 503-508 6 Raithel HJ, Ebner G, Schaller KH, Schellmann B, Valentin H (1987) Problems in establishing norm values for nickel and chromium concentrations in human pulmonary tissue. Am J Ind Med 12: 55-70 7 Raithel HJ, Schaller KH, Reith A, Svenes KB, Valentin H (1988) Investigations on the quantitative determination of nickel and chromium in human lung tissue. Industrial medical, toxicological, and occupational medical expertise aspects. Int Arch Occup Environ Health 60: 55-66 8 Seemann J, Wittig P, Kollmeier H, Rothe G (1985) Analytische Bestimmung von Cd, Pb, Zn, Cr und Ni in Humangewebe. Lab med 9: 294-299 9 Turhan U, Wollburg C, Angerer J, Szadkowski D (1985) Der Nickelgehalt menschlicher Lungen und seine Bedeutung fur die Beurteilung berufsbedingter Bronchialkarzinome. Arbeitsmed Sozialmed Praventivmed 20: 277-281 10 Zober A (1979) On the problems of evaluating bronchial carcinoma after exposure to chromium compounds. Int Arch Occup Environ Health 43: 107-121
Received June 7, 1989 . Accepted July 28, 1989
Key words: Lung tissue - Formalin-fixed tissue - Chromium/nickel concentration - Atomic absorption spectrometry (AAS) Dr. J. Seemann, Bundesanstalt fur Arbeitsschutz, Vogelpothsweg 50-52,4600 Dortmund 1, FRG
Trace Metal Analysis of Chromium and Nickel in Lung Tissue Fixed and Stored in Formalin 1 J. Seemann, P. Wittig and H. Kollmeier Bundesanstalt fur Arbeitsschutz, Dortmund, FRG
K.-M. Muller and V. Schejbal Institut fUr Pathologie der Berufsgenossenschaftlichen Krankenanstalten "Bergmannsheil Bochum'; Universitatsklinik, Bochum, FRG
SUMMARY
In an attempt to answer the question whether the determination of chromium and nickel concentration in lung tissue, fixed and stored in formalin is still tolerable despite the related sources oferror, the entire system (tissue, tissue abrasion, formalin, plastic container) was analysed by means of f/ameless atomic absorption spectrometry. It appears that such a procedure can be justified for practical purposes. In order to reduce possible errors, well defined rules are to be observed during specimen collection and processing and specialities have to be taken into account.
Introduction Tissue residues of toxic/carcinogenic material in organs (in which uptake and/or - maximal - deposition take place) serve as an indicator for a preceding total load and are used as important additional information for the determination of causality between exposition and disease. Since trace analysis is almost exclusively performed in order to obtain these results, all sources of error have serious consequences. This is true for exogenous contaminations in particular, especially through material which is more or less ubiquitous. We examined whether under the circumstances mentioned above trace element measurements (in this case of chromium (Cr) and nickel (Ni)) can still be carried out on lung tissue specimens being fixed and stored in formalin. For that purpose the mass distribution of these metals in the entire system must be determined: tissue specimen, tissue abrasion, formalin, plastic contain-
er. 1 Dedicated to Prof. Dr. Dr. h.c. Franz Biichner on the occasion of his 95th birthday.
© 1990 by Gustav Fischer Verlag, Stuttgart
Material and Methods The Cr and Ni contents of 8 postmortem lung specimens were determined, which belonged to 4 men who had died in 1988/89. The autopsies were carried out at the Institute of Pathology (Berufsgenossenschaftliche Krankenanstalten "Bergmannsheil Bochum" - Universitiitsklinik). Table 3 presents the data of the cases. 1. Place of residence (pIRe): Hattingen, 40 cigarettes per day for many years, cigars only about 5 years before his death, coal miner between 1936 and 1975, metastasising small cell bronchial carcinoma, condition after irradiation, very high pulmonary total dust content and silicosis II. 2. Oer-Erkenschwick and Datteln (pIRe), non-smoker, coal miner for 32 years, metastasising squamous cell carcinoma of the right lower lobe bronchus, very high pulmonary total dust content and fibrous anthraco-silicosis III. 3. Bochum (pIRe), 40 cigarettes per day since 1948, chronic alcoholic, plumber (soldering and welding) from 1944-1985, metastasising small cell bronchial carcinoma. 4. Liinen (pIRe), presumably heavy smoker, metastasising squamous cell carcinoma of the 2nd right bronchus segment. The postmortem specimens were taken in the usual manner with common metal alloy instruments and stored in plastic containers, filled with 5% formalin. 0344-0338/90/0186-0197$3.50/0
198 .
J. Seemann et al.
The Cr and Ni concentrations were determined by means of flameless atomic absorption spectrometry (AAS) after Iyophilisation, wet ashing under pressure, chelating, and extraction. The analytical method used has been described elsewhere 3,8. Four specimens of 100 mg were analysed from each tissue, applying standard additions before ashing. Measured or mean values that vary considerably were rejected. The following exclusion criteria were employed: coefficient of variation (CV) ;:: 3% with 4 measured values, as well as r::; 0.997 or CV of the slope and axial section of the linear correlation;:: 10%.
Table 1. Recovery in percent of metal ions in 5% formalin 50 ng/ml
5 hours
24 hours
CrH
96% 96% 107%
66% 89% 105%
CrO~
NiH
Table 2. Specimens fixed and stored in formalin and Cr and Ni concentration of the conservation solution No.
tissue g
Quantityl tissuesolution abrasion ml g
Difference to primary solution2 chromium nickel ng/ml ng/ml
1.
5.7
0.10
40
-0.3
+ 13.7
2a. 2b.
29.3 14.9
0.35 0.01
16 4
-0.1 +0.5
+ 0.4 + 1.1
3a. 3b.
44.1 26.9
0.04 0.15
12 31
-0.1 -0.5
3.6 4.8
4a. 4b. 4c.
8.9 15.3 18.3
0.05 0.01 0.05
15 16 16
-1.0
3.1 2.2 + 1.0
-1.1
-1.0
1 Before Iyophilisation. - 2 In the primary formalin-solution: 0.6 ng Crimi and 6.3 ng Ni/ml.
The analytical characteristics were: - detection limit (threefold variation from five blind values): Cr 8 ng/g, Ni 5 ng/g dry weight; - precision: Cr 1.9%, Ni 11.7%; - accuracy (checked with NBS SRM 1577 bovine liver) Cr 106 ng/g dry weight (certification value: 88 ± 12 ng/g) (the National Bureau of Standards (NBS), Washington D.C., does not provide Standard Reference Material (SRM) for Ni determination). Metal ion concentrations in tissue abrasions were determined in the following way: after removal of the tissue the solution containing tissue abrasions was centrifuged, the jutting-out material pipetted, the residue lyophilised. After weighing the dried abrasion material, it was analysed in the same way as the tissue specimens. To prove the validity of the method the question arises, how much of minor concentrations of metal ions in formalin can be recovered with time. To determine the amount of metal ions in the conservation solutions, they were centrifuged and measured directly. They were calibrated in the graphite tube applying standard additions before ashing. Metal ions at the inner surface of the plastic container were removed by exposure to 6% nitric acid for half an hour and then measuring them directly. Mass balance analyses were carried out in cases 4a-c (Tables 2-4).
Results The movement and the related recovery of small amounts of Cr3+-, CrO~- and NiH-ions in 5% formalin are fairly limited (Table 1). The Cr and Ni concentration of the primary formalin solution2 is more or less identical with that of the conservation solutions of the individual specimens (Table 2); minor deviations do only exist in relation to the Ni concentration, and this more evidently in only one case (Table 2: 1). 2 By Fa. "Sealand Recycling" (Braunschweig, FRG) 5% in a 10 Liter plastic container.
Table 3. Cr and Ni concentration of 8 lung tissue specimens from 4 autopsies No.
Age
Specimen from
1.
72
1 L(ower} L(obe}
4.39
2a. 2b.
81
rt lung I lung
7.51 2.77
3a. 3b.
58
1 U(pper}L 1 LL
4a. 4b. 4c.
76
rt UL UL UL
rt rt
dev. 2 %
Cf3
dry I-tg/g
Nickel form. l I-tg/g
dev. 2 %
0.529
33
8.3
0.547
0.066
-9
1.25 0.318
82 -33
6.0 8.7
0.846 0.628
0.141 0.072
6 -21
13.4 12.8
2.39 2.03
480 450
5.6 6.3
2.98 3.94
0.523 0.625
670 910
1.58 2.18 2.71
0.255 0.427 0.437
-57 -40 -26
6.2 5.1 6.2
0.297 0.264 0.414
0.048 0.052 0.067
-57 -61 -39
dry I-tg/g
Chromium form. l I-tg/g
1 Formalin-fixed tissue before lyophilisation. - 2 Dev.: % deviation from "expected value". weight after and before lyophilisation: x = 6.8, CV = 19%.
3
CF: conversion factor: ratio of
Chromium and Nickel in Lung Tissue . 199 Table 4. Mass balance of the specimens 4a-e Tissue, dry 4a: weight
(g)
Abrasion dry
1.43
Formalin solution)
0.0088
14.6
Container adhesion
Sum
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
1.58 2.26 99.6
2.41 0.02 0.9
-0.001 -0.02 -0.9
0.01 0.4
2.27 100.0
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.297 0.425 43.1
1.67 0.015 1.5
- 0.003 - 0.044 -4.5
0.590 59.8
0.986 100.0
0.0006
16.3 -0.001 -0.02 -0.3
0.01 0.02
6.50 100.0
0.013 1.7
0.784 100.0
4b: weight
(g)
2.98
16.0 0.01 0.2
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
2.18 6.50 100.0
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.264 0.787 100.4
28.2 0.017 2.2
-0.002 -0.033 -4.2
3.00
0.0078
16.3
7.91 0.06 0.7
-0.001 -0.02 -0.2
0.01 0.01
8.18 100.0
10.8 0.084 6.2
+0.001 +0.016 +1.2
0.012 0.9
1.352 100.0
4e: weight
(g)
Cr cone. (f-tg/g) Cr abs. (f-tg) Cr reI. (%)
2.71 8.13 99.4
Ni cone. (f-tg/g) Ni abs. (f-tg) Ni reI. (%)
0.414 1.240 91.7
) In relation to the primary solution.
Table 5. Measured values (x + s) of previous test groups3,4 n
Chromium dry x±s f-tg/g 0.001
wet) x±s f-tg/g 0.0002
Nickel dry x ± s f-tg/g 0.002
wet) x±s f-tg/g 0.0003
A.
Extreme min. stillbirth
1
B.
Extreme max. Bronehial-Ca
1
1210.0
219.0
20.6
3.75
C.
Bronehial-Ca without B. (only men)
5
6.37 ± 3.25
1.14 ± 0.56
1.38 ± 0.93
0.26 ± 0.18
D.
All remaining Ca.
5
3.57 ± 1.56
0.58 ± 0.31
1.03 ± 0.50
0.17 ± 0.10
E.
Whole group (BOIDO) without extreme min. and max. a) men b) women
85
3.17 ± 3.09
0.57 ± 0.56
0.67 ± 0.94
0.12 ± 0.17
53 32
4.04 ± 3.47 1.74 ± 1.50
0.73 ± 0.62 0.31 ± 0.32
0.87 ± 1.13 0.35 ± 0.31
0.16 ± 0.21 0.06 ± 0.06
E.a without Bronehial-Ca
48
3.80 ± 3.44
0.69 ± 0.61
0.81 ± 1.15
0.15 ± 0.21
F.
) "Individual" conversion factor.
200 .
J. Seemann et al.
Table 3 shows the Cr and Ni concentrations of the examined lung tissue specimens. The percentage deviation of an age-, sex- and regional-adjusted "expected value" is also given in order to provide information about the proportions. The former were determined in previous measurement series (Table 5)4. The ratio of the specimen weight in a formalin-fixed condition to that of dry weight amounts to a mean-value x = 6.8 and the deviation is characterized by a CV of 19% (Table 3). The metal content in the tissue abrasion is higher than in the tissue itself. Due to the abrasion quantity, that characteristic is insignificant for the mass balance. The measured Cr and Ni concentrations of the conservation solutions are mostly lower than those of the primary solution (therefore marked with a minus sign). The adsorption at the inner surface of the container is considerable in one case, otherwise relatively small (Table 4). Discussion The increase in pulmonary Cr and Ni content is caused by certain working places (local) but also by a steady low level exposure to air pollution (regional) and by smoking habits with its uptake of Cr and Ni 3 • Probably a greater portion of the chromium added to the atmosphere by man is initially present in the hexavalent oxidation state; some of this will be reduced by organic matter in the air and some will be deposited on vegetation, soils and in the water2. With regard to the oxidation state and chemical binding form during uptake by inhalation, little is known as to where and to which amount pulmonary accumulated Cr and Ni originate from. Pitfalls of trace metal analyses are numerous. This particularly holds true for Cr and Ni measurements of human lung tissue 3,8. Thus, the value of those measurements is not better than their validity and reliability. However, control of accuracy is difficult due to the lack of appropriate standard reference material. Once more efforts have recently been made to provide this material, including that for nickel l . The prevention and control of extrinsic contamination by chromium and nickel during specimen collection and processing are discussed in full detail elsewhere3, 8. The very complex problems and possibilities of errors of the actual chemical analysis can not be discussed here. The metal concentrations in the primary and conservation solutions are very low. Usually certifical aqueous standard solutions are relatively unstable over a long period of time, because ions then move out of the solution to the container wall and therefore can not be measured any longer. This effect arises particularly with aqueous solutions near a neutral ph-valueS. The formalin in use has a ph-value of 4-4.5 and is therefore within that range. The measured effect of ion movement can be tolerated but has to be taken into account (Table 1). Concerning the validity of the measurements it is important to be able to estimate the shifts of the metal contents of different sections of the total system and to ascertain which contamination effects are possible.
Tissue: Under these circumstances it can not be determined exactly, whether and how much the tissue specimens are contaminated (through common metal alloy instruments, dirty container and contaminated solution, handling etc.). However, the comparison with a previous test group from the same residential area, the Ruhr District, shows that the pulmonary Cr and Ni concentrations are within the same range (Tables 3 and 5). Previous measurements revealed that age-, sex- and regionally adjusted "expected values" can be calculated for chromium and nickel (which in part are obviously accumulated in the lung tissue with a long biological half-time4 • In this respect, the difference between the observed and expected values is interesting, too (Table 3: % dev.). Load dependent interindividual differences of course still occur. It must also be taken into account that there is a considerable intraindividual variability of Cr and Ni concentrations in lung tissue3, 6; in our measurement series it was sectoral less than 10% for chromium and less than 15% for nickel at the lateral base of the right upper lobe 3 • It was evidently higher between remote bronchopulmonary segments, for instance between the apex and the base of the lung6• Therefore, a standardised procedure for specimen collection is a prerequisite for measured values to be comparable and interpretable. Furthermore, lyophilisation as well as the concentration reference to dry weight are necessary, because the ratio of formalin-fixed to dry lung tissue before and after lyophilisation interindividually resulted in a considerable deviation (Table 3: UF), depending upon (patho-)physiological conditions like hypostases etc.; this was similar in wet specimens (n = 258): x = 6.65, CV = 16%3. Tissue abrasion: The Cr/Ni concentration of the abrasion material is relatively high due to its surface area (Table 4). This is a consequence of metal adsorption on the one hand, on the other hand, it can be assumed that the abrasion material has mainly come off the contaminated surfaces of cut. The Cr/Ni content of the abrasion material was of little importance for the mass balance (Table 4). Thus, it may be neglected in practice. Besides, the determination of its metal content requires a lot of skill and time. Formalin: For the conservation of the examined specimens the 5% formalin filled in 10 I plastic containers from Fa. "Sealand Recycling" (Braunschweig/FRG) was used. It contained 0.6 ng Cr and 6.3 ng Ni/ml and with this (accidentially) a Cr and Ni content similar to the drinking water in Bochum (Gelsenwasser, water purification area Bochum-Stiepel, in late 1988): 1 ng Cr and 6 ng Ni/ml. The maximal total concentration of heavy metals in 37% formalin of Merck (stabilised with 10% methanol) is 2 ppm (the catalogue does not provide any information about formalin for histology). In connection with production-, dilution-, filling- and storage-procedures and container material remarkably higher Cr and Ni concentrations of the primary solution are possible. The maximum possible concentrations of CrH , CrO~- and NiH in water are higher by many orders of magnitude. In particular, it depends upon the presence of precipitant, e.g. with Cr H depending upon N~N02, or
Chromium and Nickel in Lung Tissue . 201
other substances disturbing the hydrolysis-equilibrium. For example, in the primary formalin solution in use 1 gil of the ions mentioned above was still well soluble. Therefore if there is any doubt, Cr and Ni concentrations of the formalin charges used for these measurements must be known or determined. The fact that there are almost equal Cr and Ni concentrations in the primary formalin solution and each conservation solution cannot be interpreted to the effect that formalin does not become contaminated in the process. It is rather more probable that all possible effects of Cr/Ni contamination (elution out of the specimens, wash off from the contaminated surface of cut, through common metal alloy instruments, further impurities through handling) are masked through adsorption on all existing surfaces in formalin (specimens, "natural" abrasion, plastic container) with the effect that low Cr and Ni concentrations similar to the primary solution remain (Table 2). Furthermore, the content of these metal traces is subject to their specific solubility-time pattern (Table 1). Plastic container: Container adhesions are of little consequence for the mass balance. This holds true with one exception, namely when it makes up more than one half the Ni content of a specimen of 1.43 g dry weight (Table 4: 4a). The distribution of Cr and Ni masses in all parts of the total system, the Cr/Ni concentration in formalin before and after tissue conservation and the seemingly realistic Cr/Ni concentrations in the individual postmortem specimens of lung provide good reasons to state that measurements of trace elements in formalin-fixed tissue can be justified even in normal cases. Singular relevant exogenous contaminations, as they occurred with Ni adhesions on the container surface of specimen 4a, can only be detected when the whole system is analysed, which requires a great deal of asservation and measurement procedures. The trace metal analysis of Cr and Ni in specimens fixed and stored in formalin can be justified for practical purposes, e.g. for questions concerning causality in the field of occupational diseases and workers' compensation 7,9, 10, since those usually are based on a relatively wide range of threshold values, as far as the Cr and Ni content of lung tissue serves as an indicator for preceding expositions to the causal factor. This procedure does not suffice for scientific measurement. Certain rules (••) must be followed in order to attain comparability of measured values and to reduce possible sources of error (.) to an acceptable degree: • Exogenous contaminations of specimen through specimen-collection, -transport, and -storage. •• Avoidance of contact with contaminated common metal alloy instruments (through autopsy instruments made of quartz-glass, titan etc.), with hands (by means
of contamination-free gloves), with surfaces (through contamination-free plates, packing material, etc.), with fluids like transudate and exsudate and conservation solutions as well as pollution (dust)3, 8. • Intrapulmonary variability of metal concentrations. •• Always collecting the specimen from the same area (at least exact description of the area of specimen-collection), measurement of many specimens and quantification of the pulmonary variability3, 8. • Different water content of (lung-)tissue. •• Standardised specimen preparation: freezing, lyophilisation and concentration reference to dry weight.3, 4, 8. • Dependence of tissue metal concentration on age, sex, occupation, place of residence and smoking habits (total inhalative dust load)3,4. •• Adequate personal data recording. References 1 Belliardo 11, Wagstaffe PJ (1988) BCR reference materials for food and agricultural analysis: an overview. Fresenius Z Anal Chern 332: 533-538 2 Cary EE (1982) Chromium in air, soil and natural waters. In: Langards S (Ed) Biological and Environmental Aspects of Chromium, pp 49-64. Elsevier Biomedical Press, Amsterdam 3 Kollmeier H, Muller KM, Seemann J, Rothe G, Wittig P, Schejbal V, Hummelsheim G (1988) Untersuchungen wr Chromund Nickel-Belastung der Lunge. Wirtschaftsverlag NW, Bremerhaven (BAU-Forschungsbericht Nr 548, pp 1-222) 4 Kollmeier H, Seemann J, Rothe G, Muller KM, Wittig P (1989) Age-, sex-, and regional-adjusted concentrations of chromium and nickel in lung tissue. Br J Int Med (submitted) 5 Nackowski SB, Putnam RD, Robbins DA, Varner MO, White LD, Nelson KW (1977) Trace metal contamination of evacuated blood collection tubes. Am Ind Hyg Assoc J 38: 503-508 6 Raithel HJ, Ebner G, Schaller KH, Schellmann B, Valentin H (1987) Problems in establishing norm values for nickel and chromium concentrations in human pulmonary tissue. Am J Ind Med 12: 55-70 7 Raithel HJ, Schaller KH, Reith A, Svenes KB, Valentin H (1988) Investigations on the quantitative determination of nickel and chromium in human lung tissue. Industrial medical, toxicological, and occupational medical expertise aspects. Int Arch Occup Environ Health 60: 55-66 8 Seemann J, Wittig P, Kollmeier H, Rothe G (1985) Analytische Bestimmung von Cd, Pb, Zn, Cr und Ni in Humangewebe. Lab med 9: 294-299 9 Turhan U, Wollburg C, Angerer J, Szadkowski D (1985) Der Nickelgehalt menschlicher Lungen und seine Bedeutung fur die Beurteilung berufsbedingter Bronchialkarzinome. Arbeitsmed Sozialmed Praventivmed 20: 277-281 10 Zober A (1979) On the problems of evaluating bronchial carcinoma after exposure to chromium compounds. Int Arch Occup Environ Health 43: 107-121
Received June 7, 1989 . Accepted July 28, 1989
Key words: Lung tissue - Formalin-fixed tissue - Chromium/nickel concentration - Atomic absorption spectrometry (AAS) Dr. J. Seemann, Bundesanstalt fur Arbeitsschutz, Vogelpothsweg 50-52,4600 Dortmund 1, FRG
