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[9] Determination of metallothionein in biological materials
[9]
DETERMINATION OF M T IN BIOLOGICAL MATERIALS
57
[9] Determination of Metallothionein in Biological Materials By K A R L H . S U M M E R a n d D O M I N I K K L E I N Current methods for the quantification o f metallothionein ( M T ) in biological materials are based either on the direct determination of the protein moiety or the indirect determination via the metal and SH content o f M T (Table I). Some forms of MT, such as polymeric forms, hardly can be quantitatively analyzed at present. Several parameters m u s t be taken into account when deciding which m e t h o d is most appropriate for the quantification of M T in a particular sample. The m o s t i m p o r t a n t are outlined in detail below, but basic procedural steps o f sample preparation and handling, such as homogenization ( P o t t e r - E l v e h j e m , Ultra-Turrax, sonification), the buffer system (pH, ionic strength), and reductive conditions (2-mercaptoethanol, dithiothreitol, inert gas atmosphere), should also be considered. C r i t e r i a for S e l e c t i o n o f M e t a l l o t h i o n e i n D e t e r m i n a t i o n M e t h o d s Sensitivity
A m a j o r criterion for choosing a particular m e t h o d is the a m o u n t o f M T to be determined. As shown by comparative studies, I-3 immunological methods 4-7 are the m o s t sensitive. As little as 100 pg of M T can be determined using radioimmunological s and enzyme-linked i m m u n o s o r bent (ELISA) 9,1° procedures. The limits of detection with the thiomolybdate, ~1 silver staining, 1z,13 and c a d m i u m - C h e l e x assay TM are 15, 50, and M. P. Waalkes, J. S. Garvey, and C. D. Klaassen, Toxicol. AppL PharmacoL 79, 524 (1985). 2 H. H. Dieter, U MOiler, J. Abel, and K. H. Summer, ToxicoL AppL PharmacoL 85, 380 (1986). 3 C. N. Nolan and Z. A. Shaikh, Anal. Biochem. 154, 213 (1986). 4 R. J. Vander Mallie and J. S. Garvey, J. BioL Chem. 254, 8416 (1979). 5 F. O. Brady and R. L. Kafka, AnaL Biochem. 98, 89 (1979). 6 C. Tohyama and Z. A. Shaikh, Fundam. AppL ToxicoL 1, 1 (1981). 7 R. K. Mehra and I. Bremner, Biochem. J. 213, 459 (1983). 8j. S. Garvey, R. J. Vander Mallie, and C. C. Chang, this series, Vol. 84, p. 121. 9 D. G. Thomas, H. J. Linton, and J. S. Garvey, J. ImmunoL Methods 89, 239 (1986). ~oj. S. Garvey, D. G. Thomas, H., and J. Linton, Experentia, SuppL 52, 335 (1987). H D. Klein, R. Bartsch, and K. H. Summer, AnaL Biochem. 189, 35 (1991). ~2F. Otsuka, S. Koizumi, M. Kimura, and M. Ohsawa, AnaL Biochem. 168, 184 (1988). ~3L.-Y. Lin, and C. C. McCormick, Comp. Biochem. PhysioL C." Comp. PharmacoL ToxicoL 85C, 75 (1986).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright© 1991 by AcademicPress, Inc. All rightsofreprtxluctionin any form reserved.
58
[9]
QUANTIFICATION IN TISSUES AND BODY FLUIDS
TABLE I QUANTIFICATION OF METALLOTHIONEIN IN BIOLOGICAL MATERIALS
Required sample treatment
MT determination Direct
Indirect
Homogenization
Immunological methods RIA ELISA
Preparation of cytosolic fraction followed by denaturing high-molecular-weight proteins (heat, TCA, organic solvents)a
Immunological methods RIA ELISA
Metal affinity methods Cd saturation Hg saturation Ag saturation SH determination Electrochemistry Photometry
Preparation of cytosolic fraction followed by chromatography (Sephadex G-75, HPLC) Preparation of cytosolic fraction followed by electrophoresis (SDS-PAGE)
Absorption
Metal determination
Western blot Protein staining
a TCA, Trichloroacetic acid. 100 ng o f M T , respectively. F o r other methods, e.g., h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) / a t o m i c absorption spectroscopy (AAS),15-17 electrochemicaP a,~9 a n d metal affinity assays [ c a d m i u m - h e m e , 2,2°,2~ m e r c u r y - t r i c h l o r o a c e t i c acid (TCA), 22,23 a n d silver saturation assay24], a m i n i m u m a m o u n t o f a b o u t 1 #g M T is required. T h e latter m e t h o d s are nevertheless suited to determine basal M T levels in m o s t tissues, but at present quantification o f M T in b o d y fluids such as p l a s m a a n d urine 7,25-27 ~4R. Bartsch, D. Klein, and K. H. Summer, Arch. ToxicoL 64, 177 (1990). ~sL. D. Lehman and C. D. Klaassen, Anal. Biochem. 153, 305 (1986). 16M. P. Riehards, J. Chromatogr. 482, 87 (1989). 17K. T. Suzuki, AnaL Biochem. 102, 31 (1980). 18R. W. Olafson and R. G. Sim, Anal Biochem. 100, 343 (1979). 19R. W. Olafson, Experentia, SuppL 52, 329 (1987). 2oS. Onosaka and M. G. Cherian, ToxicoL AppL PharmacoL 63, 270 (1982). 21D. L. Eaton and B. F. Toal, Toxicol. Appl. Pharmacol. 66, 134 (1982). 22j. R. Piotrowski, W. Bolanowska, and A. Saporta, Acta Biochem. Pol. 211,207 (1973). 23p. B. Lobel and J. F. Payne, Comp. Biochem. PhysioL C: Comp. PharmacoL Toxicol. 86(2, 37 (1987). 24A. M. Seheuhammer and M. G. Cherian, ToxicoL AppL PharmacoL 82, 417 (1986). 25G. F. Nordberg, J. S. Garvey, and C. C. Chang, Environ. Res. 28, 179 (1982).
[9]
DETERMINATION OF M T IN BIOLOGICAL MATERIALS
59
is possible only with immunological methods. The detection limits are only approximate because the linear range of a particular assay also depends on the type of sample. The linearity of the assay, therefore, must be determined separately for each method and kind of sample. Nonmetallothionein Proteins
High amounts of non-MT proteins and a high non-MT protein-to-MT ratio may interfere with the quantification of MT. For example, when using immunological methods, antibodies may bind nonspecifically to non-MT proteins. With chromatographic methods, resolution of the separation is adversely affected by high non-MT protein-to-MT ratios. Nonspecific binding of cadmium, mercury, and silver to non-MT proteins is of minor importance for metal-affinity assays, since metal-binding high-molecular-weight proteins are denatured by heat, acid, or organic solvents. However, the results of these assays may be influenced by low-molecularweight thiols [e.g., cysteine, glutathione (GSH)], which are stable toward heat, acid, and organic solvents, particularly if present at high concentrations. ~4,21 SH-containing compounds other than MT additionally can cause adverse effects on MT quantification via its SH groups. Metallothionein Isoforms and Metals Bound to Metallothionein
The presence of MT isoforms in biological material may affect the results of immunological methods due to the different antigenicity of the isoforms against a particular antibody. This is of special importance in induction experiments leading to different patterns of MT isoforms. 28'29 Thus when assessing total amounts of MT it is necessary to ensure that the antibodies raised against MT do not differ in their specificities toward MT isoforms or, although normally not practicable, the standard MT used must represent the isoform composition of MT in the sample. Clearly, the use of standard MT from other species may also lead to erroneous results. The metals bound to MT should be considered when selecting a method for quantification, since the metal composition of MT influences its heat stability, sensitivity to oxidation, and the pH-dependent dissocia26 C. C. Chang, R. J. Vander Mallie, and J. S. Garvey, Toxicol. Appl. Pharmacol. 55, 490 (1980). 27 C. Tohyama, Z. A. Shaikh, K. Nogawa, E. Kobayashi, and R. Honda, Toxicology 20, 289 (1981). 28 K. T. Suzuki and M. Yamamura, Biochem. Pharmacol. 29, 2407 (1980). 29 L. D. Lehman-MeKeeman, G. K. Andrews, and C. D. Klaassen, Biochem. J. 249, 429 (1988).
60
QUANTIFICATION IN TISSUES AND BODY FLUIDS
[ 10]
tion of metals. For example, heating of the sample should be avoided when determining copper-containing MT. Immunological methods are less likely to be affected by oxidation of MT than the indirect methods (SH determination and metal affinity assays), provided that the epitopes remain unchanged. The metal composition of MT also affects its absorption spectrum, 3° which can have consequences for the quantification of MT by HPLC/photometric analysis. Furthermore, due to the different affinities of the metals to the protein, the use of metal saturation and SH determination methods is limited. Copper-containing MT, for example, cannot be determined with the cadmium- heme assay. In general, indirect methods cause only minor problems when measuring MT with different isoforms, whereas the direct immunological methods cause only minor problems when measuring MT with different metal composition. Both types of analysis, however, can result in a loss of information, namely, the isoform pattern (indirect methods) or the metal composition of MT (direct methods). Clearly there is no universal method for the quantification of MT, so that the parallel use of different methods is considered to be most appropriate. Comment When several methods are applicable, one should also bear in mind that the various methods require different laboratory skills and procedures. In general, immunological methods are more time consuming and difficult to perform than metal affinity assays. Comparative interpretation of results obtained from different methods seems to be the optimal approach for deciding which of the MT assays is most suitable for a particular sample. J0 M. Vasak and J. H. R. K~gi, Met. Ions Biol. Syst. 15, 213 (1983).
[ 10] A s s a y o f E x t r a c e l l u l a r M e t a l l o t h i o n e i n By IAN BREMNER
and RAJESH K.
MEHRA
Introduction Although metallothionein (MT) is rightly regarded mainly as an intracellular protein located in the cytoplasm and nucleus of the cell, it also occurs in small amounts in extracellular fluids. This was first reported in
METHODS IN ENZYMOLOGY, VOL. 205
Copyright© 1991 by Academic Press,Inc. All fightsof reproductionin any formreserved.
DETERMINATION OF M T IN BIOLOGICAL MATERIALS
57
[9] Determination of Metallothionein in Biological Materials By K A R L H . S U M M E R a n d D O M I N I K K L E I N Current methods for the quantification o f metallothionein ( M T ) in biological materials are based either on the direct determination of the protein moiety or the indirect determination via the metal and SH content o f M T (Table I). Some forms of MT, such as polymeric forms, hardly can be quantitatively analyzed at present. Several parameters m u s t be taken into account when deciding which m e t h o d is most appropriate for the quantification of M T in a particular sample. The m o s t i m p o r t a n t are outlined in detail below, but basic procedural steps o f sample preparation and handling, such as homogenization ( P o t t e r - E l v e h j e m , Ultra-Turrax, sonification), the buffer system (pH, ionic strength), and reductive conditions (2-mercaptoethanol, dithiothreitol, inert gas atmosphere), should also be considered. C r i t e r i a for S e l e c t i o n o f M e t a l l o t h i o n e i n D e t e r m i n a t i o n M e t h o d s Sensitivity
A m a j o r criterion for choosing a particular m e t h o d is the a m o u n t o f M T to be determined. As shown by comparative studies, I-3 immunological methods 4-7 are the m o s t sensitive. As little as 100 pg of M T can be determined using radioimmunological s and enzyme-linked i m m u n o s o r bent (ELISA) 9,1° procedures. The limits of detection with the thiomolybdate, ~1 silver staining, 1z,13 and c a d m i u m - C h e l e x assay TM are 15, 50, and M. P. Waalkes, J. S. Garvey, and C. D. Klaassen, Toxicol. AppL PharmacoL 79, 524 (1985). 2 H. H. Dieter, U MOiler, J. Abel, and K. H. Summer, ToxicoL AppL PharmacoL 85, 380 (1986). 3 C. N. Nolan and Z. A. Shaikh, Anal. Biochem. 154, 213 (1986). 4 R. J. Vander Mallie and J. S. Garvey, J. BioL Chem. 254, 8416 (1979). 5 F. O. Brady and R. L. Kafka, AnaL Biochem. 98, 89 (1979). 6 C. Tohyama and Z. A. Shaikh, Fundam. AppL ToxicoL 1, 1 (1981). 7 R. K. Mehra and I. Bremner, Biochem. J. 213, 459 (1983). 8j. S. Garvey, R. J. Vander Mallie, and C. C. Chang, this series, Vol. 84, p. 121. 9 D. G. Thomas, H. J. Linton, and J. S. Garvey, J. ImmunoL Methods 89, 239 (1986). ~oj. S. Garvey, D. G. Thomas, H., and J. Linton, Experentia, SuppL 52, 335 (1987). H D. Klein, R. Bartsch, and K. H. Summer, AnaL Biochem. 189, 35 (1991). ~2F. Otsuka, S. Koizumi, M. Kimura, and M. Ohsawa, AnaL Biochem. 168, 184 (1988). ~3L.-Y. Lin, and C. C. McCormick, Comp. Biochem. PhysioL C." Comp. PharmacoL ToxicoL 85C, 75 (1986).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright© 1991 by AcademicPress, Inc. All rightsofreprtxluctionin any form reserved.
58
[9]
QUANTIFICATION IN TISSUES AND BODY FLUIDS
TABLE I QUANTIFICATION OF METALLOTHIONEIN IN BIOLOGICAL MATERIALS
Required sample treatment
MT determination Direct
Indirect
Homogenization
Immunological methods RIA ELISA
Preparation of cytosolic fraction followed by denaturing high-molecular-weight proteins (heat, TCA, organic solvents)a
Immunological methods RIA ELISA
Metal affinity methods Cd saturation Hg saturation Ag saturation SH determination Electrochemistry Photometry
Preparation of cytosolic fraction followed by chromatography (Sephadex G-75, HPLC) Preparation of cytosolic fraction followed by electrophoresis (SDS-PAGE)
Absorption
Metal determination
Western blot Protein staining
a TCA, Trichloroacetic acid. 100 ng o f M T , respectively. F o r other methods, e.g., h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) / a t o m i c absorption spectroscopy (AAS),15-17 electrochemicaP a,~9 a n d metal affinity assays [ c a d m i u m - h e m e , 2,2°,2~ m e r c u r y - t r i c h l o r o a c e t i c acid (TCA), 22,23 a n d silver saturation assay24], a m i n i m u m a m o u n t o f a b o u t 1 #g M T is required. T h e latter m e t h o d s are nevertheless suited to determine basal M T levels in m o s t tissues, but at present quantification o f M T in b o d y fluids such as p l a s m a a n d urine 7,25-27 ~4R. Bartsch, D. Klein, and K. H. Summer, Arch. ToxicoL 64, 177 (1990). ~sL. D. Lehman and C. D. Klaassen, Anal. Biochem. 153, 305 (1986). 16M. P. Riehards, J. Chromatogr. 482, 87 (1989). 17K. T. Suzuki, AnaL Biochem. 102, 31 (1980). 18R. W. Olafson and R. G. Sim, Anal Biochem. 100, 343 (1979). 19R. W. Olafson, Experentia, SuppL 52, 329 (1987). 2oS. Onosaka and M. G. Cherian, ToxicoL AppL PharmacoL 63, 270 (1982). 21D. L. Eaton and B. F. Toal, Toxicol. Appl. Pharmacol. 66, 134 (1982). 22j. R. Piotrowski, W. Bolanowska, and A. Saporta, Acta Biochem. Pol. 211,207 (1973). 23p. B. Lobel and J. F. Payne, Comp. Biochem. PhysioL C: Comp. PharmacoL Toxicol. 86(2, 37 (1987). 24A. M. Seheuhammer and M. G. Cherian, ToxicoL AppL PharmacoL 82, 417 (1986). 25G. F. Nordberg, J. S. Garvey, and C. C. Chang, Environ. Res. 28, 179 (1982).
[9]
DETERMINATION OF M T IN BIOLOGICAL MATERIALS
59
is possible only with immunological methods. The detection limits are only approximate because the linear range of a particular assay also depends on the type of sample. The linearity of the assay, therefore, must be determined separately for each method and kind of sample. Nonmetallothionein Proteins
High amounts of non-MT proteins and a high non-MT protein-to-MT ratio may interfere with the quantification of MT. For example, when using immunological methods, antibodies may bind nonspecifically to non-MT proteins. With chromatographic methods, resolution of the separation is adversely affected by high non-MT protein-to-MT ratios. Nonspecific binding of cadmium, mercury, and silver to non-MT proteins is of minor importance for metal-affinity assays, since metal-binding high-molecular-weight proteins are denatured by heat, acid, or organic solvents. However, the results of these assays may be influenced by low-molecularweight thiols [e.g., cysteine, glutathione (GSH)], which are stable toward heat, acid, and organic solvents, particularly if present at high concentrations. ~4,21 SH-containing compounds other than MT additionally can cause adverse effects on MT quantification via its SH groups. Metallothionein Isoforms and Metals Bound to Metallothionein
The presence of MT isoforms in biological material may affect the results of immunological methods due to the different antigenicity of the isoforms against a particular antibody. This is of special importance in induction experiments leading to different patterns of MT isoforms. 28'29 Thus when assessing total amounts of MT it is necessary to ensure that the antibodies raised against MT do not differ in their specificities toward MT isoforms or, although normally not practicable, the standard MT used must represent the isoform composition of MT in the sample. Clearly, the use of standard MT from other species may also lead to erroneous results. The metals bound to MT should be considered when selecting a method for quantification, since the metal composition of MT influences its heat stability, sensitivity to oxidation, and the pH-dependent dissocia26 C. C. Chang, R. J. Vander Mallie, and J. S. Garvey, Toxicol. Appl. Pharmacol. 55, 490 (1980). 27 C. Tohyama, Z. A. Shaikh, K. Nogawa, E. Kobayashi, and R. Honda, Toxicology 20, 289 (1981). 28 K. T. Suzuki and M. Yamamura, Biochem. Pharmacol. 29, 2407 (1980). 29 L. D. Lehman-MeKeeman, G. K. Andrews, and C. D. Klaassen, Biochem. J. 249, 429 (1988).
60
QUANTIFICATION IN TISSUES AND BODY FLUIDS
[ 10]
tion of metals. For example, heating of the sample should be avoided when determining copper-containing MT. Immunological methods are less likely to be affected by oxidation of MT than the indirect methods (SH determination and metal affinity assays), provided that the epitopes remain unchanged. The metal composition of MT also affects its absorption spectrum, 3° which can have consequences for the quantification of MT by HPLC/photometric analysis. Furthermore, due to the different affinities of the metals to the protein, the use of metal saturation and SH determination methods is limited. Copper-containing MT, for example, cannot be determined with the cadmium- heme assay. In general, indirect methods cause only minor problems when measuring MT with different isoforms, whereas the direct immunological methods cause only minor problems when measuring MT with different metal composition. Both types of analysis, however, can result in a loss of information, namely, the isoform pattern (indirect methods) or the metal composition of MT (direct methods). Clearly there is no universal method for the quantification of MT, so that the parallel use of different methods is considered to be most appropriate. Comment When several methods are applicable, one should also bear in mind that the various methods require different laboratory skills and procedures. In general, immunological methods are more time consuming and difficult to perform than metal affinity assays. Comparative interpretation of results obtained from different methods seems to be the optimal approach for deciding which of the MT assays is most suitable for a particular sample. J0 M. Vasak and J. H. R. K~gi, Met. Ions Biol. Syst. 15, 213 (1983).
[ 10] A s s a y o f E x t r a c e l l u l a r M e t a l l o t h i o n e i n By IAN BREMNER
and RAJESH K.
MEHRA
Introduction Although metallothionein (MT) is rightly regarded mainly as an intracellular protein located in the cytoplasm and nucleus of the cell, it also occurs in small amounts in extracellular fluids. This was first reported in
METHODS IN ENZYMOLOGY, VOL. 205
Copyright© 1991 by Academic Press,Inc. All fightsof reproductionin any formreserved.