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Analytical Methodology for Biological Monitoring of Chromium
REGULATORY TOXICOLOGY AND PHARMACOLOGY ARTICLE NO.
26, S86–S93 (1997)
RT971145
Analytical Methodology for Biological Monitoring of Chromium C. Harzdorf* and J. Lewalter† *Business Group Inorganics and †Medical Department, Bayer AG, D-51368 Leverkusen, Germany Received May 20, 1997
Analytical aspects associated with biological and ambient monitoring are outlined with an emphasis on processing samples at very low concentrations and optimization of analytical methods. Verification of chromium determination in body fluids by means of ‘‘Round Robin’’ tests is discussed. In addition, an approach for indirect valency discrimination of chromium species relevant in biological monitoring is presented. Application of voltammetry for model tests is described. This is aimed at studying the reaction of hexavalent chromium in human plasma. As a result, a natural reducing capacity of plasma of up to 2 ppm chromium(VI) was detected and quantified as an individual biological index. Furthermore, the effect of ascorbic acid on the reduction of chromium(VI) in plasma is demonstrated and quantitative bases for therapy in cases of accidental intoxication are discussed. q 1997 Academic Press
INTRODUCTION
Biological monitoring of inorganic compounds has a long tradition, and numerous basic and special publications have been produced. Of these, particular attention has been paid to chromium compounds because of the significantly different effects that species of both main valency states exert on man. Consequently, attempts have been made to distinguish between chromium(III) and chromium(VI) compounds. In practice, however, direct analytical determination of both types of chromium species is difficult to achieve in biological matter, because the chromium valency states are unstable in redox-active matrices. Therefore, indirect methods of evaluating chromium valencies in biological monitoring have been considered. Moreover, determination of the total chromium content of biological matter at very low concentrations is of primary importance in analytical methodology for biological monitoring. In view of the comprehensive literature available in the field of biological monitoring of chromium and associated analytical work, we will try not to present a
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DETERMINATION OF CHROMIUM IN BIOLOGICAL SAMPLES AT LOW CONCENTRATION LEVELS
The basic tool for the determination of chromium in biological material is electrothermal atomic absorption spectrometry (ET-AAS). Although chromium is considered an ‘‘easy’’ parameter in AAS, various difficulties arise at very low concentration levels and in complex matrices loaded with organic matter and/or electrolytes. These samples give rise to interfering effects during electrothermal processing in the graphite tube comprising primarily nonspecific background absorption due to matrix components and formation of chromium carbide, thus preventing chromium from atomization. To overcome interference like this, application of an efficient background compensation system and selection of the adequate type of graphite tube are of primary importance. Apart from interference occurring directly during measurement, peripheral effects resulting from sampling and sample handling also play an important role. The obvious analytical problems in this field are reflected by a number of publications on chromium determination in biological samples and by strikingly inconsistent results of Round Robin tests with biological material. Relevant studies and reviews have been published by Fishbein (1987), Versieck and Cornelis (1980), Guthrie et al. (1978), Slavin (1981), Parr (1977), and U.S. EPA (1983). In the following text, we report on our attempts to eliminate sources of error from analytical methodology. Sampling and sample handling. In our processing scheme, errors by contamination during sampling and handling prior to measurement are essentially minimized in that samples are taken and preserved for analysis in the medical department, away from where inorganic materials such as chromium compounds are handled. Thus, the risk of contamination during sampling and preservation is very low. The samples enter
S86
0273-2300/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
survey but to focus primarily on selected aspects in analytical methodology and interpretation of results. An outline of this presentation is given in Table 1.
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TABLE 1 Disposition and Primary Aspects • Determination of chromium in biological samples at low concentration levels Optimization of AAS techniques/verification of methods • Approaches for indirect valence discrimination of chromium species in biological monitoring • Correlation between ambient and biological monitoring • Behavior of hexavalent chromium in plasma Reducing capacity of plasma/detoxification
the analytical laboratory area immediately prior to measurement, where they are processed under the usual conditions of trace analysis. Sample preservation of whole blood and plasma is done with EDTA. In addition, a surfactant (Triton 100) is added to prevent flocculation and to inhibit nonhomogeneity. Urine samples are stabilized by adding small amounts of hydrochloric acid. Once preserved, samples are stable for at least 24 hr. Nevertheless, measurement of the samples should be attempted as soon as possible. Optimization of ET-AAS conditions. As mentioned above, errors in ET-AAS result primarily from undesired chemical reactions during heating in the graphite furnace and from incorrect compensation of nonspecific spectral effects stemming from matrix components. Thus, careful optimization of experimental conditions is essential. In Fig. 1, the effect of the type of graphite tube is demonstrated with samples of whole blood containing 2 ng/ml chromium. The graphs of both the net signal (specific absorption) and the background signal (nonspecific absorption) reveal that only the pyrolytically coated tube yields a good result. The effect of background compensation was studied with both the conventional tungsten/halogen lamp and the magnetic Zeeman system. Whole blood containing 5 ng/ml chromium was tested. The resulting signal traces for specific and nonspecific absorption are shown in Fig. 2. On qualitative evaluation, both modes of background compensation work satisfactorily. On quantitative evaluation of results from routine measurements, however, a slight advantage for the conventional tungsten/ halogen system can be observed for the lowest chromium concentrations. This finding is likely due to bigger net signals, inherently produced by conventional background compensation systems. In this context, it should be emphasized that the widespread deuterium background compensation, not presented in the figures, is clearly inferior to the systems studied here, because of its insufficient energy output in the range of the chromium wavelength. Unfortunately, a number of atomic absorption spectro-
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meters are exclusively equipped with the deuterium system. Optimal instrumental ET-AAS conditions for chromium in body fluids are summarized in Table 2. Optimization was carried out with samples of whole blood, representing the most difficult analytical matrix. Preparations of erythrocyte fractions, plasma, and urine samples can be processed under the same conditions. Verification of methods. Complementing optimization of analytical methodology, verification and validation of methods are important tasks in analytical work for biological monitoring. In addition to internal laboratory work aimed at determining the performance data and checking the methods with certified standards, participation in Round Robin quality control tests is a valuable measure to check the reliability of analytical laboratory work. The experience of 11 years of Bayer’s participation in such Round Robin tests for whole blood and urine is listed in Tables 3 and 4. The tolerance ranges of the Round Robin tests have gradually become smaller in the course of time. This tendency clearly reflects better analytical performance of the participating laboratories due to optimization of methods and improved techniques in instrumentation. In addition to the measures for checking the reliability of analytical methods, the most effective way of checking the validity of analytical results is the application of an additional method based on a different analytical technique. For chromium determination, we prefer ICP–MS with 51Cr. Performance characteristics of this method are equivalent to ET-AAS; thus, an authentic verification of especially important data is possible. APPROACHES TO INDIRECT VALENCY DISCRIMINATION OF CHROMIUM SPECIES IN BIOLOGICAL MONITORING
Despite excellent performance of atomic spectrometric methods, these techniques suffer from the inherent limitation that only element concentrations can be determined. Valency discrimination is impossible to achieve unless additional analytical steps are applied prior to spectrometric measurement. Typical valency-specific methods such as spectrophotometry and polarography fail in determining chromium in body fluids directly, partly because of matrix interference (spectroscopy) and partly because of insufficient sensitivity for physiological concentration levels (polarography). Chelation/extraction methods have been proposed in this field. These methods are based on valency-specific reactions to form a chromium complex, followed by extraction and ET-AAS measurement of the extract. In our experience, these methods do not work reliably in biological matter due to side reactions with matrix components.
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FIG. 1.
Comparison of types of graphite tube: whole blood, 2 ng/ml Cr.
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FIG. 2. Comparison of mode of background compensation: whole blood, 5 ng/ml Cr.
In view of the lack of direct methods for valency discrimination, it was necessary to develop measures for indirect estimation of valency states of chromium species occurring in biological monitoring. A model was established based on the following concept. It is well known that hexavalent chromium anions, after having entered the blood circulation, rapidly pass the cell membranes of erythrocytes via the anion channel mechanism. Inside the cell, chromium(VI) is reduced by cell constituents to form chromium(III) complexes. These complexes, owing to their size, are unable to leave the cell in a reverse direction. Thus, a trapping mechanism for chromium(VI) exists with the erythrocytes representing a specific target organ for hexavalent chromium. Hence, the chromium concentration in the erythrocyte fraction, carefully prepared from whole
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blood, provides a valuable indicator for estimating both the external exposure and the internal stress from hexavalent chromium. Thus, this mode of biomonitoring is of important diagnostic value (Lewalter et al., 1985, 1990; Wiegand et al., 1985). However, it must be taken into consideration that chromium, once trapped in the erythrocyte, persists there for the lifetime of the cell, i.e., for about 115 days. Therefore, chromium concentration in the erythrocyte fraction reflects a mean level over a certain period of time rather than a momentary situation. In this context, it must be emphasized that exclusively soluble chromates of small particle size are able to undergo the mechanism described. Particles bigger than 5 mm are ‘‘filtered’’ in the lung and do not reach the blood circulation. Also, chromium trioxide is essentially
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TABLE 2 Instrumental Conditions in ET-AAS for Biological Samples Furnace
TABLE 4 Results of Annual Round Robin Tests Organized by the Institute for Occupational and Environmental Medicine, Erlangen, Germany
Pyrolytically coated tube (no platform) Temperature program
Function
Step
Temp
Ramp
Hold
Drying
1 2 3
90 140 350
15 25 20
5 15 10
Ashing
4 5
500 1250
20 25
10 15
Atomization
6
2300
0
5
Boost
7
2650
2
2
Spectrometer
Background compensation Zeeman or tungsten/halogen lamp Wavelength: 357.9 nm Spectral band width: 0.7 nm
Year
Nominal value
Result Bayer
Tolerance range
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995
2.8 7.4 41.1 44.0 5.7 5.3 13.7 29.3 39.8 12.1 5.4
3.0 7.0 42.0 48.0 5.0 5.0 12.6 29.1 40.8 12.0 4.9
1.5–4.1 5.4–9.4 18.7–53.5 31.6–56.4 4.3–7.0 4.0–6.7 10.1–17.3 24.4–34.3 37.7–45.9 10.0–14.2 4.3–6.5
Note. Results in mg/liter Cr. Samples: human urine (pool).
CORRELATION BETWEEN AMBIENT AND BIOLOGICAL MONITORING
not transported to the blood circulation because it is rapidly reduced due to its acidic nature and the reducing environment in the lung. Compared to the high specificity for Cr(VI) obtained from the chromium concentration in the erythrocyte fraction, chromium determination in urine is of less selective significance because it reflects the entire renal excretion resulting from several external sources of exposure and from different pathways through the organism. Nonetheless, urinary chromium provides an additional measure of estimating overall exposure, because most of the chromium intake is finally removed by renal excretion. Moreover, urinary chromium can give an answer on actual exposure because delay between exposure and excretion is comparatively small in most cases.
TABLE 3 Results of Annual Round Robin Tests Organized by the Institute for Occupational and Environmental Medicine, Erlangen, Germany Year
Nominal value
Result Bayer
Tolerance range
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995
10.5 5.7 17.7 21.2 10.6 6.6 9.7 21.6 12.0 5.9 9.8
12.0 6.0 16.5 25.0 12.0 7.9 10.6 20.2 12.8 6.8 8.9
7.8–13.2 3.9–7.4 14.6–20.9 17.6–24.8 8.5–12.8 4.8–8.3 7.3–12.1 17.6–25.6 10.1–13.8 4.8–6.9 8.4–11.2
Note. Results in mg/liter Cr. Samples: whole blood (pool).
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Although biological monitoring is of primary importance in evaluating occupational health effects, ambient monitoring, reflecting exposure, represents a complementary measure in occupational health care. In many countries, there is a requirement to carry out exposure measurements in order to comply with legislation. In addition to biological monitoring, direct, valencyspecific determination of chromium species is also possible in ambient monitoring, although analysis is difficult since redox effects cannot be completely avoided if sampling, sample handling, and analytical finish are not done with extreme care. At present, filter methods have been established for air sampling at the workplace (fixed-site sampling) and in man (personal sampling). For analytical processing, the filter is extracted and the leach and the residual filter load are analyzed separately. Common methods, also the officially recommended ones in the United States and Europe, use an aqueous sodium hydroxide/sodium carbonate solution for extraction and it is assumed that with this method, no redox reactions occur during leaching. Unfortunately, this assumption does not hold true. Model tests revealed that during alkaline leaching considerable amounts of trivalent chromium compounds may be oxidized, thus simulating positive results for hexavalent chromium in air (see Table 5). The amount of chromium(VI) produced during filter leaching depends on the solubility of chromium(III) compounds in the alkaline extraction solution. The problem can be overcome by leaching the filter load with water or with diluted sodium hydrogen carbonate solution at pH 8.4. In this case no chromium oxidation could be detected (see Table 6). However, in
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TABLE 5 Common Method for Extracting Air Sampling Filters Loaded with Airborne Cr(III) and Cr(VI) Particles Extraction solution: 2% NaOH (w/w); 3% Na2CO3 (w/w) Extraction procedure: filter / 5.0 ml extraction solution are heated near the boiling point, with occasional swirling, for 30–45 min; after filtration through 5 mm, the filtrate is used for measurement Filter load (mg) Cr(OH)3
Cr(VI)
2.0 0.2 0.02
— — —
Cr(VI) found after extraction (mg) 1.3, 0.9, 0.9 1.2, 1.4, 1.4 1.4, 1.5, 1.3
Mean Mean Mean
1.1 1.3 1.4
contrast to alkaline extraction, sparingly soluble chromates are not covered by neutral leaching. Since the chromates are not present at the Leverkusen sampling sites, neutral leaching is preferred in our methods for ambient monitoring. In addition to oxidation of chromium(III), chromium(VI) compounds of the filter load may react with cosampled reductants. Moreover, the filter itself may contribute to reduction of chromium(VI) if the type of filter is not adequately selected. The main sources of error are conventional membrane filters and common analytical glass fiber filters coated with organics. Meanwhile, methods using PVC filters or preignited glass fiber filters have been widely established and are specified in most official methods for air monitoring of chromium. In Table 7, sampling and measuring conditions at the Leverkusen sampling sites are compiled. They aim in particular at avoiding redox reactions of sampled chromium compounds as far as possible.
TABLE 6 Leverkusen Method for Extracting Air Sampling Filters Loaded with Airborne Cr(III) and Cr(VI) Particles Extraction solution: distilled water Extraction procedure: filter / 50 ml extraction solution are heated on a water bath, with occasional swirling, for 5 hr; after filtration through 0.45 mm, the filtrate is used for measurement Filter load (mg) Cr(OH)3
Cr(VI)
Cr(VI) found after extraction (mg)
20.0 2.0 0.2 0.02 —
— — — — 0.01
õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 10.4, 10.4, 10.3
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TABLE 7 Conditions for Ambient Monitoring of Chromium Compounds in Air Sampling mode: Sampling volume/time: Fixed site Personal Filter material:
Extraction: Measurement: Soluble fraction, filtrate
Insoluble fraction, filter
Filter 20 m3/1 hr 1 m3/8 hr Glass fiber, preignited Neutral leaching with water or sodium hydrogen carbonate solution Cr(VI) determination by spectrophotometry Determination of total soluble chromium by ET-AAS Wet ashing with HNO3 /H2SO4 /HF followed by ET-AAS
In addition to applying proven methods, it was interesting to find the correlation between ambient and biological monitoring, i.e., between exposure and biological parameters. Systematic investigations revealed a comparatively straightforward relationship between both types of data, provided the biological target medium is chosen adequately. An example is given in Fig. 3 for alkali chromate exposure and the corresponding threshold value levels of chromium in the erythrocytes. The relationship between exposure and the biological parameter is almost linear, and as a quantitative measure, biological exposure equivalents (BEEs) can be calculated from the experimental data. An additional relationship can be derived from chromium trioxide in welding fumes and chromium excretion in urine. As evident from the concept of the BEE, correct selection of the biological target is a prerequisite (Bolt and Lewalter, 1988). BEHAVIOR OF HEXAVALENT CHROMIUM IN PLASMA
The last subject of this presentation is not directly related to biological monitoring. It comprises model tests aimed at the reaction of hexavalent chromium compounds in human plasma or whole blood. It is well documented that hexavalent chromium compounds are not stable in biological matrices due to the presence of various reductants such as ascorbic acid, glutathione, and cysteine (deFlora and Wetterhahn, 1989). Moreover, model tests were made on the reaction of potassium chromate with several reductants in aqueous solution at the physiological pH of 7.4. In these investigations the decay in chromium(VI) concentration was monitored spectrophotometrically and kinetic data were derived from the tests (Connett and Wetterhahn, 1985).
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FIG. 3. Correlation between exposure and biological monitoring data.
We carried out similar experiments in plasma and whole blood, thus observing the reduction characteristics of chromium directly in the biological system (Korallus et al., 1984). Spectrophotometry for monitoring the chromium(VI) concentration could not be applied in these tests because of the colored samples. Instead, voltammetry was used and the reaction was followed point by point by sequential polarographic measurements. In a first set of experiments, we studied the reaction of chromium(VI) in pool plasma without addition of any further agents. The measurements were made at the ‘‘ppm’’ concentration level. As can be seen from Fig. 4, chromium(VI) concentra-
tion fades gradually and has decreased by about 1 ppm within the initial 60 min. The natural reducing capacity of plasma can be determined quantitatively by an extrapolation method. Studies with individual plasma samples yielded substantially different results for a number of workers, and it may be concluded that so-called ‘‘strong’’ and ‘‘weak’’ reducers exist. Examples are listed in Table 8. More detailed studies revealed that reducing capacity is essentially independent of short-term influences such as nutrition but appears to represent an individual biological index (Lewalter et al., 1985). In further experiments, ascorbic acid was added to samples of pooled plasma spiked with chromium(VI). These tests were aimed at application of ascorbic acid as an antidote in the case of accidental intoxication with hexavalent chromium compounds. For such events, the therapeutic effect of ascorbic acid has been observed in clinical and experimental studies (Samitz et al., 1962; Poppel et al., 1963; Petrilli and deFlora,
TABLE 8 Individual Reducing Capacity of Plasma Samples
FIG. 4. Reaction rate of chromium(VI) reduction in plasma.
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Sample
Reducing capacity [ppm chromium(VI)]
1 2 3 4 5 6
1.5 1.9 1.1 1.5 1.2 1.8
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FIG. 5. Reaction rate of chromium(VI) reduction in plasma at different ascorbic acid concentrations.
1978). However, there was no quantitative information available on the dose–effect relationship. Figure 5 represents the course of decay in chromium(VI) concentration in the presence of different concentrations of ascorbic acid. It can be recognized from the diagram that comparatively high concentrations of ascorbic acid are required to achieve rapid reduction, as a prerequisite for detoxification, in order to prevent the chromium(VI) ions from entering the cell. Only 1000 ppm ascorbic acid proved to be effective in reducing chromium(VI) concentrations at the ppm level (Korallus et al., 1984). Based on these results, early application of high doses of ascorbic acid, preferably by intravenous injection, is recommended for cases of overexposure to chromium(VI). REFERENCES Bolt, H. M., and Lewalter, J. (1988). Alkali chromates. In Biological Exposure Values for Occupational Toxicants, pp. 187–203. Deutsche Forschungsgemeinschaft. Connett, P. H., and Wetterhahn, K. E. (1985). In vitro reaction of chromate with thiols and carboxylic acids. J. Am. Chem. Soc. 107, 4282–4288. Fishbein, L. (1987). Perspectives of analysis of carcinogenic and mu-
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tagenic metals in biological samples. Int. J. Environ. Anal. Chem. 28, 21–96. deFlora, S., and Wetterhahn, K. E. (1989). Mechanism of chromium metabolism and genotoxicity. Life. Chem. Rep. 7, 169–244. Guthrie, B. E., et al. (1987). Background correction and related problems in the determination of chromium in urine by graphite furnace atomic absorption spectrometry. Anal. Chem. 50, 1900–1902. Korallus, U., et al. (1984). Experimental bases for ascorbic acid therapy of poisoning by hexavalent chromium compounds. Int. Arch. Occup. Health 53, 247–256. Lewalter, J., et al. (1991). Chromium determination in whole blood, plasma and erythrocytes. Anal. Hazard. Subst. Biol. Mater. 3, 109– 113. Lewalter, J., et al. (1985). Chromium bond detection in isolated erythrocytes: A new principle of biological monitoring of exposure to hexavalent chromium. Int. Arch. Occup. Environ. Health 55, 305– 318. Parr, R. M. (1977). Problems of chromium analysis in biological materials: An international perspective with special reference to results for analytical quality control samples. J. Radioanal. Chem. 39, 421–433. Petrilli, F., and deFlora, S. (1978). Metabolic deactivation of hexavalent chromium mutagenicity. Mutat. Res. 54, 139–147. Poppel, W. J., et al. (1963). Effect of ascorbic acid in vivo on labeling of red cells by radioactive sodium chromate. Blood 22, 351–356. Samitz, U. H., et al. (1962). Studies on the prevention of injurious effects of chromates in industry. Int. Med. Surg. 31, 427–432. Slavin, W. (1981). Determination of chromium in the environment and in the workplace. At. Spectros. 2, 8–12. U.S. Environmental Protection Agency (1983). Assessment Document for Chromium, Doc. No. 600/8-83-014A, Chap. 3, pp. 35–36. Versieck, J. M. J., and Cornelis, R. (1980). Normal levels of trace elements in human blood plasma and serum. Anal. Chim. Acta 116, 217–254. Wiegand, H. J., et al. (1985). Fast uptake kinetics in vitro of chromium-51(VI) by red blood cells of man and rat. Arch. Toxicol. 57, 31–34.
QUESTIONS AND ANSWERS
A, DR. CLAUS HARZDORF (SPEAKER) Q, QUESTIONER (AUDIENCE) Q: Do you have any feel for chrome(VI) air concentration versus urine concentration? Is there some minimum chrome(VI) air concentration that will influence urinary chrome? A: We carried out all those tests at various ppm levels. I showed this diagram with the 5 ppm level. We also carried out tests at 1 ppm and 2 ppm. Q: What is the retention time of chrome(VI)? I know it depends from one species to the other, from one chromium compound to the other in the lung but, how long before it’s released into the bloodstream? A: I figure that is more of a medical question. And it should be answered by our medical experts.
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26, S86–S93 (1997)
RT971145
Analytical Methodology for Biological Monitoring of Chromium C. Harzdorf* and J. Lewalter† *Business Group Inorganics and †Medical Department, Bayer AG, D-51368 Leverkusen, Germany Received May 20, 1997
Analytical aspects associated with biological and ambient monitoring are outlined with an emphasis on processing samples at very low concentrations and optimization of analytical methods. Verification of chromium determination in body fluids by means of ‘‘Round Robin’’ tests is discussed. In addition, an approach for indirect valency discrimination of chromium species relevant in biological monitoring is presented. Application of voltammetry for model tests is described. This is aimed at studying the reaction of hexavalent chromium in human plasma. As a result, a natural reducing capacity of plasma of up to 2 ppm chromium(VI) was detected and quantified as an individual biological index. Furthermore, the effect of ascorbic acid on the reduction of chromium(VI) in plasma is demonstrated and quantitative bases for therapy in cases of accidental intoxication are discussed. q 1997 Academic Press
INTRODUCTION
Biological monitoring of inorganic compounds has a long tradition, and numerous basic and special publications have been produced. Of these, particular attention has been paid to chromium compounds because of the significantly different effects that species of both main valency states exert on man. Consequently, attempts have been made to distinguish between chromium(III) and chromium(VI) compounds. In practice, however, direct analytical determination of both types of chromium species is difficult to achieve in biological matter, because the chromium valency states are unstable in redox-active matrices. Therefore, indirect methods of evaluating chromium valencies in biological monitoring have been considered. Moreover, determination of the total chromium content of biological matter at very low concentrations is of primary importance in analytical methodology for biological monitoring. In view of the comprehensive literature available in the field of biological monitoring of chromium and associated analytical work, we will try not to present a
RTP 1145
/
6e10$$$741
DETERMINATION OF CHROMIUM IN BIOLOGICAL SAMPLES AT LOW CONCENTRATION LEVELS
The basic tool for the determination of chromium in biological material is electrothermal atomic absorption spectrometry (ET-AAS). Although chromium is considered an ‘‘easy’’ parameter in AAS, various difficulties arise at very low concentration levels and in complex matrices loaded with organic matter and/or electrolytes. These samples give rise to interfering effects during electrothermal processing in the graphite tube comprising primarily nonspecific background absorption due to matrix components and formation of chromium carbide, thus preventing chromium from atomization. To overcome interference like this, application of an efficient background compensation system and selection of the adequate type of graphite tube are of primary importance. Apart from interference occurring directly during measurement, peripheral effects resulting from sampling and sample handling also play an important role. The obvious analytical problems in this field are reflected by a number of publications on chromium determination in biological samples and by strikingly inconsistent results of Round Robin tests with biological material. Relevant studies and reviews have been published by Fishbein (1987), Versieck and Cornelis (1980), Guthrie et al. (1978), Slavin (1981), Parr (1977), and U.S. EPA (1983). In the following text, we report on our attempts to eliminate sources of error from analytical methodology. Sampling and sample handling. In our processing scheme, errors by contamination during sampling and handling prior to measurement are essentially minimized in that samples are taken and preserved for analysis in the medical department, away from where inorganic materials such as chromium compounds are handled. Thus, the risk of contamination during sampling and preservation is very low. The samples enter
S86
0273-2300/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
survey but to focus primarily on selected aspects in analytical methodology and interpretation of results. An outline of this presentation is given in Table 1.
08-27-97 12:23:28
rtpa
AP: RTP
CHROMIUM SYMPOSIUM
TABLE 1 Disposition and Primary Aspects • Determination of chromium in biological samples at low concentration levels Optimization of AAS techniques/verification of methods • Approaches for indirect valence discrimination of chromium species in biological monitoring • Correlation between ambient and biological monitoring • Behavior of hexavalent chromium in plasma Reducing capacity of plasma/detoxification
the analytical laboratory area immediately prior to measurement, where they are processed under the usual conditions of trace analysis. Sample preservation of whole blood and plasma is done with EDTA. In addition, a surfactant (Triton 100) is added to prevent flocculation and to inhibit nonhomogeneity. Urine samples are stabilized by adding small amounts of hydrochloric acid. Once preserved, samples are stable for at least 24 hr. Nevertheless, measurement of the samples should be attempted as soon as possible. Optimization of ET-AAS conditions. As mentioned above, errors in ET-AAS result primarily from undesired chemical reactions during heating in the graphite furnace and from incorrect compensation of nonspecific spectral effects stemming from matrix components. Thus, careful optimization of experimental conditions is essential. In Fig. 1, the effect of the type of graphite tube is demonstrated with samples of whole blood containing 2 ng/ml chromium. The graphs of both the net signal (specific absorption) and the background signal (nonspecific absorption) reveal that only the pyrolytically coated tube yields a good result. The effect of background compensation was studied with both the conventional tungsten/halogen lamp and the magnetic Zeeman system. Whole blood containing 5 ng/ml chromium was tested. The resulting signal traces for specific and nonspecific absorption are shown in Fig. 2. On qualitative evaluation, both modes of background compensation work satisfactorily. On quantitative evaluation of results from routine measurements, however, a slight advantage for the conventional tungsten/ halogen system can be observed for the lowest chromium concentrations. This finding is likely due to bigger net signals, inherently produced by conventional background compensation systems. In this context, it should be emphasized that the widespread deuterium background compensation, not presented in the figures, is clearly inferior to the systems studied here, because of its insufficient energy output in the range of the chromium wavelength. Unfortunately, a number of atomic absorption spectro-
AID
RTP 1145
/
6e10$$$741
08-27-97 12:23:28
S87
meters are exclusively equipped with the deuterium system. Optimal instrumental ET-AAS conditions for chromium in body fluids are summarized in Table 2. Optimization was carried out with samples of whole blood, representing the most difficult analytical matrix. Preparations of erythrocyte fractions, plasma, and urine samples can be processed under the same conditions. Verification of methods. Complementing optimization of analytical methodology, verification and validation of methods are important tasks in analytical work for biological monitoring. In addition to internal laboratory work aimed at determining the performance data and checking the methods with certified standards, participation in Round Robin quality control tests is a valuable measure to check the reliability of analytical laboratory work. The experience of 11 years of Bayer’s participation in such Round Robin tests for whole blood and urine is listed in Tables 3 and 4. The tolerance ranges of the Round Robin tests have gradually become smaller in the course of time. This tendency clearly reflects better analytical performance of the participating laboratories due to optimization of methods and improved techniques in instrumentation. In addition to the measures for checking the reliability of analytical methods, the most effective way of checking the validity of analytical results is the application of an additional method based on a different analytical technique. For chromium determination, we prefer ICP–MS with 51Cr. Performance characteristics of this method are equivalent to ET-AAS; thus, an authentic verification of especially important data is possible. APPROACHES TO INDIRECT VALENCY DISCRIMINATION OF CHROMIUM SPECIES IN BIOLOGICAL MONITORING
Despite excellent performance of atomic spectrometric methods, these techniques suffer from the inherent limitation that only element concentrations can be determined. Valency discrimination is impossible to achieve unless additional analytical steps are applied prior to spectrometric measurement. Typical valency-specific methods such as spectrophotometry and polarography fail in determining chromium in body fluids directly, partly because of matrix interference (spectroscopy) and partly because of insufficient sensitivity for physiological concentration levels (polarography). Chelation/extraction methods have been proposed in this field. These methods are based on valency-specific reactions to form a chromium complex, followed by extraction and ET-AAS measurement of the extract. In our experience, these methods do not work reliably in biological matter due to side reactions with matrix components.
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S88
FIG. 1.
Comparison of types of graphite tube: whole blood, 2 ng/ml Cr.
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FIG. 2. Comparison of mode of background compensation: whole blood, 5 ng/ml Cr.
In view of the lack of direct methods for valency discrimination, it was necessary to develop measures for indirect estimation of valency states of chromium species occurring in biological monitoring. A model was established based on the following concept. It is well known that hexavalent chromium anions, after having entered the blood circulation, rapidly pass the cell membranes of erythrocytes via the anion channel mechanism. Inside the cell, chromium(VI) is reduced by cell constituents to form chromium(III) complexes. These complexes, owing to their size, are unable to leave the cell in a reverse direction. Thus, a trapping mechanism for chromium(VI) exists with the erythrocytes representing a specific target organ for hexavalent chromium. Hence, the chromium concentration in the erythrocyte fraction, carefully prepared from whole
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blood, provides a valuable indicator for estimating both the external exposure and the internal stress from hexavalent chromium. Thus, this mode of biomonitoring is of important diagnostic value (Lewalter et al., 1985, 1990; Wiegand et al., 1985). However, it must be taken into consideration that chromium, once trapped in the erythrocyte, persists there for the lifetime of the cell, i.e., for about 115 days. Therefore, chromium concentration in the erythrocyte fraction reflects a mean level over a certain period of time rather than a momentary situation. In this context, it must be emphasized that exclusively soluble chromates of small particle size are able to undergo the mechanism described. Particles bigger than 5 mm are ‘‘filtered’’ in the lung and do not reach the blood circulation. Also, chromium trioxide is essentially
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TABLE 2 Instrumental Conditions in ET-AAS for Biological Samples Furnace
TABLE 4 Results of Annual Round Robin Tests Organized by the Institute for Occupational and Environmental Medicine, Erlangen, Germany
Pyrolytically coated tube (no platform) Temperature program
Function
Step
Temp
Ramp
Hold
Drying
1 2 3
90 140 350
15 25 20
5 15 10
Ashing
4 5
500 1250
20 25
10 15
Atomization
6
2300
0
5
Boost
7
2650
2
2
Spectrometer
Background compensation Zeeman or tungsten/halogen lamp Wavelength: 357.9 nm Spectral band width: 0.7 nm
Year
Nominal value
Result Bayer
Tolerance range
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995
2.8 7.4 41.1 44.0 5.7 5.3 13.7 29.3 39.8 12.1 5.4
3.0 7.0 42.0 48.0 5.0 5.0 12.6 29.1 40.8 12.0 4.9
1.5–4.1 5.4–9.4 18.7–53.5 31.6–56.4 4.3–7.0 4.0–6.7 10.1–17.3 24.4–34.3 37.7–45.9 10.0–14.2 4.3–6.5
Note. Results in mg/liter Cr. Samples: human urine (pool).
CORRELATION BETWEEN AMBIENT AND BIOLOGICAL MONITORING
not transported to the blood circulation because it is rapidly reduced due to its acidic nature and the reducing environment in the lung. Compared to the high specificity for Cr(VI) obtained from the chromium concentration in the erythrocyte fraction, chromium determination in urine is of less selective significance because it reflects the entire renal excretion resulting from several external sources of exposure and from different pathways through the organism. Nonetheless, urinary chromium provides an additional measure of estimating overall exposure, because most of the chromium intake is finally removed by renal excretion. Moreover, urinary chromium can give an answer on actual exposure because delay between exposure and excretion is comparatively small in most cases.
TABLE 3 Results of Annual Round Robin Tests Organized by the Institute for Occupational and Environmental Medicine, Erlangen, Germany Year
Nominal value
Result Bayer
Tolerance range
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995
10.5 5.7 17.7 21.2 10.6 6.6 9.7 21.6 12.0 5.9 9.8
12.0 6.0 16.5 25.0 12.0 7.9 10.6 20.2 12.8 6.8 8.9
7.8–13.2 3.9–7.4 14.6–20.9 17.6–24.8 8.5–12.8 4.8–8.3 7.3–12.1 17.6–25.6 10.1–13.8 4.8–6.9 8.4–11.2
Note. Results in mg/liter Cr. Samples: whole blood (pool).
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Although biological monitoring is of primary importance in evaluating occupational health effects, ambient monitoring, reflecting exposure, represents a complementary measure in occupational health care. In many countries, there is a requirement to carry out exposure measurements in order to comply with legislation. In addition to biological monitoring, direct, valencyspecific determination of chromium species is also possible in ambient monitoring, although analysis is difficult since redox effects cannot be completely avoided if sampling, sample handling, and analytical finish are not done with extreme care. At present, filter methods have been established for air sampling at the workplace (fixed-site sampling) and in man (personal sampling). For analytical processing, the filter is extracted and the leach and the residual filter load are analyzed separately. Common methods, also the officially recommended ones in the United States and Europe, use an aqueous sodium hydroxide/sodium carbonate solution for extraction and it is assumed that with this method, no redox reactions occur during leaching. Unfortunately, this assumption does not hold true. Model tests revealed that during alkaline leaching considerable amounts of trivalent chromium compounds may be oxidized, thus simulating positive results for hexavalent chromium in air (see Table 5). The amount of chromium(VI) produced during filter leaching depends on the solubility of chromium(III) compounds in the alkaline extraction solution. The problem can be overcome by leaching the filter load with water or with diluted sodium hydrogen carbonate solution at pH 8.4. In this case no chromium oxidation could be detected (see Table 6). However, in
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TABLE 5 Common Method for Extracting Air Sampling Filters Loaded with Airborne Cr(III) and Cr(VI) Particles Extraction solution: 2% NaOH (w/w); 3% Na2CO3 (w/w) Extraction procedure: filter / 5.0 ml extraction solution are heated near the boiling point, with occasional swirling, for 30–45 min; after filtration through 5 mm, the filtrate is used for measurement Filter load (mg) Cr(OH)3
Cr(VI)
2.0 0.2 0.02
— — —
Cr(VI) found after extraction (mg) 1.3, 0.9, 0.9 1.2, 1.4, 1.4 1.4, 1.5, 1.3
Mean Mean Mean
1.1 1.3 1.4
contrast to alkaline extraction, sparingly soluble chromates are not covered by neutral leaching. Since the chromates are not present at the Leverkusen sampling sites, neutral leaching is preferred in our methods for ambient monitoring. In addition to oxidation of chromium(III), chromium(VI) compounds of the filter load may react with cosampled reductants. Moreover, the filter itself may contribute to reduction of chromium(VI) if the type of filter is not adequately selected. The main sources of error are conventional membrane filters and common analytical glass fiber filters coated with organics. Meanwhile, methods using PVC filters or preignited glass fiber filters have been widely established and are specified in most official methods for air monitoring of chromium. In Table 7, sampling and measuring conditions at the Leverkusen sampling sites are compiled. They aim in particular at avoiding redox reactions of sampled chromium compounds as far as possible.
TABLE 6 Leverkusen Method for Extracting Air Sampling Filters Loaded with Airborne Cr(III) and Cr(VI) Particles Extraction solution: distilled water Extraction procedure: filter / 50 ml extraction solution are heated on a water bath, with occasional swirling, for 5 hr; after filtration through 0.45 mm, the filtrate is used for measurement Filter load (mg) Cr(OH)3
Cr(VI)
Cr(VI) found after extraction (mg)
20.0 2.0 0.2 0.02 —
— — — — 0.01
õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 õ0.2, õ0.2, õ0.2 10.4, 10.4, 10.3
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TABLE 7 Conditions for Ambient Monitoring of Chromium Compounds in Air Sampling mode: Sampling volume/time: Fixed site Personal Filter material:
Extraction: Measurement: Soluble fraction, filtrate
Insoluble fraction, filter
Filter 20 m3/1 hr 1 m3/8 hr Glass fiber, preignited Neutral leaching with water or sodium hydrogen carbonate solution Cr(VI) determination by spectrophotometry Determination of total soluble chromium by ET-AAS Wet ashing with HNO3 /H2SO4 /HF followed by ET-AAS
In addition to applying proven methods, it was interesting to find the correlation between ambient and biological monitoring, i.e., between exposure and biological parameters. Systematic investigations revealed a comparatively straightforward relationship between both types of data, provided the biological target medium is chosen adequately. An example is given in Fig. 3 for alkali chromate exposure and the corresponding threshold value levels of chromium in the erythrocytes. The relationship between exposure and the biological parameter is almost linear, and as a quantitative measure, biological exposure equivalents (BEEs) can be calculated from the experimental data. An additional relationship can be derived from chromium trioxide in welding fumes and chromium excretion in urine. As evident from the concept of the BEE, correct selection of the biological target is a prerequisite (Bolt and Lewalter, 1988). BEHAVIOR OF HEXAVALENT CHROMIUM IN PLASMA
The last subject of this presentation is not directly related to biological monitoring. It comprises model tests aimed at the reaction of hexavalent chromium compounds in human plasma or whole blood. It is well documented that hexavalent chromium compounds are not stable in biological matrices due to the presence of various reductants such as ascorbic acid, glutathione, and cysteine (deFlora and Wetterhahn, 1989). Moreover, model tests were made on the reaction of potassium chromate with several reductants in aqueous solution at the physiological pH of 7.4. In these investigations the decay in chromium(VI) concentration was monitored spectrophotometrically and kinetic data were derived from the tests (Connett and Wetterhahn, 1985).
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FIG. 3. Correlation between exposure and biological monitoring data.
We carried out similar experiments in plasma and whole blood, thus observing the reduction characteristics of chromium directly in the biological system (Korallus et al., 1984). Spectrophotometry for monitoring the chromium(VI) concentration could not be applied in these tests because of the colored samples. Instead, voltammetry was used and the reaction was followed point by point by sequential polarographic measurements. In a first set of experiments, we studied the reaction of chromium(VI) in pool plasma without addition of any further agents. The measurements were made at the ‘‘ppm’’ concentration level. As can be seen from Fig. 4, chromium(VI) concentra-
tion fades gradually and has decreased by about 1 ppm within the initial 60 min. The natural reducing capacity of plasma can be determined quantitatively by an extrapolation method. Studies with individual plasma samples yielded substantially different results for a number of workers, and it may be concluded that so-called ‘‘strong’’ and ‘‘weak’’ reducers exist. Examples are listed in Table 8. More detailed studies revealed that reducing capacity is essentially independent of short-term influences such as nutrition but appears to represent an individual biological index (Lewalter et al., 1985). In further experiments, ascorbic acid was added to samples of pooled plasma spiked with chromium(VI). These tests were aimed at application of ascorbic acid as an antidote in the case of accidental intoxication with hexavalent chromium compounds. For such events, the therapeutic effect of ascorbic acid has been observed in clinical and experimental studies (Samitz et al., 1962; Poppel et al., 1963; Petrilli and deFlora,
TABLE 8 Individual Reducing Capacity of Plasma Samples
FIG. 4. Reaction rate of chromium(VI) reduction in plasma.
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Sample
Reducing capacity [ppm chromium(VI)]
1 2 3 4 5 6
1.5 1.9 1.1 1.5 1.2 1.8
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FIG. 5. Reaction rate of chromium(VI) reduction in plasma at different ascorbic acid concentrations.
1978). However, there was no quantitative information available on the dose–effect relationship. Figure 5 represents the course of decay in chromium(VI) concentration in the presence of different concentrations of ascorbic acid. It can be recognized from the diagram that comparatively high concentrations of ascorbic acid are required to achieve rapid reduction, as a prerequisite for detoxification, in order to prevent the chromium(VI) ions from entering the cell. Only 1000 ppm ascorbic acid proved to be effective in reducing chromium(VI) concentrations at the ppm level (Korallus et al., 1984). Based on these results, early application of high doses of ascorbic acid, preferably by intravenous injection, is recommended for cases of overexposure to chromium(VI). REFERENCES Bolt, H. M., and Lewalter, J. (1988). Alkali chromates. In Biological Exposure Values for Occupational Toxicants, pp. 187–203. Deutsche Forschungsgemeinschaft. Connett, P. H., and Wetterhahn, K. E. (1985). In vitro reaction of chromate with thiols and carboxylic acids. J. Am. Chem. Soc. 107, 4282–4288. Fishbein, L. (1987). Perspectives of analysis of carcinogenic and mu-
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tagenic metals in biological samples. Int. J. Environ. Anal. Chem. 28, 21–96. deFlora, S., and Wetterhahn, K. E. (1989). Mechanism of chromium metabolism and genotoxicity. Life. Chem. Rep. 7, 169–244. Guthrie, B. E., et al. (1987). Background correction and related problems in the determination of chromium in urine by graphite furnace atomic absorption spectrometry. Anal. Chem. 50, 1900–1902. Korallus, U., et al. (1984). Experimental bases for ascorbic acid therapy of poisoning by hexavalent chromium compounds. Int. Arch. Occup. Health 53, 247–256. Lewalter, J., et al. (1991). Chromium determination in whole blood, plasma and erythrocytes. Anal. Hazard. Subst. Biol. Mater. 3, 109– 113. Lewalter, J., et al. (1985). Chromium bond detection in isolated erythrocytes: A new principle of biological monitoring of exposure to hexavalent chromium. Int. Arch. Occup. Environ. Health 55, 305– 318. Parr, R. M. (1977). Problems of chromium analysis in biological materials: An international perspective with special reference to results for analytical quality control samples. J. Radioanal. Chem. 39, 421–433. Petrilli, F., and deFlora, S. (1978). Metabolic deactivation of hexavalent chromium mutagenicity. Mutat. Res. 54, 139–147. Poppel, W. J., et al. (1963). Effect of ascorbic acid in vivo on labeling of red cells by radioactive sodium chromate. Blood 22, 351–356. Samitz, U. H., et al. (1962). Studies on the prevention of injurious effects of chromates in industry. Int. Med. Surg. 31, 427–432. Slavin, W. (1981). Determination of chromium in the environment and in the workplace. At. Spectros. 2, 8–12. U.S. Environmental Protection Agency (1983). Assessment Document for Chromium, Doc. No. 600/8-83-014A, Chap. 3, pp. 35–36. Versieck, J. M. J., and Cornelis, R. (1980). Normal levels of trace elements in human blood plasma and serum. Anal. Chim. Acta 116, 217–254. Wiegand, H. J., et al. (1985). Fast uptake kinetics in vitro of chromium-51(VI) by red blood cells of man and rat. Arch. Toxicol. 57, 31–34.
QUESTIONS AND ANSWERS
A, DR. CLAUS HARZDORF (SPEAKER) Q, QUESTIONER (AUDIENCE) Q: Do you have any feel for chrome(VI) air concentration versus urine concentration? Is there some minimum chrome(VI) air concentration that will influence urinary chrome? A: We carried out all those tests at various ppm levels. I showed this diagram with the 5 ppm level. We also carried out tests at 1 ppm and 2 ppm. Q: What is the retention time of chrome(VI)? I know it depends from one species to the other, from one chromium compound to the other in the lung but, how long before it’s released into the bloodstream? A: I figure that is more of a medical question. And it should be answered by our medical experts.
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